Ancestral Relationships Using Metafounders: Finite Ancestral Populations and Across Population Relationships.

Science.gov (United States)

Legarra, Andres; Christensen, Ole F; Vitezica, Zulma G; Aguilar, Ignacio; Misztal, Ignacy

2015-06-01

Recent use of genomic (marker-based) relationships shows that relationships exist within and across base population (breeds or lines). However, current treatment of pedigree relationships is unable to consider relationships within or across base populations, although such relationships must exist due to finite size of the ancestral population and connections between populations. This complicates the conciliation of both approaches and, in particular, combining pedigree with genomic relationships. We present a coherent theoretical framework to consider base population in pedigree relationships. We suggest a conceptual framework that considers each ancestral population as a finite-sized pool of gametes. This generates across-individual relationships and contrasts with the classical view which each population is considered as an infinite, unrelated pool. Several ancestral populations may be connected and therefore related. Each ancestral population can be represented as a "metafounder," a pseudo-individual included as founder of the pedigree and similar to an "unknown parent group." Metafounders have self- and across relationships according to a set of parameters, which measure ancestral relationships, i.e., homozygozities within populations and relationships across populations. These parameters can be estimated from existing pedigree and marker genotypes using maximum likelihood or a method based on summary statistics, for arbitrarily complex pedigrees. Equivalences of genetic variance and variance components between the classical and this new parameterization are shown. Segregation variance on crosses of populations is modeled. Efficient algorithms for computation of relationship matrices, their inverses, and inbreeding coefficients are presented. Use of metafounders leads to compatibility of genomic and pedigree relationship matrices and to simple computing algorithms. Examples and code are given. Copyright © 2015 by the Genetics Society of America.

Eclipse Phase of Herpes Simplex Virus Type 1 Infection: Efficient Dynein-Mediated Capsid Transport without the Small Capsid Protein VP26

Science.gov (United States)

Döhner, Katinka; Radtke, Kerstin; Schmidt, Simone; Sodeik, Beate

2006-01-01

Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-ΔVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-ΔVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-ΔVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin. PMID:16873277

Properties and Functions of the Dengue Virus Capsid Protein.

Science.gov (United States)

Byk, Laura A; Gamarnik, Andrea V

2016-09-29

Dengue virus affects hundreds of millions of people each year around the world, causing a tremendous social and economic impact on affected countries. The aim of this review is to summarize our current knowledge of the functions, structure, and interactions of the viral capsid protein. The primary role of capsid is to package the viral genome. There are two processes linked to this function: the recruitment of the viral RNA during assembly and the release of the genome during infection. Although particle assembly takes place on endoplasmic reticulum membranes, capsid localizes in nucleoli and lipid droplets. Why capsid accumulates in these locations during infection remains unknown. In this review, we describe available data and discuss new ideas on dengue virus capsid functions and interactions. We believe that a deeper understanding of how the capsid protein works during infection will create opportunities for novel antiviral strategies, which are urgently needed to control dengue virus infections.

Ancestral sequence alignment under optimal conditions

Directory of Open Access Journals (Sweden)

Brown Daniel G

2005-11-01

Full Text Available Abstract Background Multiple genome alignment is an important problem in bioinformatics. An important subproblem used by many multiple alignment approaches is that of aligning two multiple alignments. Many popular alignment algorithms for DNA use the sum-of-pairs heuristic, where the score of a multiple alignment is the sum of its induced pairwise alignment scores. However, the biological meaning of the sum-of-pairs of pairs heuristic is not obvious. Additionally, many algorithms based on the sum-of-pairs heuristic are complicated and slow, compared to pairwise alignment algorithms. An alternative approach to aligning alignments is to first infer ancestral sequences for each alignment, and then align the two ancestral sequences. In addition to being fast, this method has a clear biological basis that takes into account the evolution implied by an underlying phylogenetic tree. In this study we explore the accuracy of aligning alignments by ancestral sequence alignment. We examine the use of both maximum likelihood and parsimony to infer ancestral sequences. Additionally, we investigate the effect on accuracy of allowing ambiguity in our ancestral sequences. Results We use synthetic sequence data that we generate by simulating evolution on a phylogenetic tree. We use two different types of phylogenetic trees: trees with a period of rapid growth followed by a period of slow growth, and trees with a period of slow growth followed by a period of rapid growth. We examine the alignment accuracy of four ancestral sequence reconstruction and alignment methods: parsimony, maximum likelihood, ambiguous parsimony, and ambiguous maximum likelihood. Additionally, we compare against the alignment accuracy of two sum-of-pairs algorithms: ClustalW and the heuristic of Ma, Zhang, and Wang. Conclusion We find that allowing ambiguity in ancestral sequences does not lead to better multiple alignments. Regardless of whether we use parsimony or maximum likelihood, the

A theory for viral capsid assembly around electrostatic cores

Science.gov (United States)

Hagan, Michael F.

2009-03-01

We develop equilibrium and kinetic theories that describe the assembly of viral capsid proteins on a charged central core, as seen in recent experiments in which brome mosaic virus capsids assemble around nanoparticles functionalized with polyelectrolyte. We model interactions between capsid proteins and nanoparticle surfaces as the interaction of polyelectrolyte brushes with opposite charge using the nonlinear Poisson Boltzmann equation. The models predict that there is a threshold density of functionalized charge, above which capsids efficiently assemble around nanoparticles, and that light scatter intensity increases rapidly at early times without the lag phase characteristic of empty capsid assembly. These predictions are consistent with and enable interpretation of preliminary experimental data. However, the models predict a stronger dependence of nanoparticle incorporation efficiency on functionalized charge density than measured in experiments and do not completely capture a logarithmic growth phase seen in experimental light scatter. These discrepancies may suggest the presence of metastable disordered states in the experimental system. In addition to discussing future experiments for nanoparticle-capsid systems, we discuss broader implications for understanding assembly around charged cores such as nucleic acids.

The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers.

Science.gov (United States)

Newman, Joseph; Asfor, Amin S; Berryman, Stephen; Jackson, Terry; Curry, Stephen; Tuthill, Tobias J

2018-03-01

Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug. IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity

Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

Science.gov (United States)

Roos, Wouter H.; Radtke, Kerstin; Kniesmeijer, Edward; Geertsema, Hylkje; Sodeik, Beate; Wuite, Gijs J. L.

2009-01-01

Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA. PMID:19487681

Antimicrobial peptide capsids of de novo design.

Science.gov (United States)

De Santis, Emiliana; Alkassem, Hasan; Lamarre, Baptiste; Faruqui, Nilofar; Bella, Angelo; Noble, James E; Micale, Nicola; Ray, Santanu; Burns, Jonathan R; Yon, Alexander R; Hoogenboom, Bart W; Ryadnov, Maxim G

2017-12-22

The spread of bacterial resistance to antibiotics poses the need for antimicrobial discovery. With traditional search paradigms being exhausted, approaches that are altogether different from antibiotics may offer promising and creative solutions. Here, we introduce a de novo peptide topology that-by emulating the virus architecture-assembles into discrete antimicrobial capsids. Using the combination of high-resolution and real-time imaging, we demonstrate that these artificial capsids assemble as 20-nm hollow shells that attack bacterial membranes and upon landing on phospholipid bilayers instantaneously (seconds) convert into rapidly expanding pores causing membrane lysis (minutes). The designed capsids show broad antimicrobial activities, thus executing one primary function-they destroy bacteria on contact.

Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

International Nuclear Information System (INIS)

Hespenheide, B M; Jacobs, D J; Thorpe, M F

2004-01-01

The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations

Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

Science.gov (United States)

Hespenheide, B. M.; Jacobs, D. J.; Thorpe, M. F.

2004-11-01

The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

Energy Technology Data Exchange (ETDEWEB)

Hespenheide, B M [Department of Physics and Astronomy, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504 (United States); Jacobs, D J [Department of Physics and Astronomy, California State University, 18111 Nordhoff Street, Northridge, CA 91330-8268 (United States); Thorpe, M F [Department of Physics and Astronomy, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504 (United States)

2004-11-10

The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

REGEN: Ancestral Genome Reconstruction for Bacteria

OpenAIRE

Yang, Kuan; Heath, Lenwood S.; Setubal, João C.

2012-01-01

Ancestral genome reconstruction can be understood as a phylogenetic study with more details than a traditional phylogenetic tree reconstruction. We present a new computational system called REGEN for ancestral bacterial genome reconstruction at both the gene and replicon levels. REGEN reconstructs gene content, contiguous gene runs, and replicon structure for each ancestral genome. Along each branch of the phylogenetic tree, REGEN infers evolutionary events, including gene creation and deleti...

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

Science.gov (United States)

Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J; Valente, Susana T

2016-04-01

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

Science.gov (United States)

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty

Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

Science.gov (United States)

Brault, V.; Bergdoll, M.; Mutterer, J.; Prasad, V.; Pfeffer, S.; Erdinger, M.; Richards, K. E.; Ziegler-Graff, V.

2003-01-01

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid. PMID:12584348

Robustness of ancestral sequence reconstruction to phylogenetic uncertainty.

Science.gov (United States)

Hanson-Smith, Victor; Kolaczkowski, Bryan; Thornton, Joseph W

2010-09-01

Ancestral sequence reconstruction (ASR) is widely used to formulate and test hypotheses about the sequences, functions, and structures of ancient genes. Ancestral sequences are usually inferred from an alignment of extant sequences using a maximum likelihood (ML) phylogenetic algorithm, which calculates the most likely ancestral sequence assuming a probabilistic model of sequence evolution and a specific phylogeny--typically the tree with the ML. The true phylogeny is seldom known with certainty, however. ML methods ignore this uncertainty, whereas Bayesian methods incorporate it by integrating the likelihood of each ancestral state over a distribution of possible trees. It is not known whether Bayesian approaches to phylogenetic uncertainty improve the accuracy of inferred ancestral sequences. Here, we use simulation-based experiments under both simplified and empirically derived conditions to compare the accuracy of ASR carried out using ML and Bayesian approaches. We show that incorporating phylogenetic uncertainty by integrating over topologies very rarely changes the inferred ancestral state and does not improve the accuracy of the reconstructed ancestral sequence. Ancestral state reconstructions are robust to uncertainty about the underlying tree because the conditions that produce phylogenetic uncertainty also make the ancestral state identical across plausible trees; conversely, the conditions under which different phylogenies yield different inferred ancestral states produce little or no ambiguity about the true phylogeny. Our results suggest that ML can produce accurate ASRs, even in the face of phylogenetic uncertainty. Using Bayesian integration to incorporate this uncertainty is neither necessary nor beneficial.

Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages.

Science.gov (United States)

Callaway, Heather M; Feng, Kurtis H; Lee, Donald W; Allison, Andrew B; Pinard, Melissa; McKenna, Robert; Agbandje-McKenna, Mavis; Hafenstein, Susan; Parrish, Colin R

2017-01-15

Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction

Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages

Science.gov (United States)

Callaway, Heather M.; Feng, Kurtis H.; Lee, Donald W.; Pinard, Melissa; McKenna, Robert; Agbandje-McKenna, Mavis; Hafenstein, Susan

2016-01-01

ABSTRACT Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. IMPORTANCE Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in

All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

Energy Technology Data Exchange (ETDEWEB)

Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S., E-mail: okazaki@apchem.nagoya-u.ac.jp [Department of Applied Chemistry, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan); Fujimoto, K. [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Nakagawa, A. [Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871 (Japan); Nomoto, A. [Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan)

2014-10-28

Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10{sup 6} all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

International Nuclear Information System (INIS)

Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S.; Fujimoto, K.; Nakagawa, A.; Nomoto, A.

2014-01-01

Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10 6 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it

All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

Science.gov (United States)

Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

2014-10-01

Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

Ancestral sequence reconstruction with Maximum Parsimony

OpenAIRE

Herbst, Lina; Fischer, Mareike

2017-01-01

One of the main aims in phylogenetics is the estimation of ancestral sequences based on present-day data like, for instance, DNA alignments. One way to estimate the data of the last common ancestor of a given set of species is to first reconstruct a phylogenetic tree with some tree inference method and then to use some method of ancestral state inference based on that tree. One of the best-known methods both for tree inference as well as for ancestral sequence inference is Maximum Parsimony (...

Assessing the Accuracy of Ancestral Protein Reconstruction Methods

OpenAIRE

Williams, Paul D; Pollock, David D; Blackburne, Benjamin P; Goldstein, Richard A

2006-01-01

The phylogenetic inference of ancestral protein sequences is a powerful technique for the study of molecular evolution, but any conclusions drawn from such studies are only as good as the accuracy of the reconstruction method. Every inference method leads to errors in the ancestral protein sequence, resulting in potentially misleading estimates of the ancestral protein's properties. To assess the accuracy of ancestral protein reconstruction methods, we performed computational population evolu...

The ancestral chromosomes of Dromiciops gliroides (Microbiotheridae), and its bearings on the karyotypic evolution of American marsupials.

Science.gov (United States)

Suárez-Villota, Elkin Y; Haro, Ronie E; Vargas, Rodrigo A; Gallardo, Milton H

2016-01-01

The low-numbered 14-chromosome karyotype of marsupials has falsified the fusion hypothesis claiming ancestrality from a 22-chromosome karyotype. Since the 14-chromosome condition of the relict Dromiciops gliroides is reminecent of ancestrality, its interstitial traces of past putative fusions and heterochromatin banding patterns were studied and added to available marsupials' cytogenetic data. Fluorescent in situ hybridization (FISH) and self-genomic in situ hybridization (self-GISH) were used to detect telomeric and repetitive sequences, respectively. These were complemented with C-, fluorescent banding, and centromere immunodetection over mitotic spreads. The presence of interstitial telomeric sequences (ITS) and diploid numbers were reconstructed and mapped onto the marsupial phylogenetic tree. No interstitial, fluorescent signals, but clearly stained telomeric regions were detected by FISH and self-GISH. Heterochromatin distribution was sparse in the telomeric/subtelomeric regions of large submetacentric chromosomes. Large AT-rich blocks were detected in the long arm of four submetacentrics and CG-rich block in the telomeric regions of all chromosomes. The ancestral reconstructions both ITS presence and diploid numbers suggested that ITS are unrelated to fusion events. Although the lack of interstitial signals in D. gliroides' karyotype does not prove absence of past fusions, our data suggests its non-rearranged plesiomorphic condition.

Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

Energy Technology Data Exchange (ETDEWEB)

Lebrun, Marielle [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Thelen, Nicolas; Thiry, Marc [University of Liege (ULg), GIGA-Neurosciences, Laboratory of Cellular and Tissular Biology, Liege (Belgium); Riva, Laura; Ote, Isabelle; Condé, Claude; Vandevenne, Patricia [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Di Valentin, Emmanuel [University of Liege (ULg), GIGA-Viral Vectors Platform, Liege (Belgium); Bontems, Sébastien [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Sadzot-Delvaux, Catherine, E-mail: csadzot@ulg.ac.be [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium)

2014-04-15

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.

Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

International Nuclear Information System (INIS)

Kim, Yoon Sik; Seo, Hyun Wook; Jung, Guhung

2015-01-01

Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H 2 O 2 and GSH modulate HBV capsid assembly. • H 2 O 2 facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H 2 O 2 and GSH induce conformation change of Hsp90

Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

Energy Technology Data Exchange (ETDEWEB)

Kim, Yoon Sik, E-mail: yumshak@naver.com; Seo, Hyun Wook, E-mail: suruk@naver.com; Jung, Guhung, E-mail: drjung@snu.ac.kr

2015-02-13

Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.

The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

International Nuclear Information System (INIS)

Shen, Peter S.; Enderlein, Dirk; Nelson, Christian D.S.; Carter, Weston S.; Kawano, Masaaki; Xing Li; Swenson, Robert D.; Olson, Norman H.; Baker, Timothy S.; Cheng, R. Holland; Atwood, Walter J.; Johne, Reimar; Belnap, David M.

2011-01-01

Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting β-hairpin observed in other polyomaviruses. We postulate that the terminal β-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

Analysis of SAT Type Foot-And-Mouth Disease Virus Capsid Proteins and the Identification of Putative Amino Acid Residues Affecting Virus Stability

Science.gov (United States)

Maree, Francois F.; Blignaut, Belinda; de Beer, Tjaart A. P.; Rieder, Elizabeth

2013-01-01

Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. PMID:23717387

Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of putative amino acid residues affecting virus stability.

Directory of Open Access Journals (Sweden)

Francois F Maree

Full Text Available Foot-and-mouth disease virus (FMDV initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces.

Assessing the accuracy of ancestral protein reconstruction methods.

Directory of Open Access Journals (Sweden)

Paul D Williams

2006-06-01

Full Text Available The phylogenetic inference of ancestral protein sequences is a powerful technique for the study of molecular evolution, but any conclusions drawn from such studies are only as good as the accuracy of the reconstruction method. Every inference method leads to errors in the ancestral protein sequence, resulting in potentially misleading estimates of the ancestral protein's properties. To assess the accuracy of ancestral protein reconstruction methods, we performed computational population evolution simulations featuring near-neutral evolution under purifying selection, speciation, and divergence using an off-lattice protein model where fitness depends on the ability to be stable in a specified target structure. We were thus able to compare the thermodynamic properties of the true ancestral sequences with the properties of "ancestral sequences" inferred by maximum parsimony, maximum likelihood, and Bayesian methods. Surprisingly, we found that methods such as maximum parsimony and maximum likelihood that reconstruct a "best guess" amino acid at each position overestimate thermostability, while a Bayesian method that sometimes chooses less-probable residues from the posterior probability distribution does not. Maximum likelihood and maximum parsimony apparently tend to eliminate variants at a position that are slightly detrimental to structural stability simply because such detrimental variants are less frequent. Other properties of ancestral proteins might be similarly overestimated. This suggests that ancestral reconstruction studies require greater care to come to credible conclusions regarding functional evolution. Inferred functional patterns that mimic reconstruction bias should be reevaluated.

Assessing the accuracy of ancestral protein reconstruction methods.

Science.gov (United States)

Williams, Paul D; Pollock, David D; Blackburne, Benjamin P; Goldstein, Richard A

2006-06-23

The phylogenetic inference of ancestral protein sequences is a powerful technique for the study of molecular evolution, but any conclusions drawn from such studies are only as good as the accuracy of the reconstruction method. Every inference method leads to errors in the ancestral protein sequence, resulting in potentially misleading estimates of the ancestral protein's properties. To assess the accuracy of ancestral protein reconstruction methods, we performed computational population evolution simulations featuring near-neutral evolution under purifying selection, speciation, and divergence using an off-lattice protein model where fitness depends on the ability to be stable in a specified target structure. We were thus able to compare the thermodynamic properties of the true ancestral sequences with the properties of "ancestral sequences" inferred by maximum parsimony, maximum likelihood, and Bayesian methods. Surprisingly, we found that methods such as maximum parsimony and maximum likelihood that reconstruct a "best guess" amino acid at each position overestimate thermostability, while a Bayesian method that sometimes chooses less-probable residues from the posterior probability distribution does not. Maximum likelihood and maximum parsimony apparently tend to eliminate variants at a position that are slightly detrimental to structural stability simply because such detrimental variants are less frequent. Other properties of ancestral proteins might be similarly overestimated. This suggests that ancestral reconstruction studies require greater care to come to credible conclusions regarding functional evolution. Inferred functional patterns that mimic reconstruction bias should be reevaluated.

Identification of Factors Promoting HBV Capsid Self-Assembly by Assembly-Promoting Antivirals.

Science.gov (United States)

Rath, Soumya Lipsa; Liu, Huihui; Okazaki, Susumu; Shinoda, Wataru

2018-02-26

Around 270 million individuals currently live with hepatitis B virus (HBV) infection. Heteroaryldihydropyrimidines (HAPs) are a family of antivirals that target the HBV capsid protein and induce aberrant self-assembly. The capsids formed resemble the native capsid structure but are unable to propagate the virus progeny because of a lack of RNA/DNA. Under normal conditions, self-assembly is initiated by the viral genome. The mode of action of HAPs, however, remains largely unknown. In this work, using molecular dynamics simulations, we attempted to understand the action of HAP by comparing the dynamics of capsid proteins with and without HAPs. We found that the inhibitor is more stable in higher oligomers. It retains its stability in the hexamer throughout 1 μs of simulation. Our results also show that the inhibitor might help in stabilizing the C-terminus, the HBc 149-183 arginine-rich domain of the capsid protein. The C-termini of dimers interact with each other, assisted by the HAP inhibitor. During capsid assembly, the termini are supposed to directly interact with the viral genome, thereby suggesting that the viral genome might work in a similar way to stabilize the capsid protein. Our results may help in understanding the underlying molecular mechanism of HBV capsid self-assembly, which should be crucial for exploring new drug targets and structure-based drug design.

KLONING GEN PUTATIVE CLEAVAGE PROTEIN 1 (PCP-1 PADA UDANG VANAME (Litopenaeus vannamei YANG TERSERANG INFECTIOUS MYONECROSIS VIRUS

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Hessy Novita

2016-12-01

Full Text Available Penanggulangan penyakit ikan dapat dilakukan dengan cara meningkatkan kekebalan tubuh ikan melalui program vaksinasi. Namun vaksinasi tidak tepat untuk udang, karena udang tidak mempunyai immunological memory seperti ikan. Oleh karena itu, perlindungan udang terhadap serangan penyakit viral dengan menggunakan RNA interference (RNAi. Teknologi RNAi digunakan untuk menghalangi (interfere proses replikasi infectious myonecrosis virus (IMNV pada udang vaname dengan cara menon-aktifkan gen putative cleavage protein 1 (PCP-1, yang berfungsi dalam pembentukan capsid dan proses transkripsi RNA IMNV. Penelitian ini bertujuan untuk melakukan kloning gen putative cleavage protein 1 dalam rangka perakitan teknologi RNAi untuk pengendalian penyakit IMNV pada udang vaname. Tahapan penelitian meliputi koleksi sampel, isolasi RNA, sintesis cDNA, amplifikasi PCR, purifikasi DNA, transformasi, isolasi plasmid, serta sekuensing dan analisis data. Hasil isolasi plasmid cDNA PCP-1 memperlihatkan semua koloni bakteri terseleksi ternyata membawa plasmid hasil insersi DNA gen PCP–1, hasil sekuen dengan nilai homologinya mencapai 100% dan 99% yang dibandingkan dengan sekuen di Genebank. Hasil penelitian menunjukkan bahwa kloning gen putative cleavage protein 1 (PCP-1 dari udang vaname yang terserang Infectious Myonecrosis Virus berhasil dikloning yang nantinya digunakan untuk perakitan RNAi. The prevention of fish diseases can be done by increasing of the fish immune through vaccination programs. However, the vaccination can not be done for the shrimp,due to the absence of  immunological memory. Therefore, the protection of shrimp against viral diseases was done by using of RNA interference (RNAi. RNAi technology is used to interfere infectious myonecrosis virus (IMNV replication process on white shrimp by disabling of putative cleavage protein 1 (PCP-1gene, which functions in capsid formation and RNA transcription process. The study was conducted to perform putative

Integrating Principles Underlying Ancestral Spirits Belief in ...

African Journals Online (AJOL)

, associated with ancestral spirits and its use as powerful therapeutic agent for influencing behavior or lifestyle changes. Explanatory models of attachment to ancestral spirits by living descendants are first discussed, followed by a discussion ...

Determining the Epitope Dominance on the Capsid of a Serotype SAT2 Foot-and-Mouth Disease Virus by Mutational Analyses

Science.gov (United States)

Opperman, Pamela A.; Rotherham, Lia S.; Esterhuysen, Jan; Charleston, Bryan; Juleff, Nicholas; Capozzo, Alejandra V.; Theron, Jacques

2014-01-01

ABSTRACT Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible βG-βH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the βG-βH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. IMPORTANCE Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been

Quantum dot-induced viral capsid assembling in dissociation buffer

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Gao D

2013-06-01

Full Text Available Ding Gao,1,2 Zhi-Ping Zhang,1 Feng Li,3 Dong Men,1 Jiao-Yu Deng,1 Hong-Ping Wei,1 Xian-En Zhang,1 Zong-Qiang Cui1 1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 2Graduate University of Chinese Academy of Sciences, Beijing, 3Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, People's Republic of China Abstract: Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs are still unknown. In this article, it was found that quantum dots (QDs can induce simian virus 40 (SV40 capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1 can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD = 2.19E-10 M, which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles. Keywords: quantum dots, simian virus 40, self-assembly, encapsulation, virus-based nanoparticles

Breadth of T cell responses after immunization with adenovirus vectors encoding ancestral antigens or polyvalent papillomavirus antigens

DEFF Research Database (Denmark)

Ragonnaud, Emeline; Pedersen, Anders Gorm; Holst, Peter Johannes

2017-01-01

to the other PV proteins. The PV sequences were fused to a T cell adjuvant, the murine invariant chain and encoded in a recombinant adenoviral vector which was administered to naïve outbred mice. By measuring T cell responses induced by these different vaccines and towards peptide pools representing 3...... circulating strains and a putative ancestor of oncogenic HPVs, we showed that the ancestral vaccine antigen has to be approximately 90% identical to the circulating PVs before a marked drop of ~90% mean CD8+ T cell responses ensues. Interestingly, the combination of two or three type-specific PV vaccines did...

Human rhinovirus capsid dynamics is controlled by canyon flexibility

International Nuclear Information System (INIS)

Reisdorph, Nichole; Thomas, John J.; Katpally, Umesh; Chase, Elaine; Harris, Ken; Siuzdak, Gary; Smith, Thomas J.

2003-01-01

Quantitative enzyme accessibility experiments using nano liquid chromatography electrospray mass spectrometry combined with limited proteolysis and isotope-labeling was used to examine the dynamic nature of the human rhinovirus (HRV) capsid in the presence of three antiviral compounds, a neutralizing Fab, and drug binding cavity mutations. Using these methods, it was found that the antivirals WIN 52084 and picovir (pleconaril) stabilized the capsid, while dansylaziridine caused destabilization. Site-directed mutations in the drug-binding cavity were found to stabilize the HRV14 capsid against proteolytic digestion in a manner similar to WIN 52084 and pleconaril. Antibodies that bind to the NIm-IA antigenic site and penetrate the canyon were also observed to protect the virion against proteolytic cleavage. These results demonstrate that quantifying the effects of antiviral ligands on protein 'breathing' can be used to compare their mode of action and efficacy. In this case, it is apparent that hydrophobic antiviral agents, antibodies, or mutations in the canyon region block viral breathing. Therefore, these studies demonstrate that mobility in the canyon region is a major determinant in capsid breathing

Investigating the thermal dissociation of viral capsid by lattice model

Science.gov (United States)

Chen, Jingzhi; Chevreuil, Maelenn; Combet, Sophie; Lansac, Yves; Tresset, Guillaume

2017-11-01

The dissociation of icosahedral viral capsids was investigated by a homogeneous and a heterogeneous lattice model. In thermal dissociation experiments with cowpea chlorotic mottle virus and probed by small-angle neutron scattering, we observed a slight shrinkage of viral capsids, which can be related to the strengthening of the hydrophobic interaction between subunits at increasing temperature. By considering the temperature dependence of hydrophobic interaction in the homogeneous lattice model, we were able to give a better estimate of the effective charge. In the heterogeneous lattice model, two sets of lattice sites represented different capsid subunits with asymmetric interaction strengths. In that case, the dissociation of capsids was found to shift from a sharp one-step transition to a gradual two-step transition by weakening the hydrophobic interaction between AB and CC subunits. We anticipate that such lattice models will shed further light on the statistical mechanics underlying virus assembly and disassembly.

Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

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Suzan-Monti Marie

2009-05-01

Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

Ancestral Sequence Reconstruction with Maximum Parsimony.

Science.gov (United States)

Herbst, Lina; Fischer, Mareike

2017-12-01

One of the main aims in phylogenetics is the estimation of ancestral sequences based on present-day data like, for instance, DNA alignments. One way to estimate the data of the last common ancestor of a given set of species is to first reconstruct a phylogenetic tree with some tree inference method and then to use some method of ancestral state inference based on that tree. One of the best-known methods both for tree inference and for ancestral sequence inference is Maximum Parsimony (MP). In this manuscript, we focus on this method and on ancestral state inference for fully bifurcating trees. In particular, we investigate a conjecture published by Charleston and Steel in 1995 concerning the number of species which need to have a particular state, say a, at a particular site in order for MP to unambiguously return a as an estimate for the state of the last common ancestor. We prove the conjecture for all even numbers of character states, which is the most relevant case in biology. We also show that the conjecture does not hold in general for odd numbers of character states, but also present some positive results for this case.

Studies towards the sex pheromone of the green capsid bug

NARCIS (Netherlands)

Drijfhout, F.P.

2001-01-01

The green capsid bug, Lygocoris pabulinus (L.) (Heteroptera: Miridae) is a serious pest in fruit orchards, which is difficult to control. Because it is difficult to determine the actual population density, fruit growers apply insecticides against the green capsid bug on

AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

International Nuclear Information System (INIS)

Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M.

2014-01-01

Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8

AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

Energy Technology Data Exchange (ETDEWEB)

Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail: wilsonjm@mail.med.upenn.edu

2014-04-15

Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

Directory of Open Access Journals (Sweden)

Philip Serwer

2017-05-01

Full Text Available Adenosine triphosphate (ATP cleavage powers packaging of a double-stranded DNA (dsDNA molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid.

Science.gov (United States)

Lee, Lye Siang; Brunk, Nicholas; Haywood, Daniel G; Keifer, David; Pierson, Elizabeth; Kondylis, Panagiotis; Wang, Joseph Che-Yen; Jacobson, Stephen C; Jarrold, Martin F; Zlotnick, Adam

2017-11-01

Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re-engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive-pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90-mers in the disassembly of uncrosslinked HBV capsids. 90-mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations. © 2017 The Protein Society.

Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization.

Directory of Open Access Journals (Sweden)

Anna D Koromyslova

2017-11-01

Full Text Available Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42 were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14, allosteric interference (Nano-32, and violation of normal capsid morphology (Nano-26 and Nano-85. Finally, we showed that two Nanobodies (Nano-26 and Nano-85 not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great

Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization

Science.gov (United States)

2017-01-01

Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to

L2, the minor capsid protein of papillomavirus

Energy Technology Data Exchange (ETDEWEB)

Wang, Joshua W. [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Roden, Richard B.S., E-mail: roden@jhmi.edu [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Oncology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, MD 21287 (United States)

2013-10-15

The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies.

L2, the minor capsid protein of papillomavirus

International Nuclear Information System (INIS)

Wang, Joshua W.; Roden, Richard B.S.

2013-01-01

The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies

High Relaxivity Gadolinium Hydroxypyridonate-Viral Capsid Conjugates: Nano-sized MRI Contrast Agents

Energy Technology Data Exchange (ETDEWEB)

Meux, Susan C.; Datta, Ankona; Hooker, Jacob M.; Botta, Mauro; Francis, Matthew B.; Aime, Silvio; Raymond, Kenneth N.

2007-08-29

High relaxivity macromolecular contrast agents based on the conjugation of gadolinium chelates to the interior and exterior surfaces of MS2 viral capsids are assessed. The proton nuclear magnetic relaxation dispersion (NMRD) profiles of the conjugates show up to a five-fold increase in relaxivity, leading to a peak relaxivity (per Gd{sup 3+} ion) of 41.6 mM{sup -1}s{sup -1} at 30 MHz for the internally modified capsids. Modification of the exterior was achieved through conjugation to flexible lysines, while internal modification was accomplished by conjugation to relatively rigid tyrosines. Higher relaxivities were obtained for the internally modified capsids, showing that (1) there is facile diffusion of water to the interior of capsids and (2) the rigidity of the linker attaching the complex to the macromolecule is important for obtaining high relaxivity enhancements. The viral capsid conjugated gadolinium hydroxypyridonate complexes appear to possess two inner-sphere water molecules (q = 2) and the NMRD fittings highlight the differences in the local motion for the internal ({tau}{sub RI} = 440 ps) and external ({tau}{sub RI} = 310 ps) conjugates. These results indicate that there are significant advantages of using the internal surface of the capsids for contrast agent attachment, leaving the exterior surface available for the installation of tissue targeting groups.

Why Meillassoux’s Speculative Materialism Struggles with Ancestrality

Directory of Open Access Journals (Sweden)

Ciprian Jeler

2014-12-01

Full Text Available This paper shows that Quentin Meillassoux’s speculative materialism doesn’t offer us the means to account for the ancestral statements that the modern sciences produce, i.e. for the scientific statements about events preceding all forms of life. An analysis of the reasons why Meillassoux thinks that the problem of ancestrality problematizes the contemporary self-evidence of correlationism is first offered. The results of this analysis are then applied to speculative materialism itself and the consequences are not very promising: very much like correlationism, speculative materialism explicitly denies what I call the “generalized version of the realistic assumption of science” and, in so doing, renders scientific ancestral statements de jure unverifiable. Therefore, if correlationism is rendered suspicious by the issue of ancestrality, the same can be said of speculative materialism.

REGEN: Ancestral Genome Reconstruction for Bacteria.

Science.gov (United States)

Yang, Kuan; Heath, Lenwood S; Setubal, João C

2012-07-18

Ancestral genome reconstruction can be understood as a phylogenetic study with more details than a traditional phylogenetic tree reconstruction. We present a new computational system called REGEN for ancestral bacterial genome reconstruction at both the gene and replicon levels. REGEN reconstructs gene content, contiguous gene runs, and replicon structure for each ancestral genome. Along each branch of the phylogenetic tree, REGEN infers evolutionary events, including gene creation and deletion and replicon fission and fusion. The reconstruction can be performed by either a maximum parsimony or a maximum likelihood method. Gene content reconstruction is based on the concept of neighboring gene pairs. REGEN was designed to be used with any set of genomes that are sufficiently related, which will usually be the case for bacteria within the same taxonomic order. We evaluated REGEN using simulated genomes and genomes in the Rhizobiales order.

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

Science.gov (United States)

Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

2015-01-01

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

Structure of the capsid of Kilham rat virus from small-angle neutron scattering

International Nuclear Information System (INIS)

Wobbe, C.R.; Mitra, S.; Ramakrishnan, V.

1984-01-01

The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 x 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H 2 O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, the authors propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H 2 O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H 2 O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H 2 O. The authors propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions

Intracellular cargo delivery by virus capsid protein-based vehicles: From nano to micro.

Science.gov (United States)

Gao, Ding; Lin, Xiu-Ping; Zhang, Zhi-Ping; Li, Wei; Men, Dong; Zhang, Xian-En; Cui, Zong-Qiang

2016-02-01

Cellular delivery is an important concern for the efficiency of medicines and sensors for disease diagnoses and therapy. However, this task is quite challenging. Self-assembly virus capsid proteins might be developed as building blocks for multifunctional cellular delivery vehicles. In this work, we found that SV40 VP1 (Simian virus 40 major capsid protein) could function as a new cell-penetrating protein. The VP1 protein could carry foreign proteins into cells in a pentameric structure. A double color structure, with red QDs (Quantum dots) encapsulated by viral capsids fused with EGFP, was created for imaging cargo delivery and release from viral capsids. The viral capsids encapsulating QDs were further used for cellular delivery of micron-sized iron oxide particles (MPIOs). MPIOs were efficiently delivered into live cells and controlled by a magnetic field. Therefore, our study built virus-based cellular delivery systems for different sizes of cargos: protein molecules, nanoparticles, and micron-sized particles. Much research is being done to investigate methods for efficient and specific cellular delivery of drugs, proteins or genetic material. In this article, the authors describe their approach in using self-assembly virus capsid proteins SV40 VP1 (Simian virus 40 major capsid protein). The cell-penetrating behavior provided excellent cellular delivery and should give a new method for biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

REGEN: Ancestral Genome Reconstruction for Bacteria

Directory of Open Access Journals (Sweden)

João C. Setubal

2012-07-01

Full Text Available Ancestral genome reconstruction can be understood as a phylogenetic study with more details than a traditional phylogenetic tree reconstruction. We present a new computational system called REGEN for ancestral bacterial genome reconstruction at both the gene and replicon levels. REGEN reconstructs gene content, contiguous gene runs, and replicon structure for each ancestral genome. Along each branch of the phylogenetic tree, REGEN infers evolutionary events, including gene creation and deletion and replicon fission and fusion. The reconstruction can be performed by either a maximum parsimony or a maximum likelihood method. Gene content reconstruction is based on the concept of neighboring gene pairs. REGEN was designed to be used with any set of genomes that are sufficiently related, which will usually be the case for bacteria within the same taxonomic order. We evaluated REGEN using simulated genomes and genomes in the Rhizobiales order.

Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly.

Science.gov (United States)

Hasek, Mary L; Maurer, Joshua B; Hendrix, Roger W; Duda, Robert L

2017-08-04

Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the "E-loop," that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

The mammary gland-specific marsupial ELP and eutherian CTI share a common ancestral gene.

Science.gov (United States)

Pharo, Elizabeth A; De Leo, Alison A; Renfree, Marilyn B; Thomson, Peter C; Lefèvre, Christophe M; Nicholas, Kevin R

2012-06-08

The marsupial early lactation protein (ELP) gene is expressed in the mammary gland and the protein is secreted into milk during early lactation (Phase 2A). Mature ELP shares approximately 55.4% similarity with the colostrum-specific bovine colostrum trypsin inhibitor (CTI) protein. Although ELP and CTI both have a single bovine pancreatic trypsin inhibitor (BPTI)-Kunitz domain and are secreted only during the early lactation phases, their evolutionary history is yet to be investigated. Tammar ELP was isolated from a genomic library and the fat-tailed dunnart and Southern koala ELP genes cloned from genomic DNA. The tammar ELP gene was expressed only in the mammary gland during late pregnancy (Phase 1) and early lactation (Phase 2A). The opossum and fat-tailed dunnart ELP and cow CTI transcripts were cloned from RNA isolated from the mammary gland and dog CTI from cells in colostrum. The putative mature ELP and CTI peptides shared 44.6%-62.2% similarity. In silico analyses identified the ELP and CTI genes in the other species examined and provided compelling evidence that they evolved from a common ancestral gene. In addition, whilst the eutherian CTI gene was conserved in the Laurasiatherian orders Carnivora and Cetartiodactyla, it had become a pseudogene in others. These data suggest that bovine CTI may be the ancestral gene of the Artiodactyla-specific, rapidly evolving chromosome 13 pancreatic trypsin inhibitor (PTI), spleen trypsin inhibitor (STI) and the five placenta-specific trophoblast Kunitz domain protein (TKDP1-5) genes. Marsupial ELP and eutherian CTI evolved from an ancestral therian mammal gene before the divergence of marsupials and eutherians between 130 and 160 million years ago. The retention of the ELP gene in marsupials suggests that this early lactation-specific milk protein may have an important role in the immunologically naïve young of these species.

A molecular thermodynamic model for the stability of hepatitis B capsids

Science.gov (United States)

Kim, Jehoon; Wu, Jianzhong

2014-06-01

Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

A molecular thermodynamic model for the stability of hepatitis B capsids

Energy Technology Data Exchange (ETDEWEB)

Kim, Jehoon; Wu, Jianzhong, E-mail: jwu@engr.ucr.edu [Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521 (United States)

2014-06-21

Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

Science.gov (United States)

Jacobs, Susan C; Taylor, Adam; Herrero, Lara J; Mahalingam, Suresh; Fazakerley, John K

2017-10-20

Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

How viral capsids adapt to mismatched cargoes—identifying mechanisms of morphology control with simulations

Science.gov (United States)

Elrad, Oren

2009-03-01

During the replication of many viruses, hundreds to thousands of protein subunits assemble around the viral nucleic acid to form a protein shell called a capsid. Most viruses form one particular structure with astonishing fidelity; yet, recent experiments demonstrate that capsids can assemble with different sizes and morphologies to accommodate nucleic acids or other cargoes such as functionalized nanoparticles. In this talk, we will explore the mechanisms of simultaneous assembly and cargo encapsidation with a computational model that describes the assembly of icosahedral capsids around functionalized nanoparticles. With this model, we find parameter values for which subunits faithfully form empty capsids with a single morphology, but adaptively assemble into different icosahedral morphologies around nanoparticles with different diameters. Analyzing trajectories in which adaptation is or is not successful sheds light on the mechanisms by which capsid morphology may be controlled in vitro and in vivo, and suggests experiments to test these mechanisms. We compare the simulation results to recent experiments in which Brome Mosaic Virus capsid proteins assemble around functionalized nanoparticles, and describe how future experiments can test the model predictions.

Resolution effects in reconstructing ancestral genomes.

Science.gov (United States)

Zheng, Chunfang; Jeong, Yuji; Turcotte, Madisyn Gabrielle; Sankoff, David

2018-05-09

The reconstruction of ancestral genomes must deal with the problem of resolution, necessarily involving a trade-off between trying to identify genomic details and being overwhelmed by noise at higher resolutions. We use the median reconstruction at the synteny block level, of the ancestral genome of the order Gentianales, based on coffee, Rhazya stricta and grape, to exemplify the effects of resolution (granularity) on comparative genomic analyses. We show how decreased resolution blurs the differences between evolving genomes, with respect to rate, mutational process and other characteristics.

Cleavage sites within the poliovirus capsid protein precursors

International Nuclear Information System (INIS)

Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

1982-01-01

Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein

Overcoming preexisting humoral immunity to AAV using capsid decoys.

Science.gov (United States)

Mingozzi, Federico; Anguela, Xavier M; Pavani, Giulia; Chen, Yifeng; Davidson, Robert J; Hui, Daniel J; Yazicioglu, Mustafa; Elkouby, Liron; Hinderer, Christian J; Faella, Armida; Howard, Carolann; Tai, Alex; Podsakoff, Gregory M; Zhou, Shangzhen; Basner-Tschakarjan, Etiena; Wright, John Fraser; High, Katherine A

2013-07-17

Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

General Model for Retroviral Capsid Pattern Recognition by TRIM5 Proteins.

Science.gov (United States)

Wagner, Jonathan M; Christensen, Devin E; Bhattacharya, Akash; Dawidziak, Daria M; Roganowicz, Marcin D; Wan, Yueping; Pumroy, Ruth A; Demeler, Borries; Ivanov, Dmitri N; Ganser-Pornillos, Barbie K; Sundquist, Wesley I; Pornillos, Owen

2018-02-15

Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity ( K D of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity ( K D of ∼10 μM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition. IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly

Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus

DEFF Research Database (Denmark)

Martinez-Torrecuadrada, Jorge L.; Cortes, Elena; Vela, Carmen

1998-01-01

Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal...

Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

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Halder, Sujata; Nam, Hyun-Joo; Govindasamy, Lakshmanan; Vogel, Michèle; Dinsart, Christiane; Salomé, Nathalie; McKenna, Robert

2013-01-01

The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel β-barrel with large loop regions between the strands. The β-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ∼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors. PMID:23449783

Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

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Reed, Jonathan C; Westergreen, Nick; Barajas, Brook C; Ressler, Dylan T B; Phuong, Daryl J; Swain, John V; Lingappa, Vishwanath R; Lingappa, Jaisri R

2018-05-01

FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we showed that FIV Gag forms complexes that resemble HIV-1 capsid assembly intermediates in size and in their association with ABCE1 and DDX6, two host facilitators of HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies also showed that known and novel assembly-defective FIV Gag mutants fail to progress past putative intermediates in a pattern resembling that observed for HIV-1 Gag mutants. Finally, we used imaging to demonstrate colocalization of FIV Gag with ABCE1 and with the RNA granule protein DCP2. Thus, we conclude that formation of assembly intermediates derived from host RNA granules is likely conserved between primate and nonprimate lentiviruses and could provide targets for future antiviral strategies. Copyright © 2018 American Society for Microbiology.

Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis

International Nuclear Information System (INIS)

Sathish, Narayanan; Yuan Yan

2010-01-01

A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-Δ65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.

Pt, Co–Pt and Fe–Pt alloy nanoclusters encapsulated in virus capsids

International Nuclear Information System (INIS)

Okuda, M; Eloi, J-C; Jones, S E Ward; Schwarzacher, W; Verwegen, M; Cornelissen, J J L M

2016-01-01

Nanostructured Pt-based alloys show great promise, not only for catalysis but also in medical and magnetic applications. To extend the properties of this class of materials, we have developed a means of synthesizing Pt and Pt-based alloy nanoclusters in the capsid of a virus. Pure Pt and Pt-alloy nanoclusters are formed through the chemical reduction of [PtCl 4 ] − by NaBH 4 with/without additional metal ions (Co or Fe). The opening and closing of the ion channels in the virus capsid were controlled by changing the pH and ionic strength of the solution. The size of the nanoclusters is limited to 18 nm by the internal diameter of the capsid. Their magnetic properties suggest potential applications in hyperthermia for the Co–Pt and Fe–Pt magnetic alloy nanoclusters. This study introduces a new way to fabricate size-restricted nanoclusters using virus capsid. (paper)

Protruding Features of Viral Capsids Are Clustered on Icosahedral Great Circles.

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David P Wilson

Full Text Available Spherical viruses are remarkably well characterized by the Triangulation (T number developed by Casper and Klug. The T-number specifies how many viral capsid proteins are required to cover the virus, as well as how they are further subdivided into pentamer and hexamer subunits. The T-number however does not constrain the orientations of these proteins within the subunits or dictate where the proteins should place their protruding features. These protrusions often take the form of loops, spires and helices, and are significant because they aid in stability of the capsid as well as recognition by the host organism. Until now there has be no overall understanding of the placement of protrusions for spherical viruses, other than they have icosahedral symmetry. We constructed a set of gauge points based upon the work affine extensions of Keef and Twarock, which have fixed relative angular locations with which to measure the locations of these features. This work adds a new element to our understanding of the geometric arrangement of spherical viral capsid proteins; chiefly that the locations of protruding features are not found stochastically distributed in an icosahedral manner across the viral surface, but instead these features are found only in specific locations along the 15 icosahedral great circles. We have found that this result holds true as the T number and viral capsids size increases, suggesting an underlying geometric constraint on their locations. This is in spite of the fact that the constraints on the pentamers and hexamer orientations change as a function of T-number, as you need to accommodate more hexamers in the same solid angle between pentamers. The existence of this angular constraint of viral capsids suggests that there is a fitness or energetic benefit to the virus placing its protrusions in this manner. This discovery may have profound impacts on identifying and eliminating viral pathogens, understanding evolutionary

Relevance of capsid structure in the buckling and maturation of spherical viruses

International Nuclear Information System (INIS)

Aznar, María; Luque, Antoni; Reguera, David

2012-01-01

The shape and mechanical properties of viral capsids play an important role in several biological processes during the virus life cycle. In particular, to become infective, many viruses require a maturation stage where the capsid undergoes a buckling transition, from an initial spherical procapsid into a final icosahedral faceted shell. Here we study, using a minimal physical model, how the capsid shape and the buckling transition depend on the triangulation number T and the icosahedral class P of the virus structure. We find that, for small shells, capsids with P = 1 are most likely to produce polyhedral shapes that minimize their energy and accumulated stress, whereas viruses with P = 3 prefer to remain spherical. For big capsids, all shells are more stable adopting an icosahedral shape, in agreement with continuum elastic theory. Moreover, spherical viruses show a buckling transition to polyhedral shells under expansion, in consonance with virus maturation. The resulting icosahedral shell is mechanically stiffer, tolerates larger expansions and withstands higher internal pressures before failing, which could explain why some dsDNA viruses, which rely on the pressurization of their genetic material to facilitate the infection, undergo a buckling transition. We emphasize that the results are general and could also be applied to non-biological systems. (paper)

Assembly of recombinant Israeli Acute Paralysis Virus capsids.

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Junyuan Ren

Full Text Available The dicistrovirus Israeli Acute Paralysis Virus (IAPV has been implicated in the worldwide decline of honey bees. Studies of IAPV and many other bee viruses in pure culture are restricted by available isolates and permissive cell culture. Here we show that coupling the IAPV major structural precursor protein ORF2 to its cognate 3C-like processing enzyme results in processing of the precursor to the individual structural proteins in a number of insect cell lines following expression by a recombinant baculovirus. The efficiency of expression is influenced by the level of IAPV 3C protein and moderation of its activity is required for optimal expression. The mature IAPV structural proteins assembled into empty capsids that migrated as particles on sucrose velocity gradients and showed typical dicistrovirus like morphology when examined by electron microscopy. Monoclonal antibodies raised to recombinant capsids were configured into a diagnostic test specific for the presence of IAPV. Recombinant capsids for each of the many bee viruses within the picornavirus family may provide virus specific reagents for the on-going investigation of the causes of honeybee loss.

Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

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Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting

Science.gov (United States)

Müller, Oliver; Ivanova, Lyudmila; Bialy, Dagmara; Pohlmann, Anja; Binz, Anne; Hegemann, Maike; Viejo-Borbolla, Abel; Rosenhahn, Bodo; Bauerfeind, Rudolf; Sodeik, Beate

2017-01-01

Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells. PMID:29284065

Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

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Victoria A. Meliopoulos

2016-11-01

Full Text Available The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1 capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2 capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3 from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

A triclinic crystal structure of the carboxy-terminal domain of HIV-1 capsid protein with four molecules in the asymmetric unit reveals a novel packing interface

International Nuclear Information System (INIS)

Lampel, Ayala; Yaniv, Oren; Berger, Or; Bacharach, Eran; Gazit, Ehud; Frolow, Felix

2013-01-01

The triclinic structure of the HIV-1 capsid protein contains four molecules in the asymmetric unit that form a novel packing interface that could conceivably resemble an intermediate structure that is involved in the early steps of HIV-1 assembly. The Gag precursor is the major structural protein of the virion of human immunodeficiency virus-1 (HIV-1). Capsid protein (CA), a cleavage product of Gag, plays an essential role in virus assembly both in Gag-precursor multimerization and in capsid core formation. The carboxy-terminal domain (CTD) of CA contains 20 residues that are highly conserved across retroviruses and constitute the major homology region (MHR). Genetic evidence implies a role for the MHR in interactions between Gag precursors during the assembly of the virus, but the structural basis for this role remains elusive. This paper describes a novel triclinic structure of the HIV-1 CA CTD at 1.6 Å resolution with two canonical dimers of CA CTD in the asymmetric unit. The canonical dimers form a newly identified packing interface where interactions of four conserved MHR residues take place. This is the first structural indication that these MHR residues participate in the putative CTD–CTD interactions. These findings suggest that the molecules forming this novel interface resemble an intermediate structure that participates in the early steps of HIV-1 assembly. This interface may therefore provide a novel target for antiviral drugs

Functional requirements of the yellow fever virus capsid protein.

Science.gov (United States)

Patkar, Chinmay G; Jones, Christopher T; Chang, Yu-hsuan; Warrier, Ranjit; Kuhn, Richard J

2007-06-01

Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.

Science.gov (United States)

Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael; Zlotnick, Adam

2018-01-29

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. © 2017, Schlicksup et al.

Inferring genome-wide patterns of admixture in Qataris using fifty-five ancestral populations

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Omberg Larsson

2012-06-01

Full Text Available Abstract Background Populations of the Arabian Peninsula have a complex genetic structure that reflects waves of migrations including the earliest human migrations from Africa and eastern Asia, migrations along ancient civilization trading routes and colonization history of recent centuries. Results Here, we present a study of genome-wide admixture in this region, using 156 genotyped individuals from Qatar, a country located at the crossroads of these migration patterns. Since haplotypes of these individuals could have originated from many different populations across the world, we have developed a machine learning method "SupportMix" to infer loci-specific genomic ancestry when simultaneously analyzing many possible ancestral populations. Simulations show that SupportMix is not only more accurate than other popular admixture discovery tools but is the first admixture inference method that can efficiently scale for simultaneous analysis of 50-100 putative ancestral populations while being independent of prior demographic information. Conclusions By simultaneously using the 55 world populations from the Human Genome Diversity Panel, SupportMix was able to extract the fine-scale ancestry of the Qatar population, providing many new observations concerning the ancestry of the region. For example, as well as recapitulating the three major sub-populations in Qatar, composed of mainly Arabic, Persian, and African ancestry, SupportMix additionally identifies the specific ancestry of the Persian group to populations sampled in Greater Persia rather than from China and the ancestry of the African group to sub-Saharan origin and not Southern African Bantu origin as previously thought.

The mammary gland-specific marsupial ELP and eutherian CTI share a common ancestral gene

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Pharo Elizabeth A

2012-06-01

Full Text Available Abstract Background The marsupial early lactation protein (ELP gene is expressed in the mammary gland and the protein is secreted into milk during early lactation (Phase 2A. Mature ELP shares approximately 55.4% similarity with the colostrum-specific bovine colostrum trypsin inhibitor (CTI protein. Although ELP and CTI both have a single bovine pancreatic trypsin inhibitor (BPTI-Kunitz domain and are secreted only during the early lactation phases, their evolutionary history is yet to be investigated. Results Tammar ELP was isolated from a genomic library and the fat-tailed dunnart and Southern koala ELP genes cloned from genomic DNA. The tammar ELP gene was expressed only in the mammary gland during late pregnancy (Phase 1 and early lactation (Phase 2A. The opossum and fat-tailed dunnart ELP and cow CTI transcripts were cloned from RNA isolated from the mammary gland and dog CTI from cells in colostrum. The putative mature ELP and CTI peptides shared 44.6%-62.2% similarity. In silico analyses identified the ELP and CTI genes in the other species examined and provided compelling evidence that they evolved from a common ancestral gene. In addition, whilst the eutherian CTI gene was conserved in the Laurasiatherian orders Carnivora and Cetartiodactyla, it had become a pseudogene in others. These data suggest that bovine CTI may be the ancestral gene of the Artiodactyla-specific, rapidly evolving chromosome 13 pancreatic trypsin inhibitor (PTI, spleen trypsin inhibitor (STI and the five placenta-specific trophoblast Kunitz domain protein (TKDP1-5 genes. Conclusions Marsupial ELP and eutherian CTI evolved from an ancestral therian mammal gene before the divergence of marsupials and eutherians between 130 and 160 million years ago. The retention of the ELP gene in marsupials suggests that this early lactation-specific milk protein may have an important role in the immunologically naïve young of these species.

Ancestrality and evolution of trait syndromes in finches (Fringillidae).

Science.gov (United States)

Ponge, Jean-François; Zuccon, Dario; Elias, Marianne; Pavoine, Sandrine; Henry, Pierre-Yves; Théry, Marc; Guilbert, Éric

2017-12-01

Species traits have been hypothesized by one of us (Ponge, 2013) to evolve in a correlated manner as species colonize stable, undisturbed habitats, shifting from "ancestral" to "derived" strategies. We predicted that generalism, r-selection, sexual monomorphism, and migration/gregariousness are the ancestral states (collectively called strategy A) and evolved correlatively toward specialism, K-selection, sexual dimorphism, and residence/territoriality as habitat stabilized (collectively called B strategy). We analyzed the correlated evolution of four syndromes, summarizing the covariation between 53 traits, respectively, involved in ecological specialization, r-K gradient, sexual selection, and dispersal/social behaviors in 81 species representative of Fringillidae, a bird family with available natural history information and that shows variability for all these traits. The ancestrality of strategy A was supported for three of the four syndromes, the ancestrality of generalism having a weaker support, except for the core group Carduelinae (69 species). It appeared that two different B-strategies evolved from the ancestral state A, both associated with highly predictable environments: one in poorly seasonal environments, called B1, with species living permanently in lowland tropics, with "slow pace of life" and weak sexual dimorphism, and one in highly seasonal environments, called B2, with species breeding out-of-the-tropics, migratory, with a "fast pace of life" and high sexual dimorphism.

Specific cross-linking of capsid proteins to virus RNA by ultraviolet irradiation of polio virus

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Wetz, K.; Habermehl, K.O. (Freie Univ. Berlin (Germany, F.R.))

1982-04-01

Poliovirus was irradiated with u.v. light under conditions causing approx. 5% cross-linking of capsid protein to virus RNA. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease, and then analysed on SDS-polyacrylamide gels. The smallest capsid polypeptide VP4 was found to be associated with the RNA to the greatest degree, followed by VP2 and VP1, while VP3 was attached only in trace amounts. Low radiation doses, which produced cross-linking of RNA to protein, did not cause breakdown of the virus particles or conformational changes of the capsid as examined physically and serologically. However, higher doses caused structural alterations of the virus capsid.

Specific cross-linking of capsid proteins to virus RNA by ultraviolet irradiation of polio virus

International Nuclear Information System (INIS)

Wetz, K.; Habermehl, K.-O.

1982-01-01

Poliovirus was irradiated with u.v. light under conditions causing approx. 5% cross-linking of capsid protein to virus RNA. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease, and then analysed on SDS-polyacrylamide gels. The smallest capsid polypeptide VP4 was found to be associated with the RNA to the greatest degree, followed by VP2 and VP1, while VP3 was attached only in trace amounts. Low radiation doses, which produced cross-linking of RNA to protein, did not cause breakdown of the virus particles or conformational changes of the capsid as examined physically and serologically. However, higher doses caused structural alterations of the virus capsid. (author)

Vast diversity of prokaryotic virus genomes encoding double jelly-roll major capsid proteins uncovered by genomic and metagenomic sequence analysis.

Science.gov (United States)

Yutin, Natalya; Bäckström, Disa; Ettema, Thijs J G; Krupovic, Mart; Koonin, Eugene V

2018-04-10

Analysis of metagenomic sequences has become the principal approach for the study of the diversity of viruses. Many recent, extensive metagenomic studies on several classes of viruses have dramatically expanded the visible part of the virosphere, showing that previously undetected viruses, or those that have been considered rare, actually are important components of the global virome. We investigated the provenance of viruses related to tail-less bacteriophages of the family Tectiviridae by searching genomic and metagenomics sequence databases for distant homologs of the tectivirus-like Double Jelly-Roll major capsid proteins (DJR MCP). These searches resulted in the identification of numerous genomes of virus-like elements that are similar in size to tectiviruses (10-15 kilobases) and have diverse gene compositions. By comparison of the gene repertoires, the DJR MCP-encoding genomes were classified into 6 distinct groups that can be predicted to differ in reproduction strategies and host ranges. Only the DJR MCP gene that is present by design is shared by all these genomes, and most also encode a predicted DNA-packaging ATPase; the rest of the genes are present only in subgroups of this unexpectedly diverse collection of DJR MCP-encoding genomes. Only a minority encode a DNA polymerase which is a hallmark of the family Tectiviridae and the putative family "Autolykiviridae". Notably, one of the identified putative DJR MCP viruses encodes a homolog of Cas1 endonuclease, the integrase involved in CRISPR-Cas adaptation and integration of transposon-like elements called casposons. This is the first detected occurrence of Cas1 in a virus. Many of the identified elements are individual contigs flanked by inverted or direct repeats and appear to represent complete, extrachromosomal viral genomes, whereas others are flanked by bacterial genes and thus can be considered as proviruses. These contigs come from metagenomes of widely different environments, some dominated by

Ancestral gene reconstruction and synthesis of ancient rhodopsins in the laboratory.

Science.gov (United States)

Chang, Belinda S W

2003-08-01

Laboratory synthesis of ancestral proteins offers an intriguing opportunity to study the past directly. The development of Bayesian methods to infer ancestral sequences, combined with advances in models of molecular evolution, and synthetic gene technology make this an increasingly promising approach in evolutionary studies of molecular function. Visual pigments form the first step in the biochemical cascade of events in the retina in all animals known to possess visual capabilities. In vertebrates, the necessity of spanning a dynamic range of light intensities of many orders of magnitude has given rise to two different types of photoreceptors, rods specialized for dim-light conditions, and cones for daylight and color vision. These photoreceptors contain different types of visual pigment genes. Reviewed here are methods of inferring ancestral sequences, chemical synthesis of artificial ancestral genes in the laboratory, and applications to the evolution of vertebrate visual systems and the experimental recreation of an archosaur rod visual pigment. The ancestral archosaurs gave rise to several notable lineages of diapsid reptiles, including the birds and the dinosaurs, and would have existed over 200 MYA. What little is known of their physiology comes from fossil remains, and inference based on the biology of their living descendants. Despite its age, an ancestral archosaur pigment was successfully recreated in the lab, and showed interesting properties of its wavelength sensitivity that may have implications for the visual capabilities of the ancestral archosaurs in dim light.

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

Science.gov (United States)

Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael

2018-01-01

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. PMID:29377794

The two capsid proteins of maize rayado fino virus contain common peptide sequences.

Science.gov (United States)

Falk, B W; Tsai, J H

1986-01-01

Virions of maize rayado fino virus (MRFV) were purified and two major capsid proteins (ca. Mr 29,000 and 22,000) were resolved by SDS-PAGE. When the two major capsid proteins were isolated from gels and compared by one-dimensional peptide mapping after digestion with Staphylococcus aureus V-8 protease, indistinguishable peptide maps were obtained, suggesting that these two proteins contain common peptide sequences. Some preparations also showed minor protein components that were intermediate between the Mr 22,000 and Mr 29,000 capsid proteins. One of the minor proteins, ca. Mr 27,000, gave a peptide map indistinguishable from the major capsid proteins. In vitro ageing of partially purified preparations or virion treatment with proteolytic enzymes failed to show conversion of the Mr 29,000 protein to a Mr 22,000. Protease inhibitors added to the buffers used for virion purification did not affect the apparent 1:3 ratio of 29,000 to 22,000 proteins in the purified preparations.

Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

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Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M. (UCSC)

2016-11-02

Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

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Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

2012-09-17

The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

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Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

2013-12-20

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

Poliovirus-associated protein kinase: Destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn2+

International Nuclear Information System (INIS)

Ratka, M.; Lackmann, M.; Ueckermann, C.; Karlins, U.; Koch, G.

1989-01-01

The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg 2+ . In this paper, the effect of Zn 2+ on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg 2+ . In the presence of Zn 2+ , phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. The results indicate the activation of more than one virus-associated protein kinase by Zn 2+ . The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn 2+ . This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. The authors suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus

A putative SUMO interacting motif in the B30.2/SPRY domain of rhesus macaque TRIM5α important for NF-κB/AP-1 signaling and HIV-1 restriction

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Marie-Édith Nepveu-Traversy

2016-01-01

Full Text Available TRIM5α from the rhesus macaque (TRIM5αRh is a restriction factor that shows strong activity against HIV-1. TRIM5αRh binds specifically to HIV-1 capsid (CA through its B30.2/PRYSPRY domain shortly after entry of the virus into the cytoplasm. Recently, three putative SUMO interacting motifs (SIMs have been identified in the PRYSPRY domain of human and macaque TRIM5α. However, structural modeling of this domain suggested that two of them were buried in the hydrophobic core of the protein, implying that interaction with SUMO was implausible, while the third one was not relevant to restriction. In light of these results, we re-analyzed the TRIM5αRh PRYSPRY sequence and identified an additional putative SIM (435VIIC438 which we named SIM4. This motif is exposed at the surface of the PRYSPRY domain, allowing potential interactions with SUMO or SUMOylated proteins. Introducing a double mutation in SIM4 (V435K, I436K did not alter stability, unlike mutations in SIM1. SIM4-mutated TRIM5αRh failed to bind HIV-1CA and lost the ability to restrict this virus. Accordingly, SIM4 undergoes significant variation among primates and substituting this motif with naturally occurring SIM4 variants affected HIV-1 restriction by TRIM5αRh, suggesting a direct role in capsid recognition. Interestingly, SIM4-mutated TRIM5αRh also failed to activate NF-κB and AP-1-mediated transcription. Although there is no direct evidence that SIM4 is involved in direct interaction with SUMO or a SUMOylated protein, mutating this motif strongly reduced co-localization of TRIM5αRh with SUMO-1 and with PML, a SUMOylated nuclear protein. In conclusion, this new putative SIM is crucial for both direct interaction with incoming capsids and for NF-κB/AP-1 signaling. We speculate that the latter function is mediated by interactions of SIM4 with a SUMOylated protein involved in the NF-κB/AP-1 signaling pathways.

Characterization of the mode of action of a potent dengue virus capsid inhibitor.

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Scaturro, Pietro; Trist, Iuni Margaret Laura; Paul, David; Kumar, Anil; Acosta, Eliana G; Byrd, Chelsea M; Jordan, Robert; Brancale, Andrea; Bartenschlager, Ralf

2014-10-01

Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts to develop vaccine candidates and antiviral drugs, there is a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small-molecule inhibitor exhibiting broad and potent antiviral activity against DV in vitro and in vivo (C. M. Byrd et al., Antimicrob. Agents Chemother. 57:15-25, 2013, doi:10 .1128/AAC.01429-12). In the present study, we investigated the mode of action of this promising compound by using a combination of biochemical, virological, and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of bimodal antiviral activity affecting both assembly/release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay, we observed an ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids, probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept that also is applicable to flaviviruses. Dengue viruses are arthropod-borne viruses representing a significant global health burden. They infect up to 400 million people and are endemic to subtropical and tropical areas of the world. Currently, there are neither vaccines nor approved therapeutics for the prophylaxis or treatment of DV infections, respectively. This study reports the characterization of the

WARACS: Wrappers to Automate the Reconstruction of Ancestral Character States.

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Gruenstaeudl, Michael

2016-02-01

Reconstructions of ancestral character states are among the most widely used analyses for evaluating the morphological, cytological, or ecological evolution of an organismic lineage. The software application Mesquite remains the most popular application for such reconstructions among plant scientists, even though its support for automating complex analyses is limited. A software tool is needed that automates the reconstruction and visualization of ancestral character states with Mesquite and similar applications. A set of command line-based Python scripts was developed that (a) communicates standardized input to and output from the software applications Mesquite, BayesTraits, and TreeGraph2; (b) automates the process of ancestral character state reconstruction; and (c) facilitates the visualization of reconstruction results. WARACS provides a simple tool that streamlines the reconstruction and visualization of ancestral character states over a wide array of parameters, including tree distribution, character state, and optimality criterion.

Why Meillassoux’s Speculative Materialism Struggles with Ancestrality

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Ciprian Jeler

2014-01-01

This paper shows that Quentin Meillassoux’s speculative materialism doesn’t offer us the means to account for the ancestral statements that the modern sciences produce, i.e. for the scientific statements about events preceding all forms of life. An analysis of the reasons why Meillassoux thinks that the problem of ancestrality problematizes the contemporary self-evidence of correlationism is first offered. The results of this analysis are then applied to speculative materialism itself and the...

Enzyme functional evolution through improved catalysis of ancestrally nonpreferred substrates

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Huang, Ruiqi; Hippauf, Frank; Rohrbeck, Diana; Haustein, Maria; Wenke, Katrin; Feike, Janie; Sorrelle, Noah; Piechulla, Birgit; Barkman, Todd J.

2012-01-01

In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (kcat/KM) for competing substrates, even though adaptive substitutions may affect KM and kcat separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities. PMID:22315396

Functional dissection of the alphavirus capsid protease: sequence requirements for activity.

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Thomas, Saijo; Rai, Jagdish; John, Lijo; Günther, Stephan; Drosten, Christian; Pützer, Brigitte M; Schaefer, Stephan

2010-11-18

The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

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Kononova, Olga; Snijder, Joost; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I.; Marx, Kenneth A.; Wuite, Gijs J. L.; Roos, Wouter H.; Barsegov, Valeri

2013-10-01

Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Virus shells can have applications as nanocontainers and delivery vehicles in biotechnology and medicine. Combined AFM experiments and computational modeling on sub-second timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus (CCMV) capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which depend on the magnitude and geometry of mechanical input. Surprisingly, under large deformations the CCMV capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state dH = 11.5 - 12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending, and the entropy change TdS = 5.1 - 5.8 MJ/mol is mostly due to coherent in-plane rearrangements of protein chains, which result in the capsid stiffening. Dynamic coupling of these modes defines the extent of elasticity and reversibility of capsid mechanical deformation. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses' biological function.

Facilitating the use of alternative capsid control methods towards sustainable production of organic cocoa in Ghana

OpenAIRE

Ayenor, G.K.; Huis, van, A.; Obeng-Ofori, D.; Padi, B.; Röling, N.G.

2007-01-01

Cocoa (Theobroma cacao L.) is an important foreign exchange earner for Ghana. However, production is constrained by a high incidence of pests and diseases. Based on farmers' needs, this study focused on the control of capsids, mainly Sahlbergella singularis Haglund and Distantiella theobroma (Distant) (both Hemiptera: Miridae). Annual crop loss caused by capsids is estimated at 25¿30%. To control capsids, formal research recommends application of synthetic insecticides four times between Augu...

Animal regeneration: ancestral character or evolutionary novelty?

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Slack, Jonathan Mw

2017-09-01

An old question about regeneration is whether it is an ancestral character which is a general property of living matter, or whether it represents a set of specific adaptations to the different circumstances faced by different types of animal. In this review, some recent results on regeneration are assessed to see if they can throw any new light on this question. Evidence in favour of an ancestral character comes from the role of Wnt and bone morphogenetic protein signalling in controlling the pattern of whole-body regeneration in acoels, which are a basal group of bilaterian animals. On the other hand, there is some evidence for adaptive acquisition or maintenance of the regeneration of appendages based on the occurrence of severe non-lethal predation, the existence of some novel genes in regenerating organisms, and differences at the molecular level between apparently similar forms of regeneration. It is tentatively concluded that whole-body regeneration is an ancestral character although has been lost from most animal lineages. Appendage regeneration is more likely to represent a derived character resulting from many specific adaptations. © 2017 The Author.

Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids.

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Nagesh Pulicherla

Full Text Available The capsid proteins of adeno-associated viruses (AAV have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.

Residues of the UL25 Protein of Herpes Simplex Virus That Are Required for Its Stable Interaction with Capsids ▿

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Cockrell, Shelley K.; Huffman, Jamie B.; Toropova, Katerina; Conway, James F.; Homa, Fred L.

2011-01-01

The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes. PMID:21411517

Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

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Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

2018-01-13

The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97

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Jens Milbradt

2018-01-01

Full Text Available The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

Virus Capsids as Targeted Nanoscale Delivery Vessels of Photoactive Compounds for Site-Specific Photodynamic Therapy

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Cohen, Brian A.

The research presented in this work details the use of a viral capsid as an addressable delivery vessel of photoactive compounds for use in photodynamic therapy. Photodynamic therapy is a treatment that involves the interaction of light with a photosensitizing molecule to create singlet oxygen, a reactive oxygen species. Overproduction of singlet oxygen in cells can cause oxidative damage leading to cytotoxicity and eventually cell death. Challenges with the current generation of FDA-approved photosensitizers for photodynamic therapy primarily stem from their lack of tissue specificity. This work describes the packaging of photoactive cationic porphyrins inside the MS2 bacteriophage capsid, followed by external modification of the capsid with cancer cell-targeting G-quadruplex DNA aptamers to generate a tumor-specific photosensitizing agent. First, a cationic porphyrin is loaded into the capsids via nucleotide-driven packaging, a process that involves charge interaction between the porphyrin and the RNA inside the capsid. Results show that over 250 porphyrin molecules associate with the RNA within each MS2 capsid. Removal of RNA from the capsid severely inhibits the packaging of the cationic porphyrins. Porphyrin-virus constructs were then shown to photogenerate singlet oxygen, and cytotoxicity in non-targeted photodynamic treatment experiments. Next, each porphyrin-loaded capsid is externally modified with approximately 60 targeting DNA aptamers by employing a heterobifunctional crosslinking agent. The targeting aptamer is known to bind the protein nucleolin, a ubiquitous protein that is overexpressed on the cell surface by many cancer cell types. MCF-7 human breast carcinoma cells and MCF-10A human mammary epithelial cells were selected as an in vitro model for breast cancer and normal tissue, respectively. Fluorescently tagged virus-aptamer constructs are shown to selectively target MCF-7 cells versus MCF-10A cells. Finally, results are shown in which porphyrin

Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation.

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Kant, Ravi; Llauró, Aida; Rayaprolu, Vamseedhar; Qazi, Shefah; de Pablo, Pedro J; Douglas, Trevor; Bothner, Brian

2018-03-15

The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared. Copyright © 2018 Elsevier B.V. All rights reserved.

Reproductive function in mice exposed to ancestral and direct irradiation

International Nuclear Information System (INIS)

Nash, D.J.; Sprackling, L.S.

1978-01-01

Reproduction was studied in 13 inbred strains of mice that had been exposed continuously to 60 Co gamma radiation for varying numbers of generations. At weaning the mice were removed from the irradiation chamber and were tested for reproductive performance. Ancestral and direct levels of irradiation were determined for each animal. Each irradiated or control female was scored as fertile or sterile, and in utero litter counts were made in pregnant females that were dissected past the 10th day of pregnancy. The number of resorptions, dead embryos, and live embryos were counted, and the ratio of living embryos to the total number of embryos was determined for each litter. The overall fertility curves were sigmoid in the range of doses below those which caused complete sterility, which indicated some sort of cumulative damage. In 11 of the 13 strains studied, an increase in ancestral and/or direct irradiation led to significant decreases in fertility. The means of the number alive in the litters for the control and irradiated mice in each strain showed a definite trend toward fewer live mice in utero after irradiation. Least-squares analyses of variance were made to detect possible effects of any of six irradiation variables (ancestral linear, ancestral quadratic, ancestral cubic, direct linear, direct quadratic, or direct cubic) or of strain differences on total litter size and on ratio. Strain effects were significant in each instance. Litter size was more likely to be affected by radiation variables than ratios were

Characterization of Reconstructed Ancestral Proteins Suggests a Change in Temperature of the Ancient Biosphere.

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Akanuma, Satoshi

2017-08-06

Understanding the evolution of ancestral life, and especially the ability of some organisms to flourish in the variable environments experienced in Earth's early biosphere, requires knowledge of the characteristics and the environment of these ancestral organisms. Information about early life and environmental conditions has been obtained from fossil records and geological surveys. Recent advances in phylogenetic analysis, and an increasing number of protein sequences available in public databases, have made it possible to infer ancestral protein sequences possessed by ancient organisms. However, the in silico studies that assess the ancestral base content of ribosomal RNAs, the frequency of each amino acid in ancestral proteins, and estimate the environmental temperatures of ancient organisms, show conflicting results. The characterization of ancestral proteins reconstructed in vitro suggests that ancient organisms had very thermally stable proteins, and therefore were thermophilic or hyperthermophilic. Experimental data supports the idea that only thermophilic ancestors survived the catastrophic increase in temperature of the biosphere that was likely associated with meteorite impacts during the early history of Earth. In addition, by expanding the timescale and including more ancestral proteins for reconstruction, it appears as though the Earth's surface temperature gradually decreased over time, from Archean to present.

Porcine circovirus-2 capsid protein induces cell death in PK15 cells

Energy Technology Data Exchange (ETDEWEB)

Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark; Czub, Markus

2014-11-15

Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

Musculature in sipunculan worms: ontogeny and ancestral states.

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Schulze, Anja; Rice, Mary E

2009-01-01

Molecular phylogenetics suggests that the Sipuncula fall into the Annelida, although they are morphologically very distinct and lack segmentation. To understand the evolutionary transformations from the annelid to the sipunculan body plan, it is important to reconstruct the ancestral states within the respective clades at all life history stages. Here we reconstruct the ancestral states for the head/introvert retractor muscles and the body wall musculature in the Sipuncula using Bayesian statistics. In addition, we describe the ontogenetic transformations of the two muscle systems in four sipunculan species with different developmental modes, using F-actin staining with fluorescent-labeled phalloidin in conjunction with confocal laser scanning microscopy. All four species, which have smooth body wall musculature and less than the full set of four introvert retractor muscles as adults, go through developmental stages with four retractor muscles that are eventually reduced to a lower number in the adult. The circular and sometimes the longitudinal body wall musculature are split into bands that later transform into a smooth sheath. Our ancestral state reconstructions suggest with nearly 100% probability that the ancestral sipunculan had four introvert retractor muscles, longitudinal body wall musculature in bands and circular body wall musculature arranged as a smooth sheath. Species with crawling larvae have more strongly developed body wall musculature than those with swimming larvae. To interpret our findings in the context of annelid evolution, a more solid phylogenetic framework is needed for the entire group and more data on ontogenetic transformations of annelid musculature are desirable.

Rapid maximum likelihood ancestral state reconstruction of continuous characters: A rerooting-free algorithm.

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Goolsby, Eric W

2017-04-01

Ancestral state reconstruction is a method used to study the evolutionary trajectories of quantitative characters on phylogenies. Although efficient methods for univariate ancestral state reconstruction under a Brownian motion model have been described for at least 25 years, to date no generalization has been described to allow more complex evolutionary models, such as multivariate trait evolution, non-Brownian models, missing data, and within-species variation. Furthermore, even for simple univariate Brownian motion models, most phylogenetic comparative R packages compute ancestral states via inefficient tree rerooting and full tree traversals at each tree node, making ancestral state reconstruction extremely time-consuming for large phylogenies. Here, a computationally efficient method for fast maximum likelihood ancestral state reconstruction of continuous characters is described. The algorithm has linear complexity relative to the number of species and outperforms the fastest existing R implementations by several orders of magnitude. The described algorithm is capable of performing ancestral state reconstruction on a 1,000,000-species phylogeny in fewer than 2 s using a standard laptop, whereas the next fastest R implementation would take several days to complete. The method is generalizable to more complex evolutionary models, such as phylogenetic regression, within-species variation, non-Brownian evolutionary models, and multivariate trait evolution. Because this method enables fast repeated computations on phylogenies of virtually any size, implementation of the described algorithm can drastically alleviate the computational burden of many otherwise prohibitively time-consuming tasks requiring reconstruction of ancestral states, such as phylogenetic imputation of missing data, bootstrapping procedures, Expectation-Maximization algorithms, and Bayesian estimation. The described ancestral state reconstruction algorithm is implemented in the Rphylopars

WARACS: Wrappers to Automate the Reconstruction of Ancestral Character States1

Science.gov (United States)

Gruenstaeudl, Michael

2016-01-01

Premise of the study: Reconstructions of ancestral character states are among the most widely used analyses for evaluating the morphological, cytological, or ecological evolution of an organismic lineage. The software application Mesquite remains the most popular application for such reconstructions among plant scientists, even though its support for automating complex analyses is limited. A software tool is needed that automates the reconstruction and visualization of ancestral character states with Mesquite and similar applications. Methods and Results: A set of command line–based Python scripts was developed that (a) communicates standardized input to and output from the software applications Mesquite, BayesTraits, and TreeGraph2; (b) automates the process of ancestral character state reconstruction; and (c) facilitates the visualization of reconstruction results. Conclusions: WARACS provides a simple tool that streamlines the reconstruction and visualization of ancestral character states over a wide array of parameters, including tree distribution, character state, and optimality criterion. PMID:26949580

Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses.

Science.gov (United States)

Ekström, Jens-Ola; Habayeb, Mazen S; Srivastava, Vaibhav; Kieselbach, Thomas; Wingsle, Gunnar; Hultmark, Dan

2011-09-01

The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales. Copyright © 2011 Elsevier B.V. All rights reserved.

Estimation of the ancestral effective population sizes of African great apes under different selection regimes.

Science.gov (United States)

Schrago, Carlos G

2014-08-01

Reliable estimates of ancestral effective population sizes are necessary to unveil the population-level phenomena that shaped the phylogeny and molecular evolution of the African great apes. Although several methods have previously been applied to infer ancestral effective population sizes, an analysis of the influence of the selective regime on the estimates of ancestral demography has not been thoroughly conducted. In this study, three independent data sets under different selective regimes were used were composed to tackle this issue. The results showed that selection had a significant impact on the estimates of ancestral effective population sizes of the African great apes. The inference of the ancestral demography of African great apes was affected by the selection regime. The effects, however, were not homogeneous along the ancestral populations of great apes. The effective population size of the ancestor of humans and chimpanzees was more impacted by the selection regime when compared to the same parameter in the ancestor of humans, chimpanzees and gorillas. Because the selection regime influenced the estimates of ancestral effective population size, it is reasonable to assume that a portion of the discrepancy found in previous studies that inferred the ancestral effective population size may be attributable to the differential action of selection on the genes sampled.

Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons

Science.gov (United States)

Anderson, Fenja; Rother, Franziska; Rudolph, Kathrin; Prank, Ute; Binz, Anne; Hügel, Stefanie; Hartmann, Enno; Bader, Michael; Bauerfeind, Rudolf; Sodeik, Beate

2018-01-01

Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons. PMID:29304174

Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex

Science.gov (United States)

Cappadona, Ilaria; Villinger, Clarissa; Schutzius, Gabi; Mertens, Thomas

2015-01-01

ABSTRACT Human cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids. IMPORTANCE Generation of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for

Labeling and localization of the herpes simplex virus capsid protein UL25 and its interaction with the two triplexes closest to the penton

Science.gov (United States)

Conway, James F.; Cockrell, Shelley K.; Copeland, Anna Maria; Newcomb, William W.; Brown, Jay C.; Homa, Fred L.

2010-01-01

The herpes simplex virus type 1 (HSV-1) UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component or CCSC. Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid binding domain of UL25 located on the surface of the mature DNA-containing capsid. PMID:20109467

Exploring the role of genome and structural ions in preventing viral capsid collapse during dehydration

Science.gov (United States)

Martín-González, Natalia; Guérin Darvas, Sofía M.; Durana, Aritz; Marti, Gerardo A.; Guérin, Diego M. A.; de Pablo, Pedro J.

2018-03-01

Even though viruses evolve mainly in liquid milieu, their horizontal transmission routes often include episodes of dry environment. Along their life cycle, some insect viruses, such as viruses from the Dicistroviridae family, withstand dehydrated conditions with presently unknown consequences to their structural stability. Here, we use atomic force microscopy to monitor the structural changes of viral particles of Triatoma virus (TrV) after desiccation. Our results demonstrate that TrV capsids preserve their genome inside, conserving their height after exposure to dehydrating conditions, which is in stark contrast with other viruses that expel their genome when desiccated. Moreover, empty capsids (without genome) resulted in collapsed particles after desiccation. We also explored the role of structural ions in the dehydration process of the virions (capsid containing genome) by chelating the accessible cations from the external solvent milieu. We observed that ion suppression helps to keep the virus height upon desiccation. Our results show that under drying conditions, the genome of TrV prevents the capsid from collapsing during dehydration, while the structural ions are responsible for promoting solvent exchange through the virion wall.

Invasion of Ancestral Mammals into Dim-light Environments Inferred from Adaptive Evolution of the Phototransduction Genes.

Science.gov (United States)

Wu, Yonghua; Wang, Haifeng; Hadly, Elizabeth A

2017-04-20

Nocturnality is a key evolutionary innovation of mammals that enables mammals to occupy relatively empty nocturnal niches. Invasion of ancestral mammals into nocturnality has long been inferred from the phylogenetic relationships of crown Mammalia, which is primarily nocturnal, and crown Reptilia, which is primarily diurnal, although molecular evidence for this is lacking. Here we used phylogenetic analyses of the vision genes involved in the phototransduction pathway to predict the diel activity patterns of ancestral mammals and reptiles. Our results demonstrated that the common ancestor of the extant Mammalia was dominated by positive selection for dim-light vision, supporting the predominate nocturnality of the ancestral mammals. Further analyses showed that the nocturnality of the ancestral mammals was probably derived from the predominate diurnality of the ancestral amniotes, which featured strong positive selection for bright-light vision. Like the ancestral amniotes, the common ancestor of the extant reptiles and various taxa in Squamata, one of the main competitors of the temporal niches of the ancestral mammals, were found to be predominate diurnality as well. Despite this relatively apparent temporal niche partitioning between ancestral mammals and the relevant reptiles, our results suggested partial overlap of their temporal niches during crepuscular periods.

Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells

DEFF Research Database (Denmark)

Gullberg, Maria; Muszynski, Bartosz; Organtini, Lindsey J.

2013-01-01

The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3Cpro) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3Cpro can be toxic...... (from serotypes O and A) and 3Cpro were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3Cpro expression dependent on a mutant FMDV internal ribosome entry site (IRES) (designated P1-2A-mIRES-3C). The effects of using a mutant 3Cpro with reduced catalytic....... These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD...

The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

Science.gov (United States)

Blutt, Sarah E.; Crawford, Sue E.; Warfield, Kelly L.; Lewis, Dorothy E.; Estes, Mary K.; Conner, Margaret E.

2004-01-01

The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection. PMID:15194774

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

Science.gov (United States)

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard; Grimm, Dirk

2017-10-15

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus

Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus.

Science.gov (United States)

Chen, Yiyang; Liu, Baoyuan; Sun, Yani; Li, Huixia; Du, Taofeng; Nan, Yuchen; Hiscox, Julian A; Zhou, En-Min; Zhao, Qin

2018-04-18

Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection with domestic animals being a reservoir including swine and rabbits. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs); three novel; 1E4, 2C7, 2G9, and one previously characterized (1B5), were evaluated for binding to the capsid protein from genotype 4 (swine) hepatitis E virus (HEV). The results indicated that 625 DFCP 628 , 458 PSRPF 462 , and 407 EPTV 410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368-606), can exist in multimeric forms. Pre-incubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and anti-viral design. IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within

Effects of immunosuppression on circulating adeno-associated virus capsid-specific T cells in humans.

Science.gov (United States)

Parzych, Elizabeth M; Li, Hua; Yin, Xiangfan; Liu, Qin; Wu, Te-Lang; Podsakoff, Gregory M; High, Katherine A; Levine, Matthew H; Ertl, Hildegund C J

2013-04-01

In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4⁺ T cells, whereas numbers of circulating CD8⁺ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients.

Ancestral effect on HOMA-IR levels quantitated in an American population of Mexican origin.

Science.gov (United States)

Qu, Hui-Qi; Li, Quan; Lu, Yang; Hanis, Craig L; Fisher-Hoch, Susan P; McCormick, Joseph B

2012-12-01

An elevated insulin resistance index (homeostasis model assessment of insulin resistance [HOMA-IR]) is more commonly seen in the Mexican American population than in European populations. We report quantitative ancestral effects within a Mexican American population, and we correlate ancestral components with HOMA-IR. We performed ancestral analysis in 1,551 participants of the Cameron County Hispanic Cohort by genotyping 103 ancestry-informative markers (AIMs). These AIMs allow determination of the percentage (0-100%) ancestry from three major continental populations, i.e., European, African, and Amerindian. We observed that predominantly Amerindian ancestral components were associated with increased HOMA-IR (β = 0.124, P = 1.64 × 10(-7)). The correlation was more significant in males (Amerindian β = 0.165, P = 5.08 × 10(-7)) than in females (Amerindian β = 0.079, P = 0.019). This unique study design demonstrates how genomic markers for quantitative ancestral information can be used in admixed populations to predict phenotypic traits such as insulin resistance.

The structured ancestral selection graph and the many-demes limit.

Science.gov (United States)

Slade, Paul F; Wakeley, John

2005-02-01

We show that the unstructured ancestral selection graph applies to part of the history of a sample from a population structured by restricted migration among subpopulations, or demes. The result holds in the limit as the number of demes tends to infinity with proportionately weak selection, and we have also made the assumptions of island-type migration and that demes are equivalent in size. After an instantaneous sample-size adjustment, this structured ancestral selection graph converges to an unstructured ancestral selection graph with a mutation parameter that depends inversely on the migration rate. In contrast, the selection parameter for the population is independent of the migration rate and is identical to the selection parameter in an unstructured population. We show analytically that estimators of the migration rate, based on pairwise sequence differences, derived under the assumption of neutrality should perform equally well in the presence of weak selection. We also modify an algorithm for simulating genealogies conditional on the frequencies of two selected alleles in a sample. This permits efficient simulation of stronger selection than was previously possible. Using this new algorithm, we simulate gene genealogies under the many-demes ancestral selection graph and identify some situations in which migration has a strong effect on the time to the most recent common ancestor of the sample. We find that a similar effect also increases the sensitivity of the genealogy to selection.

Reconstruction of Ancestral Genomes in Presence of Gene Gain and Loss.

Science.gov (United States)

Avdeyev, Pavel; Jiang, Shuai; Aganezov, Sergey; Hu, Fei; Alekseyev, Max A

2016-03-01

Since most dramatic genomic changes are caused by genome rearrangements as well as gene duplications and gain/loss events, it becomes crucial to understand their mechanisms and reconstruct ancestral genomes of the given genomes. This problem was shown to be NP-complete even in the "simplest" case of three genomes, thus calling for heuristic rather than exact algorithmic solutions. At the same time, a larger number of input genomes may actually simplify the problem in practice as it was earlier illustrated with MGRA, a state-of-the-art software tool for reconstruction of ancestral genomes of multiple genomes. One of the key obstacles for MGRA and other similar tools is presence of breakpoint reuses when the same breakpoint region is broken by several different genome rearrangements in the course of evolution. Furthermore, such tools are often limited to genomes composed of the same genes with each gene present in a single copy in every genome. This limitation makes these tools inapplicable for many biological datasets and degrades the resolution of ancestral reconstructions in diverse datasets. We address these deficiencies by extending the MGRA algorithm to genomes with unequal gene contents. The developed next-generation tool MGRA2 can handle gene gain/loss events and shares the ability of MGRA to reconstruct ancestral genomes uniquely in the case of limited breakpoint reuse. Furthermore, MGRA2 employs a number of novel heuristics to cope with higher breakpoint reuse and process datasets inaccessible for MGRA. In practical experiments, MGRA2 shows superior performance for simulated and real genomes as compared to other ancestral genome reconstruction tools.

Four levels of hierarchical organization, including noncovalent chainmail, brace the mature tumor herpesvirus capsid against pressurization.

Science.gov (United States)

Zhou, Z Hong; Hui, Wong Hoi; Shah, Sanket; Jih, Jonathan; O'Connor, Christine M; Sherman, Michael B; Kedes, Dean H; Schein, Stan

2014-10-07

Like many double-stranded DNA viruses, tumor gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus withstand high internal pressure. Bacteriophage HK97 uses covalent chainmail for this purpose, but how this is achieved noncovalently in the much larger gammaherpesvirus capsid is unknown. Our cryoelectron microscopy structure of a gammaherpesvirus capsid reveals a hierarchy of four levels of organization: (1) Within a hexon capsomer, each monomer of the major capsid protein (MCP), 1,378 amino acids and six domains, interacts with its neighboring MCPs at four sites. (2) Neighboring capsomers are linked in pairs by MCP dimerization domains and in groups of three by heterotrimeric triplex proteins. (3) Small (∼280 amino acids) HK97-like domains in MCP monomers alternate with triplex heterotrimers to form a belt that encircles each capsomer. (4) One hundred sixty-two belts concatenate to form noncovalent chainmail. The triplex heterotrimer orchestrates all four levels and likely drives maturation to an angular capsid that can withstand pressurization. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ancestral sequence reconstruction in primate mitochondrial DNA: compositional bias and effect on functional inference.

Science.gov (United States)

Krishnan, Neeraja M; Seligmann, Hervé; Stewart, Caro-Beth; De Koning, A P Jason; Pollock, David D

2004-10-01

Reconstruction of ancestral DNA and amino acid sequences is an important means of inferring information about past evolutionary events. Such reconstructions suggest changes in molecular function and evolutionary processes over the course of evolution and are used to infer adaptation and convergence. Maximum likelihood (ML) is generally thought to provide relatively accurate reconstructed sequences compared to parsimony, but both methods lead to the inference of multiple directional changes in nucleotide frequencies in primate mitochondrial DNA (mtDNA). To better understand this surprising result, as well as to better understand how parsimony and ML differ, we constructed a series of computationally simple "conditional pathway" methods that differed in the number of substitutions allowed per site along each branch, and we also evaluated the entire Bayesian posterior frequency distribution of reconstructed ancestral states. We analyzed primate mitochondrial cytochrome b (Cyt-b) and cytochrome oxidase subunit I (COI) genes and found that ML reconstructs ancestral frequencies that are often more different from tip sequences than are parsimony reconstructions. In contrast, frequency reconstructions based on the posterior ensemble more closely resemble extant nucleotide frequencies. Simulations indicate that these differences in ancestral sequence inference are probably due to deterministic bias caused by high uncertainty in the optimization-based ancestral reconstruction methods (parsimony, ML, Bayesian maximum a posteriori). In contrast, ancestral nucleotide frequencies based on an average of the Bayesian set of credible ancestral sequences are much less biased. The methods involving simpler conditional pathway calculations have slightly reduced likelihood values compared to full likelihood calculations, but they can provide fairly unbiased nucleotide reconstructions and may be useful in more complex phylogenetic analyses than considered here due to their speed and

Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures.

Directory of Open Access Journals (Sweden)

Kerstin Radtke

2010-07-01

Full Text Available Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1 show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

Science.gov (United States)

White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

2008-12-01

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

Science.gov (United States)

Nance, Michael E; Duan, Dongsheng

2015-12-01

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

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Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

2015-01-01

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

Science.gov (United States)

Prokop, Pavol

2015-08-01

A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

Prenatal effects of ancestral irradiation in inbred mice

International Nuclear Information System (INIS)

Sprackling, L.E.S.

1975-01-01

Mice from 13 inbred strains (S, Z, E, Bab, BaB, BrR, C, K, N, Q, G, CFW, CF1) received continuous cobalt 60 irradiation at low dose rates for varying numbers of consecutive generations. Some Bab and BaB mice had received continuous irradiation for from 24 to 31 generations and the other mice had up to six generations of continuous irradiation in their ancestry. At weaning, the mice were removed from the irradiation room and were mated within strains either to sibs or nonsibs. Ancestral and direct irradiation doses were calculated. The ancestral dose was the effective accumulated dose to the progeny of the mated mice. The direct dose was the amount of irradiation received by any mated female from her conception to her weaning. Each irradiated or control female was scored as fertile or sterile and in utero litter counts were made in pregnant females that were dissected past the tenth day of pregnancy; the sum of moles, dead embryos, and live embryos was the total in utero litter size. A ratio of the living embryos to the total number of embryos in utero was determined for each litter. An increase in ancestral or direct irradiation dose significantly decreased fertility in 11 of the 13 strains. The fertility curves for the pooled data were sigmoid in the area of the doses below those that caused complete sterility. Among the controls, there were significant strain differences in total litter size and in the ratio. Strain X--Y plots, with ancestral or direct doses plotted against total litter size or ratio, revealed the tendency for litter size to decrease as dose increased. The only trend shown for ratio was for the litters with ratios of 0.50 or less to appear more frequently among the irradiated mice. The few corpora lutea counts revealed nothing of significance. Generally, there was a definite trend toward fewer mice alive in utero among the irradiated mice

In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

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Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

2016-12-01

HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

The ancestral selection graph under strong directional selection.

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Pokalyuk, Cornelia; Pfaffelhuber, Peter

2013-08-01

The ancestral selection graph (ASG) was introduced by  Neuhauser and Krone (1997) in order to study populations of constant size which evolve under selection. Coalescence events, which occur at rate 1 for every pair of lines, lead to joint ancestry. In addition, splitting events in the ASG at rate α, the scaled selection coefficient, produce possible ancestors, such that the real ancestor depends on the ancestral alleles. Here, we use the ASG in the case without mutation in order to study fixation of a beneficial mutant. Using our main tool, a reversibility property of the ASG, we provide a new proof of the fact that a beneficial allele fixes roughly in time (2logα)/α if α is large. Copyright © 2012 Elsevier Inc. All rights reserved.

Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

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Albertha R. van Zyl

2016-03-01

Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

Viral capsid is a pathogen-associated molecular pattern in adenovirus keratitis.

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Ashish V Chintakuntlawar

2010-04-01

Full Text Available Human adenovirus (HAdV infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9 signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9(-/- mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD. These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.

Molecular characterization of capsid protein gene of potato virus X ...

African Journals Online (AJOL)

Molecular characterization of capsid protein gene of potato virus X from Pakistan. Arshad Jamal, Idrees Ahmad Nasir, Bushra Tabassum, Muhammad Tariq, Abdul Munim Farooq, Zahida Qamar, Mohsin Ahmad Khan, Nadeem Ahmad, Muhammad Shafiq, Muhammad Saleem Haider, M. Arshad Javed, Tayyab Husnain ...

Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design.

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Taylor, Adam; Liu, Xiang; Zaid, Ali; Goh, Lucas Y H; Hobson-Peters, Jody; Hall, Roy A; Merits, Andres; Mahalingam, Suresh

2017-02-21

Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design. IMPORTANCE CHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain

A comparison of ancestral state reconstruction methods for quantitative characters.

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Royer-Carenzi, Manuela; Didier, Gilles

2016-09-07

Choosing an ancestral state reconstruction method among the alternatives available for quantitative characters may be puzzling. We present here a comparison of seven of them, namely the maximum likelihood, restricted maximum likelihood, generalized least squares under Brownian, Brownian-with-trend and Ornstein-Uhlenbeck models, phylogenetic independent contrasts and squared parsimony methods. A review of the relations between these methods shows that the maximum likelihood, the restricted maximum likelihood and the generalized least squares under Brownian model infer the same ancestral states and can only be distinguished by the distributions accounting for the reconstruction uncertainty which they provide. The respective accuracy of the methods is assessed over character evolution simulated under a Brownian motion with (and without) directional or stabilizing selection. We give the general form of ancestral state distributions conditioned on leaf states under the simulation models. Ancestral distributions are used first, to give a theoretical lower bound of the expected reconstruction error, and second, to develop an original evaluation scheme which is more efficient than comparing the reconstructed and the simulated states. Our simulations show that: (i) the distributions of the reconstruction uncertainty provided by the methods generally make sense (some more than others); (ii) it is essential to detect the presence of an evolutionary trend and to choose a reconstruction method accordingly; (iii) all the methods show good performances on characters under stabilizing selection; (iv) without trend or stabilizing selection, the maximum likelihood method is generally the most accurate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Infant and juvenile growth in ancestral Pueblo Indians.

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Schillaci, Michael A; Nikitovic, Dejana; Akins, Nancy J; Tripp, Lianne; Palkovich, Ann M

2011-06-01

The present study examines patterns of infant and juvenile growth in a diachronic sample of ancestral Pueblo Indians (AD 1300-1680) from the American Southwest. An assessment of growth patterns is accompanied by an evaluation of pathological conditions often considered to be indicators of nutritional deficiencies and/or gastrointestinal infections. Growth patterns and the distribution of pathological conditions are interpreted relative to culturally relevant age categories defined by Puebloan rites of passage described in the ethnographic literature. A visual comparison of growth distance curves revealed that relative to a modern comparative group our sample of ancestral Pueblo infant and juveniles exhibited faltering growth beginning soon after birth to about 5 years of age. A comparison of curves describing growth relative to adult femoral length, however, indicated reduced growth occurring later, by around 2 years of age. Similar to previous studies, we observed a high proportion of nonsurvivors exhibiting porotic cranial lesions during the first 2 years of life. Contrary to expectations, infants and juveniles without evidence of porotic cranial lesions exhibited a higher degree of stunting. Our study is generally consistent with previous research reporting poor health and high mortality for ancestral Pueblo Indian infants and juveniles. Through use of a culturally relevant context defining childhood, we argue that the observed poor health and high mortality in our sample occur before the important transition from young to older child and the concomitant initial incorporation into tribal ritual organization. Copyright © 2011 Wiley-Liss, Inc.

Choosing the best ancestral character state reconstruction method.

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Royer-Carenzi, Manuela; Pontarotti, Pierre; Didier, Gilles

2013-03-01

Despite its intrinsic difficulty, ancestral character state reconstruction is an essential tool for testing evolutionary hypothesis. Two major classes of approaches to this question can be distinguished: parsimony- or likelihood-based approaches. We focus here on the second class of methods, more specifically on approaches based on continuous-time Markov modeling of character evolution. Among them, we consider the most-likely-ancestor reconstruction, the posterior-probability reconstruction, the likelihood-ratio method, and the Bayesian approach. We discuss and compare the above-mentioned methods over several phylogenetic trees, adding the maximum-parsimony method performance in the comparison. Under the assumption that the character evolves according a continuous-time Markov process, we compute and compare the expectations of success of each method for a broad range of model parameter values. Moreover, we show how the knowledge of the evolution model parameters allows to compute upper bounds of reconstruction performances, which are provided as references. The results of all these reconstruction methods are quite close one to another, and the expectations of success are not so far from their theoretical upper bounds. But the performance ranking heavily depends on the topology of the studied tree, on the ancestral node that is to be inferred and on the parameter values. Consequently, we propose a protocol providing for each parameter value the best method in terms of expectation of success, with regard to the phylogenetic tree and the ancestral node to infer. Copyright © 2012 Elsevier Inc. All rights reserved.

Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity

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Justin Judd

2012-01-01

Full Text Available Adeno-associated virus (AAV-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase. The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.

Imaging and Quantitation of a Succession of Transient Intermediates Reveal the Reversible Self-Assembly Pathway of a Simple Icosahedral Virus Capsid.

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Medrano, María; Fuertes, Miguel Ángel; Valbuena, Alejandro; Carrillo, Pablo J P; Rodríguez-Huete, Alicia; Mateu, Mauricio G

2016-11-30

Understanding the fundamental principles underlying supramolecular self-assembly may facilitate many developments, from novel antivirals to self-organized nanodevices. Icosahedral virus particles constitute paradigms to study self-assembly using a combination of theory and experiment. Unfortunately, assembly pathways of the structurally simplest virus capsids, those more accessible to detailed theoretical studies, have been difficult to study experimentally. We have enabled the in vitro self-assembly under close to physiological conditions of one of the simplest virus particles known, the minute virus of mice (MVM) capsid, and experimentally analyzed its pathways of assembly and disassembly. A combination of electron microscopy and high-resolution atomic force microscopy was used to structurally characterize and quantify a succession of transient assembly and disassembly intermediates. The results provided an experiment-based model for the reversible self-assembly pathway of a most simple (T = 1) icosahedral protein shell. During assembly, trimeric capsid building blocks are sequentially added to the growing capsid, with pentamers of building blocks and incomplete capsids missing one building block as conspicuous intermediates. This study provided experimental verification of many features of self-assembly of a simple T = 1 capsid predicted by molecular dynamics simulations. It also demonstrated atomic force microscopy imaging and automated analysis, in combination with electron microscopy, as a powerful single-particle approach to characterize at high resolution and quantify transient intermediates during supramolecular self-assembly/disassembly reactions. Finally, the efficient in vitro self-assembly achieved for the oncotropic, cell nucleus-targeted MVM capsid may facilitate its development as a drug-encapsidating nanoparticle for anticancer targeted drug delivery.

AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

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Daniel J Hui

Full Text Available Adeno-associated virus (AAV has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC class I epitopes for common human leukocyte antigen (HLA types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX

International Nuclear Information System (INIS)

Zakhartchouk, Alexander; Connors, Wayne; Van Kessel, Andrew; Tikoo, Suresh Kumar

2004-01-01

Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions. Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion. This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins. We constructed recombinant BAV950 containing a small peptide (21 amino acid), including the RGD motif or recombinant BAV951 containing enhanced yellow-green fluorescent protein (EYFP) fused to the C-terminus of pIX. Western blot analysis demonstrated that the chimeric pIX-RGD was incorporated into virion capsids. Incorporation of the RGD motif into the pIX resulted in significant augmentation of BAdV-3 fiber knob-independent infection of the integrin-positive cells, suggesting that RGD motifs are displayed on the surface of virion capsids and are accessible for binding to integrins. Analysis of BAV951 revealed that the chimeric pIX is incorporated into virion capsids and EYFP containing the C-terminus of pIX is exposed on the surface of the virion. Moreover, insertion of chimeric pIXs was maintained without change through successive rounds of viral replication. These results suggested that in contrast to major capsid proteins (hexon, penton, fiber), the minor capsid protein IX can be use for the incorporation of targeting ligands based on either small peptides or longer polypeptides

Electrostatic potential of human immunodeficiency virus type 2 and rhesus macaque simian immunodeficiency virus capsid proteins

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Katarzyna eBozek

2012-06-01

Full Text Available Human immunodeficiency virus type 2 (HIV-2 and simian immunodeficiency virus isolated from a macaque monkey (SIVmac are assumed to have originated from simian immunodeficiency virus isolated from sooty mangabey (SIVsm. Despite their close similarity in genome structure, HIV-2 and SIVmac show different sensitivities to TRIM5α, a host restriction factor against retroviruses. The replication of HIV-2 strains is potently restricted by rhesus (Rh monkey TRIM5α, while that of SIVmac strain 239 (SIVmac239 is not. Viral capsid protein is the determinant of this differential sensitivity to TRIM5α, as the HIV-2 mutant carrying SIVmac239 capsid protein evaded Rh TRIM5α-mediated restriction. However, the molecular determinants of this restriction mechanism are unknown. Electrostatic potential on the protein-binding site is one of the properties regulating protein-protein interactions. In this study, we investigated the electrostatic potential on the interaction surface of capsid protein of HIV-2 strain GH123 and SIVmac239. Although HIV-2 GH123 and SIVmac239 capsid proteins share more than 87% amino acid identity, we observed a large difference between the two molecules with the HIV-2 GH123 molecule having predominantly positive and SIVmac239 predominantly negative electrostatic potential on the surface of the loop between α-helices 4 and 5 (L4/5. As L4/5 is one of the major determinants of Rh TRIM5α sensitivity of these viruses, the present results suggest that the binding site of the Rh TRIM5α may show complementarity to the HIV-2 GH123 capsid surface charge distribution.

Solid-to-fluid DNA transition inside HSV-1 capsid close to the temperature of infection

Energy Technology Data Exchange (ETDEWEB)

Sae-Ueng, Udom; Li, Dong; Zuo, Xiaobing; Huffman, Jamie B.; Homa, Fred L.; Rau, Donald; Evilevitch, Alex

2014-10-01

DNA in the human Herpes simplex virus type 1 (HSV-1) capsid is packaged to a tight density. This leads to tens of atmospheres of internal pressure responsible for the delivery of the herpes genome into the cell nucleus. In this study we show that, despite its liquid crystalline state inside the capsid, the DNA is fluid-like, which facilitates its ejection into the cell nucleus during infection. We found that the sliding friction between closely packaged DNA strands, caused by interstrand repulsive interactions, is reduced by the ionic environment of epithelial cells and neurons susceptible to herpes infection. However, variations in the ionic conditions corresponding to neuronal activity can restrict DNA mobility in the capsid, making it more solid-like. This can inhibit intranuclear DNA release and interfere with viral replication. In addition, the temperature of the human host (37 °C) induces a disordering transition of the encapsidated herpes genome, which reduces interstrand interactions and provides genome mobility required for infection.

Indigenous ancestral sayings contribute to modern conservation partnerships: examples using Phormium tenax.

Science.gov (United States)

Wehi, Priscilla M

2009-01-01

Traditional ecological knowledge (TEK) is central to indigenous worldviews and practices and is one of the most important contributions that indigenous people can bring to conservation management partnerships. However, researchers and managers may have difficulty accessing such knowledge, particularly where knowledge transmission has been damaged. A new methodological approach analyzes ancestral sayings from Maori oral traditions for ecological information about Phormium tenax, a plant with high cultural value that is a dominant component in many threatened wetland systems, and frequently used in restoration plantings in New Zealand. Maori ancestral sayings record an association with nectar-feeding native parrots that has only rarely been reported, as well as indications of important environmental parameters (rainfall and drought) for this species. These sayings provide evidence of indigenous management that has not been reported from interviews with elders, including evidence of fire use to create Phormium cultivations. TEK in Maori ancestral sayings imply landscape-scale processes in comparison to intensive, small-scale management methods often reported in interviews. TEK in ancestral sayings can be used to generate new scientific hypotheses, negotiate collaborative pathways, and identify ecological management strategies that support biodiversity retention. TEK can inform restoration ecology, historical ecology, and conservation management of species and ecosystems, especially where data from pollen records and archaeological artifacts are incomplete.

Chronic hepatitis B infection and HBV DNA-containing capsids: Modeling and analysis

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Manna, Kalyan; Chakrabarty, Siddhartha P.

2015-05-01

We analyze the dynamics of chronic HBV infection taking into account both uninfected and infected hepatocytes along with the intracellular HBV DNA-containing capsids and the virions. While previous HBV models have included either the uninfected hepatocytes or the intracellular HBV DNA-containing capsids, our model accounts for both these two populations. We prove the conditions for local and global stability of both the uninfected and infected steady states in terms of the basic reproduction number. Further, we incorporate a time lag in the model to encompass the intracellular delay in the production of the infected hepatocytes and find that this delay does not affect the overall dynamics of the system. The results for the model and the delay model are finally numerically illustrated.

α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

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Mayim E. Wiens

2017-01-01

Full Text Available α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5 blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses.

Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane.

Science.gov (United States)

Bailer, Susanne M.

2017-11-25

Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3.

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Iverson, Eric A; Goodman, David A; Gorchels, Madeline E; Stedman, Kenneth M

2017-05-15

Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae , where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3 , allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of

Dengue Virus Uses a Non-Canonical Function of the Host GBF1-Arf-COPI System for Capsid Protein Accumulation on Lipid Droplets.

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Iglesias, Nestor G; Mondotte, Juan A; Byk, Laura A; De Maio, Federico A; Samsa, Marcelo M; Alvarez, Cecilia; Gamarnik, Andrea V

2015-09-01

Dengue viruses cause the most important human viral disease transmitted by mosquitoes. In recent years, a great deal has been learned about molecular details of dengue virus genome replication; however, little is known about genome encapsidation and the functions of the viral capsid protein. During infection, dengue virus capsid progressively accumulates around lipid droplets (LDs) by an unknown mechanism. Here, we examined the process by which the viral capsid is transported from the endoplasmic reticulum (ER) membrane, where the protein is synthesized, to LDs. Using different methods of intervention, we found that the GBF1-Arf1/Arf4-COPI pathway is necessary for capsid transport to LDs, while the process is independent of both COPII components and Golgi integrity. The transport was sensitive to Brefeldin A, while a drug resistant form of GBF1 was sufficient to restore capsid subcellular distribution in infected cells. The mechanism by which LDs gain or lose proteins is still an open question. Our results support a model in which the virus uses a non-canonical function of the COPI system for capsid accumulation on LDs, providing new ideas for antiviral strategies. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Modeling X-linked ancestral origins in multiparental populations

NARCIS (Netherlands)

Zheng, Chaozhi

2015-01-01

The models for the mosaic structure of an individual's genome from multiparental populations have been developed primarily for autosomes, whereas X chromosomes receive very little attention. In this paper, we extend our previous approach to model ancestral origin processes along two X chromosomes

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid.

Directory of Open Access Journals (Sweden)

Neil J Ball

2016-11-01

Full Text Available The Spumaretrovirinae, or foamy viruses (FVs are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV. The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA and C-terminal domains (CtDCA of archetypal orthoretroviral capsid protein (CA. Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN-CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold.

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid

Science.gov (United States)

Dutta, Moumita; Pollard, Dominic J.; Goldstone, David C.; Ramos, Andres; Müllers, Erik; Stirnnagel, Kristin; Stanke, Nicole; Lindemann, Dirk; Taylor, William R.; Rosenthal, Peter B.

2016-01-01

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold. PMID:27829070

Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds

Czech Academy of Sciences Publication Activity Database

Machara, A.; Lux, V.; Kožíšek, Milan; Grantz Šašková, Klára; Štěpánek, O.; Kotora, M.; Parkan, Kamil; Pávová, Marcela; Glass, B.; Sehr, P.; Lewis, J.; Müller, B.; Kräusslich, H. G.; Konvalinka, Jan

2016-01-01

Roč. 59, č. 2 (2016), s. 545-558 ISSN 0022-2623 R&D Projects: GA ČR GA13-19561S EU Projects: European Commission(XE) 201095 - HIV ACE Institutional support: RVO:61388963 Keywords : HIV -1 assembly * capsid * high-throughput screening * AlphaScreen assay Subject RIV: CE - Biochemistry Impact factor: 6.259, year: 2016

On the Accuracy of Ancestral Sequence Reconstruction for Ultrametric Trees with Parsimony.

Science.gov (United States)

Herbst, Lina; Fischer, Mareike

2018-04-01

We examine a mathematical question concerning the reconstruction accuracy of the Fitch algorithm for reconstructing the ancestral sequence of the most recent common ancestor given a phylogenetic tree and sequence data for all taxa under consideration. In particular, for the symmetric four-state substitution model which is also known as Jukes-Cantor model, we answer affirmatively a conjecture of Li, Steel and Zhang which states that for any ultrametric phylogenetic tree and a symmetric model, the Fitch parsimony method using all terminal taxa is more accurate, or at least as accurate, for ancestral state reconstruction than using any particular terminal taxon or any particular pair of taxa. This conjecture had so far only been answered for two-state data by Fischer and Thatte. Here, we focus on answering the biologically more relevant case with four states, which corresponds to ancestral sequence reconstruction from DNA or RNA data.

Development and validation of novel AAV2 random libraries displaying peptides of diverse lengths and at diverse capsid positions.

Science.gov (United States)

Naumer, Matthias; Ying, Ying; Michelfelder, Stefan; Reuter, Antje; Trepel, Martin; Müller, Oliver J; Kleinschmidt, Jürgen A

2012-05-01

Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

International Nuclear Information System (INIS)

Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.; Sinning, Allan; Henegar, Jeffrey; Norcross, Erin; Chinchar, V. Gregory

2010-01-01

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

Outer capsid proteins induce the formation of pores in epithelial cells

International Nuclear Information System (INIS)

Ruiz, M; Abad M; Michelangely, F; Charpilienne, A; Cohen, J

1995-01-01

Two mechanisms of entrance in cell of the rotavirus, during the infection, were proposed: a direct entrance through the plasmatic membrane or by means of endocytosis. In the two cases, a permeabilization mechanism of the membrane (cellular or of the endocytic vesicle, respectively) should occur. It has been shown that the rotavirus induces permeabilization of liposomes and of membrane vesicles. In this work, are studied the changes of intact cells permeability, measuring the entrance of e tide bromides. Viral particles of double capsid of the RF stump produce an increase of the cells membrane MA104 permeability, while the simple capsid ones don't induce effect. This phenomenon requires the particles trypsinization, and occurs in a means where the concentration of free Ca is lower to 1 micromolar. The temporary course of the fluorescence increase is sigmoid. The latency, the speed and the width depend on the relationship of virus / cell, and it can be observed up to 100% of permeabilization in relation to the effect of digitonin. The pores induced in the membrane by the rotavirus are irreversible. The permeabilizer effect of the rotavirus on the membrane was observed in other cellular lines as Hela and HT29, but not in the L929 ones. These results suggest that one or more proteins of the external capsid are responsible s of the effect. These could be involved in the penetration process of the virus towards the cytoplasm and could be one of the restrictive factor of the cell infection by means of the virus [es

Environmental enrichment mitigates the impact of ancestral stress on motor skill and corticospinal tract plasticity.

Science.gov (United States)

McCreary, J Keiko; Erickson, Zachary T; Metz, Gerlinde A S

2016-10-06

An adverse fetal environment in utero has been associated with long-term alterations in brain structure and function, and a higher risk of neurological disorders in later life. A common consequence of early adverse experience is impaired motor system function. A causal relationship for stress-associated impairments and a suitable therapy, however, have not been determined yet. To investigate the impact of ancestral stress on corticospinal tract (CST) morphology and fine motor performance in rats, and to determine if adverse programming by ancestral stress can be mitigated by environmental enrichment therapy in rats. The study examined F3 offspring generated by three lineages; one with prenatal stress only in the F1 generation, one with compounding effects of multigenerational prenatal stress, and a non-stress control lineage. F3 offspring from each lineage were injected with biotinylated dextran amine (BDA) into the motor cortex for anterograde tracing of the CST. Examination of the CST revealed reduced axonal density in the ancestrally stressed lineages. These anatomical changes were associated with significant impairments in skilled walking, as indicated by reduced foot placement accuracy and disturbed inter-limb coordination. Therapeutic intervention by environmental enrichment reduced the neuromorphological consequences of ancestral stress and restored skilled walking ability. The data suggest a causal relationship between stress-induced abnormal CST function and loss of fine motor performance. Thus, ancestral stress may be a determinant of motor system development and motor skill. Environmental enrichment may represent an effective intervention for the adverse programming by ancestral stress and trauma. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Foot-and-mouth disease virus capsid proteins; analysis of protein processing, assembly and utility as vaccines

DEFF Research Database (Denmark)

Belsham, Graham

Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. The infection is caused by foot-and-mouth disease virus (FMDV), a member of the picornavirus family. The positive sense RNA genome of the virus includes a single, large......, open reading frame that encodes a polyprotein. The intact polyprotein is never observed as it is processed, both during and after translation, to 15 different mature proteins plus a variety of precursors. The FMDV capsid protein precursor, P1-2A, is cleaved by the virus encoded 3C protease (3Cpro......) to generate VP0, VP3, VP1 and the peptide 2A. Sixty copies of each of the capsid proteins “self-assemble” into empty capsid particles or with the RNA genome into infectious viruses. These particles normally lack 2A but it is possible to construct and isolate mutant FMDVs in which the cleavage of the VP1/2A...

MADS goes genomic in conifers: towards determining the ancestral set of MADS-box genes in seed plants.

Science.gov (United States)

Gramzow, Lydia; Weilandt, Lisa; Theißen, Günter

2014-11-01

MADS-box genes comprise a gene family coding for transcription factors. This gene family expanded greatly during land plant evolution such that the number of MADS-box genes ranges from one or two in green algae to around 100 in angiosperms. Given the crucial functions of MADS-box genes for nearly all aspects of plant development, the expansion of this gene family probably contributed to the increasing complexity of plants. However, the expansion of MADS-box genes during one important step of land plant evolution, namely the origin of seed plants, remains poorly understood due to the previous lack of whole-genome data for gymnosperms. The newly available genome sequences of Picea abies, Picea glauca and Pinus taeda were used to identify the complete set of MADS-box genes in these conifers. In addition, MADS-box genes were identified in the growing number of transcriptomes available for gymnosperms. With these datasets, phylogenies were constructed to determine the ancestral set of MADS-box genes of seed plants and to infer the ancestral functions of these genes. Type I MADS-box genes are under-represented in gymnosperms and only a minimum of two Type I MADS-box genes have been present in the most recent common ancestor (MRCA) of seed plants. In contrast, a large number of Type II MADS-box genes were found in gymnosperms. The MRCA of extant seed plants probably possessed at least 11-14 Type II MADS-box genes. In gymnosperms two duplications of Type II MADS-box genes were found, such that the MRCA of extant gymnosperms had at least 14-16 Type II MADS-box genes. The implied ancestral set of MADS-box genes for seed plants shows simplicity for Type I MADS-box genes and remarkable complexity for Type II MADS-box genes in terms of phylogeny and putative functions. The analysis of transcriptome data reveals that gymnosperm MADS-box genes are expressed in a great variety of tissues, indicating diverse roles of MADS-box genes for the development of gymnosperms. This study is

Historian: accurate reconstruction of ancestral sequences and evolutionary rates.

Science.gov (United States)

Holmes, Ian H

2017-04-15

Reconstruction of ancestral sequence histories, and estimation of parameters like indel rates, are improved by using explicit evolutionary models and summing over uncertain alignments. The previous best tool for this purpose (according to simulation benchmarks) was ProtPal, but this tool was too slow for practical use. Historian combines an efficient reimplementation of the ProtPal algorithm with performance-improving heuristics from other alignment tools. Simulation results on fidelity of rate estimation via ancestral reconstruction, along with evaluations on the structurally informed alignment dataset BAliBase 3.0, recommend Historian over other alignment tools for evolutionary applications. Historian is available at https://github.com/evoldoers/historian under the Creative Commons Attribution 3.0 US license. ihholmes+historian@gmail.com. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.

Science.gov (United States)

Pascual, Elena; Mata, Carlos P; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L; Castón, José R

2015-03-01

Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign

Reconstructed Ancestral Enzymes Impose a Fitness Cost upon Modern Bacteria Despite Exhibiting Favourable Biochemical Properties.

Science.gov (United States)

Hobbs, Joanne K; Prentice, Erica J; Groussin, Mathieu; Arcus, Vickery L

2015-10-01

Ancestral sequence reconstruction has been widely used to study historical enzyme evolution, both from biochemical and cellular perspectives. Two properties of reconstructed ancestral proteins/enzymes are commonly reported--high thermostability and high catalytic activity--compared with their contemporaries. Increased protein stability is associated with lower aggregation rates, higher soluble protein abundance and a greater capacity to evolve, and therefore, these proteins could be considered "superior" to their contemporary counterparts. In this study, we investigate the relationship between the favourable in vitro biochemical properties of reconstructed ancestral enzymes and the organismal fitness they confer in vivo. We have previously reconstructed several ancestors of the enzyme LeuB, which is essential for leucine biosynthesis. Our initial fitness experiments revealed that overexpression of ANC4, a reconstructed LeuB that exhibits high stability and activity, was only able to partially rescue the growth of a ΔleuB strain, and that a strain complemented with this enzyme was outcompeted by strains carrying one of its descendants. When we expanded our study to include five reconstructed LeuBs and one contemporary, we found that neither in vitro protein stability nor the catalytic rate was correlated with fitness. Instead, fitness showed a strong, negative correlation with estimated evolutionary age (based on phylogenetic relationships). Our findings suggest that, for reconstructed ancestral enzymes, superior in vitro properties do not translate into organismal fitness in vivo. The molecular basis of the relationship between fitness and the inferred age of ancestral LeuB enzymes is unknown, but may be related to the reconstruction process. We also hypothesise that the ancestral enzymes may be incompatible with the other, contemporary enzymes of the metabolic network.

Ancestral Rocky Mountian Tectonics: A Sedimentary Record of Ancestral Front Range and Uncompahgre Exhumation

Science.gov (United States)

Smith, T. M.; Saylor, J. E.; Lapen, T. J.

2015-12-01

The Ancestral Rocky Mountains (ARM) encompass multiple crustal provinces with characteristic crystallization ages across the central and western US. Two driving mechanisms have been proposed to explain ARM deformation. (1) Ouachita-Marathon collision SE of the ARM uplifts has been linked to an E-to-W sequence of uplift and is consistent with proposed disruption of a larger Paradox-Central Colorado Trough Basin by exhumation of the Uncompahgre Uplift. Initial exhumation of the Amarillo-Wichita Uplift to the east would provide a unique ~530 Ma signal absent from source areas to the SW, and result in initial exhumation of the Ancestral Front Range. (2) Alternatively, deformation due to flat slab subduction along a hypothesized plate boundary to the SW suggests a SW-to-NE younging of exhumation. This hypothesis suggests a SW-derived Grenville signature, and would trigger uplift of the Uncompahgre first. We analyzed depositional environments, sediment dispersal patterns, and sediment and basement zircon U-Pb and (U-Th)/He ages in 3 locations in the Paradox Basin and Central Colorado Trough (CCT). The Paradox Basin exhibits an up-section transition in fluvial style that suggests a decrease in overbank stability and increased lateral migration. Similarly, the CCT records a long-term progradation of depositional environments from marginal marine to fluvial, indicating that sediment supply in both basins outpaced accommodation. Preliminary provenance results indicate little to no input from the Amarillo-Wichita uplift in either basin despite uniformly westward sediment dispersal systems in both basins. Results also show that the Uncompahgre Uplift was the source for sediment throughout Paradox Basin deposition. These observations are inconsistent with the predictions of scenario 1 above. Rather, they suggest either a synchronous response to tectonic stress across the ARM provinces or an SW-to-NE pattern of deformation.

The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion.

Directory of Open Access Journals (Sweden)

Shu-Fan Chou

2015-10-01

Full Text Available The Endosomal Sorting Complex Required for Transport (ESCRT is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV, we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1-147 containing no arginine-rich domain (ARD failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1-147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex.

The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion

Science.gov (United States)

Chou, Shu-Fan; Tsai, Ming-Lin; Huang, Jyun-Yuan; Chang, Ya-Shu; Shih, Chiaho

2015-01-01

The Endosomal Sorting Complex Required for Transport (ESCRT) is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV), we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1–147) containing no arginine-rich domain (ARD) failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1–147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex. PMID

Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

NARCIS (Netherlands)

Roos, W.H.; Radtke, K.; Kniesmeijer, E.G.R.; Geertsema, H.J.; Sodeik, B.; Wuite, G.J.L.

2009-01-01

Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze

Scaffold expulsion and genome packaging trigger stabilization of Herpes Simplex Virus capsids

NARCIS (Netherlands)

Roos, W.H.; Radtke, K.; Kniesmeijer, E.; Geertsema, H.J.; Sodeik, B.; Wuite, G.J.L.

2009-01-01

Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze

Ancestral TCDD exposure promotes epigenetic transgenerational inheritance of imprinted gene Igf2: Methylation status and DNMTs

International Nuclear Information System (INIS)

Ma, Jing; Chen, Xi; Liu, Yanan; Xie, Qunhui; Sun, Yawen; Chen, Jingshan; Leng, Ling; Yan, Huan; Zhao, Bin; Tang, Naijun

2015-01-01

Ancestral TCDD exposure could induce epigenetic transgenerational phenotypes, which may be mediated in part by imprinted gene inheritance. The aim of our study was to evaluate the transgenerational effects of ancestral TCDD exposure on the imprinted gene insulin-like growth factor-2 (Igf2) in rat somatic tissue. TCDD was administered daily by oral gavage to groups of F0 pregnant SD rats at dose levels of 0 (control), 200 or 800 ng/kg bw during gestation day 8–14. Animal transgenerational model of ancestral exposure to TCDD was carefully built, avoiding sibling inbreeding. Hepatic Igf2 expression of the TCDD male progeny was decreased concomitantly with hepatic damage and increased activities of serum hepatic enzymes both in the F1 and F3 generation. Imprinted Control Region (ICR) of Igf2 manifested a hypermethylated pattern, whereas methylation status in the Differentially Methylated Region 2 (DMR2) showed a hypomethylated manner in the F1 generation. These epigenetic alterations in these two regions maintained similar trends in the F3 generation. Meanwhile, the expressions of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) changed in a non-monotonic manner both in the F1 and F3 generation. This study provides evidence that ancestral TCDD exposure may promote epigenetic transgenerational alterations of imprinted gene Igf2 in adult somatic tissue. - Highlights: • Ancestral TCDD exposure induces epigenetic transgenerational inheritance. • Ancestral TCDD exposure affects methylation status in ICR and DMR2 region of Igf2. • DNMTs play a role in TCDD induced epigenetic transgenerational changes of Igf2.

Ancestral TCDD exposure promotes epigenetic transgenerational inheritance of imprinted gene Igf2: Methylation status and DNMTs

Energy Technology Data Exchange (ETDEWEB)

Ma, Jing; Chen, Xi; Liu, Yanan [Department of Occupational and Environmental Health, School of Public Health, Tianjin Medical University, Tianjin 300070 (China); Xie, Qunhui [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Sun, Yawen; Chen, Jingshan; Leng, Ling; Yan, Huan [Department of Occupational and Environmental Health, School of Public Health, Tianjin Medical University, Tianjin 300070 (China); Zhao, Bin, E-mail: binzhao@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Tang, Naijun, E-mail: tangnaijun@tijmu.edu.cn [Department of Occupational and Environmental Health, School of Public Health, Tianjin Medical University, Tianjin 300070 (China)

2015-12-01

Ancestral TCDD exposure could induce epigenetic transgenerational phenotypes, which may be mediated in part by imprinted gene inheritance. The aim of our study was to evaluate the transgenerational effects of ancestral TCDD exposure on the imprinted gene insulin-like growth factor-2 (Igf2) in rat somatic tissue. TCDD was administered daily by oral gavage to groups of F0 pregnant SD rats at dose levels of 0 (control), 200 or 800 ng/kg bw during gestation day 8–14. Animal transgenerational model of ancestral exposure to TCDD was carefully built, avoiding sibling inbreeding. Hepatic Igf2 expression of the TCDD male progeny was decreased concomitantly with hepatic damage and increased activities of serum hepatic enzymes both in the F1 and F3 generation. Imprinted Control Region (ICR) of Igf2 manifested a hypermethylated pattern, whereas methylation status in the Differentially Methylated Region 2 (DMR2) showed a hypomethylated manner in the F1 generation. These epigenetic alterations in these two regions maintained similar trends in the F3 generation. Meanwhile, the expressions of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) changed in a non-monotonic manner both in the F1 and F3 generation. This study provides evidence that ancestral TCDD exposure may promote epigenetic transgenerational alterations of imprinted gene Igf2 in adult somatic tissue. - Highlights: • Ancestral TCDD exposure induces epigenetic transgenerational inheritance. • Ancestral TCDD exposure affects methylation status in ICR and DMR2 region of Igf2. • DNMTs play a role in TCDD induced epigenetic transgenerational changes of Igf2.

Asymptotic distributions of coalescence times and ancestral lineage numbers for populations with temporally varying size.

Science.gov (United States)

Chen, Hua; Chen, Kun

2013-07-01

The distributions of coalescence times and ancestral lineage numbers play an essential role in coalescent modeling and ancestral inference. Both exact distributions of coalescence times and ancestral lineage numbers are expressed as the sum of alternating series, and the terms in the series become numerically intractable for large samples. More computationally attractive are their asymptotic distributions, which were derived in Griffiths (1984) for populations with constant size. In this article, we derive the asymptotic distributions of coalescence times and ancestral lineage numbers for populations with temporally varying size. For a sample of size n, denote by Tm the mth coalescent time, when m + 1 lineages coalesce into m lineages, and An(t) the number of ancestral lineages at time t back from the current generation. Similar to the results in Griffiths (1984), the number of ancestral lineages, An(t), and the coalescence times, Tm, are asymptotically normal, with the mean and variance of these distributions depending on the population size function, N(t). At the very early stage of the coalescent, when t → 0, the number of coalesced lineages n - An(t) follows a Poisson distribution, and as m → n, $$n\\left(n-1\\right){T}_{m}/2N\\left(0\\right)$$ follows a gamma distribution. We demonstrate the accuracy of the asymptotic approximations by comparing to both exact distributions and coalescent simulations. Several applications of the theoretical results are also shown: deriving statistics related to the properties of gene genealogies, such as the time to the most recent common ancestor (TMRCA) and the total branch length (TBL) of the genealogy, and deriving the allele frequency spectrum for large genealogies. With the advent of genomic-level sequencing data for large samples, the asymptotic distributions are expected to have wide applications in theoretical and methodological development for population genetic inference.

Social capital and health: evidence that ancestral trust promotes health among children of immigrants.

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Ljunge, Martin

2014-12-01

This paper presents evidence that generalized trust promotes health. Children of immigrants in a broad set of European countries with ancestry from across the world are studied. Individuals are examined within country of residence using variation in trust across countries of ancestry. The approach addresses reverse causality and concerns that the trust measure picks up institutional factors in the individual's contextual setting. There is a significant positive estimate of ancestral trust in explaining self-assessed health. The finding is robust to accounting for individual, parental, and extensive ancestral country characteristics. Individuals with higher ancestral trust are also less likely to be hampered by health problems in their daily life, providing evidence of trust influencing real life outcomes. Individuals with high trust feel and act healthier, enabling a more productive life.

Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17-36.

Science.gov (United States)

Bywaters, Stephanie M; Brendle, Sarah A; Tossi, Kerstin P; Biryukov, Jennifer; Meyers, Craig; Christensen, Neil D

2017-11-10

The currently available nonavalent human papillomavirus (HPV) vaccine exploits the highly antigenic L1 major capsid protein to promote high-titer neutralizing antibodies, but is limited to the HPV types included in the vaccine since the responses are highly type-specific. The limited cross-protection offered by the L1 virus-like particle (VLP) vaccine warrants further investigation into cross-protective L2 epitopes. The L2 proteins are yet to be fully characterized as to their precise placement in the virion. Adding to the difficulties in localizing L2, studies have suggested that L2 epitopes are not well exposed on the surface of the mature capsid prior to cellular engagement. Using a series of competition assays between previously mapped anti-L1 monoclonal antibodies (mAbs) (H16.V5, H16.U4 and H16.7E) and novel anti-L2 mAbs, we probed the capsid surface for the location of an L2 epitope (aa17-36). The previously characterized L1 epitopes together with our competition data is consistent with a proposed L2 epitope within the canyons of pentavalent capsomers.

An Epistemological Analysis of the African Ontology of `Ancestral ...

African Journals Online (AJOL)

The paper explores the contemporary debate surrounding the idea of ancestral reincarnation in African society and philosophy. It analyzes various problem areas having to do with the physical and spiritual status of ancestors, their relationship with their societies of orientation, the philosophical contexts of their existence, ...

Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance

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Asunción Delgado

2017-05-01

Full Text Available Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance.

Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals.

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Broce, Sean; Hensley, Lisa; Sato, Tomoharu; Lehrer-Graiwer, Joshua; Essrich, Christian; Edwards, Katie J; Pajda, Jacqueline; Davis, Christopher J; Bhadresh, Rami; Hurt, Clarence R; Freeman, Beverly; Lingappa, Vishwanath R; Kelleher, Colm A; Karpuj, Marcela V

2016-05-14

Viral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus. Here, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures. These results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner. Reviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers' comments section.

Processing of the VP1/2A junction is not necessary for production of foot-and-mouth disease virus empty capsids and infectious viruses: characterization of "self-tagged" particles.

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Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette; Belsham, Graham J

2013-11-01

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

Herpes Simplex Virus 1 Us3 Deletion Mutant is Infective Despite Impaired Capsid Translocation to the Cytoplasm

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Peter Wild

2015-01-01

Full Text Available Herpes simplex virus 1 (HSV-1 capsids are assembled in the nucleus bud at the inner nuclear membrane into the perinuclear space, acquiring envelope and tegument. In theory, these virions are de-enveloped by fusion of the envelope with the outer nuclear membrane and re-enveloped by Golgi membranes to become infective. Us3 enables the nucleus to cytoplasm capsid translocation. Nevertheless, Us3 is not essential for the production of infective progeny viruses. Determination of phenotype distribution by quantitative electron microscopy, and calculation per mean nuclear or cell volume revealed the following: (i The number of R7041(∆US3 capsids budding at the inner nuclear membrane was significantly higher than that of wild type HSV-1; (ii The mean number of R7041(∆US3 virions per mean cell volume was 2726, that of HSV-1 virions 1460 by 24 h post inoculation; (iii 98% of R7041(∆US3 virions were in the perinuclear space; (iv The number of R7041(∆US3 capsids in the cytoplasm, including those budding at Golgi membranes, was significantly reduced. Cell associated R7041(∆US3 yields were 2.37 × 108 and HSV-1 yields 1.57 × 108 PFU/mL by 24 h post inoculation. We thus conclude that R7041(∆US3 virions, which acquire envelope and tegument by budding at the inner nuclear membrane into the perinuclear space, are infective.

Evidence that the ancestral haplotype in Australian hemochromatosis patients may be associated with a common mutation in the gene

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Crawford, D.H.G.; Powell, L.W.; Leggett, B.A. [Univ. of Queensland (Australia)] [and others

1995-08-01

Hemochromatosis (HC) is a common inherited disorder of iron metabolism for which neither the gene nor biochemical defect have yet been identified. The aim of this study was to look for clinical evidence that the predominant ancestral haplotype in Australian patients is associated with a common mutation in the gene. We compared indices of iron metabolism and storage in three groups of HC patients categorized according to the presence of the ancestral haplotype (i.e., patients with two copies, one copy, and no copies of the ancestral haplotype). We also examined iron indices in two groups of HC heterozygotes (those with the ancestral haplotype and those without) and in age-matched controls. These analyses indicate that (i) HC patients with two copies of the ancestral haplotype show significantly more severe expression of the disorder than those with one copy or those without, (ii) HC heterozygotes have partial clinical expression, which may be influenced by the presence of the ancestral haplotype in females but not in males, and (iii) the high population frequency of the HC gene may be the result of the selective advantage conferred by protecting heterozygotes against iron deficiency. 18 refs., 3 tabs.

Novel Infectivity-Enhanced Oncolytic Adenovirus with a Capsid-Incorporated Dual-Imaging Moiety for Monitoring Virotherapy in Ovarian Cancer

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Kristopher J. Kimball

2009-09-01

Full Text Available We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for non-invasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk and monomeric red fluorescent protein 1 (mRFP1 into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

Cross-serotype immunity induced by immunization with a conserved rhinovirus capsid protein.

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Nicholas Glanville

Full Text Available Human rhinovirus (RV infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2 capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.

Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity

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Höglund Stefan

2007-09-01

Full Text Available Abstract Background The mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which ultimately leads to substantial rearrangements of the capsid (CAp24 molecule involving both inter- and intra-molecular contacts of the CAp24 molecules. In this aspect, Asp51 which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro1 following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. Thus, previous substitution mutation of Asp51 to alanine (D51A has shown to be lethal and that this invariable residue was found essential for tube formation in vitro, virus replication and virus capsid formation. Results We extended the above investigation by introducing three different D51 substitution mutations (D51N, D51E, and D51Q into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro capsid assembly and virus infectivity. Two substitution mutations (D51E and D51N had no substantial effect on in vitro capsid assembly, yet they impaired viral infectivity and particle production. In contrast, the D51Q mutant was defective both for in vitro capsid assembly and for virus replication in cell culture. Conclusion These results show that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could partially rescue both in vitro capsid assembly and intra-cellular CAp24 production but not replication of the virus in cultured cells.

Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

Science.gov (United States)

Bell, Peter; Vandenberghe, Luk H; Wilson, James M

2014-06-01

Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k.

Science.gov (United States)

Condezo, Gabriela N; Marabini, Roberto; Ayora, Silvia; Carazo, José M; Alba, Raúl; Chillón, Miguel; San Martín, Carmen

2015-09-01

Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the

Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17–36

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Stephanie M. Bywaters

2017-11-01

Full Text Available The currently available nonavalent human papillomavirus (HPV vaccine exploits the highly antigenic L1 major capsid protein to promote high-titer neutralizing antibodies, but is limited to the HPV types included in the vaccine since the responses are highly type-specific. The limited cross-protection offered by the L1 virus-like particle (VLP vaccine warrants further investigation into cross-protective L2 epitopes. The L2 proteins are yet to be fully characterized as to their precise placement in the virion. Adding to the difficulties in localizing L2, studies have suggested that L2 epitopes are not well exposed on the surface of the mature capsid prior to cellular engagement. Using a series of competition assays between previously mapped anti-L1 monoclonal antibodies (mAbs (H16.V5, H16.U4 and H16.7E and novel anti-L2 mAbs, we probed the capsid surface for the location of an L2 epitope (aa17–36. The previously characterized L1 epitopes together with our competition data is consistent with a proposed L2 epitope within the canyons of pentavalent capsomers.

Ancestral state reconstruction, rate heterogeneity, and the evolution of reptile viviparity.

Science.gov (United States)

King, Benedict; Lee, Michael S Y

2015-05-01

Virtually all models for reconstructing ancestral states for discrete characters make the crucial assumption that the trait of interest evolves at a uniform rate across the entire tree. However, this assumption is unlikely to hold in many situations, particularly as ancestral state reconstructions are being performed on increasingly large phylogenies. Here, we show how failure to account for such variable evolutionary rates can cause highly anomalous (and likely incorrect) results, while three methods that accommodate rate variability yield the opposite, more plausible, and more robust reconstructions. The random local clock method, implemented in BEAST, estimates the position and magnitude of rate changes on the tree; split BiSSE estimates separate rate parameters for pre-specified clades; and the hidden rates model partitions each character state into a number of rate categories. Simulations show the inadequacy of traditional models when characters evolve with both asymmetry (different rates of change between states within a character) and heterotachy (different rates of character evolution across different clades). The importance of accounting for rate heterogeneity in ancestral state reconstruction is highlighted empirically with a new analysis of the evolution of viviparity in squamate reptiles, which reveal a predominance of forward (oviparous-viviparous) transitions and very few reversals. © The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Isolation of Ancestral Sylvatic Dengue Virus Type 1, Malaysia

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Teoh, Boon-Teong; Sam, Sing-Sin; Abd-Jamil, Juraina

2010-01-01

Ancestral sylvatic dengue virus type 1, which was isolated from a monkey in 1972, was isolated from a patient with dengue fever in Malaysia. The virus is neutralized by serum of patients with endemic DENV-1 infection. Rare isolation of this virus suggests a limited spillover infection from an otherwise restricted sylvatic cycle. PMID:21029545

Ancestral Variations in the Shape and Size of the Zygoma.

Science.gov (United States)

Oettlé, Anna C; Demeter, Fabrice P; L'abbé, Ericka N

2017-01-01

The variable development of the zygoma, dictating its shape and size variations among ancestral groups, has important clinical implications and valuable anthropological and evolutionary inferences. The purpose of the study was to review the literature regarding the variations in the zygoma with ancestry. Ancestral variation in the zygoma reflects genetic variations because of genetic drift as well as natural selection and epigenetic changes to adapt to diet and climate variations with possible intensification by isolation. Prominence of the zygoma, zygomaxillary tuberosity, and malar tubercle have been associated with Eastern Asian populations in whom these features intensified. Prominence of the zygoma is also associated with groups from Eastern Europe and the rest of Asia. Diffusion of these traits occurred across the Behring Sea to the Arctic areas and to North and South America. The greatest zygomatic projections are exhibited in Arctic groups as an adaptation to extreme cold conditions, while Native South American groups also present with other features of facial robusticity. Groups from Australia, Malaysia, and Oceania show prominence of the zygoma to a certain extent, possibly because of archaic occupations by undifferentiated Southeast Asian populations. More recent interactions with Chinese groups might explain the prominent cheekbones noted in certain South African groups. Many deductions regarding evolutionary processes and diversifications of early groups have been made. Cognisance of these ancestral variations also have implications for forensic anthropological assessments as well as plastic and reconstructive surgery. More studies are needed to improve accuracy of forensic anthropological identification techniques. Anat Rec, 300:196-208, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

What was the ancestral sex-determining mechanism in amniote vertebrates?

Science.gov (United States)

Johnson Pokorná, Martina; Kratochvíl, Lukáš

2016-02-01

Amniote vertebrates, the group consisting of mammals and reptiles including birds, possess various mechanisms of sex determination. Under environmental sex determination (ESD), the sex of individuals depends on the environmental conditions occurring during their development and therefore there are no sexual differences present in their genotypes. Alternatively, through the mode of genotypic sex determination (GSD), sex is determined by a sex-specific genotype, i.e. by the combination of sex chromosomes at various stages of differentiation at conception. As well as influencing sex determination, sex-specific parts of genomes may, and often do, develop specific reproductive or ecological roles in their bearers. Accordingly, an individual with a mismatch between phenotypic (gonadal) and genotypic sex, for example an individual sex-reversed by environmental effects, should have a lower fitness due to the lack of specialized, sex-specific parts of their genome. In this case, evolutionary transitions from GSD to ESD should be less likely than transitions in the opposite direction. This prediction contrasts with the view that GSD was the ancestral sex-determining mechanism for amniote vertebrates. Ancestral GSD would require several transitions from GSD to ESD associated with an independent dedifferentiation of sex chromosomes, at least in the ancestors of crocodiles, turtles, and lepidosaurs (tuataras and squamate reptiles). In this review, we argue that the alternative theory postulating ESD as ancestral in amniotes is more parsimonious and is largely concordant with the theoretical expectations and current knowledge of the phylogenetic distribution and homology of sex-determining mechanisms. © 2014 Cambridge Philosophical Society.

Cases in which ancestral maximum likelihood will be confusingly misleading.

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Handelman, Tomer; Chor, Benny

2017-05-07

Ancestral maximum likelihood (AML) is a phylogenetic tree reconstruction criteria that "lies between" maximum parsimony (MP) and maximum likelihood (ML). ML has long been known to be statistically consistent. On the other hand, Felsenstein (1978) showed that MP is statistically inconsistent, and even positively misleading: There are cases where the parsimony criteria, applied to data generated according to one tree topology, will be optimized on a different tree topology. The question of weather AML is statistically consistent or not has been open for a long time. Mossel et al. (2009) have shown that AML can "shrink" short tree edges, resulting in a star tree with no internal resolution, which yields a better AML score than the original (resolved) model. This result implies that AML is statistically inconsistent, but not that it is positively misleading, because the star tree is compatible with any other topology. We show that AML is confusingly misleading: For some simple, four taxa (resolved) tree, the ancestral likelihood optimization criteria is maximized on an incorrect (resolved) tree topology, as well as on a star tree (both with specific edge lengths), while the tree with the original, correct topology, has strictly lower ancestral likelihood. Interestingly, the two short edges in the incorrect, resolved tree topology are of length zero, and are not adjacent, so this resolved tree is in fact a simple path. While for MP, the underlying phenomenon can be described as long edge attraction, it turns out that here we have long edge repulsion. Copyright © 2017. Published by Elsevier Ltd.

Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus

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Chakrabarti Mrinmay

2010-08-01

Full Text Available Abstract Background Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV, a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11 in its genome. Some of its genome segments (S2 and S6-S11 have been previously characterized but genome segments encoding viral capsid have not been characterized. Results In this study genome segments 1 (S1 and 3 (S3 of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV, Lymantria dispar CPV (LdCPV, and Dendrolimus punctatus CPV (DpCPV. The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein. Conclusion Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3

Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance.

Science.gov (United States)

Delgado, Asunción; Arco, Rocio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

2017-05-09

Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

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Bennett, Shauna M. [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Zhao, Linbo [Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Bosard, Catherine [Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Imperiale, Michael J., E-mail: imperial@umich.edu [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States)

2015-01-01

BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection.

Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

International Nuclear Information System (INIS)

Bennett, Shauna M.; Zhao, Linbo; Bosard, Catherine; Imperiale, Michael J.

2015-01-01

BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection

Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

DEFF Research Database (Denmark)

Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin

2015-01-01

necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from...... the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral...... evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants....

Magmatism and Epithermal Gold-Silver Deposits of the Southern Ancestral Cascade Arc, Western Nevada and Eastern California

Science.gov (United States)

John, David A.; du Bray, Edward A.; Henry, Christopher D.; Vikre, Peter

2015-01-01

Many epithermal gold-silver deposits are temporally and spatially associated with late Oligocene to Pliocene magmatism of the southern ancestral Cascade arc in western Nevada and eastern California. These deposits, which include both quartz-adularia (low- and intermediate-sulfidation; Comstock Lode, Tonopah, Bodie) and quartz-alunite (high-sulfidation; Goldfield, Paradise Peak) types, were major producers of gold and silver. Ancestral Cascade arc magmatism preceded that of the modern High Cascades arc and reflects subduction of the Farallon plate beneath North America. Ancestral arc magmatism began about 45 Ma, continued until about 3 Ma, and extended from near the Canada-United States border in Washington southward to about 250 km southeast of Reno, Nevada. The ancestral arc was split into northern and southern segments across an inferred tear in the subducting slab between Mount Shasta and Lassen Peak in northern California. The southern segment extends between 42°N in northern California and 37°N in western Nevada and was active from about 30 to 3 Ma. It is bounded on the east by the northeast edge of the Walker Lane. Ancestral arc volcanism represents an abrupt change in composition and style of magmatism relative to that in central Nevada. Large volume, caldera-forming, silicic ignimbrites associated with the 37 to 19 Ma ignimbrite flareup are dominant in central Nevada, whereas volcanic centers of the ancestral arc in western Nevada consist of andesitic stratovolcanoes and dacitic to rhyolitic lava domes that mostly formed between 25 and 4 Ma. Both ancestral arc and ignimbrite flareup magmatism resulted from rollback of the shallowly dipping slab that began about 45 Ma in northeast Nevada and migrated south-southwest with time. Most southern segment ancestral arc rocks have oxidized, high potassium, calc-alkaline compositions with silica contents ranging continuously from about 55 to 77 wt%. Most lavas are porphyritic and contain coarse plagioclase �

Detention of HPV L1 Capsid Protein and hTERC Gene in Screening of Cervical Cancer

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Huang Bin

2013-06-01

Full Text Available   Objective(s: To investigate the expression of human papilloma virus (HPV L1 capsid protein, and human telomerase RNA component (hTERC in cervical cancer and the role of detection of both genes in screening of cervical cancer.   Materials and Methods: A total of 309 patients were recruited and cervical exfoliated cells were collected. Immunocytochemistry was employed to detect HPV L1 capsid protein, and fluorescent in situ hybridization (FISH was performed to detect the hTERC. Results: The expression of HPV L1 capsid protein reduced with the increase of the histological grade of cervical cells and was negatively related to the grade of cervical lesions. However, the expression of hTERC increased with the increase of the histological grade and positively associated with the grade of cervical lesions. The proportion of patients with L1(-/hTERC(+ was higher in patients with histological grade of CIN2 or higher than that in those with histological grade of CIN1. The L1(+/hTERC(- and L1(-/hTERC(- were negatively related to the grade of cervical lesions. L1(-/hTERC(+ was positively associated with the grade of cervical lesions. The L1/hTERC ratio increased. The negative predictive value of both HPV L1 and hTERC was higher than that of HPV L1 or hTERC, but there was no marked difference in the screening efficacy of cervical cancer among HPV L1, hTERC and HPV L1+hTERC. Conclusion: HPV L1 capsid protein and hTERC gene may serve as markers for the early diagnosis and prediction of cervical lesions. The increase in L1/hTERC ratio reflects the progression of cervical lesions to a certain extent.

Structures of foot and mouth disease virus pentamers: Insight into capsid dissociation and unexpected pentamer reassociation.

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Nayab Malik

2017-09-01

Full Text Available Foot-and-mouth disease virus (FMDV belongs to the Aphthovirus genus of the Picornaviridae, a family of small, icosahedral, non-enveloped, single-stranded RNA viruses. It is a highly infectious pathogen and is one of the biggest hindrances to the international trade of animals and animal products. FMDV capsids (which are unstable below pH6.5 release their genome into the host cell from an acidic compartment, such as that of an endosome, and in the process dissociate into pentamers. Whilst other members of the family (enteroviruses have been visualized to form an expanded intermediate capsid with holes from which inner capsid proteins (VP4, N-termini (VP1 and RNA can be released, there has been no visualization of any such state for an aphthovirus, instead the capsid appears to simply dissociate into pentamers. Here we present the 8-Å resolution structure of isolated dissociated pentamers of FMDV, lacking VP4. We also found these pentamers to re-associate into a rigid, icosahedrally symmetric assembly, which enabled their structure to be solved at higher resolution (5.2 Å. In this assembly, the pentamers unexpectedly associate 'inside out', but still with their exposed hydrophobic edges buried. Stabilizing interactions occur between the HI loop of VP2 and its symmetry related partners at the icosahedral 3-fold axes, and between the BC and EF loops of VP3 with the VP2 βB-strand and the CD loop at the 2-fold axes. A relatively extensive but subtle structural rearrangement towards the periphery of the dissociated pentamer compared to that in the mature virus provides insight into the mechanism of dissociation of FMDV and the marked difference in antigenicity.

Perianth evolution in Ranunculaceae: are petals ancestral in the family?

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Sophie Nadot

2016-04-01

Full Text Available Progress has been made recently towards the elucidation of phylogenetic relationships among subfamilies and tribes of the Ranunculaceae – the most recent hypothesis was published in 2016 by our team. Although relationships among the 10 tribes of the subfamily Ranunculoideae remain incompletely supported, this hypothesis provides an interesting framework to address the key issue of the ancestral vs. derived nature of a differentiated perianth within the family, and at the level of Ranunculales as a whole. Here, we present ancestral state reconstructions for several perianth characters, such as differentiation into sepals and petals, shape of petals, presence/absence of nectaries, and petaloid or sepaloid aspect of sepals. Characters were scored using the PROTEUS database and optimized on the most recent phylogeny of Ranunculaceae using parsimony and maximum likelihood methods. The results are discussed with regard to recent evo-devo studies focused on identifying genes involved in floral organs identity (the so-called ABC model in Ranunculales.

Approximate maximum parsimony and ancestral maximum likelihood.

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Alon, Noga; Chor, Benny; Pardi, Fabio; Rapoport, Anat

2010-01-01

We explore the maximum parsimony (MP) and ancestral maximum likelihood (AML) criteria in phylogenetic tree reconstruction. Both problems are NP-hard, so we seek approximate solutions. We formulate the two problems as Steiner tree problems under appropriate distances. The gist of our approach is the succinct characterization of Steiner trees for a small number of leaves for the two distances. This enables the use of known Steiner tree approximation algorithms. The approach leads to a 16/9 approximation ratio for AML and asymptotically to a 1.55 approximation ratio for MP.

Reconstruction of ancestral RNA sequences under multiple structural constraints.

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Tremblay-Savard, Olivier; Reinharz, Vladimir; Waldispühl, Jérôme

2016-11-11

Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA) families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. In this paper, we introduce achARNement, a maximum parsimony approach that, given two alignments of homologous ncRNA families with consensus secondary structures and a phylogenetic tree, simultaneously calculates ancestral RNA sequences for these two families. We test our methodology on simulated data sets, and show that achARNement outperforms classical maximum parsimony approaches in terms of accuracy, but also reduces by several orders of magnitude the number of candidate sequences. To conclude this study, we apply our algorithms on the Glm clan and the FinP-traJ clan from the Rfam database. Our results show that our methods reconstruct small sets of high-quality candidate ancestors with better agreement to the two target structures than with classical approaches. Our program is freely available at: http://csb.cs.mcgill.ca/acharnement .

Ancestrality and evolution of trait syndromes in finches (Fringillidae)

OpenAIRE

Ponge, Jean‐François; Zuccon, Dario; Elias, Marianne; Pavoine, Sandrine; Henry, Pierre‐Yves; Théry, Marc; Guilbert, Éric

2017-01-01

International audience; Species traits have been hypothesized by one of us (Ponge, 2013) to evolve in a correlated manner as species colonize stable, undisturbed habitats, shifting from “ancestral” to “derived” strategies. We predicted that generalism, r-selection, sexual monomorphism, and migration/gregariousness are the ancestral states (collectively called strategy A) and evolved correlatively toward specialism, K-selection, sexual dimorphism, and residence/territoriality as habitat stabil...

Lack of Social Support Raises Stress Vulnerability in Rats with a History of Ancestral Stress.

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Faraji, Jamshid; Soltanpour, Nabiollah; Lotfi, Hamid; Moeeini, Reza; Moharreri, Ali-Reza; Roudaki, Shabnam; Hosseini, S Abedin; Olson, David M; Abdollahi, Ali-Akbar; Soltanpour, Nasrin; Mohajerani, Majid H; Metz, Gerlinde A S

2017-07-13

Stress is a primary risk factor for psychiatric disorders. However, it is not fully understood why some stressed individuals are more vulnerable to psychiatric disorders than others. Here, we investigated whether multigenerational ancestral stress produces phenotypes that are sensitive to depression-like symptoms in rats. We also examined whether social isolation reveals potentially latent sensitivity to depression-like behaviours. F4 female rats born to a lineage of stressed mothers (F0-F3) received stress in adulthood while housed in pairs or alone. Social isolation during stress induced cognitive and psychomotor retardation only in rats exposed to ancestral stress. Social isolation also hampered the resilience of the hypothalamic-pituitary-adrenal axis to chronic stress and reduced hippocampal volume and brain-derived neurotrophic factor (BDNF) expression. Thus, synergy between social isolation and stress may unmask a latent history of ancestral stress, and raises vulnerability to mental health conditions. The findings support the notion that social support critically promotes stress coping and resilience.

Resurrecting ancestral genes in bacteria to interpret ancient biosignatures

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Kacar, Betul; Guy, Lionel; Smith, Eric; Baross, John

2017-11-01

Two datasets, the geologic record and the genetic content of extant organisms, provide complementary insights into the history of how key molecular components have shaped or driven global environmental and macroevolutionary trends. Changes in global physico-chemical modes over time are thought to be a consistent feature of this relationship between Earth and life, as life is thought to have been optimizing protein functions for the entirety of its approximately 3.8 billion years of history on the Earth. Organismal survival depends on how well critical genetic and metabolic components can adapt to their environments, reflecting an ability to optimize efficiently to changing conditions. The geologic record provides an array of biologically independent indicators of macroscale atmospheric and oceanic composition, but provides little in the way of the exact behaviour of the molecular components that influenced the compositions of these reservoirs. By reconstructing sequences of proteins that might have been present in ancient organisms, we can downselect to a subset of possible sequences that may have been optimized to these ancient environmental conditions. How can one use modern life to reconstruct ancestral behaviours? Configurations of ancient sequences can be inferred from the diversity of extant sequences, and then resurrected in the laboratory to ascertain their biochemical attributes. One way to augment sequence-based, single-gene methods to obtain a richer and more reliable picture of the deep past, is to resurrect inferred ancestral protein sequences in living organisms, where their phenotypes can be exposed in a complex molecular-systems context, and then to link consequences of those phenotypes to biosignatures that were preserved in the independent historical repository of the geological record. As a first step beyond single-molecule reconstruction to the study of functional molecular systems, we present here the ancestral sequence reconstruction of the

Expression, purification and crystallization of the ancestral androgen receptor-DHT complex.

Science.gov (United States)

Colucci, Jennifer K; Ortlund, Eric A

2013-09-01

Steroid receptors (SRs) are a closely related family of ligand-dependent nuclear receptors that mediate the transcription of genes critical for development, reproduction and immunity. SR dysregulation has been implicated in cancer, inflammatory diseases and metabolic disorders. SRs bind their cognate hormone ligand with exquisite specificity, offering a unique system to study the evolution of molecular recognition. The SR family evolved from an estrogen-sensitive ancestor and diverged to become sensitive to progestagens, corticoids and, most recently, androgens. To understand the structural mechanisms driving the evolution of androgen responsiveness, the ancestral androgen receptor (ancAR1) was crystallized in complex with 5α-dihydrotestosterone (DHT) and a fragment of the transcriptional mediator/intermediary factor 2 (Tif2). Crystals diffracted to 2.1 Å resolution and the resulting structure will permit a direct comparison with its progestagen-sensitive ancestor, ancestral steroid receptor 2 (AncSR2).

Essential C-Terminal region of the baculovirus minor capsid protein VP80 binds DNA

NARCIS (Netherlands)

Marek, M.; Merten, O.W.; Francis-Devaraj, F.; Oers, van M.M.

2012-01-01

The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and

Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

Energy Technology Data Exchange (ETDEWEB)

Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhang, Jun; Xia, Ning-Shao [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China); Miao, Ji, E-mail: jmiao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhao, Qinjian, E-mail: qinjian_zhao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China)

2013-01-18

Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

Science.gov (United States)

Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

2011-01-01

Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

Visual system evolution and the nature of the ancestral snake.

Science.gov (United States)

Simões, B F; Sampaio, F L; Jared, C; Antoniazzi, M M; Loew, E R; Bowmaker, J K; Rodriguez, A; Hart, N S; Hunt, D M; Partridge, J C; Gower, D J

2015-07-01

The dominant hypothesis for the evolutionary origin of snakes from 'lizards' (non-snake squamates) is that stem snakes acquired many snake features while passing through a profound burrowing (fossorial) phase. To investigate this, we examined the visual pigments and their encoding opsin genes in a range of squamate reptiles, focusing on fossorial lizards and snakes. We sequenced opsin transcripts isolated from retinal cDNA and used microspectrophotometry to measure directly the spectral absorbance of the photoreceptor visual pigments in a subset of samples. In snakes, but not lizards, dedicated fossoriality (as in Scolecophidia and the alethinophidian Anilius scytale) corresponds with loss of all visual opsins other than RH1 (λmax 490-497 nm); all other snakes (including less dedicated burrowers) also have functional sws1 and lws opsin genes. In contrast, the retinas of all lizards sampled, even highly fossorial amphisbaenians with reduced eyes, express functional lws, sws1, sws2 and rh1 genes, and most also express rh2 (i.e. they express all five of the visual opsin genes present in the ancestral vertebrate). Our evidence of visual pigment complements suggests that the visual system of stem snakes was partly reduced, with two (RH2 and SWS2) of the ancestral vertebrate visual pigments being eliminated, but that this did not extend to the extreme additional loss of SWS1 and LWS that subsequently occurred (probably independently) in highly fossorial extant scolecophidians and A. scytale. We therefore consider it unlikely that the ancestral snake was as fossorial as extant scolecophidians, whether or not the latter are para- or monophyletic. © 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.

Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy

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Kristin N. Parent

2018-02-01

Full Text Available The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell’s icosahedral symmetry, providing a gateway through the capsid. In this review, we take an inventory of molecular structures observed either internally, or at the 5-fold vertices of icosahedral DNA viruses that infect bacteria, archea and eukaryotes. Taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM and building upon a wealth of crystallographic structures of individual components, we review the design principles of non-icosahedral structural components that interrupt icosahedral symmetry and discuss how these macromolecules play vital roles in genome packaging, ejection and host receptor-binding.

Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands

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Mengya eNiu

2015-12-01

Full Text Available Human noroviruses (HuNoVs are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2 and the protruding domain (P domain encoding gene (3’ terminal fragment of ORF2 of HuNoVs GI.1 and GII.4 were fused with 5’ terminal fragment of ice nucleation protein encoding gene (inaQn. The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an

Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal

International Nuclear Information System (INIS)

Patterson, Edward I.; Dombrovski, Andrew K.; Swarbrick, Crystall M.D.; Raidal, Shane R.; Forwood, Jade K.

2013-01-01

Highlights: •Circovirus capsid proteins contain large nuclear localization signals (NLS). •A method of nuclear import has not been elucidated. •Beak and feather disease virus (BFDV) capsid NLS was crystallized with importin α. •The structure showed BFDV NLS binding to the major site of importin α. •Result shows implications for mechanism of nuclear transport for all circoviruses. -- Abstract: Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediate nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9 Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface

Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

International Nuclear Information System (INIS)

Nalcacioglu, Remziye; Marks, Hendrik; Vlak, Just M.; Demirbag, Zihni; Oers, Monique M. van

2003-01-01

The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an immediate-early gene and confirmed that the major capsid protein (MCP) is a late gene. Transcription of DNApol initiated 35 nt upstream and that of MCP 14 nt upstream of the translational start site. In a luciferase reporter gene assay both promoters were active only when cells were infected with CIV. For DNApol sequences between position -27 and -6, relative to the transcriptional start site, were essential for promoter activity. Furthermore, mutation of a G within the sequence TTGTTTT located just upstream of the DNApol transcription initiation site reduced the promoter activity by 25%. Sequences crucial for MCP promoter activity are located between positions -53 and -29

Mapping the Structural Determinants Responsible for Enhanced T Cell Activation to the Immunogenic Adeno-Associated Virus Capsid from Isolate Rhesus 32.33

Science.gov (United States)

Mays, Lauren E.; Wang, Lili; Tenney, Rebeca; Bell, Peter; Nam, Hyun-Joo; Lin, Jianping; Gurda, Brittney; Van Vliet, Kim; Mikals, Kyle; Agbandje-McKenna, Mavis

2013-01-01

Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8+ T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33. PMID:23720715

Production of highly knotted DNA by means of cosmid circularization inside phage capsids

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Trigueros Sonia

2007-12-01

Full Text Available Abstract Background The formation of DNA knots is common during biological transactions. Yet, functional implications of knotted DNA are not fully understood. Moreover, potential applications of DNA molecules condensed by means of knotting remain to be explored. A convenient method to produce abundant highly knotted DNA would be highly valuable for these studies. Results We had previously shown that circularization of the 11.2 kb linear DNA of phage P4 inside its viral capsid generates complex knots by the effect of confinement. We demonstrate here that this mechanism is not restricted to the viral genome. We constructed DNA cosmids as small as 5 kb and introduced them inside P4 capsids. Such cosmids were then recovered as a complex mixture of highly knotted DNA circles. Over 250 μg of knotted cosmid were typically obtained from 1 liter of bacterial culture. Conclusion With this biological system, DNA molecules of varying length and sequence can be shaped into very complex and heterogeneous knotted forms. These molecules can be produced in preparative amounts suitable for systematic studies and applications.

Genetically Thermo-Stabilised, Immunogenic Poliovirus Empty Capsids; a Strategy for Non-replicating Vaccines.

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Helen Fox

2017-01-01

Full Text Available While wild type polio has been nearly eradicated there will be a need to continue immunisation programmes for some time because of the possibility of re-emergence and the existence of long term excreters of poliovirus. All vaccines in current use depend on growth of virus and most of the non-replicating (inactivated vaccines involve wild type viruses known to cause poliomyelitis. The attenuated vaccine strains involved in the eradication programme have been used to develop new inactivated vaccines as production is thought safer. However it is known that the Sabin vaccine strains are genetically unstable and can revert to a virulent transmissible form. A possible solution to the need for virus growth would be to generate empty viral capsids by recombinant technology, but hitherto such particles are so unstable as to be unusable. We report here the genetic manipulation of the virus to generate stable empty capsids for all three serotypes. The particles are shown to be extremely stable and to generate high levels of protective antibodies in animal models.

Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

DEFF Research Database (Denmark)

Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette

2013-01-01

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3Cpro to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm...... the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction...

Ancestral genes can control the ability of horizontally acquired loci to confer new traits.

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H Deborah Chen

2011-07-01

Full Text Available Horizontally acquired genes typically function as autonomous units conferring new abilities when introduced into different species. However, we reasoned that proteins preexisting in an organism might constrain the functionality of a horizontally acquired gene product if it operates on an ancestral pathway. Here, we determine how the horizontally acquired pmrD gene product activates the ancestral PmrA/PmrB two-component system in Salmonella enterica but not in the closely related bacterium Escherichia coli. The Salmonella PmrD protein binds to the phosphorylated PmrA protein (PmrA-P, protecting it from dephosphorylation by the PmrB protein. This results in transcription of PmrA-dependent genes, including those conferring polymyxin B resistance. We now report that the E. coli PmrD protein can activate the PmrA/PmrB system in Salmonella even though it cannot do it in E. coli, suggesting that these two species differ in an additional component controlling PmrA-P levels. We establish that the E. coli PmrB displays higher phosphatase activity towards PmrA-P than the Salmonella PmrB, and we identified a PmrB subdomain responsible for this property. Replacement of the E. coli pmrB gene with the Salmonella homolog was sufficient to render E. coli resistant to polymyxin B under PmrD-inducing conditions. Our findings provide a singular example whereby quantitative differences in the biochemical activities of orthologous ancestral proteins dictate the ability of a horizontally acquired gene product to confer species-specific traits. And they suggest that horizontally acquired genes can potentiate selection at ancestral loci.

Reconstruction of ancestral RNA sequences under multiple structural constraints

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Olivier Tremblay-Savard

2016-11-01

Full Text Available Abstract Background Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. Methods In this paper, we introduce achARNement, a maximum parsimony approach that, given two alignments of homologous ncRNA families with consensus secondary structures and a phylogenetic tree, simultaneously calculates ancestral RNA sequences for these two families. Results We test our methodology on simulated data sets, and show that achARNement outperforms classical maximum parsimony approaches in terms of accuracy, but also reduces by several orders of magnitude the number of candidate sequences. To conclude this study, we apply our algorithms on the Glm clan and the FinP-traJ clan from the Rfam database. Conclusions Our results show that our methods reconstruct small sets of high-quality candidate ancestors with better agreement to the two target structures than with classical approaches. Our program is freely available at: http://csb.cs.mcgill.ca/acharnement .

Temperature-dependent respiration-growth relations in ancestral maize cultivars

Science.gov (United States)

Bruce N. Smith; Jillian L. Walker; Rebekka L. Stone; Angela R. Jones; Lee D. Hansen

2001-01-01

Shoots from 4- to 6-day old seedlings of seven ancestral or old cultivars of Zea mays L. were placed in a calorimeter. Dark metabolic heat rate (q) and CO2 production rate (RCO2) were measured at nine temperatures (5, 10, 15, 20, 25, 30, 35, 40, and 45 °C). Temperature dependencies of q and RCO2 were used to model response of both growth and substrate carbon conversion...

Reconstructed ancestral Myo-inositol-3-phosphate synthases indicate that ancestors of the Thermococcales and Thermotoga species were more thermophilic than their descendants.

Science.gov (United States)

Butzin, Nicholas C; Lapierre, Pascal; Green, Anna G; Swithers, Kristen S; Gogarten, J Peter; Noll, Kenneth M

2013-01-01

The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants.

Reconstructed ancestral Myo-inositol-3-phosphate synthases indicate that ancestors of the Thermococcales and Thermotoga species were more thermophilic than their descendants.

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Nicholas C Butzin

Full Text Available The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS. These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants.

Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

NARCIS (Netherlands)

Nalcacioglu, R.; Marks, H.; Vlak, J.M.; Demirbag, Z.; Oers, van M.M.

2003-01-01

The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an

Thermotolerant Yeast Strains Adapted by Laboratory Evolution Show Trade-Off at Ancestral Temperatures and Preadaptation to Other Stresses

DEFF Research Database (Denmark)

Caspeta, Luis; Nielsen, Jens

2015-01-01

adaptive laboratory evolution, we previously isolated seven Saccharomyces cerevisiae strains with improved growth at 40°C. Here, we show that genetic adaptations to high temperature caused a growth trade-off at ancestral temperatures, reduced cellular functions, and improved tolerance of other stresses...... in the ancestral strain. The latter is an advantageous attribute for acquiring thermotolerance and correlates with the reduction of yeast functions associated with loss of respiration capacity. This trait caused glycerol overproduction that was associated with the growth trade-off at ancestral temperatures....... In combination with altered sterol composition of cellular membranes, glycerol overproduction was also associated with yeast osmotolerance and improved tolerance of high concentrations of glucose and ethanol. Our study shows that thermal adaptation of yeast is suitable for improving yeast resistance...

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

Science.gov (United States)

Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

2012-08-01

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.

Science.gov (United States)

Drouin, Lauren M; Lins, Bridget; Janssen, Maria; Bennett, Antonette; Chipman, Paul; McKenna, Robert; Chen, Weijun; Muzyczka, Nicholas; Cardone, Giovanni; Baker, Timothy S; Agbandje-McKenna, Mavis

2016-10-01

The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid

HCIV-1 and Other Tailless Icosahedral Internal Membrane-Containing Viruses of the Family Sphaerolipoviridae

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Tatiana A. Demina

2017-02-01

Full Text Available Members of the virus family Sphaerolipoviridae include both archaeal viruses and bacteriophages that possess a tailless icosahedral capsid with an internal membrane. The genera Alpha- and Betasphaerolipovirus comprise viruses that infect halophilic euryarchaea, whereas viruses of thermophilic Thermus bacteria belong to the genus Gammasphaerolipovirus. Both sequence-based and structural clustering of the major capsid proteins and ATPases of sphaerolipoviruses yield three distinct clades corresponding to these three genera. Conserved virion architectural principles observed in sphaerolipoviruses suggest that these viruses belong to the PRD1-adenovirus structural lineage. Here we focus on archaeal alphasphaerolipoviruses and their related putative proviruses. The highest sequence similarities among alphasphaerolipoviruses are observed in the core structural elements of their virions: the two major capsid proteins, the major membrane protein, and a putative packaging ATPase. A recently described tailless icosahedral haloarchaeal virus, Haloarcula californiae icosahedral virus 1 (HCIV-1, has a double-stranded DNA genome and an internal membrane lining the capsid. HCIV-1 shares significant similarities with the other tailless icosahedral internal membrane-containing haloarchaeal viruses of the family Sphaerolipoviridae. The proposal to include a new virus species, Haloarcula virus HCIV1, into the genus Alphasphaerolipovirus was submitted to the International Committee on Taxonomy of Viruses (ICTV in 2016.

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

Science.gov (United States)

Bloom, M E; Martin, D A; Oie, K L; Huhtanen, M E; Costello, F; Wolfinbarger, J B; Hayes, S F; Agbandje-McKenna, M

1997-01-01

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins

A new series of HAPs as anti-HBV agents targeting at capsid assembly.

Science.gov (United States)

Yang, Xiu-yan; Xu, Xiao-qian; Guan, Hua; Wang, Li-li; Wu, Qin; Zhao, Guo-ming; Li, Song

2014-09-01

A series of novel Heteroaryldihydropyrimidines (HAPs) derivatives were designed and synthesized as potent inhibitors of HBV capsid assembly. These compounds were prepared from efforts to optimize an earlier series of HAPs, and compounds Mo1, Mo7, Mo8, Mo10, Mo12, and Mo13 demonstrated potent inhibition of HBV DNA replication at submicromolar range. Copyright © 2014. Published by Elsevier Ltd.

Reconstruction of ancestral RNA sequences under multiple structural constraints

OpenAIRE

Tremblay-Savard, Olivier; Reinharz, Vladimir; Waldisp?hl, J?r?me

2016-01-01

Background Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA) families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. Methods In this paper, we introduce achARNement, a maximum parsimony approach that, given...

Accuracy of Genomic Prediction in Synthetic Populations Depending on the Number of Parents, Relatedness, and Ancestral Linkage Disequilibrium.

Science.gov (United States)

Schopp, Pascal; Müller, Dominik; Technow, Frank; Melchinger, Albrecht E

2017-01-01

Synthetics play an important role in quantitative genetic research and plant breeding, but few studies have investigated the application of genomic prediction (GP) to these populations. Synthetics are generated by intermating a small number of parents ([Formula: see text] and thereby possess unique genetic properties, which make them especially suited for systematic investigations of factors contributing to the accuracy of GP. We generated synthetics in silico from [Formula: see text]2 to 32 maize (Zea mays L.) lines taken from an ancestral population with either short- or long-range linkage disequilibrium (LD). In eight scenarios differing in relatedness of the training and prediction sets and in the types of data used to calculate the relationship matrix (QTL, SNPs, tag markers, and pedigree), we investigated the prediction accuracy (PA) of Genomic best linear unbiased prediction (GBLUP) and analyzed contributions from pedigree relationships captured by SNP markers, as well as from cosegregation and ancestral LD between QTL and SNPs. The effects of training set size [Formula: see text] and marker density were also studied. Sampling few parents ([Formula: see text]) generates substantial sample LD that carries over into synthetics through cosegregation of alleles at linked loci. For fixed [Formula: see text], [Formula: see text] influences PA most strongly. If the training and prediction set are related, using [Formula: see text] parents yields high PA regardless of ancestral LD because SNPs capture pedigree relationships and Mendelian sampling through cosegregation. As [Formula: see text] increases, ancestral LD contributes more information, while other factors contribute less due to lower frequencies of closely related individuals. For unrelated prediction sets, only ancestral LD contributes information and accuracies were poor and highly variable for [Formula: see text] due to large sample LD. For large [Formula: see text], achieving moderate accuracy requires

Ancestral informative marker selection and population structure visualization using sparse Laplacian eigenfunctions.

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Jun Zhang

Full Text Available Identification of a small panel of population structure informative markers can reduce genotyping cost and is useful in various applications, such as ancestry inference in association mapping, forensics and evolutionary theory in population genetics. Traditional methods to ascertain ancestral informative markers usually require the prior knowledge of individual ancestry and have difficulty for admixed populations. Recently Principal Components Analysis (PCA has been employed with success to select SNPs which are highly correlated with top significant principal components (PCs without use of individual ancestral information. The approach is also applicable to admixed populations. Here we propose a novel approach based on our recent result on summarizing population structure by graph laplacian eigenfunctions, which differs from PCA in that it is geometric and robust to outliers. Our approach also takes advantage of the priori sparseness of informative markers in the genome. Through simulation of a ring population and the real global population sample HGDP of 650K SNPs genotyped in 940 unrelated individuals, we validate the proposed algorithm at selecting most informative markers, a small fraction of which can recover the similar underlying population structure efficiently. Employing a standard Support Vector Machine (SVM to predict individuals' continental memberships on HGDP dataset of seven continents, we demonstrate that the selected SNPs by our method are more informative but less redundant than those selected by PCA. Our algorithm is a promising tool in genome-wide association studies and population genetics, facilitating the selection of structure informative markers, efficient detection of population substructure and ancestral inference.

Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1

International Nuclear Information System (INIS)

Diaz-Griffero, Felipe; Vandegraaff, Nick; Li Yuan; McGee-Estrada, Kathleen; Stremlau, Matthew; Welikala, Sohanya; Si Zhihai; Engelman, Alan; Sodroski, Joseph

2006-01-01

In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5α with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain

Evolution of sexes from an ancestral mating-type specification pathway.

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Sa Geng

2014-07-01

Full Text Available Male and female sexes have evolved repeatedly in eukaryotes but the origins of dimorphic sexes and their relationship to mating types in unicellular species are not understood. Volvocine algae include isogamous species such as Chlamydomonas reinhardtii, with two equal-sized mating types, and oogamous multicellular species such as Volvox carteri with sperm-producing males and egg-producing females. Theoretical work predicts genetic linkage of a gamete cell-size regulatory gene(s to an ancestral mating-type locus as a possible step in the evolution of dimorphic gametes, but this idea has not been tested. Here we show that, contrary to predictions, a single conserved mating locus (MT gene in volvocine algae-MID, which encodes a RWP-RK domain transcription factor-evolved from its ancestral role in C. reinhardtii as a mating-type specifier, to become a determinant of sperm and egg development in V. carteri. Transgenic female V. carteri expressing male MID produced functional sperm packets during sexual development. Transgenic male V. carteri with RNA interference (RNAi-mediated knockdowns of VcMID produced functional eggs, or self-fertile hermaphrodites. Post-transcriptional controls were found to regulate cell-type-limited expression and nuclear localization of VcMid protein that restricted its activity to nuclei of developing male germ cells and sperm. Crosses with sex-reversed strains uncoupled sex determination from sex chromosome identity and revealed gender-specific roles for male and female mating locus genes in sexual development, gamete fitness and reproductive success. Our data show genetic continuity between the mating-type specification and sex determination pathways of volvocine algae, and reveal evidence for gender-specific adaptations in the male and female mating locus haplotypes of Volvox. These findings will enable a deeper understanding of how a master regulator of mating-type determination in an ancestral unicellular species was

Do Père David's deer lose memories of their ancestral predators?

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Chunwang Li

Full Text Available Whether prey retains antipredator behavior after a long period of predator relaxation is an important question in predator-prey evolution. Père David's deer have been raised in enclosures for more than 1200 years and this isolation provides an opportunity to study whether Père David's deer still respond to the cues of their ancestral predators or to novel predators. We played back the sounds of crows (familiar sound and domestic dogs (familiar non-predators, of tigers and wolves (ancestral predators, and of lions (potential naïve predator to Père David's deer in paddocks, and blank sounds to the control group, and videoed the behavior of the deer during the experiment. We also showed life-size photo models of dog, leopard, bear, tiger, wolf, and lion to the deer and video taped their responses after seeing these models. Père David's deer stared at and approached the hidden loudspeaker when they heard the roars of tiger or lion. The deer listened to tiger roars longer, approached to tiger roars more and spent more time staring at the tiger model. The stags were also found to forage less in the trials of tiger roars than that of other sound playbacks. Additionally, it took longer for the deer to restore their normal behavior after they heard tiger roars, which was longer than that after the trial of other sound playbacks. Moreover, the deer were only found to walk away after hearing the sounds of tiger and wolf. Therefore, the tiger was probably the main predator for Père David's deer in ancient time. Our study implies that Père David's deer still retain the memories of the acoustic and visual cues of their ancestral predators in spite of the long term isolation from natural habitat.

Gaussian fluctuation of the diffusion exponent of virus capsid in a living cell nucleus

Science.gov (United States)

Itto, Yuichi

2018-05-01

In their work [4], Bosse et al. experimentally showed that virus capsid exhibits not only normal diffusion but also anomalous diffusion in nucleus of a living cell. There, it was found that the distribution of fluctuations of the diffusion exponent characterizing them takes the Gaussian form, which is, quite remarkably, the same form for two different types of the virus. This suggests high robustness of such fluctuations. Here, the statistical property of local fluctuations of the diffusion exponent of the virus capsid in the nucleus is studied. A maximum-entropy-principle approach (originally proposed for a different virus in a different cell) is applied for obtaining the fluctuation distribution of the exponent. Largeness of the number of blocks identified with local areas of interchromatin corrals is also examined based on the experimental data. It is shown that the Gaussian distribution of the local fluctuations can be derived, in accordance with the above form. In addition, it is quantified how the fluctuation distribution on a long time scale is different from the Gaussian distribution.

Thermodynamic characterization of the peptide assembly inhibitor binding to HIV-1 capsid protein

Czech Academy of Sciences Publication Activity Database

Kožíšek, Milan; Durčák, Jindřich; Konvalinka, Jan

2013-01-01

Roč. 10, Suppl. 1 (2013), S37-S37 ISSN 1742-4690. [Frontiers of Retrovirology: Complex retorviruses, retroelements and their hosts. 16.09.2013-18.09.2013, Cambridge] R&D Projects: GA ČR GA13-19561S Institutional support: RVO:61388963 Keywords : HIV -1 capsid protein * CAI Subject RIV: EE - Microbiology, Virology http://www.retrovirology.com/content/10/S1/P108

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

Science.gov (United States)

Diprimio, Nina; Bowles, Dawn E.; Hirsch, Matthew L.; Monahan, Paul E.; Asokan, Aravind; Rabinowitz, Joseph; Agbandje-McKenna, Mavis

2012-01-01

Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration. PMID:22593151

Facilitating the use of alternative capsid control methods towards sustainable production of organic cocoa in Ghana

NARCIS (Netherlands)

Ayenor, G.K.; Huis, van A.; Obeng-Ofori, D.; Padi, B.; Röling, N.G.

2007-01-01

Cocoa (Theobroma cacao L.) is an important foreign exchange earner for Ghana. However, production is constrained by a high incidence of pests and diseases. Based on farmers' needs, this study focused on the control of capsids, mainly Sahlbergella singularis Haglund and Distantiella theobroma

Transient gene expression in serum-free suspension-growing mammalian cells for the production of foot-and-mouth disease virus empty capsids.

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Ana Clara Mignaqui

Full Text Available Foot-and-mouth disease (FMD is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV. The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

Pollination and reproduction of an invasive plant inside and outside its ancestral range

Science.gov (United States)

Petanidou, Theodora; Price, Mary V.; Bronstein, Judith L.; Kantsa, Aphrodite; Tscheulin, Thomas; Kariyat, Rupesh; Krigas, Nikos; Mescher, Mark C.; De Moraes, Consuelo M.; Waser, Nickolas M.

2018-05-01

Comparing traits of invasive species within and beyond their ancestral range may improve our understanding of processes that promote aggressive spread. Solanum elaeagnifolium (silverleaf nightshade) is a noxious weed in its ancestral range in North America and is invasive on other continents. We compared investment in flowers and ovules, pollination success, and fruit and seed set in populations from Arizona, USA ("AZ") and Greece ("GR"). In both countries, the populations we sampled varied in size and types of present-day disturbance. Stature of plants increased with population size in AZ samples whereas GR plants were uniformly tall. Taller plants produced more flowers, and GR plants produced more flowers for a given stature and allocated more ovules per flower. Similar functional groups of native bees pollinated in AZ and GR populations, but visits to flowers decreased with population size and we observed no visits in the largest GR populations. As a result, plants in large GR populations were pollen-limited, and estimates of fecundity were lower on average in GR populations despite the larger allocation to flowers and ovules. These differences between plants in our AZ and GR populations suggest promising directions for further study. It would be useful to sample S. elaeagnifolium in Mediterranean climates within the ancestral range (e.g., in California, USA), to study asexual spread via rhizomes, and to use common gardens and genetic studies to explore the basis of variation in allocation patterns and of relationships between visitation and fruit set.

Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93.

Science.gov (United States)

Köppen-Rung, Pánja; Dittmer, Alexandra; Bogner, Elke

2016-07-01

DNA packaging into procapsids is a common multistep process during viral maturation in herpesviruses. In human cytomegalovirus (HCMV), the proteins involved in this process are terminase subunits pUL56 and pUL89, which are responsible for site-specific cleavage and insertion of the DNA into the procapsid via portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the plasmid that encodes capsid-associated pUL77 encodes another potential player during capsid maturation. Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed in the early late part of the infectious cycle. The sequence of pUL77 was analyzed to find nuclear localization sequences (NLSs), revealing monopartite NLSm at the N terminus and bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829, which encodes a chimeric protein with β-galactosidase and green fluorescent protein. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by coimmunoprecipitation whether packaging proteins, as well as pUL93, the homologue protein of herpes simplex virus 1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins, as well as pUL93, were verified. We showed that the capsid-associated pUL77 is another potential player during capsid maturation of HCMV. Protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has early-late expression kinetics during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring of the encapsidated DNA during packaging, interaction with further DNA packaging proteins is required. We identified physical interactions with terminase subunits pUL56 and p

Inference of the ancestral vertebrate phenotype through vestiges of the whole-genome duplications.

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Onimaru, Koh; Kuraku, Shigehiro

2018-03-16

Inferring the phenotype of the last common ancestor of living vertebrates is a challenging problem because of several unresolvable factors. They include the lack of reliable out-groups of living vertebrates, poor information about less fossilizable organs and specialized traits of phylogenetically important species, such as lampreys and hagfishes (e.g. secondary loss of vertebrae in adult hagfishes). These factors undermine the reliability of ancestral reconstruction by traditional character mapping approaches based on maximum parsimony. In this article, we formulate an approach to hypothesizing ancestral vertebrate phenotypes using information from the phylogenetic and functional properties of genes duplicated by genome expansions in early vertebrate evolution. We named the conjecture as 'chronological reconstruction of ohnolog functions (CHROF)'. This CHROF conjecture raises the possibility that the last common ancestor of living vertebrates may have had more complex traits than currently thought.

RNA packaging of MRFV virus-like particles: The interplay between RNA pools and capsid coat protein

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Virus-like particles (VLPs) can be produced through self-assembly of capsid protein (CP) into particles with discrete shapes and sizes and containing different types of RNA molecules. The general principle that governs particle assembly and RNA packaging is determined by unique interactions between ...

Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

International Nuclear Information System (INIS)

Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

2011-01-01

We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: → We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. → Cre/loxP recombination was used to modify the adenovirus genome. → A targeting ligand present on capsid protein IX was removed or replaced using recombination. → Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles

DEFF Research Database (Denmark)

Gullberg, Maria; Lohse, Louise; Bøtner, Anette

2016-01-01

Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can successfully control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced...... in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a "single cycle" packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When...... the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. The products interact with anti-FMDV antibodies in an ELISA and bind to the integrin αvβ6 (a cellular receptor for FMDV). In cattle...

An allele of an ancestral transcription factor dependent on a horizontally acquired gene product.

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Chen, H Deborah; Jewett, Mollie W; Groisman, Eduardo A

2012-01-01

Changes in gene regulatory circuits often give rise to phenotypic differences among closely related organisms. In bacteria, these changes can result from alterations in the ancestral genome and/or be brought about by genes acquired by horizontal transfer. Here, we identify an allele of the ancestral transcription factor PmrA that requires the horizontally acquired pmrD gene product to promote gene expression. We determined that a single amino acid difference between the PmrA proteins from the human adapted Salmonella enterica serovar Paratyphi B and the broad host range S. enterica serovar Typhimurium rendered transcription of PmrA-activated genes dependent on the PmrD protein in the former but not the latter serovar. Bacteria harboring the serovar Typhimurium allele exhibited polymyxin B resistance under PmrA- or under PmrA- and PmrD-inducing conditions. By contrast, isogenic strains with the serovar Paratyphi B allele displayed PmrA-regulated polymyxin B resistance only when experiencing activating conditions for both PmrA and PmrD. We establish that the two PmrA orthologs display quantitative differences in several biochemical properties. Strains harboring the serovar Paratyphi B allele showed enhanced biofilm formation, a property that might promote serovar Paratyphi B's chronic infection of the gallbladder. Our findings illustrate how subtle differences in ancestral genes can impact the ability of horizontally acquired genes to confer new properties.

Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein.

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Cortines, Juliana R; Motwani, Tina; Vyas, Aashay A; Teschke, Carolyn M

2014-05-01

Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving

The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

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Burger Marieta

2011-02-01

Full Text Available Abstract Background One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1 and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1, an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.

TensorCalculator: exploring the evolution of mechanical stress in the CCMV capsid

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Kononova, Olga; Maksudov, Farkhad; Marx, Kenneth A.; Barsegov, Valeri

2018-01-01

A new computational methodology for the accurate numerical calculation of the Cauchy stress tensor, stress invariants, principal stress components, von Mises and Tresca tensors is developed. The methodology is based on the atomic stress approach which permits the calculation of stress tensors, widely used in continuum mechanics modeling of materials properties, using the output from the MD simulations of discrete atomic and C_α -based coarse-grained structural models of biological particles. The methodology mapped into the software package TensorCalculator was successfully applied to the empty cowpea chlorotic mottle virus (CCMV) shell to explore the evolution of mechanical stress in this mechanically-tested specific example of a soft virus capsid. We found an inhomogeneous stress distribution in various portions of the CCMV structure and stress transfer from one portion of the virus structure to another, which also points to the importance of entropic effects, often ignored in finite element analysis and elastic network modeling. We formulate a criterion for elastic deformation using the first principal stress components. Furthermore, we show that von Mises and Tresca stress tensors can be used to predict the onset of a viral capsid’s mechanical failure, which leads to total structural collapse. TensorCalculator can be used to study stress evolution and dynamics of defects in viral capsids and other large-size protein assemblies.

Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs.

Science.gov (United States)

Lohse, Louise; Jackson, Terry; Bøtner, Anette; Belsham, Graham J

2012-05-24

The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus.In the present study we compared the pathogenicity of different FMDVs in young pigs. In total 32 pigs, 7-weeks-old, were exposed to virus, either by direct inoculation or through contact with inoculated pigs, using cell culture adapted (O1K B64), chimeric (O1K/A-TUR and O1K/O-UKG) or field strain (O-UKG/34/2001) viruses. The O1K B64 virus and the two chimeric viruses are identical to each other except for the capsid coding region.Animals exposed to O1K B64 did not exhibit signs of disease, while pigs exposed to each of the other viruses showed typical clinical signs of foot-and-mouth disease (FMD). All pigs infected with the O1K/O-UKG chimera or the field strain (O-UKG/34/2001) developed fulminant disease. Furthermore, 3 of 4 in-contact pigs exposed to the O1K/O-UKG virus died in the acute phase of infection, likely from myocardial infection. However, in the group exposed to the O1K/A-TUR chimeric virus, only 1 pig showed symptoms of disease within the time frame of the experiment (10 days). All pigs that developed clinical disease showed a high level of viral RNA in serum and infected pigs that survived the acute phase of infection developed a serotype specific antibody response. It is concluded that the capsid coding sequences are determinants of FMDV pathogenicity in pigs.

Preliminary appraisal of ground water in and near the ancestral Missouri River Valley, northeastern Montana

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Levings, G.W.

1986-01-01

A preliminary appraisal was conducted in and near the ancestral Missouri River valley in northeastern Montana to describe the groundwater resources and to establish a data base for the area. The data base then could be used for future evaluation of possible changes in water levels or water quality. In this area, consolidated aquifers are the Upper Cretaceous Fox Hills-lower Hell Creek aquifer and the overlying Paleocene Fort Union Formation. Unconsolidated aquifers are Pleistocene terrace gravel and glacial deposits and Holocene alluvial deposits. Aquifers are recharged by precipitation, infiltration of streamflow, and possibly leakage from lakes and potholes. Groundwater moves from topographically higher areas to the ancestral valley, then along the ancestral valley to the southwest. Water is discharged from aquifers by evapotranspiration, springs and seeps, movement directly into streams and lakes, and from pumping wells. Average well yields are greatest for irrigation wells completed in outwash gravel (886 gallons/min). Eighteen wells were completed in various aquifers to monitor potential long-term changes in water levels and water quality. Measured water levels declined about 2 ft. or less during the study (1982-85). Chemical analysis of groundwater samples indicated that concentrations of some dissolved constituents exceeded U.S. Environmental Protection Agency standards for drinking water. (USGS)

When ancestral heritage is a source of discomfort: culture, pre-object relatedness, and self-alienation.

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Kradin, Richard L

2012-04-01

The ancestral claims on an individual can evoke mental conflict when they involve separating from an ethnic group whose beliefs and customs are devalued by the dominant culture. However, these claims are engraved on the psyche early in development by caretakers to the level of pre-object relatedness, where contents and affect tones are implicit and may be unavailable for later psychoanalytical interventions. In addition, as the anthropologist Clifford Geertz notes, one's culture of origin precedes the development of psyche and creates its own set of claims that must be renegotiated when one encounters a different domain of cultural symbols, a confrontation that can produce psychological dissonance and self-alienation. In this paper, three cases are examined in which mental conflicts were evoked by attempts at divesting ancestral claims in response to conscious efforts to assimilate into the dominant culture. These patients suffered from separation guilt and unstable self-esteem and reported dream imagery suggesting psychological imbalance. The requirement to carefully delineate the ancestral claims on psyche as well as those contents and affects that may not be accessible to therapeutic intervention is emphasized, and the importance of compromise and acceptance with respect to the psychological demands of the unconscious are considered. 2012, The Society of Analytical Psychology.

BI-2 destabilizes HIV-1 cores during infection and Prevents Binding of CPSF6 to the HIV-1 Capsid.

Science.gov (United States)

Fricke, Thomas; Buffone, Cindy; Opp, Silvana; Valle-Casuso, Jose; Diaz-Griffero, Felipe

2014-12-11

The recently discovered small-molecule BI-2 potently blocks HIV-1 infection. BI-2 binds to the N-terminal domain of HIV-1 capsid. BI-2 utilizes the same capsid pocket used by the small molecule PF74. Although both drugs bind to the same pocket, it has been proposed that BI-2 uses a different mechanism to block HIV-1 infection when compared to PF74. This work demonstrates that BI-2 destabilizes the HIV-1 core during infection, and prevents the binding of the cellular factor CPSF6 to the HIV-1 core. Overall this short-form paper suggests that BI-2 is using a similar mechanism to the one used by PF74 to block HIV-1 infection.

Structure of the immature HIV-1 capsid in intact virus particles at 8.8 angstrom resolution

Czech Academy of Sciences Publication Activity Database

Schur, F. K. M.; Hagen, W. J. H.; Rumlová, Michaela; Ruml, T.; Müller, B.; Kräusslich, H. G.; Briggs, J. A. G.

2015-01-01

Roč. 517, č. 7535 (2015), s. 505-508 ISSN 0028-0836 R&D Projects: GA ČR(CZ) GA14-15326S Institutional support: RVO:61388963 Keywords : retrovirus * HIV * M-PMV * capsid protein * CA * assembly * immature particles Subject RIV: CE - Biochemistry Impact factor: 38.138, year: 2015

Inferring ancestral distribution area and survival vegetation of Caragana (Fabaceae) in Tertiary

Science.gov (United States)

Mingli Zhang; Juanjuan Xue; Qiang Zhang; Stewart C. Sanderson

2015-01-01

Caragana, a leguminous genus mainly restricted to temperate Central and East Asia, occurs in arid, semiarid, and humid belts, and has forest, grassland, and desert ecotypes. Based on the previous molecular phylogenetic tree and dating, biogeographical analyses of extant species area and ecotype were conducted by means of four ancestral optimization approaches: S-DIVA,...

Reconstructing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex.

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Katherine Belov

2006-03-01

Full Text Available The first sequenced marsupial genome promises to reveal unparalleled insights into mammalian evolution. We have used the Monodelphis domestica (gray short-tailed opossum sequence to construct the first map of a marsupial major histocompatibility complex (MHC. The MHC is the most gene-dense region of the mammalian genome and is critical to immunity and reproductive success. The marsupial MHC bridges the phylogenetic gap between the complex MHC of eutherian mammals and the minimal essential MHC of birds. Here we show that the opossum MHC is gene dense and complex, as in humans, but shares more organizational features with non-mammals. The Class I genes have amplified within the Class II region, resulting in a unique Class I/II region. We present a model of the organization of the MHC in ancestral mammals and its elaboration during mammalian evolution. The opossum genome, together with other extant genomes, reveals the existence of an ancestral "immune supercomplex" that contained genes of both types of natural killer receptors together with antigen processing genes and MHC genes.

Are palaeoscolecids ancestral ecdysozoans?

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Harvey, Thomas H P; Dong, Xiping; Donoghue, Philip C J

2010-01-01

The reconstruction of ancestors is a central aim of comparative anatomy and evolutionary developmental biology, not least in attempts to understand the relationship between developmental and organismal evolution. Inferences based on living taxa can and should be tested against the fossil record, which provides an independent and direct view onto historical character combinations. Here, we consider the nature of the last common ancestor of living ecdysozoans through a detailed analysis of palaeoscolecids, an early and extinct group of introvert-bearing worms that have been proposed to be ancestral ecdysozoans. In a review of palaeoscolecid anatomy, including newly resolved details of the internal and external cuticle structure, we identify specific characters shared with various living nematoid and scalidophoran worms, but not with panarthropods. Considered within a formal cladistic context, these characters provide most overall support for a stem-priapulid affinity, meaning that palaeoscolecids are far-removed from the ecdysozoan ancestor. We conclude that previous interpretations in which palaeoscolecids occupy a deeper position in the ecdysozoan tree lack particular morphological support and rely instead on a paucity of preserved characters. This bears out a more general point that fossil taxa may appear plesiomorphic merely because they preserve only plesiomorphies, rather than the mélange of primitive and derived characters anticipated of organisms properly allocated to a position deep within animal phylogeny.

Evidence that the ancestral haplotype in Australian hemochromatosis patients may be associated with a common mutation in the gene.

OpenAIRE

Crawford, D H; Powell, L W; Leggett, B A; Francis, J S; Fletcher, L M; Webb, S I; Halliday, J W; Jazwinska, E C

1995-01-01

Hemochromatosis (HC) is a common inherited disorder of iron metabolism for which neither the gene nor biochemical defect have yet been identified. The aim of this study was to look for clinical evidence that the predominant ancestral haplotype in Australian patients is associated with a common mutation in the gene. We compared indices of iron metabolism and storage in three groups of HC patients categorized according to the presence of the ancestral haplotype (i.e., patients with two copies, ...

Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization

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Gao Fan

2012-01-01

Full Text Available Abstract Background To characterize the human humoral immune response against enterovirus 71 (EV71 infection and map human epitopes on the viral capsid proteins. Methods A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3 of BJ08 strain (genomic C4 were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl. Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel. Results A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3 were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15 was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71 were identified and mapped to VP1. Conclusion These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents.

Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella.

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Wilson, M E; Consigli, R A

1985-06-01

A cyclic-nucleotide independent protein kinase activity has been demonstrated in highly purified preparations of the granulosis virus infecting the Indian meal moth, Plodia interpunctella. A divalent cation was required for activity. Manganese was the preferred cation and a pH of 8.0 resulted in optimal incorporation of 32P radiolabel into acid-precipitable protein. Although both ATP and GTP could serve as phosphate donors, ATP was utilized more efficiently by the enzyme. The kinase activity was localized to purified capsids; and the basic, internal core protein, VP12, was found to be the predominant viral acceptor. Histones and protamine sulfate could also serve as acceptors for the capsid-associated kinase activity. Using acid hydrolysis and phosphoamino acid analysis of phosphorylated nucleocapsid protein and nuclear magnetic resonance of phosphorylated VP12, it was determined that the enzyme catalyzes the transfer of phosphate to both serine and arginine residues of acceptor proteins. We believe this kinase activity may play a significant role in the viral replication cycle.

Distinct actions of ancestral vinclozolin and juvenile stress on neural gene expression in the male rat

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Ross eGillette

2015-03-01

Full Text Available Exposure to the endocrine disrupting chemical vinclozolin during gestation of an F0 generation and/or chronic restraint stress during adolescence of the F3 descendants affects behavior, physiology, and gene expression in the brain. Genes related to the networks of growth factors, signaling peptides and receptors, steroid hormone receptors and enzymes, and epigenetic related factors were measured using quantitative polymerase chain reaction via Taqman low density arrays targeting 48 genes in the central amygdaloid nucleus, medial amygdaloid nucleus, medial preoptic area, lateral hypothalamus, and the ventromedial nucleus of the hypothalamus. We found that growth factors are particularly vulnerable to ancestral exposure in the central and medial amygdala; restraint stress during adolescence affected neural growth factors in the medial amygdala. Signaling peptides were affected by both ancestral exposure and stress during adolescence primarily in hypothalamic nuclei. Steroid hormone receptors and enzymes were strongly affected by restraint stress in the medial preoptic area. Epigenetic related genes were affected by stress in the ventromedial hypothalamus and by both ancestral exposure and stress during adolescence independently in the central amygdala. It is noteworthy that the lateral hypothalamus showed no effects of either manipulation. Gene expression is discussed in the context of behavioral and physiological measures previously published.

Distinct actions of ancestral vinclozolin and juvenile stress on neural gene expression in the male rat.

Science.gov (United States)

Gillette, Ross; Miller-Crews, Isaac; Skinner, Michael K; Crews, David

2015-01-01

Exposure to the endocrine disrupting chemical vinclozolin during gestation of an F0 generation and/or chronic restraint stress during adolescence of the F3 descendants affects behavior, physiology, and gene expression in the brain. Genes related to the networks of growth factors, signaling peptides, and receptors, steroid hormone receptors and enzymes, and epigenetic related factors were measured using quantitative polymerase chain reaction via Taqman low density arrays targeting 48 genes in the central amygdaloid nucleus, medial amygdaloid nucleus, medial preoptic area (mPOA), lateral hypothalamus (LH), and the ventromedial nucleus of the hypothalamus. We found that growth factors are particularly vulnerable to ancestral exposure in the central and medial amygdala; restraint stress during adolescence affected neural growth factors in the medial amygdala. Signaling peptides were affected by both ancestral exposure and stress during adolescence primarily in hypothalamic nuclei. Steroid hormone receptors and enzymes were strongly affected by restraint stress in the mPOA. Epigenetic related genes were affected by stress in the ventromedial nucleus and by both ancestral exposure and stress during adolescence independently in the central amygdala. It is noteworthy that the LH showed no effects of either manipulation. Gene expression is discussed in the context of behavioral and physiological measures previously published.

Structures of the major capsid proteins of the human Karolinska Institutet and Washington University polyomaviruses.

Science.gov (United States)

Neu, Ursula; Wang, Jianbo; Macejak, Dennis; Garcea, Robert L; Stehle, Thilo

2011-07-01

The Karolinska Institutet and Washington University polyomaviruses (KIPyV and WUPyV, respectively) are recently discovered human viruses that infect the respiratory tract. Although they have not yet been linked to disease, they are prevalent in populations worldwide, with initial infection occurring in early childhood. Polyomavirus capsids consist of 72 pentamers of the major capsid protein viral protein 1 (VP1), which determines antigenicity and receptor specificity. The WUPyV and KIPyV VP1 proteins are distant in evolution from VP1 proteins of known structure such as simian virus 40 or murine polyomavirus. We present here the crystal structures of unassembled recombinant WUPyV and KIPyV VP1 pentamers at resolutions of 2.9 and 2.55 Å, respectively. The WUPyV and KIPyV VP1 core structures fold into the same β-sandwich that is a hallmark of all polyomavirus VP1 proteins crystallized to date. However, differences in sequence translate into profoundly different surface loop structures in KIPyV and WUPyV VP1 proteins. Such loop structures have not been observed for other polyomaviruses, and they provide initial clues about the possible interactions of these viruses with cell surface receptors.

Perianth evolution in Ranunculaceae: are petals ancestral in the family?

OpenAIRE

Nadot S.; Sauquet H.; Damerval C.; Jabbour F.; Domenech B.

2016-01-01

Progress has been made recently towards the elucidation of phylogenetic relationships among subfamilies and tribes of the Ranunculaceae – the most recent hypothesis was published in 2016 by our team. Although relationships among the 10 tribes of the subfamily Ranunculoideae remain incompletely supported, this hypothesis provides an interesting framework to address the key issue of the ancestral vs. derived nature of a differentiated perianth within the family, and at the level of Ranunculales...

Analysis of ancestral and functionally relevant CD5 variants in systemic lupus erythematosus patients.

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Maria Carmen Cenit

Full Text Available CD5 plays a crucial role in autoimmunity and is a well-established genetic risk factor of developing RA. Recently, evidence of positive selection has been provided for the CD5 Pro224-Val471 haplotype in East Asian populations. The aim of the present work was to further analyze the functional relevance of non-synonymous CD5 polymorphisms conforming the ancestral and the newly derived haplotypes (Pro224-Ala471 and Pro224-Val471, respectively as well as to investigate the potential role of CD5 on the development of SLE and/or SLE nephritis.The CD5 SNPs rs2241002 (C/T; Pro224Leu and rs2229177 (C/T; Ala471Val were genotyped using TaqMan allelic discrimination assays in a total of 1,324 controls and 681 SLE patients of Spanish origin. In vitro analysis of CD3-mediated T cell proliferative and cytokine response profiles of healthy volunteers homozygous for the above mentioned CD5 haplotypes were also analyzed.T-cell proliferation and cytokine release were significantly increased showing a bias towards to a Th2 profile after CD3 cross-linking of peripheral mononuclear cells from healthy individuals homozygous for the ancestral Pro224-Ala471 (CC haplotype, compared to the more recently derived Pro224-Val471 (CT. The same allelic combination was statistically associated with Lupus nephritis.The ancestral Ala471 CD5 allele confers lymphocyte hyper-responsiveness to TCR/CD3 cross-linking and is associated with nephritis in SLE patients.

Expression and purification of capsid proteins of Aichi virus and in vitro reassembly of empty virion

Czech Academy of Sciences Publication Activity Database

Smola, Miroslav; Dubánková, Anna; Šilhán, Jan; Bouřa, Evžen

2017-01-01

Roč. 284, Suppl 1 (2017), s. 107 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] R&D Projects: GA ČR GJ15-21030Y; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : Aichi virus * capsid proteins Subject RIV: CE - Biochemistry

Novel antibody binding determinants on the capsid surface of serotype O foot-and-mouth disease virus

Science.gov (United States)

Asfor, Amin S.; Upadhyaya, Sasmita; Knowles, Nick J.; King, Donald P.; Paton, David J.

2014-01-01

Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside the mAb binding sites contribute to antibody-mediated in vivo neutralization of FMDV. Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17 Å away from VP2-72), located at the threefold axis and more distant from previously identified antigenic sites, exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design. PMID:24584474

Analysis of Hydraulic Flood Control Structure at Putat Boro River

OpenAIRE

Ruzziyatno, Ruhban

2015-01-01

Putat Boro River is one of the main drainage systems of Surakarta city which drains into Bengawan Solo river. The primary problem when flood occur is the higher water level of Bengawan Solo than Boro River and then backwater occur and inundates Putat Boro River. The objective of the study is to obtain operational method of Putat Boro River floodgate to control both inflows and outflows not only during flood but also normal condition. It also aims to know the Putat Boro rivers floodgate op...

Estimating Divergence Time and Ancestral Effective Population Size of Bornean and Sumatran Orangutan Subspecies Using a Coalescent Hidden Markov Model

DEFF Research Database (Denmark)

Mailund, Thomas; Dutheil, Julien; Hobolth, Asger

2011-01-01

event has occurred to split them apart. The size of these segments of constant divergence depends on the recombination rate, but also on the speciation time, the effective population size of the ancestral population, as well as demographic effects and selection. Thus, inference of these parameters may......, and the ancestral effective population size. The model is efficient enough to allow inference on whole-genome data sets. We first investigate the power and consistency of the model with coalescent simulations and then apply it to the whole-genome sequences of the two orangutan sub-species, Bornean (P. p. pygmaeus......) and Sumatran (P. p. abelii) orangutans from the Orangutan Genome Project. We estimate the speciation time between the two sub-species to be thousand years ago and the effective population size of the ancestral orangutan species to be , consistent with recent results based on smaller data sets. We also report...

Twenty putative palmitoyl-acyl transferase genes with distinct ...

African Journals Online (AJOL)

There are 20 genes containing DHHC domain predicted to encode putative palmitoyltransferase in Arabidopsis thaliana genome. However, little is known about their characteristics such as genetic relationship and expression profile. Here, we present an overview of the putative PAT genes in A. thaliana focusing on their ...

Effectiveness of ancestral irradiation on the direct and correlated responses to selection for body weight in rats

International Nuclear Information System (INIS)

Gianola, D.

1975-01-01

The effects of ancestral irradiation of rat spermatogonia (a cumulative total of 4050 r of x-rays) were studied in a highly inbred line of rats to explore the feasibility of using irradiation to enhance the effectiveness of selection. Six generations after irradiation was terminated, a selection experiment for body weight at six weeks of age was started in both ancestrally irradiated and non-irradiated populations. There were two non-contemporaneous replicates in each of the populations. Within each of the ancestral treatment-replicate combinations one line was selected for high, one for low body weight at six weeks of age, and a third line was maintained by random selection. In each line, avoidance of mating of animals with grandparents in common was attempted. Data on the first ten progeny generations of selection were included in this study. Five types of covariances among relatives were used to estimate causal components of variance for five different genetic models within the ''non-irradiated'' and ''irradiated'' randomly selected models. The parameters in the genetic models were estimated by generalized least-squares. This analysis suggested that a genetic model including direct genetic and maternal genetic effects was adequate to describe the body weights at 3, 6 and 10 weeks of age and the weight gains between these ages. Ancestral irradiation seemed to have enhanced the maternal genetic variance and the covariance between the direct genetic and the maternal genetic effects. On the basis of the above analysis, it was deduced that mass selection should have been more effective in the descendants of irradiated males than in those of the non-irradiated males as a consequence of greater phenotypic variability in their progeny and an enhancement in the regression of the genetic value on the selection criterion

Genotype-based ancestral background consistently predicts efficacy and side effects across treatments in CATIE and STAR*D.

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Daniel E Adkins

Full Text Available Only a subset of patients will typically respond to any given prescribed drug. The time it takes clinicians to declare a treatment ineffective leaves the patient in an impaired state and at unnecessary risk for adverse drug effects. Thus, diagnostic tests robustly predicting the most effective and safe medication for each patient prior to starting pharmacotherapy would have tremendous clinical value. In this article, we evaluated the use of genetic markers to estimate ancestry as a predictive component of such diagnostic tests. We first estimated each patient's unique mosaic of ancestral backgrounds using genome-wide SNP data collected in the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE (n = 765 and the Sequenced Treatment Alternatives to Relieve Depression (STAR*D (n = 1892. Next, we performed multiple regression analyses to estimate the predictive power of these ancestral dimensions. For 136/89 treatment-outcome combinations tested in CATIE/STAR*D, results indicated 1.67/1.84 times higher median test statistics than expected under the null hypothesis assuming no predictive power (p<0.01, both samples. Thus, ancestry showed robust and pervasive correlations with drug efficacy and side effects in both CATIE and STAR*D. Comparison of the marginal predictive power of MDS ancestral dimensions and self-reported race indicated significant improvements to model fit with the inclusion of MDS dimensions, but mixed evidence for self-reported race. Knowledge of each patient's unique mosaic of ancestral backgrounds provides a potent immediate starting point for developing algorithms identifying the most effective and safe medication for a wide variety of drug-treatment response combinations. As relatively few new psychiatric drugs are currently under development, such personalized medicine offers a promising approach toward optimizing pharmacotherapy for psychiatric conditions.

The lectin from Musa paradisiaca binds with the capsid protein of tobacco mosaic virus and prevents viral infection.

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Liu, Xiao-Yu; Li, Huan; Zhang, Wei

2014-05-04

It has been demonstrated that the lectin from Musa paradisiaca (BanLec-1) could inhibit the cellular entry of human immunodeficiency virus (HIV). In order to evaluate its effects on tobacco mosaic virus (TMV), the banlec-1 gene was cloned and transformed into Escherichia coli and tobacco, respectively. Recombinant BanLec-1 showed metal ions dependence, and higher thermal and pH stability. Overexpression of banlec-1 in tobacco resulted in decreased leaf size, and higher resistance to TMV infection, which includes reduced TMV cellular entry, more stable chlorophyll contents, and enhanced antioxidant enzymes. BanLec-1 was found to bind directly to the TMV capsid protein in vitro , and to inhibit TMV infection in a dose-dependent manner. In contrast to limited prevention in vivo , purified rBanLec-1 exhibited more significant effects on TMV infection in vitro . Taken together, our study indicated that BanLec-1 could prevent TMV infection in tobacco, probably through the interaction between BanLec-1 and TMV capsid protein.

Language Shift and Maintenance in Multilingual Mauritius: The Case of Indian Ancestral Languages

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Bissoonauth, Anu

2011-01-01

This paper reports on a research study conducted in Mauritius between June and July 2009. The aim of this research was to investigate the use of Indian ancestral languages in the domestic domain by the younger generations. The data were collected in the field by means of a questionnaire and interviews from a quota sample of secondary school…

Identification of the ancestral haplotype for apolipoprotein B suggests an African origin of Homo sapiens sapiens and traces their subsequent migration to Europe and the Pacific

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Rapacz, J.; Hasler-Rapacz, J.O. (Univ. of Wisconsin, Madison (United States)); Chen, L.; Wu, Mingjiuan; Schumaker, V.N. (Univ. of California, Los Angeles (United States)); Butler-Brunner, E.; Butler, R. (Swiss Red Cross Blood Transfusion Service, Bern (Switzerland))

1991-02-15

The probable ancestral haplotype for human apolipoprotein B (apoB) has been identified through immunological analysis of chimpanzee and gorilla serum and sequence analysis of their DNA. Moreover, the frequency of this ancestral apoB haplotype among different human populations provides strong support for the African origin of Homo sapiens sapiens and their subsequent migration from Africa to Europe and to the Pacific. The approach used here for the identification of the ancestral human apoB haplotype is likely to be applicable to many other genes.

Identification of the ancestral haplotype for apolipoprotein B suggests an African origin of Homo sapiens sapiens and traces their subsequent migration to Europe and the Pacific

International Nuclear Information System (INIS)

Rapacz, J.; Hasler-Rapacz, J.O.; Chen, L.; Wu, Mingjiuan; Schumaker, V.N.; Butler-Brunner, E.; Butler, R.

1991-01-01

The probable ancestral haplotype for human apolipoprotein B (apoB) has been identified through immunological analysis of chimpanzee and gorilla serum and sequence analysis of their DNA. Moreover, the frequency of this ancestral apoB haplotype among different human populations provides strong support for the African origin of Homo sapiens sapiens and their subsequent migration from Africa to Europe and to the Pacific. The approach used here for the identification of the ancestral human apoB haplotype is likely to be applicable to many other genes

Sequence Analysis of the Capsid Gene during a Genotype II.4 Dominated Norovirus Season in One University Hospital

DEFF Research Database (Denmark)

Holzknecht, Barbara Juliane; Franck, Kristina Træholt; Nielsen, Rikke Thoft

2015-01-01

Norovirus (NoV) is a leading cause of gastroenteritis and genotype II.4 (GII.4) is responsible for the majority of nosocomial NoV infections. Our objective was to examine whether sequencing of the capsid gene might be a useful tool for the hospital outbreak investigation to define possible...

Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions

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In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained several positively charged amino acids...

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy.

Science.gov (United States)

Ho, Phuong T; Reddy, Vijay S

2018-01-01

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (∼3.0 Å) and size (∼310.0 Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508 Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9 Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations. Copyright © 2017 Elsevier Inc. All rights reserved.

Evolutionary and Functional Analysis of Old World Primate TRIM5 Reveals the Ancient Emergence of Primate Lentiviruses and Convergent Evolution Targeting a Conserved Capsid Interface.

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Kevin R McCarthy

2015-08-01

Full Text Available The widespread distribution of lentiviruses among African primates, and the lack of severe pathogenesis in many of these natural reservoirs, are taken as evidence for long-term co-evolution between the simian immunodeficiency viruses (SIVs and their primate hosts. Evidence for positive selection acting on antiviral restriction factors is consistent with virus-host interactions spanning millions of years of primate evolution. However, many restriction mechanisms are not virus-specific, and selection cannot be unambiguously attributed to any one type of virus. We hypothesized that the restriction factor TRIM5, because of its unique specificity for retrovirus capsids, should accumulate adaptive changes in a virus-specific fashion, and therefore, that phylogenetic reconstruction of TRIM5 evolution in African primates should reveal selection by lentiviruses closely related to modern SIVs. We analyzed complete TRIM5 coding sequences of 22 Old World primates and identified a tightly-spaced cluster of branch-specific adaptions appearing in the Cercopithecinae lineage after divergence from the Colobinae around 16 million years ago. Functional assays of both extant TRIM5 orthologs and reconstructed ancestral TRIM5 proteins revealed that this cluster of adaptations in TRIM5 specifically resulted in the ability to restrict Cercopithecine lentiviruses, but had no effect (positive or negative on restriction of other retroviruses, including lentiviruses of non-Cercopithecine primates. The correlation between lineage-specific adaptations and ability to restrict viruses endemic to the same hosts supports the hypothesis that lentiviruses closely related to modern SIVs were present in Africa and infecting the ancestors of Cercopithecine primates as far back as 16 million years ago, and provides insight into the evolution of TRIM5 specificity.

Structure, Immunogenicity, and Protective Mechanism of an Engineered Enterovirus 71-Like Particle Vaccine Mimicking 80S Empty Capsid.

Science.gov (United States)

Wang, Xiaoli; Ku, Zhiqiang; Zhang, Xiang; Ye, Xiaohua; Chen, Jinhuan; Liu, Qingwei; Zhang, Wei; Zhang, Chao; Fu, Zhenglin; Jin, Xia; Cong, Yao; Huang, Zhong

2018-01-01

Enterovirus 71 (EV71) is the major causative agent of severe hand, foot, and mouth disease, which affects millions of young children in the Asia-Pacific region annually. In this study, we engineered a novel EV71 virus-like particle (VLP) that lacks VP4 (therefore designated VLP ΔVP4 ) and investigated its structure, antigenicity, and vaccine potential. The cryo-electron microscopy (cryo-EM) structure of VLP ΔVP4 was reconstructed to 3.71-Å resolution. Results from structural and biochemical analyses revealed that VLP ΔVP4 resembles the end product of the viral uncoating process, the 80S empty capsid. VLP ΔVP4 is able to elicit high-titer neutralizing antibodies and to fully protect mice against lethal viral challenge. Mechanistic studies showed that, at the cellular level, the anti-VLP ΔVP4 sera exert neutralization effects at both pre- and postattachment stages by inhibiting both virus attachment and internalization, and at the molecular level, the antisera can block multiple interactions between EV71 and its key receptors. Our study gives a better understanding of EV71 capsid assembly and provides important information for the design and development of new-generation vaccines for EV71, and perhaps for other enteroviruses, as well. IMPORTANCE Enterovirus 71 (EV71) infection may lead to severe hand, foot, and mouth disease, with significant morbidity and mortality. Knowledge regarding EV71 particle assembly remains limited. Here, we report the generation and characterization of a novel EV71 virus-like particle that lacks the VP4 capsid subunit protein. This particle, termed VLP ΔVP4 , structurally mimics the 80S empty capsid, which is the end stage of EV71 uncoating. We further show that VLP ΔVP4 exhibits desirable immunogenicity and protective efficacy in proof-of-concept studies. In addition, the inhibitory mechanisms of the VLP ΔVP4 -induced antibodies are unraveled at both the cellular and molecular levels. Our work provides the first evidence of

Palaeohistological Evidence for Ancestral High Metabolic Rate in Archosaurs.

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Legendre, Lucas J; Guénard, Guillaume; Botha-Brink, Jennifer; Cubo, Jorge

2016-11-01

Metabolic heat production in archosaurs has played an important role in their evolutionary radiation during the Mesozoic, and their ancestral metabolic condition has long been a matter of debate in systematics and palaeontology. The study of fossil bone histology provides crucial information on bone growth rate, which has been used to indirectly investigate the evolution of thermometabolism in archosaurs. However, no quantitative estimation of metabolic rate has ever been performed on fossils using bone histological features. Moreover, to date, no inference model has included phylogenetic information in the form of predictive variables. Here we performed statistical predictive modeling using the new method of phylogenetic eigenvector maps on a set of bone histological features for a sample of extant and extinct vertebrates, to estimate metabolic rates of fossil archosauromorphs. This modeling procedure serves as a case study for eigenvector-based predictive modeling in a phylogenetic context, as well as an investigation of the poorly known evolutionary patterns of metabolic rate in archosaurs. Our results show that Mesozoic theropod dinosaurs exhibit metabolic rates very close to those found in modern birds, that archosaurs share a higher ancestral metabolic rate than that of extant ectotherms, and that this derived high metabolic rate was acquired at a much more inclusive level of the phylogenetic tree, among non-archosaurian archosauromorphs. These results also highlight the difficulties of assigning a given heat production strategy (i.e., endothermy, ectothermy) to an estimated metabolic rate value, and confirm findings of previous studies that the definition of the endotherm/ectotherm dichotomy may be ambiguous. © The Author(s) 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Ancestral diet leads to dynamic transgenerational plasticity for five generations in Drosophila melanogaster

OpenAIRE

Mikheyev, Alexander; Emborski, Carmen

2018-01-01

Ancestral exposures can influence phenotypic expression in subsequent generations, which influence diverse biological processes ranging from phenotypic plasticity to obesity. Currently, most transgenerational studies work under the assumption of transgenerational response stability and reproducibility through time and across exposure differences, relying on short-term (i.e. 2-3 generations) single-exposure experiments. Yet, little evidence exists in the literature to validate this assumption,...

Cyclophilin A interacts with diverse lentiviral capsids

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Emerman Michael

2006-10-01

Full Text Available Abstract Background The capsid (CA protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA. This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay. Results We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA. Conclusion These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.

Evolution of stress-regulated gene expression in duplicate genes of Arabidopsis thaliana.

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Cheng Zou

2009-07-01

Full Text Available Due to the selection pressure imposed by highly variable environmental conditions, stress sensing and regulatory response mechanisms in plants are expected to evolve rapidly. One potential source of innovation in plant stress response mechanisms is gene duplication. In this study, we examined the evolution of stress-regulated gene expression among duplicated genes in the model plant Arabidopsis thaliana. Key to this analysis was reconstructing the putative ancestral stress regulation pattern. By comparing the expression patterns of duplicated genes with the patterns of their ancestors, duplicated genes likely lost and gained stress responses at a rapid rate initially, but the rate is close to zero when the synonymous substitution rate (a proxy for time is > approximately 0.8. When considering duplicated gene pairs, we found that partitioning of putative ancestral stress responses occurred more frequently compared to cases of parallel retention and loss. Furthermore, the pattern of stress response partitioning was extremely asymmetric. An analysis of putative cis-acting DNA regulatory elements in the promoters of the duplicated stress-regulated genes indicated that the asymmetric partitioning of ancestral stress responses are likely due, at least in part, to differential loss of DNA regulatory elements; the duplicated genes losing most of their stress responses were those that had lost more of the putative cis-acting elements. Finally, duplicate genes that lost most or all of the ancestral responses are more likely to have gained responses to other stresses. Therefore, the retention of duplicates that inherit few or no functions seems to be coupled to neofunctionalization. Taken together, our findings provide new insight into the patterns of evolutionary changes in gene stress responses after duplication and lay the foundation for testing the adaptive significance of stress regulatory changes under highly variable biotic and abiotic environments.

A phylogenetic Kalman filter for ancestral trait reconstruction using molecular data.

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Lartillot, Nicolas

2014-02-15

Correlation between life history or ecological traits and genomic features such as nucleotide or amino acid composition can be used for reconstructing the evolutionary history of the traits of interest along phylogenies. Thus far, however, such ancestral reconstructions have been done using simple linear regression approaches that do not account for phylogenetic inertia. These reconstructions could instead be seen as a genuine comparative regression problem, such as formalized by classical generalized least-square comparative methods, in which the trait of interest and the molecular predictor are represented as correlated Brownian characters coevolving along the phylogeny. Here, a Bayesian sampler is introduced, representing an alternative and more efficient algorithmic solution to this comparative regression problem, compared with currently existing generalized least-square approaches. Technically, ancestral trait reconstruction based on a molecular predictor is shown to be formally equivalent to a phylogenetic Kalman filter problem, for which backward and forward recursions are developed and implemented in the context of a Markov chain Monte Carlo sampler. The comparative regression method results in more accurate reconstructions and a more faithful representation of uncertainty, compared with simple linear regression. Application to the reconstruction of the evolution of optimal growth temperature in Archaea, using GC composition in ribosomal RNA stems and amino acid composition of a sample of protein-coding genes, confirms previous findings, in particular, pointing to a hyperthermophilic ancestor for the kingdom. The program is freely available at www.phylobayes.org.

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

OpenAIRE

Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino

2017-01-01

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ?procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: a...

Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis.

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Ying Liu

2010-08-01

Full Text Available In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV, is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20 and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20 were blocked in encapsidation (no virus after blind passages but could be rescued if the capsid and 2C(ATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i genome replication is known to be stringently linked to translation, (ii morphogenesis is known to be stringently linked to genome replication, (iii newly synthesized 2C(ATPase is an essential component of the replication complex, and (iv 2C(ATPase has specific affinity to capsid protein(s. These conditions lead to morphogenesis at the site where newly

Modeling capsid kinetics assembly from the steady state distribution of multi-sizes aggregates

Energy Technology Data Exchange (ETDEWEB)

Hozé, Nathanaël; Holcman, David

2014-01-24

The kinetics of aggregation for particles of various sizes depends on their diffusive arrival and fusion at a specific nucleation site. We present here a mean-field approximation and a stochastic jump model for aggregates at equilibrium. This approach is an alternative to the classical Smoluchowski equations that do not have a close form and are not solvable in general. We analyze these mean-field equations and obtain the kinetics of a cluster formation. Our approach provides a simplified theoretical framework to study the kinetics of viral capsid formation, such as HIV from the self-assembly of the structural proteins Gag.

Cyclophilin A Levels Dictate Infection Efficiency of Human Immunodeficiency Virus Type 1 Capsid Escape Mutants A92E and G94D ▿

Science.gov (United States)

Ylinen, Laura M. J.; Schaller, Torsten; Price, Amanda; Fletcher, Adam J.; Noursadeghi, Mahdad; James, Leo C.; Towers, Greg J.

2009-01-01

Cyclophilin A (CypA) is an important human immunodeficiency virus type 1 (HIV-1) cofactor in human cells. HIV-1 A92E and G94D capsid escape mutants arise during CypA inhibition and in certain cell lines are dependent on CypA inhibition. Here we show that dependence on CypA inhibition is due to high CypA levels. Restricted HIV-1 is stable, and remarkably, restriction is augmented by arresting cell division. Nuclear entry is not inhibited. We propose that high CypA levels and capsid mutations combine to disturb uncoating, leading to poor infectivity, particularly in arrested cells. Our data suggest a role for CypA in uncoating the core of HIV-1 to facilitate integration. PMID:19073742

Systematics and morphological evolution within the moss family Bryaceae: a comparison between parsimony and Bayesian methods for reconstruction of ancestral character states.

Science.gov (United States)

Pedersen, Niklas; Holyoak, David T; Newton, Angela E

2007-06-01

The Bryaceae are a large cosmopolitan moss family including genera of significant morphological and taxonomic complexity. Phylogenetic relationships within the Bryaceae were reconstructed based on DNA sequence data from all three genomic compartments. In addition, maximum parsimony and Bayesian inference were employed to reconstruct ancestral character states of 38 morphological plus four habitat characters and eight insertion/deletion events. The recovered phylogenetic patterns are generally in accord with previous phylogenies based on chloroplast DNA sequence data and three major clades are identified. The first clade comprises Bryum bornholmense, B. rubens, B. caespiticium, and Plagiobryum. This corroborates the hypothesis suggested by previous studies that several Bryum species are more closely related to Plagiobryum than to the core Bryum species. The second clade includes Acidodontium, Anomobryum, and Haplodontium, while the third clade contains the core Bryum species plus Imbribryum. Within the latter clade, B. subapiculatum and B. tenuisetum form the sister clade to Imbribryum. Reconstructions of ancestral character states under maximum parsimony and Bayesian inference suggest fourteen morphological synapomorphies for the ingroup and synapomorphies are detected for most clades within the ingroup. Maximum parsimony and Bayesian reconstructions of ancestral character states are mostly congruent although Bayesian inference shows that the posterior probability of ancestral character states may decrease dramatically when node support is taken into account. Bayesian inference also indicates that reconstructions may be ambiguous at internal nodes for highly polymorphic characters.

Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to surface of Romaine lettuce through HBGA-like molecules

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Human Noroviruses (HuNoVs) are the main cause of nonbacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein (INP) mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein (G...

Virulent variants emerging in mice infected with the apathogenic prototype strain of the parvovirus minute virus of mice exhibit a capsid with low avidity for a primary receptor.

Science.gov (United States)

Rubio, Mari-Paz; López-Bueno, Alberto; Almendral, José M

2005-09-01

The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of

Sexually dimorphic effects of ancestral exposure to vinclozolin on stress reactivity in rats.

Science.gov (United States)

Gillette, Ross; Miller-Crews, Isaac; Nilsson, Eric E; Skinner, Michael K; Gore, Andrea C; Crews, David

2014-10-01

How an individual responds to the environment depends upon both personal life history as well as inherited genetic and epigenetic factors from ancestors. Using a 2-hit, 3 generations apart model, we tested how F3 descendants of rats given in utero exposure to the environmental endocrine-disrupting chemical (EDC) vinclozolin reacted to stress during adolescence in their own lives, focusing on sexually dimorphic phenotypic outcomes. In adulthood, male and female F3 vinclozolin- or vehicle-lineage rats, stressed or nonstressed, were behaviorally characterized on a battery of tests and then euthanized. Serum was used for hormone assays, and brains were used for quantitative PCR and transcriptome analyses. Results showed that the effects of ancestral exposure to vinclozolin converged with stress experienced during adolescence in a sexually dimorphic manner. Debilitating effects were seen at all levels of the phenotype, including physiology, behavior, brain metabolism, gene expression, and genome-wide transcriptome modifications in specific brain nuclei. Additionally, females were significantly more vulnerable than males to transgenerational effects of vinclozolin on anxiety but not sociality tests. This fundamental transformation occurs in a manner not predicted by the ancestral exposure or the proximate effects of stress during adolescence, an interaction we refer to as synchronicity.

Travelling in time with networks: Revealing present day hybridization versus ancestral polymorphism between two species of brown algae, Fucus vesiculosus and F. spiralis

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Pearson Gareth A

2011-01-01

Full Text Available Abstract Background Hybridization or divergence between sympatric sister species provides a natural laboratory to study speciation processes. The shared polymorphism in sister species may either be ancestral or derive from hybridization, and the accuracy of analytic methods used thus far to derive convincing evidence for the occurrence of present day hybridization is largely debated. Results Here we propose the application of network analysis to test for the occurrence of present day hybridization between the two species of brown algae Fucus spiralis and F. vesiculosus. Individual-centered networks were analyzed on the basis of microsatellite genotypes from North Africa to the Pacific American coast, through the North Atlantic. Two genetic distances integrating different time steps were used, the Rozenfeld (RD; based on alleles divergence and the Shared Allele (SAD; based on alleles identity distances. A diagnostic level of genotype divergence and clustering of individuals from each species was obtained through RD while screening for exchanges through putative hybridization was facilitated using SAD. Intermediate individuals linking both clusters on the RD network were those sampled at the limits of the sympatric zone in Northwest Iberia. Conclusion These results suggesting rare hybridization were confirmed by simulation of hybrids and F2 with directed backcrosses. Comparison with the Bayesian method STRUCTURE confirmed the usefulness of both approaches and emphasized the reliability of network analysis to unravel and study hybridization

QA prime-boost vaccination strategy in prevent serotype O FMDV infection using a "single-cycle" alphavirus vector and empty capsid particles

DEFF Research Database (Denmark)

Gullberg, Maria; Lohse, Louise; Bøtner, Anette

Introduction Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can help to control this disease, however, current vaccines based on chemically inactivated FMDV, are imperfect and there is a need for new, safe...... and effective vaccines to control FMD. There is no cross protection between the 7 serotypes but serotype O is the most abundant globally. Material and methods The FMDV capsid protein precursor (P1-2A) of strain O1 Manisa has been expressed with the FMDV 3C protease (3Cpro) using a “single cycle” packaged...... alphavirus self-replicating RNA based on Semliki Forest virus (SFV). Purified O1 Manisa empty capsid particles (ECs) have been prepared using a recombinant vaccinia virus expression system. Cattle have been vaccinated with the SFV-FMDV vectors and boosted subsequently with the ECs and then challenged...

Ancestral state reconstruction infers phytopathogenic origins of sooty blotch and flyspeck fungi on apple.

Science.gov (United States)

Ismail, Siti Izera; Batzer, Jean Carlson; Harrington, Thomas C; Crous, Pedro W; Lavrov, Dennis V; Li, Huanyu; Gleason, Mark L

2016-01-01

Members of the sooty blotch and flyspeck (SBFS) complex are epiphytic fungi in the Ascomycota that cause economically damaging blemishes of apples worldwide. SBFS fungi are polyphyletic, but approx. 96% of SBFS species are in the Capnodiales. Evolutionary origins of SBFS fungi remain unclear, so we attempted to infer their origins by means of ancestral state reconstruction on a phylogenetic tree built utilizing genes for the nuc 28S rDNA (approx. 830 bp from near the 59 end) and the second largest subunit of RNA polymerase II (RPB2). The analyzed taxa included the well-known genera of SBFS as well as non-SBFS fungi from seven families within the Capnodiales. The non-SBFS taxa were selected based on their distinct ecological niches, including plant-parasitic and saprophytic species. The phylogenetic analyses revealed that most SBFS species in the Capnodiales are closely related to plant-parasitic fungi. Ancestral state reconstruction provided strong evidence that plant-parasitic fungi were the ancestors of the major SBFS lineages. Knowledge gained from this study may help to better understand the ecology and evolution of epiphytic fungi. © 2016 by The Mycological Society of America.

Bearing the unbearable: ancestral transmission through dreams and moving metaphors in the analtyic field.

Science.gov (United States)

Pickering, Judith

2012-11-01

This paper explores how untold and unresolved intergenerational trauma may be transmitted through unconscious channels of communication, manifesting in the dreams of descendants. Unwitting carriers for that which was too horrific for their ancestors to bear, descendants may enter analysis through an unconscious need to uncover past secrets, piece together ancestral histories before the keys to comprehending their terrible inheritance die with their forebears. They seek the relational containment of the analytic relationship to provide psychological conditions to bear the unbearable, know the unknowable, speak the unspeakable and redeem the unredeemable. In the case of 'Rachael', initial dreams gave rise to what Hobson (1984) called 'moving metaphors of self' in the analytic field. Dream imagery, projective and introjective processes in the transference-countertransference dynamics gradually revealed an unknown ancestral history. I clarify the back and forth process from dream to waking dream thoughts to moving metaphors and differentiate the moving metaphor from a living symbol. I argue that the containment of the analytic relationship nested within the security of the analytic space is a necessary precondition for such healing processes to occur. © 2012, The Society of Analytical Psychology.

Ancestral dichlorodiphenyltrichloroethane (DDT) exposure promotes epigenetic transgenerational inheritance of obesity

Science.gov (United States)

2013-01-01

Background Ancestral environmental exposures to a variety of environmental factors and toxicants have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. The present work examined the potential transgenerational actions of the insecticide dichlorodiphenyltrichloroethane (DDT) on obesity and associated disease. Methods Outbred gestating female rats were transiently exposed to a vehicle control or DDT and the F1 generation offspring bred to generate the F2 generation and F2 generation bred to generate the F3 generation. The F1 and F3 generation control and DDT lineage rats were aged and various pathologies investigated. The F3 generation male sperm were collected to investigate methylation between the control and DDT lineage male sperm. Results The F1 generation offspring (directly exposed as a fetus) derived from the F0 generation exposed gestating female rats were not found to develop obesity. The F1 generation DDT lineage animals did develop kidney disease, prostate disease, ovary disease and tumor development as adults. Interestingly, the F3 generation (great grand-offspring) had over 50% of males and females develop obesity. Several transgenerational diseases previously shown to be associated with metabolic syndrome and obesity were observed in the testis, ovary and kidney. The transgenerational transmission of disease was through both female (egg) and male (sperm) germlines. F3 generation sperm epimutations, differential DNA methylation regions (DMR), induced by DDT were identified. A number of the genes associated with the DMR have previously been shown to be associated with obesity. Conclusions Observations indicate ancestral exposure to DDT can promote obesity and associated disease transgenerationally. The etiology of disease such as obesity may be in part due to environmentally induced epigenetic transgenerational inheritance. PMID:24228800

Low levels of foot-and-mouth disease virus 3C protease expression are required to achieve optimal capsid protein expression and processing in mammalian cells

DEFF Research Database (Denmark)

Polacek, Charlotta; Gullberg, Maria; Li, Jiong

2013-01-01

transient-expression assays, within mammalian cells, it is possible to modify the relative amounts of the substrate and protease. It has now been shown that optimal production of the processed capsid proteins from P1-2A is achieved with reduced levels of 3Cpro expression, relative to the P1-2A, compared...... detected by FMDV antigen detection assays. Furthermore, the P1-2A and the processed forms each bind to the integrin αvβ6, the major FMDV receptor. These results contribute to the development of systems which efficiently express the components of empty capsid particles and may represent the basis for safer...... production of diagnostic reagents and improved vaccines against foot-and-mouth disease....

Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

International Nuclear Information System (INIS)

Rumlova, Michaela; Ruml, Tomas; Pohl, Jan; Pichova, Iva

2003-01-01

Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration

Effect of the assignment of ancestral CpG state on the estimation of nucleotide substitution rates in mammals

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Keightley Peter D

2008-09-01

Full Text Available Abstract Background Molecular evolutionary studies in mammals often estimate nucleotide substitution rates within and outside CpG dinucleotides separately. Frequently, in alignments of two sequences, the division of sites into CpG and non-CpG classes is based simply on the presence or absence of a CpG dinucleotide in either sequence, a procedure that we refer to as CpG/non-CpG assignment. Although it likely that this procedure is biased, it is generally assumed that the bias is negligible if species are very closely related. Results Using simulations of DNA sequence evolution we show that assignment of the ancestral CpG state based on the simple presence/absence of the CpG dinucleotide can seriously bias estimates of the substitution rate, because many true non-CpG changes are misassigned as CpG. Paradoxically, this bias is most severe between closely related species, because a minimum of two substitutions are required to misassign a true ancestral CpG site as non-CpG whereas only a single substitution is required to misassign a true ancestral non-CpG site as CpG in a two branch tree. We also show that CpG misassignment bias differentially affects fourfold degenerate and noncoding sites due to differences in base composition such that fourfold degenerate sites can appear to be evolving more slowly than noncoding sites. We demonstrate that the effects predicted by our simulations occur in a real evolutionary setting by comparing substitution rates estimated from human-chimp coding and intronic sequence using CpG/non-CpG assignment with estimates derived from a method that is largely free from bias. Conclusion Our study demonstrates that a common method of assigning sites into CpG and non CpG classes in pairwise alignments is seriously biased and recommends against the adoption of ad hoc methods of ancestral state assignment.

Phylogenetic analysis of glycerol 3-phosphate acyltransferases in opisthokonts reveals unexpected ancestral complexity and novel modern biosynthetic components.

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Heather C Smart

Full Text Available Glycerolipid synthesis represents a central metabolic process of all forms of life. In the last decade multiple genes coding for enzymes responsible for the first step of the pathway, catalyzed by glycerol 3-phosphate acyltransferase (GPAT, have been described, and characterized primarily in model organisms like Saccharomyces cerevisiae and mice. Notoriously, the fungal enzymes share low sequence identity with their known animal counterparts, and the nature of their homology is unclear. Furthermore, two mitochondrial GPAT isoforms have been described in animal cells, while no such enzymes have been identified in Fungi. In order to determine if the yeast and mammalian GPATs are representative of the set of enzymes present in their respective groups, and to test the hypothesis that metazoan orthologues are indeed absent from the fungal clade, a comparative genomic and phylogenetic analysis was performed including organisms spanning the breadth of the Opisthokonta supergroup. Surprisingly, our study unveiled the presence of 'fungal' orthologs in the basal taxa of the holozoa and 'animal' orthologues in the basal holomycetes. This includes a novel clade of fungal homologues, with putative peroxisomal targeting signals, of the mitochondrial/peroxisomal acyltransferases in Metazoa, thus potentially representing an undescribed metabolic capacity in the Fungi. The overall distribution of GPAT homologues is suggestive of high relative complexity in the ancestors of the opisthokont clade, followed by loss and sculpting of the complement in the descendent lineages. Divergence from a general versatile metabolic model, present in ancestrally deduced GPAT complements, points to distinctive contributions of each GPAT isoform to lipid metabolism and homeostasis in contemporary organisms like humans and their fungal pathogens.

A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

Science.gov (United States)

Hughes, J.F.; Brown, G.K.

2004-01-01

Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

Science.gov (United States)

Balinsky, Corey A.; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C.

2013-01-01

Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics. PMID:24027323

Evidence for Ancestral Programming of Resilience in a Two-Hit Stress Model

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Jamshid Faraji

2017-05-01

Full Text Available In a continuously stressful environment, the effects of recurrent prenatal stress (PS may accumulate across generations and alter stress vulnerability and resilience. Here, we report in female rats that a family history of recurrent ancestral PS facilitates certain aspects of movement performance, and that these benefits are abolished by the experience of a second hit, induced by a silent ischemia during adulthood. Female F4-generation rats with and without a family history of cumulative multigenerational PS (MPS were tested for skilled motor function before and after the induction of a minor ischemic insult by endothelin-1 infusion into the primary motor cortex. MPS resulted in improved skilled motor abilities and blunted hypothalamic-pituitary-adrenal (HPA axis function compared to non-stressed rats. Deep sequencing revealed downregulation of miR-708 in MPS rats along with upregulation of its predicted target genes Mapk10 and Rasd2. Through miR-708 stress may regulate mitogen-activated protein kinase (MAPK pathway activity. Hair trace elemental analysis revealed an increased Na/K ratio, which suggests a chronic shift in adrenal gland function. The ischemic lesion activated the HPA axis in MPS rats only; the lesion, however, abolished the advantage of MPS in skilled reaching. The findings indicate that MPS generates adaptive flexibility in movement, which is challenged by a second stressor, such as a neuropathological condition. Thus, a second “hit” by a stressor may limit behavioral flexibility and neural plasticity associated with ancestral stress.

A skull might lie: modelling ancestral ranges and diet from genes and shape of tree squirrels

Czech Academy of Sciences Publication Activity Database

Pečnerová, Patrícia; Moravec, Jiří C.; Martínková, Natália

2015-01-01

Roč. 64, č. 6 (2015), s. 1074-1088 ISSN 1063-5157 EU Projects: European Commission(XE) CZ.1.07/2.4.00/17.0138 Institutional support: RVO:68081766 Keywords : Sciurini * multilocus phylogeny * geometric morphometry * speciation * ancestral range reconstruction * diet modelling Subject RIV: EG - Zoology Impact factor: 8.225, year: 2015

Human Genetic Ancestral Composition Correlates with the Origin of Mycobacterium leprae Strains in a Leprosy Endemic Population.

Science.gov (United States)

Cardona-Castro, Nora; Cortés, Edwin; Beltrán, Camilo; Romero, Marcela; Badel-Mogollón, Jaime E; Bedoya, Gabriel

2015-01-01

Recent reports have suggested that leprosy originated in Africa, extended to Asia and Europe, and arrived in the Americas during European colonization and the African slave trade. Due to colonization, the contemporary Colombian population is an admixture of Native-American, European and African ancestries. Because microorganisms are known to accompany humans during migrations, patterns of human migration can be traced by examining genomic changes in associated microbes. The current study analyzed 118 leprosy cases and 116 unrelated controls from two Colombian regions endemic for leprosy (Atlantic and Andean) in order to determine possible associations of leprosy with patient ancestral background (determined using 36 ancestry informative markers), Mycobacterium leprae genotype and/or patient geographical origin. We found significant differences between ancestral genetic composition. European components were predominant in Andean populations. In contrast, African components were higher in the Atlantic region. M. leprae genotypes were then analyzed for cluster associations and compared with the ancestral composition of leprosy patients. Two M. leprae principal clusters were found: haplotypes C54 and T45. Haplotype C54 associated with African origin and was more frequent in patients from the Atlantic region with a high African component. In contrast, haplotype T45 associated with European origin and was more frequent in Andean patients with a higher European component. These results suggest that the human and M. leprae genomes have co-existed since the African and European origins of the disease, with leprosy ultimately arriving in Colombia during colonization. Distinct M. leprae strains followed European and African settlement in the country and can be detected in contemporary Colombian populations.

Gammasphaerolipovirus, a newly proposed bacteriophage genus, unifies viruses of halophilic archaea and thermophilic bacteria within the novel family Sphaerolipoviridae.

Science.gov (United States)

Pawlowski, Alice; Rissanen, Ilona; Bamford, Jaana K H; Krupovic, Mart; Jalasvuori, Matti

2014-06-01

A new family of viruses named Sphaerolipoviridae has been proposed recently. It comprises icosahedral, tailless haloarchaeal viruses with an internal lipid membrane located between the protein capsid and the dsDNA genome. The proposed family Sphaerolipoviridae was divided into two genera: Alphasphaerolipovirus, including Haloarcula hispanica viruses SH1, PH1 and HHIV-2, and Betasphaerolipovirus, including Natrinema virus SNJ1. Here, we propose to expand the family Sphaerolipoviridae to include a group of bacteriophages infecting extreme thermophilic Thermus thermophilus and sharing a number of structural and genomic properties with archaeal sphaerolipoviruses. This new group comprises two members, lytic phage P23-77 and temperate phage IN93, as well as putative members P23-72 and P23-65H. In addition, several related proviruses have been discovered as integrated elements in bacterial genomes of the families Thermus and Meiothermus. Morphology of the virus particles and the overall capsid architecture of these bacteriophages resembles that of archaeal members of the Sphaerolipoviridae, including an unusual capsid arrangement in a T = 28 dextro lattice. Alpha- and betasphaerolipoviruses share with P23-77-like bacteriophages a conserved block of core genes that encode a putative genome-packaging ATPase and the two major capsid proteins (MCPs). The recently determined X-ray structure of the small and large MCPs of P23-77 revealed a single beta-barrel (jelly-roll) fold that is superimposable with the cryo-EM density maps of the SH1 capsomers. Given the common features of these viruses, we propose to include the so far unclassified P23-77-like bacteriophages into a new genus, "Gammasphaerolipovirus", within the family Sphaerolipoviridae.

Ancestral origins and invasion pathways in a globally invasive bird correlate with climate and influences from bird trade.

Science.gov (United States)

Jackson, Hazel; Strubbe, Diederik; Tollington, Simon; Prys-Jones, Robert; Matthysen, Erik; Groombridge, Jim J

2015-08-01

Invasive species present a major threat to global biodiversity. Understanding genetic patterns and evolutionary processes that reinforce successful establishment is paramount for elucidating mechanisms underlying biological invasions. Among birds, the ring-necked parakeet (Psittacula krameri) is one of the most successful invasive species, established in over 35 countries. However, little is known about the evolutionary genetic origins of this species and what population genetic signatures tell us about patterns of invasion. We reveal the ancestral origins of populations across the invasive range and explore the potential influence of climate and propagule pressure from the pet trade on observed genetic patterns. Ring-necked parakeet samples representing the ancestral native range (n = 96) were collected from museum specimens, and modern samples from the invasive range (n = 855) were gathered from across Europe, Mauritius and Seychelles, and sequenced for two mitochondrial DNA markers comprising 868 bp of cytochrome b and control region, and genotyped at 10 microsatellite loci. Invasive populations comprise birds that originate predominantly from Pakistan and northern areas of India. Haplotypes associated with more northerly distribution limits in the ancestral native range were more prevalent in invasive populations in Europe, and the predominance of Asian haplotypes in Europe is consistent with the higher number of Asian birds transported by the pet trade outside the native range. Successful establishment of invasive species is likely to be underpinned by a combination of environmental and anthropogenic influences. © 2015 John Wiley & Sons Ltd.

Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study

OpenAIRE

Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

2016-01-01

Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis ...

Emergence, Retention and Selection: A Trilogy of Origination for Functional De Novo Proteins from Ancestral LncRNAs in Primates.

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Jia-Yu Chen

2015-07-01

Full Text Available While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.

Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

Science.gov (United States)

Martini, Sabrina V; Silva, Adriana L; Ferreira, Debora; Rabelo, Rafael; Ornellas, Felipe M; Gomes, Karina; Rocco, Patricia R M; Petrs-Silva, Hilda; Morales, Marcelo M

2016-01-01

Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy. © 2016 The Author(s) Published by S. Karger AG, Basel.

Ancestral state reconstructions require biological evidence to test evolutionary hypotheses: A case study examining the evolution of reproductive mode in squamate reptiles.

Science.gov (United States)

Griffith, Oliver W; Blackburn, Daniel G; Brandley, Matthew C; Van Dyke, James U; Whittington, Camilla M; Thompson, Michael B

2015-09-01

To understand evolutionary transformations it is necessary to identify the character states of extinct ancestors. Ancestral character state reconstruction is inherently difficult because it requires an accurate phylogeny, character state data, and a statistical model of transition rates and is fundamentally constrained by missing data such as extinct taxa. We argue that model based ancestral character state reconstruction should be used to generate hypotheses but should not be considered an analytical endpoint. Using the evolution of viviparity and reversals to oviparity in squamates as a case study, we show how anatomical, physiological, and ecological data can be used to evaluate hypotheses about evolutionary transitions. The evolution of squamate viviparity requires changes to the timing of reproductive events and the successive loss of features responsible for building an eggshell. A reversal to oviparity requires that those lost traits re-evolve. We argue that the re-evolution of oviparity is inherently more difficult than the reverse. We outline how the inviability of intermediate phenotypes might present physiological barriers to reversals from viviparity to oviparity. Finally, we show that ecological data supports an oviparous ancestral state for squamates and multiple transitions to viviparity. In summary, we conclude that the first squamates were oviparous, that frequent transitions to viviparity have occurred, and that reversals to oviparity in viviparous lineages either have not occurred or are exceedingly rare. As this evidence supports conclusions that differ from previous ancestral state reconstructions, our paper highlights the importance of incorporating biological evidence to evaluate model-generated hypotheses. © 2015 Wiley Periodicals, Inc.

The mechanistic basis of hemoglobin adaptation in the high-flying barheaded goose: insights from ancestral protein resurrection

DEFF Research Database (Denmark)

Natarajan, Chandrasekhar; Kumar, Amit; Moriyama, Hideaki

2016-01-01

the functional effects of causative mutations on the genetic background in which they actually occurred during evolution (i.e., in the BHG ancestor). An alternative ‘vertical’ approach is to reconstruct and resurrect ancestral proteins to test the effects of historical mutations on the genetic background...

Human Genetic Ancestral Composition Correlates with the Origin of Mycobacterium leprae Strains in a Leprosy Endemic Population.

Directory of Open Access Journals (Sweden)

Nora Cardona-Castro

Full Text Available Recent reports have suggested that leprosy originated in Africa, extended to Asia and Europe, and arrived in the Americas during European colonization and the African slave trade. Due to colonization, the contemporary Colombian population is an admixture of Native-American, European and African ancestries. Because microorganisms are known to accompany humans during migrations, patterns of human migration can be traced by examining genomic changes in associated microbes. The current study analyzed 118 leprosy cases and 116 unrelated controls from two Colombian regions endemic for leprosy (Atlantic and Andean in order to determine possible associations of leprosy with patient ancestral background (determined using 36 ancestry informative markers, Mycobacterium leprae genotype and/or patient geographical origin. We found significant differences between ancestral genetic composition. European components were predominant in Andean populations. In contrast, African components were higher in the Atlantic region. M. leprae genotypes were then analyzed for cluster associations and compared with the ancestral composition of leprosy patients. Two M. leprae principal clusters were found: haplotypes C54 and T45. Haplotype C54 associated with African origin and was more frequent in patients from the Atlantic region with a high African component. In contrast, haplotype T45 associated with European origin and was more frequent in Andean patients with a higher European component. These results suggest that the human and M. leprae genomes have co-existed since the African and European origins of the disease, with leprosy ultimately arriving in Colombia during colonization. Distinct M. leprae strains followed European and African settlement in the country and can be detected in contemporary Colombian populations.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

Science.gov (United States)

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the genome of the dairy Lactobacillus helveticus bacteriophage {Phi}AQ113.

Science.gov (United States)

Zago, Miriam; Scaltriti, Erika; Rossetti, Lia; Guffanti, Alessandro; Armiento, Angelarita; Fornasari, Maria Emanuela; Grolli, Stefano; Carminati, Domenico; Brini, Elena; Pavan, Paolo; Felsani, Armando; D'Urzo, Annalisa; Moles, Anna; Claude, Jean-Baptiste; Grandori, Rita; Ramoni, Roberto; Giraffa, Giorgio

2013-08-01

The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism.

EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR AND HUMAN PAPILLOMAVIRUS (HPV L1 CAPSID PROTEIN IN CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS

Directory of Open Access Journals (Sweden)

Balan Raluca

2010-09-01

Full Text Available We analyzed the immunohistochemical pattern of epidermal growth factor receptor (EGFR in cervical squamous intraepithelial lesions (SILs in correlation with L1 HPV capsid protein, in order to determine the relationship between EGFR expression and the infection status of human papillomavirus (HPV. The study included 40 cases, 24 LSIL (low grade SIL (CIN1, cervical intraepithelial neoplasia and 16 HSIL (high grade SIL (6 cases of CIN2 and 10 cases of CIN3. The immunoexpression of L1 HPV protein was assessed on conventional cervico-vaginal smears and EGFR was immunohistochemically evaluated on the corresponding cervical biopsies. The HPV L1 capsid protein was expressed in 45.83% of LSIL and 25% of HSIL. EGFR was overexpressed in 62,4% of HSIL (58,4% CIN2 and 41,6% CIN3 and 37,6% LSIL. The immunoexpression of L1 HPV has clinical application in the progression assessment of the cervical precancerous lesions without a correlation to the grade of the cervical SIL. EGFR is expressed by all proliferating squamous epithelial cells, thus corresponding with the grade of SIL. The evaluation of EGFR status, correlated with L1 HPV protein expression, can provide useful data of progression risk of cervical squamous intraepithelial lesions

Founder haplotype analysis of Fanconi anemia in the Korean population finds common ancestral haplotypes for a FANCG variant.

Science.gov (United States)

Park, Joonhong; Kim, Myungshin; Jang, Woori; Chae, Hyojin; Kim, Yonggoo; Chung, Nack-Gyun; Lee, Jae-Wook; Cho, Bin; Jeong, Dae-Chul; Park, In Yang; Park, Mi Sun

2015-05-01

A common ancestral haplotype is strongly suggested in the Korean and Japanese patients with Fanconi anemia (FA), because common mutations have been frequently found: c.2546delC and c.3720_3724delAAACA of FANCA; c.307+1G>C, c.1066C>T, and c.1589_1591delATA of FANCG. Our aim in this study was to investigate the origin of these common mutations of FANCA and FANCG. We genotyped 13 FA patients consisting of five FA-A patients and eight FA-G patients from the Korean FA population. Microsatellite markers used for haplotype analysis included four CA repeat markers which are closely linked with FANCA and eight CA repeat markers which are contiguous with FANCG. As a result, Korean FA-A patients carrying c.2546delC or c.3720_3724delAAACA did not share the same haplotypes. However, three unique haplotypes carrying c.307+1G>C, c.1066C > T, or c.1589_1591delATA, that consisted of eight polymorphic loci covering a flanking region were strongly associated with Korean FA-G, consistent with founder haplotypes reported previously in the Japanese FA-G population. Our finding confirmed the common ancestral haplotypes on the origins of the East Asian FA-G patients, which will improve our understanding of the molecular population genetics of FA-G. To the best of our knowledge, this is the first report on the association between disease-linked mutations and common ancestral haplotypes in the Korean FA population. © 2015 John Wiley & Sons Ltd/University College London.

Estimation of the net acid load of the diet of ancestral preagricultural Homo sapiens and their hominid ancestors.

Science.gov (United States)

Sebastian, Anthony; Frassetto, Lynda A; Sellmeyer, Deborah E; Merriam, Renée L; Morris, R Curtis

2002-12-01

Natural selection has had diet resulting from the inventions of agriculture and animal husbandry. The objective was to estimate the net systemic load of acid (net endogenous acid production; NEAP) from retrojected ancestral preagricultural diets and to compare it with that of contemporary diets, which are characterized by an imbalance of nutrient precursors of hydrogen and bicarbonate ions that induces a lifelong, low-grade, pathogenically significant systemic metabolic acidosis. Using established computational methods, we computed NEAP for a large number of retrojected ancestral preagricultural diets and compared them with computed and measured values for typical American diets. The mean (+/- SD) NEAP for 159 retrojected preagricultural diets was -88 +/- 82 mEq/d; 87% were net base-producing. The computational model predicted NEAP for the average American diet (as recorded in the third National Health and Nutrition Examination Survey) as 48 mEq/d, within a few percentage points of published measured values for free-living Americans; the model, therefore, was not biased toward generating negative NEAP values. The historical shift from negative to positive NEAP was accounted for by the displacement of high-bicarbonate-yielding plant foods in the ancestral diet by cereal grains and energy-dense, nutrient-poor foods in the contemporary diet-neither of which are net base-producing. The findings suggest that diet-induced metabolic acidosis and its sequelae in humans eating contemporary diets reflect a mismatch between the nutrient composition of the diet and genetically determined nutritional requirements for optimal systemic acid-base status.

Time-Dependent-Asymmetric-Linear-Parsimonious Ancestral State Reconstruction.

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Didier, Gilles

2017-10-01

The time-dependent-asymmetric-linear parsimony is an ancestral state reconstruction method which extends the standard linear parsimony (a.k.a. Wagner parsimony) approach by taking into account both branch lengths and asymmetric evolutionary costs for reconstructing quantitative characters (asymmetric costs amount to assuming an evolutionary trend toward the direction with the lowest cost). A formal study of the influence of the asymmetry parameter shows that the time-dependent-asymmetric-linear parsimony infers states which are all taken among the known states, except for some degenerate cases corresponding to special values of the asymmetry parameter. This remarkable property holds in particular for the Wagner parsimony. This study leads to a polynomial algorithm which determines, and provides a compact representation of, the parametric reconstruction of a phylogenetic tree, that is for all the unknown nodes, the set of all the possible reconstructed states associated with the asymmetry parameters leading to them. The time-dependent-asymmetric-linear parsimony is finally illustrated with the parametric reconstruction of the body size of cetaceans.

Nanofluidic Devices with Two Pores in Series for Resistive-Pulse Sensing of Single Virus Capsids

DEFF Research Database (Denmark)

Harms, Zachary D.; Mogensen, Klaus Bo; Rodrigues de Sousa Nunes, Pedro André

2011-01-01

We report fabrication and characterization of nanochannel devices with two nanopores in series for resistive-pulse sensing of hepatitis B virus (HBV) capsids. The nanochannel and two pores are patterned by electron beam lithography between two microchannels and etched by reactive ion etching....... The two nanopores are 50-nm wide, 50-nm deep, and 40-nm long and are spaced 2.0-μm apart. The nanochannel that brackets the two pores is 20 wider (1 μm) to reduce the electrical resistance adjacent to the two pores and to ensure the current returns to its baseline value between resistive-pulse events...

Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: The Alteration of the product by an affinity tag

Czech Academy of Sciences Publication Activity Database

Rumlová, Michaela; Benedíková, Jitka; Cubínková, Romana; Pichová, Iva; Ruml, Tomáš

2001-01-01

Roč. 23, - (2001), s. 75-83 ISSN 1046-5928 R&D Projects: GA ČR GA203/00/1005 Institutional research plan: CEZ:AV0Z4055905 Keywords : Mason-Pfizer monkey virus * capsid protein Subject RIV: CE - Biochemistry Impact factor: 1.497, year: 2001

A branch-heterogeneous model of protein evolution for efficient inference of ancestral sequences.

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Groussin, M; Boussau, B; Gouy, M

2013-07-01

Most models of nucleotide or amino acid substitution used in phylogenetic studies assume that the evolutionary process has been homogeneous across lineages and that composition of nucleotides or amino acids has remained the same throughout the tree. These oversimplified assumptions are refuted by the observation that compositional variability characterizes extant biological sequences. Branch-heterogeneous models of protein evolution that account for compositional variability have been developed, but are not yet in common use because of the large number of parameters required, leading to high computational costs and potential overparameterization. Here, we present a new branch-nonhomogeneous and nonstationary model of protein evolution that captures more accurately the high complexity of sequence evolution. This model, henceforth called Correspondence and likelihood analysis (COaLA), makes use of a correspondence analysis to reduce the number of parameters to be optimized through maximum likelihood, focusing on most of the compositional variation observed in the data. The model was thoroughly tested on both simulated and biological data sets to show its high performance in terms of data fitting and CPU time. COaLA efficiently estimates ancestral amino acid frequencies and sequences, making it relevant for studies aiming at reconstructing and resurrecting ancestral amino acid sequences. Finally, we applied COaLA on a concatenate of universal amino acid sequences to confirm previous results obtained with a nonhomogeneous Bayesian model regarding the early pattern of adaptation to optimal growth temperature, supporting the mesophilic nature of the Last Universal Common Ancestor.

Viable adenovirus vaccine prototypes: High-level production of a papillomavirus capsid antigen from the major late transcriptional unit

OpenAIRE

Berg, Michael; DiFatta, Julie; Hoiczyk, Egbert; Schlegel, Richard; Ketner, Gary

2005-01-01

Safe, effective, orally delivered, live adenovirus vaccines have been in use for three decades. Recombinant derivatives of the live adenovirus vaccines may prove an economical alternative to current vaccines for a variety of diseases. To explore that possibility, we constructed a series of recombinants that express the major capsid protein (L1) of canine oral papillomavirus (COPV), a model for mucosal human papillomavirus (HPV) infection. Vaccination with virus-like particles (VLPs) composed ...

NMR structure of the N-terminal domain of capsid protein from the Mason-Pfizer monkey virus

Czech Academy of Sciences Publication Activity Database

Macek, Pavel; Chmelík, Josef; Křížová, Ivana; Kadeřávek, P.; Padrta, P.; Žídek, L.; Wildová, Marcela; Hadravová, Romana; Chaloupková, R.; Pichová, Iva; Ruml, T.; Rumlová, Michaela; Sklenář, V.

2009-01-01

Roč. 392, č. 1 (2009), s. 100-114 ISSN 0022-2836 R&D Projects: GA MŠk LC545; GA MŠk 1M0508; GA ČR GA204/09/1388; GA ČR GESCO/06/E001 Grant - others:GA MŠk(CZ) 1M0520; MŠk(CZ) LC06030 Program:1M; LC Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50200510 Keywords : M-PMV * betaretroviruses * capsid protein * NMR structure * internal dynamics Subject RIV: CE - Biochemistry Impact factor: 3.871, year: 2009

ReAS: Recovery of ancestral sequences for transposable elements from the unassembled reads of a whole genome shotgun

DEFF Research Database (Denmark)

Li, Ruiqiang; Ye, Jia; Li, Songgang

2005-01-01

in comparison to their ancestral sequences. Tested on the japonica rice genome, ReAS was able to reconstruct all of the high copy sequences in the Repbase repository of known TEs, and increase the effectiveness of RepeatMasker in identifying TEs from genome sequences. Udgivelsesdato: 2005-Sep...

Rare natural type 3/type 2 intertypic capsid recombinant vaccine-related poliovirus isolated from a case of acute flaccid paralysis in Brazil, 2015.

Science.gov (United States)

Cassemiro, Klécia M S M; Burlandy, Fernanda M; da Silva, Edson E

2016-07-01

A natural type 3/type 2 intertypic capsid recombinant vaccine-related poliovirus was isolated from an acute flaccid paralytic case in Brazil. Genome sequencing revealed the uncommon location of the crossover site in the VP1 coding region (nucleotides 3251-3258 of Sabin 3 genome). The Sabin 2 donor sequence replaced the last 118 nt of VP1, resulting in the substitution of the complete antigenic site IIIa by PV2-specific amino acids. The low overall number of nucleotide substitutions in P1 region indicated that the predicted replication time of the isolate was about 8-9 weeks. Two of the principal determinants of attenuation in Sabin 3 genomes were mutated (U472C and C2493U), but the temperature-sensitive phenotype of the isolate was preserved. Our results support the theory that there exists a PV3/PV2 recombination hotspot site in the tail region of the VP1 capsid protein and that the recombination may occur soon after oral poliovirus vaccine administration.

Reestablishment of the Ancestral Cascades Arc in Western Nevada and Eastern California by Rollback of the Shallow Farallon Slab

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Henry, C. D.; Cousens, B.; John, D. A.; Colgan, J. P.

2009-12-01

The character and even existence of an ancestral Tertiary Cascades arc in western Nevada and eastern California south of the modern arc are controversial. Based on extensive published and new data on the regional distribution, timing, style, and composition of magmatism, we conclude that an ancestral arc was established by WSW migration of magmatism into western NV and the northeastern Sierra Nevada in the Oligocene and Miocene as a result of progressive rollback of the shallow subducted slab. Magma migration started with the well-known southward sweep through NE NV and NW UT between ~46 and 36 Ma. By ~30 Ma, migration of the leading edge and central belt of activity was much more WSW, especially after removing younger ~E-W extension. Locally sourced, initially dispersed and small volume, intermediate to mafic lavas erupted in western NV and northeastern CA by ~30 Ma and the eastern Sierra Nevada by ~28 Ma, contemporaneous with the much more voluminous ignimbrite flare-up in central NV. As migration continued, the ignimbrite flare-up tapered off. A voluminous, NNW-trending, dominantly effusive volcanic belt developed by ~22-18 Ma in western NV and was continuous from the Bodie Hills (CA/NV) to the Warner Range (northeast CA) by ~16-15 Ma. The volcanic belt was dominated by intermediate to mafic magmas compositionally similar to those of the modern south Cascades arc but reflecting melting of an old, subduction-modified lithosphere (Cousens et al. 2008; Geosphere). Extensive middle Miocene bimodal rocks related to the Yellowstone hotspot cover these rocks in NW NV, NE CA, and SE OR, but 30-23 Ma, intermediate to mafic and lesser silicic rocks are voluminous wherever older rocks are exposed below the middle Miocene rocks. Between ~25 Ma and the present, magmatism migrated WSW at an average rate of ~8 km/Ma but was at least partly stepwise, as exemplified by an ~50 km westward step at 2 Ma in the Lassen area (Guffanti et al. 1990, JGR). The magmatic belt was as much

Computational analysis and functional expression of ancestral copepod luciferase.

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Takenaka, Yasuhiro; Noda-Ogura, Akiko; Imanishi, Tadashi; Yamaguchi, Atsushi; Gojobori, Takashi; Shigeri, Yasushi

2013-10-10

We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species (Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species. © 2013.

Identification of structural similarities between putative transmission proteins of Polymyxa and Spongospora transmitted bymoviruses and furoviruses.

Science.gov (United States)

Dessens, J T; Meyer, M

1996-01-01

Comparison of amino acid sequence and hydropathy profiles shows conserved, structural similarities between the capsid readthrough protein of potato mop top virus (transmitted by Spongospora subterranea) and furovirus and bymovirus proteins implicated in transmission by Polymyxa spp. This suggests that these proteins have a common ancestry and are involved in a common biological process: virus transmission by plasmodiophorid fungi.

Structure of deformed wing virus, a major honey bee pathogen.

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Škubník, Karel; Nováček, Jiří; Füzik, Tibor; Přidal, Antonín; Paxton, Robert J; Plevka, Pavel

2017-03-21

The worldwide population of western honey bees ( Apis mellifera ) is under pressure from habitat loss, environmental stress, and pathogens, particularly viruses that cause lethal epidemics. Deformed wing virus (DWV) from the family Iflaviridae , together with its vector, the mite Varroa destructor , is likely the major threat to the world's honey bees. However, lack of knowledge of the atomic structures of iflaviruses has hindered the development of effective treatments against them. Here, we present the virion structures of DWV determined to a resolution of 3.1 Å using cryo-electron microscopy and 3.8 Å by X-ray crystallography. The C-terminal extension of capsid protein VP3 folds into a globular protruding (P) domain, exposed on the virion surface. The P domain contains an Asp-His-Ser catalytic triad that is, together with five residues that are spatially close, conserved among iflaviruses. These residues may participate in receptor binding or provide the protease, lipase, or esterase activity required for entry of the virus into a host cell. Furthermore, nucleotides of the DWV RNA genome interact with VP3 subunits. The capsid protein residues involved in the RNA binding are conserved among honey bee iflaviruses, suggesting a putative role of the genome in stabilizing the virion or facilitating capsid assembly. Identifying the RNA-binding and putative catalytic sites within the DWV virion structure enables future analyses of how DWV and other iflaviruses infect insect cells and also opens up possibilities for the development of antiviral treatments.

Stabilization of the beta-hairpin in Mason-Pfizer monkey virus capsid protein- a critical step for infectivity

Czech Academy of Sciences Publication Activity Database

Obr, M.; Hadravová, Romana; Doležal, Michal; Křížová, Ivana; Papoušková, V.; Žídek, L.; Hrabal, R.; Ruml, T.; Rumlová, Michaela

2014-01-01

Roč. 11, Oct 30 (2014), 94/1-94/14 ISSN 1742-4690 R&D Projects: GA ČR(CZ) GA14-15326S; GA MŠk LO1302 Grant - others:GA MŠk(CZ) ED1.1.00/02.0068; Seventh Framework Programme of the European Union(XE) FP7-261863 Program:ED Institutional support: RVO:61388963 Keywords : retrovirus * assembly * M-PMV * capsid protein * maturation * beta-hairpin Subject RIV: EE - Microbiology, Virology Impact factor: 4.185, year: 2014 http://www.retrovirology.com/content/11/1/94

The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

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Lucas Y. H. Goh

2015-06-01

Full Text Available Chikungunya virus (CHIKV is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs previously generated towards the capsid protein (CP of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

Impact of reducing and oxidizing agents on the infectivity of Qβ phage and the overall structure of its capsid.

Science.gov (United States)

Loison, Pauline; Majou, Didier; Gelhaye, Eric; Boudaud, Nicolas; Gantzer, Christophe

2016-11-01

Qβ phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Qβ phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cyclophilin A potentiates TRIM5α inhibition of HIV-1 nuclear import without promoting TRIM5α binding to the viral capsid.

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Mallori Burse

Full Text Available The host immunophilin cyclophilin A (CypA binds to the capsid protein (CA of HIV-1 and regulates its infectivity. Depending on the target cell type, CypA can either promote or inhibit HIV-1 infection. The ability of CypA to promote HIV-1 infection has been extensively studied and linked to several steps in early replication including uncoating, reverse transcription and nuclear import. By contrast, the mechanism by which CypA inhibits infection is less well understood. We investigated the mechanism by which CypA potentiates restriction of HIV-1 by the tripartite motif-containing protein 5 (TRIM5α. Depletion of TRIM5α in the African green monkey cell line Vero, resulted in a loss of inhibition of infection by CypA, demonstrating that inhibition by CypA is mediated by TRIM5α. Complementary genetic and biochemical assays failed to demonstrate an ability of CypA to promote binding of TRIM5α to the viral capsid. TRIM5α inhibits HIV-1 reverse transcription in a proteasome-dependent manner; however, we observed that inhibition of proteasome activity did not reduce the ability of CypA to inhibit infection, suggesting that CypA acts at a step after reverse transcription. Accordingly, we observed a CypA-dependent reduction in the accumulation of nuclear HIV-1 DNA, indicating that CypA specifically promotes TRIM5α inhibition of HIV-1 nuclear import. We also observed that the ability of CypA to inhibit HIV-1 infection is abolished by amino acid substitutions within the conserved CPSF6-binding surface in CA. Our results indicate that CypA inhibits HIV-1 infection in Vero cells not by promoting TRIM5α binding to the capsid but by blocking nuclear import of the HIV-1 preintegration complex.

Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies

Czech Academy of Sciences Publication Activity Database

Marčeková, Zuzana; Psikal, P.; Kosinová, E.; Benada, Oldřich; Šebo, Peter; Bumba, Ladislav

2009-01-01

Roč. 162, 1-2 (2009), s. 133-141 ISSN 0166-0934 R&D Projects: GA ČR GP310/07/P115; GA MŠk 2B06161 Institutional research plan: CEZ:AV0Z50200510 Keywords : PCV 2 * Porcine circovirus * Capsid protein Subject RIV: EE - Microbiology, Virology Impact factor: 2.133, year: 2009

Enhanced sensitivity in detection of antiviral antibody responses using biotinylation of foot-and-mouth disease virus (FMDV) capsids.

Science.gov (United States)

Kenney, Mary; Waters, Ryan A; Rieder, Elizabeth; Pega, Juan; Perez-Filguera, Mariano; Golde, William T

2017-11-01

Analysis of the immune response to infection of livestock by foot-and-mouth disease virus (FMDV) is most often reported as the serum antibody response to the virus. While measurement of neutralizing antibody has been sensitive and specific, measurements of the quality of the antibody response are less robust. Determining the immunoglobulin (Ig) isotype of the serum antibody response provides a deeper understanding of the biology of the response and more sensitive methods for these assays will facilitate analyses of B cell mediated immunity. We tested the hypothesis that using the virus as the molecular probe could be achieved by adding tags to the surface of the FMDV capsid, and that would enhance sensitivity in assays for anti-FMDV antibody responses. The use of a FLAG-tagged virus in these assays failed to yield improvement whereas chemically biotinylating the virus capsid resulted in significant enhancement of the signal. Here we describe methods using biotinylated virus for measuring anti-viral antibody in serum and antibody secreting cells (ASCs) in blood that are sensitive and specific. Finally, we describe using the biotinylated virus in flow cytometry where such assays should greatly enhance the analysis of anti-virus antibody producing B cells, allowing the investigator to focus on only the FMDV specific B cells when analyzing the development of the B cell response to either infection or vaccination. Published by Elsevier B.V.

B-cell depletion is protective against anti-AAV capsid immune response: a human subject case study

Directory of Open Access Journals (Sweden)

M Corti

2014-01-01

Full Text Available Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV vectors due to aspects of the clinical application, such as: (i administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; (ii progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and (iii a possibly faster depletion of pathogenic myofibers in this patient population. Immune response triggered by the first vector administration, and to subsequent doses, represents a major obstacle for successful gene transfer in young patients. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or enzyme replacement therapy (ERT for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. In this study, we evaluated the immune response of a Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate reactions against ERT. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.

Down-Regulation of Na+/K+ ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

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Ahmad Almilaji

2013-05-01

Full Text Available Background/Aims: Human parvovirus B19 (B19V may cause inflammatory cardiomyopathy (iCMP which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na+/K+ ATPase. The present study explored whether VP1 modifies Na+/K+ ATPase activity. Methods: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A and K+ induced pump current (Ipump as well as ouabain-inhibited current (Iouabain both reflecting Na+/K+-ATPase activity were determined by dual electrode voltage clamp. Results: Injection of cRNA encoding VP1, but not of VP1(H153A or water, was followed by a significant decrease of both, Ipump and Iouabain in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml. According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml similarly decreased Ipump in human microvascular endothelial cells (HMEC. Conclusion: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na+/K+ ATPase, an effect at least partially due to phospholipase A2 (PLA2 dependent formation of lysophosphatidylcholine.

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein

OpenAIRE

Bertagnoli, Stéphane; Gelfi, Jacqueline; Le Gall, Ghislaine; Boilletot, Eric; Vautherot, Jean-François; Rasschaert, Denis; Laurent, Sylvie; Petit, Frédérique; Boucraut-Baralon, Corine; Milon, Alain

1996-01-01

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma vir...

Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

Science.gov (United States)

Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O,A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutral...

Sustainability of ancestral methods of agricultural production in Perú: ¿keep or replace?

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Dani Eduardo Vargas Huanca

2016-09-01

Full Text Available Based on the success of some Andean products such as quinoa, potatoes or maca in international food trade and the growing environmental degradation facing developing countries, resulting from intensive exploitation activities; Our research seeks to show the trend that is assumed from the academic / scientific community and public officials in the food sector in Peru, against the need to maintain sustainable various ancestral modes of agricultural production (case quinoa, for it analyze quantitative and qualitative obtained from public institutions and Peruvian universities.

Influence of minor displacements in loops of the porcine parvovirus VP2 capsid on virus-like particles assembly and the induction of antibody responses.

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Pan, Qunxing; He, Kongwang; Wang, Yongshan; Wang, Xiaoli; Ouyang, Wei

2013-06-01

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine parvovirus (PPV) and expressing foreign peptides offers an alternative method for vaccination. In this study, the three-dimensional structure of the PPV capsid protein and surface loops deletion mutants were analyzed to define essential domains in PPV VP2 for the assembly of VLPs. Electron microscopic analysis and SDS-PAGE analysis confirmed the presence of abundant VLPs in a loop2 deletion mutant of expected size and appropriate morphology. Loop4 and loop2-loop4 deletion mutants, however, resulted in a lower number of particles and the morphology of the particles was not well preserved. Furthermore, the green fluorescent protein (gfp) gene was used as a model. GFP was observed at the same level in displacements mutants. However, GFP displacement mutants in loop2 construct allowed better adaptation for the fusion GFP to be further displayed on the surface of the capsid-like structure. Immunogenicity study showed that there is no obvious difference in mice inoculated with rAd-VP2(Δloop2), rAd-VP2(Δloop4), rAd-VP2(Δloop2-Δloop4), and PPV inactivated vaccine. The results suggested the possibility of inserting simultaneously B and T cell epitopes in the surface loop2 and the N-terminus. The combination of different types of epitopes (B, CD4+, and CD8+) in different positions of the PPV particles opens the way to the development of highly efficient vaccines, able to stimulate at the same time the different branches of the immune system.

Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat.

Directory of Open Access Journals (Sweden)

Barbara Bogner

Full Text Available Adeno associated virus (AAV is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV's ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc genomes in the anterior segment of the eye.AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE, iris and chamber angle including trabecular meshwork, with scAAV2(Y444F and scAAV2(triple being the most efficient.This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene

Derived (mutated)-types of TRPV6 channels elicit greater Ca²+ influx into the cells than ancestral-types of TRPV6: evidence from Xenopus oocytes and mammalian cell expression system.

Science.gov (United States)

Sudo, Yuka; Matsuo, Kiyotaka; Tetsuo, Tomoyuki; Tsutsumi, Satoshi; Ohkura, Masamichi; Nakai, Junichi; Uezono, Yasuhito

2010-01-01

The frequency of the allele containing three derived nonsynonymous SNPs (157C, 378M, 681M) of the gene encoding calcium permeable TRPV6 channels expressed in the intestine has been increased by positive selection in non-African populations. To understand the nature of these SNPs, we compared the properties of Ca²+ influx of ancestral (in African populations) and derived-TRPV6 (in non-African populations) channels with electrophysiological, Ca²+-imaging, and morphological methods using both the Xenopus oocyte and mammalian cell expression systems. Functional electrophysiological and Ca²+-imaging analyses indicated that the derived-TRPV6 elicited more Ca²+ influx than the ancestral one in TRPV6-expressing cells where both channels were equally expressed in the cells. Ca²+-inactivation properties in the ancestral- and derived-TRPV6 were almost the same. Furthermore, fluorescence resonance energy transfer (FRET) analysis showed that both channels have similar multimeric formation properties, suggesting that derived-TRPV6 itself could cause higher Ca²+ influx. These findings suggest that populations having derived-TRPV6 in non-African areas may absorb higher Ca²+ from the intestine than ancestral-TRPV6 in the African area.

Metamorphosis Is Ancestral for Crown Euarthropods, and Evolved in the Cambrian or Earlier.

Science.gov (United States)

Wolfe, Joanna M

2017-09-01

Macroevolutionary developmental biology employs fossilized ontogenetic data and phylogenetic comparative methods to probe the evolution of development at ancient nodes. Despite the prevalence of ecologically differentiated larval forms in marine invertebrates, it has been frequently presumed that the ancestors of arthropods were direct developers, and that metamorphosis may not have evolved until the Ordovician or later. Using fossils and new dated phylogenies, I infer that metamorphosis was likely ancestral for crown arthropods, contradicting this assumption. Based on a published morphological dataset encompassing 217 exceptionally preserved fossil and 96 extant taxa, fossils were directly incorporated into both the topology and age estimates, as in "tip dating" analyses. Using data from post-embryonic fossils representing 25 species throughout stem and crown arthropod lineages (as well as most of the 96 extant taxa), characters for metamorphosis were assigned based on inferred ecological changes in development (e.g., changes in habitat and adaptive landscape). Under all phylogenetic hypotheses, metamorphosis was supported as most likely ancestral to both ecdysozoans and euarthropods. Care must be taken to account for potential drastic post-embryonic morphological changes in evolutionary analyses. Many stem group euarthrpods may have had ecologically differentiated larval stages that did not preserve in the fossil record. Moreover, a complex life cycle and planktonic ecology may have evolved in the Ediacaran or earlier, and may have typified the pre-Cambrian explosion "wormworld" prior to the origin of crown group euarthropods. © The Author 2017. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Vaccination of mice with plasmids expressing processed capsid protein of foot-and-mouth disease virus - Importance of dominant and subdominant epitopes for antigenicity and protection

DEFF Research Database (Denmark)

Frimann, Tine; Barfoed, Annette Malene; Aasted, Bent

2007-01-01

The capsid of foot-and-mouth disease virus (FMDV) displays several independent B cell epitopes, which stimulate the production of neutralising antibodies. Some of these epitopes are highly variable between virus strains, but dominate the immune response. The site A on VP1 is the most prominent...

Thermotolerant Yeast Strains Adapted by Laboratory Evolution Show Trade-Off at Ancestral Temperatures and Preadaptation to Other Stresses.

Science.gov (United States)

Caspeta, Luis; Nielsen, Jens

2015-07-21

A major challenge for the production of ethanol from biomass-derived feedstocks is to develop yeasts that can sustain growth under the variety of inhibitory conditions present in the production process, e.g., high osmolality, high ethanol titers, and/or elevated temperatures (≥ 40 °C). Using adaptive laboratory evolution, we previously isolated seven Saccharomyces cerevisiae strains with improved growth at 40 °C. Here, we show that genetic adaptations to high temperature caused a growth trade-off at ancestral temperatures, reduced cellular functions, and improved tolerance of other stresses. Thermotolerant yeast strains showed horizontal displacement of their thermal reaction norms to higher temperatures. Hence, their optimal and maximum growth temperatures increased by about 3 °C, whereas they showed a growth trade-off at temperatures below 34 °C. Computational analysis of the physical properties of proteins showed that the lethal temperature for yeast is around 49 °C, as a large fraction of the yeast proteins denature above this temperature. Our analysis also indicated that the number of functions involved in controlling the growth rate decreased in the thermotolerant strains compared with the number in the ancestral strain. The latter is an advantageous attribute for acquiring thermotolerance and correlates with the reduction of yeast functions associated with loss of respiration capacity. This trait caused glycerol overproduction that was associated with the growth trade-off at ancestral temperatures. In combination with altered sterol composition of cellular membranes, glycerol overproduction was also associated with yeast osmotolerance and improved tolerance of high concentrations of glucose and ethanol. Our study shows that thermal adaptation of yeast is suitable for improving yeast resistance to inhibitory conditions found in industrial ethanol production processes. Yeast thermotolerance can significantly reduce the production costs of biomass

Origin of amphibian and avian chromosomes by fission, fusion, and retention of ancestral chromosomes

Science.gov (United States)

Voss, Stephen R.; Kump, D. Kevin; Putta, Srikrishna; Pauly, Nathan; Reynolds, Anna; Henry, Rema J.; Basa, Saritha; Walker, John A.; Smith, Jeramiah J.

2011-01-01

Amphibian genomes differ greatly in DNA content and chromosome size, morphology, and number. Investigations of this diversity are needed to identify mechanisms that have shaped the evolution of vertebrate genomes. We used comparative mapping to investigate the organization of genes in the Mexican axolotl (Ambystoma mexicanum), a species that presents relatively few chromosomes (n = 14) and a gigantic genome (>20 pg/N). We show extensive conservation of synteny between Ambystoma, chicken, and human, and a positive correlation between the length of conserved segments and genome size. Ambystoma segments are estimated to be four to 51 times longer than homologous human and chicken segments. Strikingly, genes demarking the structures of 28 chicken chromosomes are ordered among linkage groups defining the Ambystoma genome, and we show that these same chromosomal segments are also conserved in a distantly related anuran amphibian (Xenopus tropicalis). Using linkage relationships from the amphibian maps, we predict that three chicken chromosomes originated by fusion, nine to 14 originated by fission, and 12–17 evolved directly from ancestral tetrapod chromosomes. We further show that some ancestral segments were fused prior to the divergence of salamanders and anurans, while others fused independently and randomly as chromosome numbers were reduced in lineages leading to Ambystoma and Xenopus. The maintenance of gene order relationships between chromosomal segments that have greatly expanded and contracted in salamander and chicken genomes, respectively, suggests selection to maintain synteny relationships and/or extremely low rates of chromosomal rearrangement. Overall, the results demonstrate the value of data from diverse, amphibian genomes in studies of vertebrate genome evolution. PMID:21482624

Wiring a periscope--ocelli, retinula axons, visual neuropils and the ancestrality of sea spiders.

Science.gov (United States)

Lehmann, Tobias; Hess, Martin; Melzer, Roland R

2012-01-01

The Pycnogonida or sea spiders are cryptic, eight-legged arthropods with four median ocelli in a 'periscope' or eye tubercle. In older attempts at reconstructing phylogeny they were Arthropoda incertae sedis, but recent molecular trees placed them as the sister group either to all other euchelicerates or even to all euarthropods. Thus, pycnogonids are among the oldest extant arthropods and hold a key position for the understanding of arthropod evolution. This has stimulated studies of new sets of characters conductive to cladistic analyses, e.g. of the chelifores and of the hox gene expression pattern. In contrast knowledge of the architecture of the visual system is cursory. A few studies have analysed the ocelli and the uncommon "pseudoinverted" retinula cells. Moreover, analyses of visual neuropils are still at the stage of Hanström's early comprehensive works. We have therefore used various techniques to analyse the visual fibre pathways and the structure of their interrelated neuropils in several species. We found that pycnogonid ocelli are innervated to first and second visual neuropils in close vicinity to an unpaired midline neuropil, i.e. possibly the arcuate body, in a way very similar to ancestral euarthropods like Euperipatoides rowelli (Onychophora) and Limulus polyphemus (Xiphosura). This supports the ancestrality of pycnogonids and sheds light on what eyes in the pycnogonid ground plan might have 'looked' like. Recently it was suggested that arthropod eyes originated from simple ocelli similar to larval eyes. Hence, pycnogonid eyes would be one of the early offshoots among the wealth of more sophisticated arthropod eyes.

Wiring a periscope--ocelli, retinula axons, visual neuropils and the ancestrality of sea spiders.

Directory of Open Access Journals (Sweden)

Tobias Lehmann

Full Text Available The Pycnogonida or sea spiders are cryptic, eight-legged arthropods with four median ocelli in a 'periscope' or eye tubercle. In older attempts at reconstructing phylogeny they were Arthropoda incertae sedis, but recent molecular trees placed them as the sister group either to all other euchelicerates or even to all euarthropods. Thus, pycnogonids are among the oldest extant arthropods and hold a key position for the understanding of arthropod evolution. This has stimulated studies of new sets of characters conductive to cladistic analyses, e.g. of the chelifores and of the hox gene expression pattern. In contrast knowledge of the architecture of the visual system is cursory. A few studies have analysed the ocelli and the uncommon "pseudoinverted" retinula cells. Moreover, analyses of visual neuropils are still at the stage of Hanström's early comprehensive works. We have therefore used various techniques to analyse the visual fibre pathways and the structure of their interrelated neuropils in several species. We found that pycnogonid ocelli are innervated to first and second visual neuropils in close vicinity to an unpaired midline neuropil, i.e. possibly the arcuate body, in a way very similar to ancestral euarthropods like Euperipatoides rowelli (Onychophora and Limulus polyphemus (Xiphosura. This supports the ancestrality of pycnogonids and sheds light on what eyes in the pycnogonid ground plan might have 'looked' like. Recently it was suggested that arthropod eyes originated from simple ocelli similar to larval eyes. Hence, pycnogonid eyes would be one of the early offshoots among the wealth of more sophisticated arthropod eyes.

CLONING, SEQUENCE ANALYSIS, AND CHARACTERIZATION OF PUTATIVE BETA-LACTAMASE OF STENOTROPHOMONAS MALTOPHILIA

Directory of Open Access Journals (Sweden)

Chong Seng Shueh

2012-10-01

Full Text Available The main objective of current study was to explore the function of chromosomal putative beta-lactamase gene (smlt 0115 in clinical Stenotrophomonas maltophilia. Antibiotic susceptibility test (AST screening for current antimicrobial drugs was done and Minimum Inhibitory Concentration (MIC level towards beta-lactams was determined by E-test. Putative beta-lactamase gene of S. maltophilia was amplified via PCR, with specific primers, then cloned into pET-15 expression plasmid and transformed into Escherichia coli BL21. The gene was sequenced and analyzed. The expressed protein was purified by affinity chromatography and the kinetic assay was performed. S. maltophilia ATCC 13637 was included in this experiment. Besides, a hospital strain which exhibited resistant to a series of beta-lactams including cefepime was identified via AST and MIC, hence it was named as S2 strain and was considered in this study. Sequencing result showed that putative beta-lactamase gene obtained from ATCC 13637 and S2 strains were predicted to have cephalosporinase activity by National Center for Biotechnology Information (NCBI blast program. Differences in the sequences of both ATCC 13637 and S2 strains were found via ClustalW alignment software. Kinetic assay proved a cephalosporinase characteristic produced by E. coli BL21 clone that overexpressed the putative beta-lactamase gene cloned under the control of an external promoter. Yet, expressed protein purified from S2 strain had high catalytic activity against beta-lactam antibiotics which was 14-fold higher than expressed protein purified from ATCC 13637 strain. This study represents the characterization analysis of putative beta-lactamase gene (smlt 0115 of S. maltophilia. The presence of the respective gene in the chromosome of S. maltophilia suggested that putative beta-lactamase gene (smlt 0115 of S. maltophilia plays a role in beta-lactamase resistance.

Evolution to pathogenicity of the parvovirus minute virus of mice in immunodeficient mice involves genetic heterogeneity at the capsid domain that determines tropism.

Science.gov (United States)

López-Bueno, Alberto; Segovia, José C; Bueren, Juan A; O'Sullivan, M Gerard; Wang, Feng; Tattersall, Peter; Almendral, José M

2008-02-01

Very little is known about the role that evolutionary dynamics plays in diseases caused by mammalian DNA viruses. To address this issue in a natural host model, we compared the pathogenesis and genetics of the attenuated fibrotropic and the virulent lymphohematotropic strains of the parvovirus minute virus of mice (MVM), and of two invasive fibrotropic MVM (MVMp) variants carrying the I362S or K368R change in the VP2 major capsid protein, in the infection of severe combined immunodeficient (SCID) mice. By 14 to 18 weeks after oronasal inoculation, the I362S and K368R viruses caused lethal leukopenia characterized by tissue damage and inclusion bodies in hemopoietic organs, a pattern of disease found by 7 weeks postinfection with the lymphohematotropic MVM (MVMi) strain. The MVMp populations emerging in leukopenic mice showed consensus sequence changes in the MVMi genotype at residues G321E and A551V of VP2 in the I362S virus infections or A551V and V575A changes in the K368R virus infections, as well as a high level of genetic heterogeneity within a capsid domain at the twofold depression where these residues lay. Amino acids forming this capsid domain are important MVM tropism determinants, as exemplified by the switch in MVMi host range toward mouse fibroblasts conferred by coordinated changes of some of these residues and by the essential character of glutamate at residue 321 for maintaining MVMi tropism toward primary hemopoietic precursors. The few viruses within the spectrum of mutants from mice that maintained the respective parental 321G and 575V residues were infectious in a plaque assay, whereas the viruses with the main consensus sequences exhibited low levels of fitness in culture. Consistent with this finding, a recombinant MVMp virus carrying the consensus sequence mutations arising in the K368R virus background in mice failed to initiate infection in cell lines of different tissue origins, even though it caused rapid-course lethal leukopenia in SCID

Next-generation AAV vectors for clinical use: an ever-accelerating race.

Science.gov (United States)

Weinmann, Jonas; Grimm, Dirk

2017-10-01

During the past five decades, it has become evident that Adeno-associated virus (AAV) represents one of the most potent, most versatile, and thus most auspicious platforms available for gene delivery into cells, animals and, ultimately, humans. Particularly attractive is the ease with which the viral capsid-the major determinant of virus-host interaction including cell specificity and antibody recognition-can be modified and optimized at will. This has motivated countless researchers to develop high-throughput technologies in which genetically engineered AAV capsid libraries are subjected to a vastly hastened emulation of natural evolution, with the aim to enrich novel synthetic AAV capsids displaying superior features for clinical application. While the power and potential of these forward genetics approaches is undisputed, they are also inherently challenging as success depends on a combination of library quality, fidelity, and complexity. Here, we will describe and discuss two original, very exciting strategies that have emerged over the last three years and that promise to alleviate at least some of these concerns, namely, (i) a reverse genetics approach termed "ancestral AAV sequence reconstruction," and (ii) AAV genome barcoding as a technology that can advance both, forward and reverse genetics stratagems. Notably, despite the conceptual differences of these two technologies, they pursue the same goal which is tailored acceleration of AAV evolution and thus winning the race for the next-generation AAV vectors for clinical use.

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

Science.gov (United States)

Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Toranzos, Gary A; Marota, Isolina; Giuffra, Valentina; Cano, Raul J

2017-11-07

Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

DEFF Research Database (Denmark)

Johannesson, Martina; Semb, Tor Henrik; Serup, Palle

2012-01-01

. Thus, the purpose of this study is to describe a method whereby the in vivo functionality of DE derived from ESCs can be assessed. Methods: By directed differentiation, putative DE was derived from human and mouse ESCs. This putative DE was subsequently transplanted into the endoderm of chick embryos...... to determine any occurrence of integration. Putative DE was analyzed by gene and protein expression prior to transplantation and 48 h post transplantation. Results: Putative DE, derived from mouse and human ESCs, was successfully integrated within the chick endoderm. Endoderm-specific genes were expressed...... result show that putative DE integrates with the chick endoderm and participate in the development of the chicken gut, indicating the generation of functional DE from ESCs. This functional assay can be used to assess the generation of functional DE derived from both human and mouse ESCs and provides...

Effects of ancestral populations on entrepreneurial founding and failure: private liquor stores in Alberta, 1994--2003

OpenAIRE

Glen Dowell; Robert J. David

2011-01-01

Until 1993, all liquor stores in the Canadian province of Alberta were government owned and run. In the fall of 1993, the provincial government exited liquor retailing, all government stores were shut down, and entrepreneurs were allowed to open private liquor stores. In this article, we take advantage of this abrupt regulatory change in the Alberta liquor-retailing industry to address two related issues that have received little empirical attention. First, we investigate how an ancestral pop...

Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

Czech Academy of Sciences Publication Activity Database

Rumlová, Michaela; Ruml, T.; Pohl, J.; Pichová, Iva

2003-01-01

Roč. 310, - (2003), s. 310-318 ISSN 0042-6822 R&D Projects: GA ČR GA203/00/1241; GA AV ČR IAB4055202 Institutional research plan: CEZ:AV0Z4055905 Keywords : M-PMV protease * HIV-1 capsid protein * HIV-1 protease Subject RIV: CE - Biochemistry Impact factor: 3.391, year: 2003

Atomic force microscopy investigation of Turnip Yellow Mosaic Virus capsid disruption and RNA extrusion

International Nuclear Information System (INIS)

Kuznetsov, Yu. G.; McPherson, Alexander

2006-01-01

Turnip Yellow Mosaic Virus (TYMV) was subjected to a variety of procedures which disrupted the protein capsids and produced exposure of the ssRNA genome. The results of the treatments were visualized by atomic force microscopy (AFM). Both in situ and ex situ freeze-thawing produced RNA emission, though at low efficiency. The RNA lost from such particles was evident, in some cases in the process of exiting the virions. More severe disruption of TYMV and extrusion of intact RNA onto the substrate were produced by drying the virus and rehydrating with neutral buffer. Similar products were also obtained by heating TYMV to 70-75 deg. C and by exposure to alkaline pH. Experiments showed the nucleic acid to have an elaborate secondary structure distributed linearly along its length

Toddlers' Duration of Attention toward Putative Threat

Science.gov (United States)

Kiel, Elizabeth J.; Buss, Kristin A.

2011-01-01

Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

Phylogenetic signal from rearrangements in 18 Anopheles species by joint scaffolding extant and ancestral genomes.

Science.gov (United States)

Anselmetti, Yoann; Duchemin, Wandrille; Tannier, Eric; Chauve, Cedric; Bérard, Sèverine

2018-05-09

Genomes rearrangements carry valuable information for phylogenetic inference or the elucidation of molecular mechanisms of adaptation. However, the detection of genome rearrangements is often hampered by current deficiencies in data and methods: Genomes obtained from short sequence reads have generally very fragmented assemblies, and comparing multiple gene orders generally leads to computationally intractable algorithmic questions. We present a computational method, ADSEQ, which, by combining ancestral gene order reconstruction, comparative scaffolding and de novo scaffolding methods, overcomes these two caveats. ADSEQ provides simultaneously improved assemblies and ancestral genomes, with statistical supports on all local features. Compared to previous comparative methods, it runs in polynomial time, it samples solutions in a probabilistic space, and it can handle a significantly larger gene complement from the considered extant genomes, with complex histories including gene duplications and losses. We use ADSEQ to provide improved assemblies and a genome history made of duplications, losses, gene translocations, rearrangements, of 18 complete Anopheles genomes, including several important malaria vectors. We also provide additional support for a differentiated mode of evolution of the sex chromosome and of the autosomes in these mosquito genomes. We demonstrate the method's ability to improve extant assemblies accurately through a procedure simulating realistic assembly fragmentation. We study a debated issue regarding the phylogeny of the Gambiae complex group of Anopheles genomes in the light of the evolution of chromosomal rearrangements, suggesting that the phylogenetic signal they carry can differ from the phylogenetic signal carried by gene sequences, more prone to introgression.

1H, 13C, and 15N resonance assignment of the N-terminal domainof Mason-Pfizer monkey virus capsid protein, CA 1-140

Czech Academy of Sciences Publication Activity Database

Macek, Pavel; Žídek, L.; Rumlová, Michaela; Pichová, Iva; Sklenář, V.

2008-01-01

Roč. 2, č. 1 (2008), s. 43-45 ISSN 1874-2718 R&D Projects: GA MŠk LC545; GA MŠk(CZ) LC06030; GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z40550506 Keywords : nmr * assignment * capsid protein Subject RIV: EE - Microbiology, Virology Impact factor: 0.015, year: 2008

Identification and characterization of putative conserved IAM ...

African Journals Online (AJOL)

Available putative AMI sequences from a wide array of monocot and dicot plants were identified and the phylogenetic tree was constructed and analyzed. We identified in this tree, a clade that contained sequences from species across the plant kingdom suggesting that AMI is conserved and may have a primary role in plant ...

Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs

DEFF Research Database (Denmark)

Lohse, Louise; Jackson, Terry; Bøtner, Anette

2012-01-01

The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus. In the present study we...... compared the pathogenicity of different FMDVs in young pigs. In total 32 pigs, 7-weeks-old, were exposed to virus, either by direct inoculation or through contact with inoculated pigs, using cell culture adapted (O1K B64), chimeric (O1K/A-TUR and O1K/O-UKG) or field strain (O-UKG/34/2001) viruses. The O1K...... coding sequences are determinants of FMDV pathogenicity in pigs....

Prognostic relevance of human papillomavirus L1 capsid protein detection within mild and moderate dysplastic lesions of the cervix uteri in combination with p16 biomarker

DEFF Research Database (Denmark)

Hilfrich, Ralf; Hariri, Jalil

2008-01-01

OBJECTIVE: To proof the prognostic relevance of HPV L1 capsid protein detection on colposcopically-guided punch biopsies in combination with p16. STUDY DESIGN: Sections of colposcopically-guided punch biopsies from 191 consecutive cases with at least 5 years of follow-up were stained with HPV L1 ...

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies

Directory of Open Access Journals (Sweden)

Tasha M. Santiago-Rodriguez

2017-11-01

Full Text Available Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

Keeping your armour intact: how HIV-1 evades detection by the innate immune system: HIV-1 capsid controls detection of reverse transcription products by the cytosolic DNA sensor cGAS.

Science.gov (United States)

Maelfait, Jonathan; Seiradake, Elena; Rehwinkel, Jan

2014-07-01

HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity, Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design. © 2014 The Authors. Bioessays published by WILEY Periodicals, Inc.

Evolutionary changes in the capsid P2 region of Australian strains of the norovirus GII.Pe_GII.4.

Science.gov (United States)

Bruggink, Leesa D; Moselen, Jean M; Roberts, Jason A; Marshall, John A

2017-07-01

The protruding (P) 2 region of the norovirus capsid is thought to include hypervariable sites involved in receptor binding. This study examines the changes that occurred in the P2 region of GII.Pe_GII.4 norovirus in the course of its evolution from a precursor phase (2008-2009), to an intermediate phase (2010) and finally to an epidemic phase (2012-2015). Twenty-two P2 region amino acid (aa) sequences (166 aa long) from all phases of the evolution of the virus were compared and the changes analysed.Results/key findings. Twenty sites in the P2 region underwent aa change and of these, 10 corresponded to previously proposed hypervariable sites and 10 to novel hypervariable sites. It was notable that aa changes at two sites, X and Y, only emerged as the epidemic phase progressed. 3D computer modelling of the P2 region indicated that neither X nor Y were in the uppermost 'crown', but further down in the 'neck' portion. The location of X and Y and the nature of aa change at Y suggest these sites were important in enhancing the structural integrity of the capsid, which in turn may have facilitated the longer term viability of the virus. The current study helps establish the validity of previously proposed hypervariable sites in the P2 region as well as indicating new ones. It also provides quantitative and qualitative data on how these sites changed over the evolutionary history of a particular norovirus strain.

Ten Putative Contributors to the Obesity Epidemic

Science.gov (United States)

McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

2010-01-01

The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

Allo-allo-triploid Sphagnum × falcatulum: single individuals contain most of the Holantarctic diversity for ancestrally indicative markers.

Science.gov (United States)

Karlin, Eric F; Smouse, Peter E

2017-08-01

Allopolyploids exhibit both different levels and different patterns of genetic variation than are typical of diploids. However, scant attention has been given to the partitioning of allelic information and diversity in allopolyploids, particularly that among homeologous monoploid components of the hologenome. Sphagnum × falcatulum is a double allopolyploid peat moss that spans a considerable portion of the Holantarctic. With monoploid genomes from three ancestral species, this organism exhibits a complex evolutionary history involving serial inter-subgeneric allopolyploidizations. Studying populations from three disjunct regions [South Island (New Zealand); Tierra de Fuego archipelago (Chile, Argentina); Tasmania (Australia)], allelic information for five highly stable microsatellite markers that differed among the three (ancestral) monoploid genomes was examined. Using Shannon information and diversity measures, the holoploid information, as well as the information within and among the three component monoploid genomes, was partitioned into separate components for individuals within and among populations and regions, and those information components were then converted into corresponding diversity measures. The majority (76 %) of alleles detected across these five markers are most likely to have been captured by hybridization, but the information within each of the three monoploid genomes varied, suggesting a history of recurrent allopolyploidization between ancestral species containing different levels of genetic diversity. Information within individuals, equivalent to the information among monoploid genomes (for this dataset), was relatively stable, and represented 83 % of the grand total information across the Holantarctic, with both inter-regional and inter-population diversification each accounting for about 5 % of the total information. Sphagnum × falcatulum probably inherited the great majority of its genetic diversity at these markers by reticulation

Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

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Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

2015-01-01

Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

Ancestral Variations of the PCDHG Gene Cluster Predispose to Dyslexia in a Multiplex Family

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Teesta Naskar

2018-02-01

Full Text Available Dyslexia is a heritable neurodevelopmental disorder characterized by difficulties in reading and writing. In this study, we describe the identification of a set of 17 polymorphisms located across 1.9 Mb region on chromosome 5q31.3, encompassing genes of the PCDHG cluster, TAF7, PCDH1 and ARHGAP26, dominantly inherited with dyslexia in a multi-incident family. Strikingly, the non-risk form of seven variations of the PCDHG cluster, are preponderant in the human lineage, while risk alleles are ancestral and conserved across Neanderthals to non-human primates. Four of these seven ancestral variations (c.460A > C [p.Ile154Leu], c.541G > A [p.Ala181Thr], c.2036G > C [p.Arg679Pro] and c.2059A > G [p.Lys687Glu] result in amino acid alterations. p.Ile154Leu and p.Ala181Thr are present at EC2: EC3 interacting interface of γA3-PCDH and γA4-PCDH respectively might affect trans-homophilic interaction and hence neuronal connectivity. p.Arg679Pro and p.Lys687Glu are present within the linker region connecting trans-membrane to extracellular domain. Sequence analysis indicated the importance of p.Ile154, p.Arg679 and p.Lys687 in maintaining class specificity. Thus the observed association of PCDHG genes encoding neural adhesion proteins reinforces the hypothesis of aberrant neuronal connectivity in the pathophysiology of dyslexia. Additionally, the striking conservation of the identified variants indicates a role of PCDHG in the evolution of highly specialized cognitive skills critical to reading.

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Science.gov (United States)

Bertagnoli, S; Gelfi, J; Le Gall, G; Boilletot, E; Vautherot, J F; Rasschaert, D; Laurent, S; Petit, F; Boucraut-Baralon, C; Milon, A

1996-08-01

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.

The pig story (tiboi sakkoko Storytelling of kinship, memories of the past, and rights to plots of ancestral land in Mentawai

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Juniator Tulius

2016-07-01

Full Text Available This paper examines some significant elements of the pig story (tiboi sakkoko. This tale contains crucial information about the collective identity, ancestors and historical events affecting particular Mentawai kin-groups. As families do not preserve their culture and traditions in written form, storytellers of kin-groups have narrated the pig story from generation to generation so as to preserve it carefully. In the course of time, storytellers establish particular ways of telling their stories so as to remember the content and plot of the stories easily. Through the pig story, members of kin groups also recollect their ancestral place of origin and plots of ancestral lands. The role of human memory is indispensable to recalling all these important elements. Therefore, this paper analyses memories of the past of different family generations. To achieve its aims, this paper also evaluates the roles of family stories in the culture and traditions of Mentawai society.

Optimizing the Targeting of Mouse Parvovirus 1 to Murine Melanoma Selects for Recombinant Genomes and Novel Mutations in the Viral Capsid Gene

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Matthew Marr

2018-01-01

Full Text Available Combining virus-enhanced immunogenicity with direct delivery of immunomodulatory molecules would represent a novel treatment modality for melanoma, and would require development of new viral vectors capable of targeting melanoma cells preferentially. Here we explore the use of rodent protoparvoviruses targeting cells of the murine melanoma model B16F10. An uncloned stock of mouse parvovirus 1 (MPV1 showed some efficacy, which was substantially enhanced following serial passage in the target cell. Molecular cloning of the genes of both starter and selected virus pools revealed considerable sequence diversity. Chimera analysis mapped the majority of the improved infectivity to the product of the major coat protein gene, VP2, in which linked blocks of amino acid changes and one or other of two apparently spontaneous mutations were selected. Intragenic chimeras showed that these represented separable components, both contributing to enhanced infection. Comparison of biochemical parameters of infection by clonal viruses indicated that the enhancement due to changes in VP2 operates after the virus has bound to the cell surface and penetrated into the cell. Construction of an in silico homology model for MPV1 allowed placement of these changes within the capsid shell, and revealed aspects of the capsid involved in infection initiation that had not been previously recognized.

Wiring a Periscope – Ocelli, Retinula Axons, Visual Neuropils and the Ancestrality of Sea Spiders

Science.gov (United States)

Lehmann, Tobias; Heß, Martin; Melzer, Roland R.

2012-01-01

The Pycnogonida or sea spiders are cryptic, eight-legged arthropods with four median ocelli in a ‘periscope’ or eye tubercle. In older attempts at reconstructing phylogeny they were Arthropoda incertae sedis, but recent molecular trees placed them as the sister group either to all other euchelicerates or even to all euarthropods. Thus, pycnogonids are among the oldest extant arthropods and hold a key position for the understanding of arthropod evolution. This has stimulated studies of new sets of characters conductive to cladistic analyses, e.g. of the chelifores and of the hox gene expression pattern. In contrast knowledge of the architecture of the visual system is cursory. A few studies have analysed the ocelli and the uncommon “pseudoinverted” retinula cells. Moreover, analyses of visual neuropils are still at the stage of Hanström's early comprehensive works. We have therefore used various techniques to analyse the visual fibre pathways and the structure of their interrelated neuropils in several species. We found that pycnogonid ocelli are innervated to first and second visual neuropils in close vicinity to an unpaired midline neuropil, i.e. possibly the arcuate body, in a way very similar to ancestral euarthropods like Euperipatoides rowelli (Onychophora) and Limulus polyphemus (Xiphosura). This supports the ancestrality of pycnogonids and sheds light on what eyes in the pycnogonid ground plan might have ‘looked’ like. Recently it was suggested that arthropod eyes originated from simple ocelli similar to larval eyes. Hence, pycnogonid eyes would be one of the early offshoots among the wealth of more sophisticated arthropod eyes. PMID:22279594

Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals.

Science.gov (United States)

Lefèvre, Christophe M; Sharp, Julie A; Nicholas, Kevin R

2009-01-01

Using a milk-cell cDNA sequencing approach we characterised milk-protein sequences from two monotreme species, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) and found a full set of caseins and casein variants. The genomic organisation of the platypus casein locus is compared with other mammalian genomes, including the marsupial opossum and several eutherians. Physical linkage of casein genes has been seen in the casein loci of all mammalian genomes examined and we confirm that this is also observed in platypus. However, we show that a recent duplication of beta-casein occurred in the monotreme lineage, as opposed to more ancient duplications of alpha-casein in the eutherian lineage, while marsupials possess only single copies of alpha- and beta-caseins. Despite this variability, the close proximity of the main alpha- and beta-casein genes in an inverted tail-tail orientation and the relative orientation of the more distant kappa-casein genes are similar in all mammalian genome sequences so far available. Overall, the conservation of the genomic organisation of the caseins indicates the early, pre-monotreme development of the fundamental role of caseins during lactation. In contrast, the lineage-specific gene duplications that have occurred within the casein locus of monotremes and eutherians but not marsupials, which may have lost part of the ancestral casein locus, emphasises the independent selection on milk provision strategies to the young, most likely linked to different developmental strategies. The monotremes therefore provide insight into the ancestral drivers for lactation and how these have adapted in different lineages.

¿Con o sin ancestros? Vigencia de lo ancestral en la Amazonía peruana

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Thomas Mouriès

2014-07-01

Full Text Available The existence —or not— of the concept of ancestors in the indigenous Amazon has been the subject of much debate. However, regional leaders do not hesitate to call upon ‘ancestral’ knowledge, customs, or territories in the sense that, from an academic point of view, could appear enigmatic. «Ancestral, but… with or without ancestors?» is the question a confused anthropologist might ask. In this article, I propose to offer elements of a response to this question,based on a case study in Peru. First I analyze how Amazonian indigenous leaders, following international law, have adopted the legal notion of ‘ancestral possession’ of their territory to adapt it to the political sphere. This approach accounts for the recent generalization and uniformization of the term ‘ancestral’, but poses the problem of how it articulates with the indigenous cosmologies that it supposes to reflect. For this reason, I explore in the second section the pertinence of the category of ‘ancestor’ in the indigenous Amazon, briefly drawing upon the academic debate in order to define inwhat way this category takes on meaning. Based on testimony from an experienced Awajún leader, we thus return in the third section more explicitly to the different meanings and planes of reference that unfold when one uses the term ‘ancestral’, showing how Amazonian indigenous people not only adopt external conceptual elements and arguments, but also transform them based on their own cosmological singularities and political perspectives.

A novel inhibitor of dengue virus replication that targets the capsid protein.

Science.gov (United States)

Byrd, Chelsea M; Dai, Dongcheng; Grosenbach, Douglas W; Berhanu, Aklile; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Wineinger, Kristin A; Page, Jessica M; Harver, Chris; Stavale, Eric; Tyavanagimatt, Shanthakumar; Stone, Melialani A; Bartenschlager, Ralf; Scaturro, Pietro; Hruby, Dennis E; Jordan, Robert

2013-01-01

Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.

Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus.

Science.gov (United States)

Soldatova, Irina; Prilepskaja, Terezie; Abrahamyan, Levon; Forstová, Jitka; Huérfano, Sandra

2018-03-31

The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.

Amplification of an ancestral mammalian L1 family of long interspersed repeated DNA occurred just before the murine radiation

International Nuclear Information System (INIS)

Pascale, E.; Valle, E.; Furano, A.V.

1990-01-01

Each mammalian genus examined so far contains 50,000-100,000 members of an L1 (LINE 1) family of long interspersed repeated DNA elements. Current knowledge on the evolution of L1 families presents a paradox because, although L1 families have been in mammalian genomes since before the mammalian radiation ∼80 million years ago, most members of the L1 families are only a few million years old. Accordingly it has been suggested either that the extensive amplification that characterizes present-day L1 families did not occur in the past or that old members were removed as new one were generated. However, the authors show here that an ancestral rodent L1 family was extensively amplified ∼10 million years ago and that the relics of this amplification have persisted in modern murine genomes. This amplification occurred just before the divergence of modern murine genera from their common ancestor and identifies the murine node in the lineage of modern muroid rodents The results suggest that repeated amplification of L1 elements is a feature of the evaluation of mammalian genomes and that ancestral amplification events could provide a useful tool for determining mammalian lineages

Mating animals by minimising the covariance between ancestral contributions generates less inbreeding without compromising genetic gain in breeding schemes with truncation selection

DEFF Research Database (Denmark)

Henryon, M; Berg, P; Sørensen, A C

2009-01-01

We reasoned that mating animals by minimising the covariance between ancestral contributions (MCAC mating) will generate less inbreeding and at least as much genetic gain as minimum-coancestry mating in breeding schemes where the animals are truncation-selected. We tested this hypothesis by stoch...

A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

International Nuclear Information System (INIS)

Valdes, Iris; Bernardo, Lidice; Gil, Lazaro; Pavon, Alekis; Lazo, Laura; Lopez, Carlos; Romero, Yaremis; Menendez, Ivon; Falcon, Viviana; Betancourt, Lazaro; Martin, Jorge; Chinea, Glay; Silva, Ricardo; Guzman, Maria G.; Guillen, Gerardo; Hermida, Lisset

2009-01-01

Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-γ secretion and protection experiments, mediated by CD4 + and CD8 + cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

Science.gov (United States)

Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A

2016-10-01

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain.

Science.gov (United States)

Matsuzaki, Yasunori; Konno, Ayumu; Mochizuki, Ryuta; Shinohara, Yoichiro; Nitta, Keisuke; Okada, Yukihiro; Hirai, Hirokazu

2018-02-05

Intravenous administration of adeno-associated virus (AAV)-PHP.B, a capsid variant of AAV9 containing seven amino acid insertions, results in a greater permeability of the blood brain barrier (BBB) than standard AAV9 in mice, leading to highly efficient and global transduction of the central nervous system (CNS). The present study aimed to examine whether the enhanced BBB penetrance of AAV-PHP.B observed in mice also occurs in non-human primates. Thus, a young adult (age, 1.6 years) and an old adult (age, 7.2 years) marmoset received an intravenous injection of AAV-PHP.B expressing enhanced green fluorescent protein (EGFP) under the control of the constitutive CBh promoter (a hybrid of cytomegalovirus early enhancer and chicken β-actin promoter). Age-matched control marmosets were treated with standard AAV9-capsid vectors. The animals were sacrificed 6 weeks after the viral injection. Based on the results, only limited transduction of neurons (0-2%) and astrocytes (0.1-2.5%) was observed in both AAV-PHP.B- and AAV9-treated marmosets. One noticeable difference between AAV-PHP.B and AAV9 was the marked transduction of the peripheral dorsal root ganglia neurons. Indeed, the soma and axons in the projection from the spinal cord to the nucleus cuneatus in the medulla oblongata were strongly labeled with EGFP by AAV-PHP.B. Thus, except for the peripheral dorsal root ganglia neurons, the AAV-PHP.B transduction efficiency in the CNS of marmosets was comparable to that of AAV9 vectors. Copyright © 2017 Elsevier B.V. All rights reserved.

Saccharomyces cerevisiae Bat1 and Bat2 aminotransferases have functionally diverged from the ancestral-like Kluyveromyces lactis orthologous enzyme.

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Maritrini Colón

Full Text Available BACKGROUND: Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism. PRINCIPAL FINDINGS: Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs. This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1, while catabolic substrates are accumulated in the cytosol (Bat2. Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition. CONCLUSIONS: Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the

Inference of Ancestral Recombination Graphs through Topological Data Analysis

Science.gov (United States)

Cámara, Pablo G.; Levine, Arnold J.; Rabadán, Raúl

2016-01-01

The recent explosion of genomic data has underscored the need for interpretable and comprehensive analyses that can capture complex phylogenetic relationships within and across species. Recombination, reassortment and horizontal gene transfer constitute examples of pervasive biological phenomena that cannot be captured by tree-like representations. Starting from hundreds of genomes, we are interested in the reconstruction of potential evolutionary histories leading to the observed data. Ancestral recombination graphs represent potential histories that explicitly accommodate recombination and mutation events across orthologous genomes. However, they are computationally costly to reconstruct, usually being infeasible for more than few tens of genomes. Recently, Topological Data Analysis (TDA) methods have been proposed as robust and scalable methods that can capture the genetic scale and frequency of recombination. We build upon previous TDA developments for detecting and quantifying recombination, and present a novel framework that can be applied to hundreds of genomes and can be interpreted in terms of minimal histories of mutation and recombination events, quantifying the scales and identifying the genomic locations of recombinations. We implement this framework in a software package, called TARGet, and apply it to several examples, including small migration between different populations, human recombination, and horizontal evolution in finches inhabiting the Galápagos Islands. PMID:27532298

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

Science.gov (United States)

Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

2009-07-01

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

Directory of Open Access Journals (Sweden)

Malgorzata Bienkowska-Haba

2009-07-01

Full Text Available Following attachment to primary receptor heparan sulfate proteoglycans (HSPG, human papillomavirus type 16 (HPV16 particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Putative Lineage of Novel African Usutu Virus, Central Europe

Centers for Disease Control (CDC) Podcasts

2015-10-15

Sarah Gregory reads an abridged version of "Putative Lineage of Novel African Usutu Virus, Central Europe.".  Created: 10/15/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 10/15/2015.

Did warfare among ancestral hunter-gatherers affect the evolution of human social behaviors?

Science.gov (United States)

Bowles, Samuel

2009-06-05

Since Darwin, intergroup hostilities have figured prominently in explanations of the evolution of human social behavior. Yet whether ancestral humans were largely "peaceful" or "warlike" remains controversial. I ask a more precise question: If more cooperative groups were more likely to prevail in conflicts with other groups, was the level of intergroup violence sufficient to influence the evolution of human social behavior? Using a model of the evolutionary impact of between-group competition and a new data set that combines archaeological evidence on causes of death during the Late Pleistocene and early Holocene with ethnographic and historical reports on hunter-gatherer populations, I find that the estimated level of mortality in intergroup conflicts would have had substantial effects, allowing the proliferation of group-beneficial behaviors that were quite costly to the individual altruist.

Molecular cloning and characterization of a putative OGG_N domain ...

African Journals Online (AJOL)

Molecular cloning and characterization of a putative OGG_N domain from the camel, Camelus dromedarius. Farid Shokry Ataya, Mohammad Saud Alanazi, Dalia Fouad, Hehsam Mahmoud Saeed, Mohammad Bazzi ...

Archaeology of the Afro-Ecuadorians in La Concepción, Ancestral Territory of the Chota-Mira Valley (Carchi-Ecuador)

OpenAIRE

Balanzategui , Daniela Catalina

2017-01-01

Historical Archaeology of the African Diaspora in the Americas urges for interdisciplinary, collaborative, and intercultural approaches to shed light on how the material culture reflects conditions of enslavement and racialization, but also process of resistance and historical reparation. This investigation is organized in five articles connected around the topic of the cultural construction of the African Diaspora identities in the Afro-Ecuadorian Ancestral Territory of the Chota-Mira Valley...

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

Science.gov (United States)

Waye, J S; Willard, H F

1986-09-01

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.

Latest Miocene-earliest Pliocene evolution of the ancestral Rio Grande at the Española-San Luis Basin boundary, northern New Mexico

Science.gov (United States)

Daniel J. Koning,; Aby, Scott B.; Grauch, V. J.; Matthew J. Zimmerer,

2016-01-01

We use stratigraphic relations, paleoflow data, and 40Ar/39Ar dating to interpret net aggradation, punctuated by at least two minor incisional events, along part of the upper ancestral Rio Grande fluvial system between 5.5 and 4.5 Ma (in northern New Mexico). The studied fluvial deposits, which we informally call the Sandlin unit of the Santa Fe Group, overlie a structural high between the San Luis and Española Basins. The Sandlin unit was deposited by two merging, west- to southwest-flowing, ancestral Rio Grande tributaries respectively sourced in the central Taos Mountains and southern Taos Mountains-northeastern Picuris Mountains. The river confluence progressively shifted southwestward (downstream) with time, and the integrated river (ancestral Rio Grande) flowed southwards into the Española Basin to merge with the ancestral Rio Chama. Just prior to the end of the Miocene, this fluvial system was incised in the southern part of the study area (resulting in an approximately 4–7 km wide paleovalley), and had sufficient competency to transport cobbles and boulders. Sometime between emplacement of two basalt flows dated at 5.54± 0.38 Ma and 4.82±0.20 Ma (groundmass 40Ar/39Ar ages), this fluvial system deposited 10–12 m of sandier sediment (lower Sandlin subunit) preserved in the northern part of this paleovalley. The fluvial system widened between 4.82±0.20 and 4.50±0.07 Ma, depositing coarse sand and fine gravel up to 14 km north of the present-day Rio Grande. This 10–25 m-thick sediment package (upper Sandlin unit) buried earlier south- to southeast-trending paleovalleys (500–800 m wide) inferred from aeromagnetic data. Two brief incisional events are recognized. The first was caused by the 4.82±0.20 Ma basalt flow impounding south-flowing paleodrainages, and the second occurred shortly after emplacement of a 4.69±0.09 Ma basalt flow in the northern study area. Drivers responsible for Sandlin unit aggradation may include climate

PhyloBot: A Web Portal for Automated Phylogenetics, Ancestral Sequence Reconstruction, and Exploration of Mutational Trajectories.

Directory of Open Access Journals (Sweden)

Victor Hanson-Smith

2016-07-01

Full Text Available The method of phylogenetic ancestral sequence reconstruction is a powerful approach for studying evolutionary relationships among protein sequence, structure, and function. In particular, this approach allows investigators to (1 reconstruct and "resurrect" (that is, synthesize in vivo or in vitro extinct proteins to study how they differ from modern proteins, (2 identify key amino acid changes that, over evolutionary timescales, have altered the function of the protein, and (3 order historical events in the evolution of protein function. Widespread use of this approach has been slow among molecular biologists, in part because the methods require significant computational expertise. Here we present PhyloBot, a web-based software tool that makes ancestral sequence reconstruction easy. Designed for non-experts, it integrates all the necessary software into a single user interface. Additionally, PhyloBot provides interactive tools to explore evolutionary trajectories between ancestors, enabling the rapid generation of hypotheses that can be tested using genetic or biochemical approaches. Early versions of this software were used in previous studies to discover genetic mechanisms underlying the functions of diverse protein families, including V-ATPase ion pumps, DNA-binding transcription regulators, and serine/threonine protein kinases. PhyloBot runs in a web browser, and is available at the following URL: http://www.phylobot.com. The software is implemented in Python using the Django web framework, and runs on elastic cloud computing resources from Amazon Web Services. Users can create and submit jobs on our free server (at the URL listed above, or use our open-source code to launch their own PhyloBot server.

The Putative Chemosignal Androstadienone Makes Women More Generous.

Science.gov (United States)

Perrotta, Valentina; Graffeo, Michele; Bonini, Nicolao; Gottfried, Jay A

2016-06-01

Putative human chemosignals have been shown to influence mood states and emotional processing, but the connection between these effects and higher-order cognitive processing is not well established. This study utilized an economic game (Dictator Game) to test whether androstadienone (AND), an odorous compound derived from testosterone, impacts on altruistic behavior. We predicted that the female participants would act more generously in the AND condition, exhibiting a significant interaction effect between gender and AND on Dictator Game contributions. We also expected that the presence of AND should increase the positive mood of the female participants, compared to a control odor condition and also compared to the mood of the male participants. The results confirm our hypotheses: for women the subliminal perception of AND led to larger monetary donations, compared to a control odor, and also increased positive mood. These effects were absent or significantly weaker in men. Our findings highlight the capacity of human putative chemosignals to influence emotions and higher cognitive processes - in particular the processes used in the context of economic decisions - in a gender-specific way.

Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

Energy Technology Data Exchange (ETDEWEB)

Jiang, Hongbing; Franz, Carl J.; Wu, Guang; Renshaw, Hilary; Zhao, Guoyan [Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (United States); Firth, Andrew E. [Department of Pathology, University of Cambridge, Cambridge CB2 1QP (United Kingdom); Wang, David, E-mail: davewang@borcim.wustl.edu [Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (United States)

2014-02-15

Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated into Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions.

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

Science.gov (United States)

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

Energy Technology Data Exchange (ETDEWEB)

Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Sun, Ya-Ni [College of Veterinary Medicine, Northwest A and F University, Shanxi, Yangling 712100 (China); Gao, Ji-Ming; Xie, Zhi-Jing [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Wang, Yu [Department of Basic Medical Sciences, Taishan Medical College, Shandong, Taian 271000 (China); Zhu, Yan-Li [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Jiang, Shi-Jin, E-mail: sjjiang@sdau.edu.cn [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China)

2013-02-05

The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

International Nuclear Information System (INIS)

Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Wang, Yu; Zhu, Yan-Li; Jiang, Shi-Jin

2013-01-01

The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1–17 and 18–36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

Specific interaction of capsid protein and importin-α/β influences West Nile virus production

International Nuclear Information System (INIS)

Bhuvanakantham, Raghavan; Chong, Mun-Keat; Ng, Mah-Lee

2009-01-01

West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-α. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-α/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-α/C protein interaction in the context of flavivirus life-cycle.

Specific interaction of capsid protein and importin-{alpha}/{beta} influences West Nile virus production

Energy Technology Data Exchange (ETDEWEB)

Bhuvanakantham, Raghavan; Chong, Mun-Keat [Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597 (Singapore); Ng, Mah-Lee, E-mail: micngml@nus.edu.sg [Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597 (Singapore)

2009-11-06

West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-{alpha}. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-{alpha}/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-{alpha}/C protein interaction in the context of flavivirus life-cycle.

The Polerovirus Minor Capsid Protein Determines Vector Specificity and Intestinal Tropism in the Aphid

Science.gov (United States)

Brault, Véronique; Périgon, Sophie; Reinbold, Catherine; Erdinger, Monique; Scheidecker, Danièle; Herrbach, Etienne; Richards, Ken; Ziegler-Graff, Véronique

2005-01-01

Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD. PMID:16014930

African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

Directory of Open Access Journals (Sweden)

Bruno Hernáez

2016-04-01

Full Text Available African swine fever virus (ASFV is a nucleocytoplasmic large DNA virus (NCLDV that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

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Frank Powilleit

Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

DEFF Research Database (Denmark)

Nilsson, Martin; Chiang, Wen-Chi; Fazli, Mustafa

2011-01-01

We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P....... putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable...

Evidence for an Ancestral Association of Human Coronavirus 229E with Bats.

Science.gov (United States)

Corman, Victor Max; Baldwin, Heather J; Tateno, Adriana Fumie; Zerbinati, Rodrigo Melim; Annan, Augustina; Owusu, Michael; Nkrumah, Evans Ewald; Maganga, Gael Darren; Oppong, Samuel; Adu-Sarkodie, Yaw; Vallo, Peter; da Silva Filho, Luiz Vicente Ribeiro Ferreira; Leroy, Eric M; Thiel, Volker; van der Hoek, Lia; Poon, Leo L M; Tschapka, Marco; Drosten, Christian; Drexler, Jan Felix

2015-12-01

We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3' end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E. The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a

Effect of capsid proteins to ICG mass ratio on fluorescent quantum yield of virus-resembling optical nano-materials

Science.gov (United States)

Gupta, Sharad; Ico, Gerardo; Matsumura, Paul; Rao, A. L. N.; Vullev, Valentine; Anvari, Bahman

2012-03-01

We recently reported construction of a new type of optical nano-construct composed of genome-depleted plant infecting brome mosaic virus (BMV) doped with Indocyanine green (ICG), an FDA-approved chromophore. We refer to these constructs as optical viral ghosts (OVGs) since only the capsid protein (CP) subunits of BMV remain to encapsulate ICG. To utilize OVGs as effective nano-probes in fluorescence imaging applications, their fluorescence quantum yield needs to be maximized. In this study, we investigate the effect of altering the CP to ICG mass ratio on the fluorescent quantum yield of OVGs. Results of this study provide the basis for construction of OVGs with optimal amounts of CP and ICG to yield maximal fluorescence quantum yield.

Loop Replacement Enhances the Ancestral Antibacterial Function of a Bifunctional Scorpion Toxin

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Shangfei Zhang

2018-06-01

Full Text Available On the basis of the evolutionary relationship between scorpion toxins targeting K+ channels (KTxs and antibacterial defensins (Zhu S., Peigneur S., Gao B., Umetsu Y., Ohki S., Tytgat J. Experimental conversion of a defensin into a neurotoxin: Implications for origin of toxic function. Mol. Biol. Evol. 2014, 31, 546–559, we performed protein engineering experiments to modify a bifunctional KTx (i.e., weak inhibitory activities on both K+ channels and bacteria via substituting its carboxyl loop with the structurally equivalent loop of contemporary defensins. As expected, the engineered peptide (named MeuTXKα3-KFGGI remarkably improved the antibacterial activity, particularly on some Gram-positive bacteria, including several antibiotic-resistant opportunistic pathogens. Compared with the unmodified toxin, its antibacterial spectrum also enlarged. Our work provides a new method to enhance the antibacterial activity of bifunctional scorpion venom peptides, which might be useful in engineering other proteins with an ancestral activity.

Exploring universal partnerships and putative marriages as tools for ...

African Journals Online (AJOL)

Following upon the Supreme Court of Appeal's judgment in Butters v Mncora 2012 4 SA 1 (SCA), which broadened the criteria and consequences of universal partnerships in cohabitation relationships, this article investigates the potential of universal partnerships and putative marriages to allocate rights to share in ...

A new putative deltapartitivirus recovered from Dianthus amurensis.

Science.gov (United States)

An, Hongliu; Tan, Guanlin; Xiong, Guihong; Li, Meirong; Fang, Shouguo; Islam, Saif Ul; Zhang, Songbai; Li, Fan

2017-09-01

Two double stranded RNAs (dsRNA), likely representing the genome of a novel deltapartitivirus, provisionally named carnation cryptic virus 3 (CCV3), were recovered from Dianthus amurensis. The two dsRNAs were 1,573 (dsRNA1) and 1,561 (dsRNA2) bp in size, each containing a single open reading frame (ORF) encoding a 475- and 411-aa protein, respectively. The 475-aa protein contains a conserved RNA dependent RNA polymerase (RdRp) domain which shows significant homology to RdRps of established or putative partitiviruses, particularly those belonging to the genus Deltapartitivirus. However, it shares an amino acid identity of 75% with its closest relative, the RdRp of the deltapartitivirus beet cryptic virus 2 (BCV2), and is <62% identical to the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRps of selected partitiviruses, CCV3 clustered with BCV2 and formed a well-supported monophyletic clade with known or putative deltapartitiviruses.

Zinc and glutamate dehydrogenase in putative glutamatergic brain structures.

Science.gov (United States)

Wolf, G; Schmidt, W

1983-01-01

A certain topographic parallelism between the distribution of histochemically (TIMM staining) identified zinc and putative glutamatergic structures in the rat brain was demonstrated. Glutamate dehydrogenase as a zinc containing protein is in consideration to be an enzyme synthesizing transmitter glutamate. In a low concentration range externally added zinc ions (10(-9) to 10(-7) M) induced an increase in the activity of glutamate dehydrogenase (GDH) originating from rat hippocampal formation, neocortex, and cerebellum up to 142.4%. With rising molarity of Zn(II) in the incubation medium, the enzyme of hippocampal formation and cerebellum showed a biphasic course of activation. Zinc ions of a concentration higher than 10(-6) M caused a strong inhibition of GDH. The effect of Zn(II) on GDH originating from spinal ganglia and liver led only to a decrease of enzyme activity. These results are discussed in connection with a functional correlation between zinc and putatively glutamatergic system.

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

Science.gov (United States)

Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A; Barry, Kerrie W; Spatafora, Joseph; Grigoriev, Igor V; Martin, Francis M; Pujade-Renaud, Valérie

2018-01-01

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species ( Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca , and Botrosphaeria dothidea ) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors

Directory of Open Access Journals (Sweden)

David Lopez

2018-03-01

Full Text Available Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

Molecular characterization of the genome of Maize rayado fino virus, the type member of the genus Marafivirus.

Science.gov (United States)

Hammond, R W; Ramirez, P

2001-04-10

The complete nucleotide sequence of the single-stranded RNA genome of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus, is 6305 nucleotides (nts) in length and contains two putative open reading frames (ORFs). The largest ORF (nt 97-6180) encodes a polyprotein of 224 kDa with sequence similarities at its N-terminus to the replication-associated proteins of other viruses with positive-strand RNA genomes and to the papainlike protease domain found in tymoviruses. The C-terminus of the 224-kDa ORF also encodes the MRFV capsid protein. A smaller, overlapping ORF (nt 302-1561) encodes a putative protein of 43 kDa with unknown function but with limited sequence similarities to putative movement proteins of tymoviruses. The nucleotide sequence and proposed genome expression strategy of MRFV is most closely related to that of oat blue dwarf virus (OBDV). Unlike OBDV, MRFV RNA does not appear to contain a poly(A) tail, and it encodes a putative second overlapping open reading frame.

Complete tribal sampling reveals basal split in Muscidae (Diptera), confirms saprophagy as ancestral feeding mode, and reveals an evolutionary correlation between instar numbers and carnivory

DEFF Research Database (Denmark)

Kutty, Sujatha Narayanan; Pont, Adrian C.; Meier, Rudolf

2014-01-01

split within this family. The ancestral larval feeding habit is reconstructed to be saprophagy with more specialised coprophagous saprophagy, phytophagy, and carnivory evolving multiple times from saprophagous ancestors. The origins of carnivory in larvae are significantly correlated with a reduction...

Genes Suggest Ancestral Colour Polymorphisms Are Shared across Morphologically Cryptic Species in Arctic Bumblebees.

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Paul H Williams

Full Text Available Our grasp of biodiversity is fine-tuned through the process of revisionary taxonomy. If species do exist in nature and can be discovered with available techniques, then we expect these revisions to converge on broadly shared interpretations of species. But for the primarily arctic bumblebees of the subgenus Alpinobombus of the genus Bombus, revisions by some of the most experienced specialists are unusual for bumblebees in that they have all reached different conclusions on the number of species present. Recent revisions based on skeletal morphology have concluded that there are from four to six species, while variation in colour pattern of the hair raised questions as to whether at least seven species might be present. Even more species are supported if we accept the recent move away from viewing species as morphotypes to viewing them instead as evolutionarily independent lineages (EILs using data from genes. EILs are recognised here in practice from the gene coalescents that provide direct evidence for their evolutionary independence. We show from fitting both general mixed Yule/coalescent (GMYC models and Poisson-tree-process (PTP models to data for the mitochondrial COI gene that there is support for nine species in the subgenus Alpinobombus. Examination of the more slowly evolving nuclear PEPCK gene shows further support for a previously unrecognised taxon as a new species in northwestern North America. The three pairs of the most morphologically similar sister species are separated allopatrically and prevented from interbreeding by oceans. We also find that most of the species show multiple shared colour patterns, giving the appearance of mimicry among parts of the different species. However, reconstructing ancestral colour-pattern states shows that speciation is likely to have cut across widespread ancestral polymorphisms, without or largely without convergence. In the particular case of Alpinobombus, morphological, colour-pattern, and

Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

Science.gov (United States)

Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

2014-01-01

Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins

International Nuclear Information System (INIS)

Zullo, J.; Stiles, C.D.; Garcea, R.L.

1987-01-01

The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP 1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP 1 protein stimulated [ 3 H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for the cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins

Mitochondrial introgression suggests extensive ancestral hybridization events among Saccharomyces species.

Science.gov (United States)

Peris, David; Arias, Armando; Orlić, Sandi; Belloch, Carmela; Pérez-Través, Laura; Querol, Amparo; Barrio, Eladio

2017-03-01

Horizontal gene transfer (HGT) in eukaryotic plastids and mitochondrial genomes is common, and plays an important role in organism evolution. In yeasts, recent mitochondrial HGT has been suggested between S. cerevisiae and S. paradoxus. However, few strains have been explored given the lack of accurate mitochondrial genome annotations. Mitochondrial genome sequences are important to understand how frequent these introgressions occur, and their role in cytonuclear incompatibilities and fitness. Indeed, most of the Bateson-Dobzhansky-Muller genetic incompatibilities described in yeasts are driven by cytonuclear incompatibilities. We herein explored the mitochondrial inheritance of several worldwide distributed wild Saccharomyces species and their hybrids isolated from different sources and geographic origins. We demonstrated the existence of several recombination points in mitochondrial region COX2-ORF1, likely mediated by either the activity of the protein encoded by the ORF1 (F-SceIII) gene, a free-standing homing endonuclease, or mostly facilitated by A+T tandem repeats and regions of integration of GC clusters. These introgressions were shown to occur among strains of the same species and among strains of different species, which suggests a complex model of Saccharomyces evolution that involves several ancestral hybridization events in wild environments. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative analysis of programmed cell death pathways in filamentous fungi

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Wortman Jennifer R

2005-12-01

Full Text Available Abstract Background Fungi can undergo autophagic- or apoptotic-type programmed cell death (PCD on exposure to antifungal agents, developmental signals, and stress factors. Filamentous fungi can also exhibit a form of cell death called heterokaryon incompatibility (HI triggered by fusion between two genetically incompatible individuals. With the availability of recently sequenced genomes of Aspergillus fumigatus and several related species, we were able to define putative components of fungi-specific death pathways and the ancestral core apoptotic machinery shared by all fungi and metazoa. Results Phylogenetic profiling of HI-associated proteins from four Aspergilli and seven other fungal species revealed lineage-specific protein families, orphan genes, and core genes conserved across all fungi and metazoa. The Aspergilli-specific domain architectures include NACHT family NTPases, which may function as key integrators of stress and nutrient availability signals. They are often found fused to putative effector domains such as Pfs, SesB/LipA, and a newly identified domain, HET-s/LopB. Many putative HI inducers and mediators are specific to filamentous fungi and not found in unicellular yeasts. In addition to their role in HI, several of them appear to be involved in regulation of cell cycle, development and sexual differentiation. Finally, the Aspergilli possess many putative downstream components of the mammalian apoptotic machinery including several proteins not found in the model yeast, Saccharomyces cerevisiae. Conclusion Our analysis identified more than 100 putative PCD associated genes in the Aspergilli, which may help expand the range of currently available treatments for aspergillosis and other invasive fungal diseases. The list includes species-specific protein families as well as conserved core components of the ancestral PCD machinery shared by fungi and metazoa.

Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes - Characterization of a Novel Phage Helper-Satellite System.

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Lukasz Dziewit

Full Text Available Two novel prophages ФAH14a and ФAH14b of a psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_H14 have been characterized. They were simultaneously induced with mitomycin C and packed into capsids of the same size and protein composition. The genome sequences of ФAH14a and ФAH14b have been determined. ФAH14b, the phage with a smaller genome (16,812 bp seems to parasitize ФAH14a (55,060 bp and utilizes its capsids, as only the latter encodes a complete set of structural proteins. Both viruses probably constitute a phage helper-satellite system, analogous to the P2-P4 duo. This study describes the architecture and function of the ФAH14a and ФAH14b genomes. Moreover, a functional analysis of a ФAH14a-encoded lytic enzyme and a DNA methyltransferase was performed. In silico analysis revealed the presence of the homologs of ФAH14a and ФAH14b in other Pseudomonas genomes, which may suggest that helper-satellite systems related to the one described in this work are common in pseudomonads.

Structure of a novel shoulder-to-shoulder p24 dimer in complex with the broad-spectrum antibody A10F9 and its implication in capsid assembly.

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Ying Gu

Full Text Available Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4 in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication.

Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry

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Sanchez-Martinez, Cristina; Grueso, Esther [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Cantoblanco, Madrid (Spain); Carroll, Miles [Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury SP4 OJG, Wilts (United Kingdom); Rommelaere, Jean [Deutsches Krebsforschungszentrum Division F010, Im Neuenheimer Feld 242, D-69120 Heidelberg (Germany); Almendral, Jose M., E-mail: jmalmendral@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Cantoblanco, Madrid (Spain)

2012-10-10

The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus. The VEGFR2-binding peptide (V1) inserted at both 2Nt and VP1-Nt efficiently assembled in virions, but V1 disrupted VP1 and VP2 entry functions. The VP2 defect correlated with restricted externalization of V1-2Nt out of the coat. The specific infectivity of MVM and wtVP-pseudotyped mosaic MVM-V1 virions, upon heating and/or partial 2Nt cleavage, demonstrated that some 2Nt domains become intracellularly translocated out of the virus shell and cleaved to initiate entry. The V1 insertion defines a VP2-driven endosomal enlargement of the channel as an essential structural rearrangement performed by the MVM virion to infect.

Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry

International Nuclear Information System (INIS)

Sánchez-Martínez, Cristina; Grueso, Esther; Carroll, Miles; Rommelaere, Jean; Almendral, José M.

2012-01-01

The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus. The VEGFR2-binding peptide (V1) inserted at both 2Nt and VP1-Nt efficiently assembled in virions, but V1 disrupted VP1 and VP2 entry functions. The VP2 defect correlated with restricted externalization of V1-2Nt out of the coat. The specific infectivity of MVM and wtVP-pseudotyped mosaic MVM-V1 virions, upon heating and/or partial 2Nt cleavage, demonstrated that some 2Nt domains become intracellularly translocated out of the virus shell and cleaved to initiate entry. The V1 insertion defines a VP2-driven endosomal enlargement of the channel as an essential structural rearrangement performed by the MVM virion to infect.

Identification of immunogenic hot spots within plum pox potyvirus capsid protein for efficient antigen presentation.

Science.gov (United States)

Fernández-Fernández, M Rosario; Martínez-Torrecuadrada, Jorge L; Roncal, Fernando; Domínguez, Elvira; García, Juan Antonio

2002-12-01

PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-gamma, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence.

Kinetics of the association of dengue virus capsid protein with the granular component of nucleolus.

Science.gov (United States)

Tiwary, Ashish Kumar; Cecilia, D

2017-02-01

Dengue virus (DENV) replicates in the cytoplasm but translocation of the capsid protein (C) to the nucleoli of infected cells has been shown to facilitate virus multiplication for DENV-2. This study demonstrates that the nucleolar localization of C occurs with all four serotypes of DENV. The interaction of C with the nucleolus was found to be dynamic with a mobile fraction of 66% by FRAP. That the C shuttled between the nucleus and cytoplasm was suggested by FLIP and translation inhibition experiments. Colocalization with B23 indicated that DENV C targeted the granular component (GC) of the nucleolus. Presence of DENV C in the nucleolus affected the recovery kinetics of B23 in infected and transfected cells. Sub-nucleolar localization of DENV C of all serotypes to the GC, its mobility in and out of the nucleolus and its affect on the dynamics of B23 is being shown for the first time. Copyright © 2016 Elsevier Inc. All rights reserved.

The Microcephalin Ancestral Allele in a Neanderthal Individual

Science.gov (United States)

Lari, Martina; Rizzi, Ermanno; Milani, Lucio; Corti, Giorgio; Balsamo, Carlotta; Vai, Stefania; Catalano, Giulio; Pilli, Elena; Longo, Laura; Condemi, Silvana; Giunti, Paolo; Hänni, Catherine; De Bellis, Gianluca; Orlando, Ludovic; Barbujani, Guido; Caramelli, David

2010-01-01

Background The high frequency (around 0.70 worlwide) and the relatively young age (between 14,000 and 62,000 years) of a derived group of haplotypes, haplogroup D, at the microcephalin (MCPH1) locus led to the proposal that haplogroup D originated in a human lineage that separated from modern humans >1 million years ago, evolved under strong positive selection, and passed into the human gene pool by an episode of admixture circa 37,000 years ago. The geographic distribution of haplogroup D, with marked differences between Africa and Eurasia, suggested that the archaic human form admixing with anatomically modern humans might have been Neanderthal. Methodology/Principal Findings Here we report the first PCR amplification and high- throughput sequencing of nuclear DNA at the microcephalin (MCPH1) locus from Neanderthal individual from Mezzena Rockshelter (Monti Lessini, Italy). We show that a well-preserved Neanderthal fossil dated at approximately 50,000 years B.P., was homozygous for the ancestral, non-D, allele. The high yield of Neanderthal mtDNA sequences of the studied specimen, the pattern of nucleotide misincorporation among sequences consistent with post-mortem DNA damage and an accurate control of the MCPH1 alleles in all personnel that manipulated the sample, make it extremely unlikely that this result might reflect modern DNA contamination. Conclusions/Significance The MCPH1 genotype of the Monti Lessini (MLS) Neanderthal does not prove that there was no interbreeding between anatomically archaic and modern humans in Europe, but certainly shows that speculations on a possible Neanderthal origin of what is now the most common MCPH1 haplogroup are not supported by empirical evidence from ancient DNA. PMID:20498832

The microcephalin ancestral allele in a Neanderthal individual.

Directory of Open Access Journals (Sweden)

Martina Lari

Full Text Available BACKGROUND: The high frequency (around 0.70 worldwide and the relatively young age (between 14,000 and 62,000 years of a derived group of haplotypes, haplogroup D, at the microcephalin (MCPH1 locus led to the proposal that haplogroup D originated in a human lineage that separated from modern humans >1 million years ago, evolved under strong positive selection, and passed into the human gene pool by an episode of admixture circa 37,000 years ago. The geographic distribution of haplogroup D, with marked differences between Africa and Eurasia, suggested that the archaic human form admixing with anatomically modern humans might have been Neanderthal. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the first PCR amplification and high-throughput sequencing of nuclear DNA at the microcephalin (MCPH1 locus from Neanderthal individual from Mezzena Rockshelter (Monti Lessini, Italy. We show that a well-preserved Neanderthal fossil dated at approximately 50,000 years B.P., was homozygous for the ancestral, non-D, allele. The high yield of Neanderthal mtDNA sequences of the studied specimen, the pattern of nucleotide misincorporation among sequences consistent with post-mortem DNA damage and an accurate control of the MCPH1 alleles in all personnel that manipulated the sample, make it extremely unlikely that this result might reflect modern DNA contamination. CONCLUSIONS/SIGNIFICANCE: The MCPH1 genotype of the Monti Lessini (MLS Neanderthal does not prove that there was no interbreeding between anatomically archaic and modern humans in Europe, but certainly shows that speculations on a possible Neanderthal origin of what is now the most common MCPH1 haplogroup are not supported by empirical evidence from ancient DNA.

Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets.

Science.gov (United States)

Marreiros, Rita; Müller-Schiffmann, Andreas; Bader, Verian; Selvarajah, Suganya; Dey, Debendranath; Lingappa, Vishwanath R; Korth, Carsten

2015-09-02

Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation

Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study.

Science.gov (United States)

Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

2016-06-15

Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis [Pg], Aggregatibacer actinomycetemcomitans [Aa], Fusobacterium nucleatum [Fn], Prevotella intermedia [Pi], Tannerella forsythia [Tf], and Treponema denticola [Td]) and three herpesviruses (Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], and herpes simplex virus [HSV]) were detected. Socio-demographic data and oral health related behaviors, and salivary estradiol and progesterone levels were also collected. The results showed no significant differences in socio-demographic background, oral health related behaviors, and salivary estradiol and progesterone levels between the two groups (all P > 0.05). The detection rates of included periodontopathic microorganisms were not significantly different between the two groups (all P > 0.05), but the coinfection rate of EBV and Pg was significantly higher in the case group than in the control group (P = 0.028). EBV and Pg coinfection may promote the development of chronic periodontitis among pregnant women.

Computational identification of putative cytochrome P450 genes in ...

African Journals Online (AJOL)

In this work, a computational study of expressed sequence tags (ESTs) of soybean was performed by data mining methods and bio-informatics tools and as a result 78 putative P450 genes were identified, including 57 new ones. These genes were classified into five clans and 20 families by sequence similarities and among ...

Detection of Weakly Conserved Ancestral Mammalian RegulatorySequences by Primate Comparisons

Energy Technology Data Exchange (ETDEWEB)

Wang, Qian-fei; Prabhakar, Shyam; Chanan, Sumita; Cheng,Jan-Fang; Rubin, Edward M.; Boffelli, Dario

2006-06-01

Genomic comparisons between human and distant, non-primatemammals are commonly used to identify cis-regulatory elements based onconstrained sequence evolution. However, these methods fail to detectcryptic functional elements, which are too weakly conserved among mammalsto distinguish from nonfunctional DNA. To address this problem, weexplored the potential of deep intra-primate sequence comparisons. Wesequenced the orthologs of 558 kb of human genomic sequence, coveringmultiple loci involved in cholesterol homeostasis, in 6 nonhumanprimates. Our analysis identified 6 noncoding DNA elements displayingsignificant conservation among primates, but undetectable in more distantcomparisons. In vitro and in vivo tests revealed that at least three ofthese 6 elements have regulatory function. Notably, the mouse orthologsof these three functional human sequences had regulatory activity despitetheir lack of significant sequence conservation, indicating that they arecryptic ancestral cis-regulatory elements. These regulatory elementscould still be detected in a smaller set of three primate speciesincluding human, rhesus and marmoset. Since the human and rhesus genomesequences are already available, and the marmoset genome is activelybeing sequenced, the primate-specific conservation analysis describedhere can be applied in the near future on a whole-genome scale, tocomplement the annotation provided by more distant speciescomparisons.

Roles of three amino acids of capsid proteins in mink enteritis parvovirus replication.

Science.gov (United States)

Mao, Yaping; Su, Jun; Wang, Jigui; Zhang, Xiaomei; Hou, Qiang; Bian, Dawei; Liu, Weiquan

2016-08-15

Virulent mink enteritis parvovirus (MEV) strain MEV-LHV replicated to higher titers in feline F81 cells than attenuated strain MEV-L. Phylogenetic and sequence analyses of the VP2 gene of MEV-LHV, MEV-L and other strains in GenBank revealed two evolutionary branches separating virulent and attenuated strains. Three residues, 101, 232 and 411, differed between virulent and attenuated strains but were conserved within the two branches. Site-directed mutagenesis of the VP2 gene of infectious plasmids of attenuated strain MEV-L respectively replacing residues 101 Ile and 411 Ala with Thr and Glu of virulent strains (MEV-L I101T and MEV-L A411E) increased replication efficiency but still to lower levels than MEV-LHV. However, viruses with mutation of residue 232 (MEV-L I232V and MEV-L I101T/I232V/A411E) decreased viral transcription and replication levels. The three VP2 residues 101, 232 and 411, located on or near the capsid surface, played different roles in the infection processes of MEV. Copyright © 2016 Elsevier B.V. All rights reserved.

The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

International Nuclear Information System (INIS)

Sun Jian; Yu Jisheng; Yu Zhiwu; Zha Xiao; Wu Yuqing

2012-01-01

Graphial abstract: The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1. Highlights: ► Several driving forces contribute to the interaction between heparin and peptides. ► C-terminal of HPV L1 is a potential candidate for the attachment to host cells. ► The C-terminal peptides of HPV-16 and -18 L1 have different heparin-binding. ► The different heparin-binding provides an explanation for the distinct prevalences. - Abstract: The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.

Putative golden proportions as predictors of facial esthetics in adolescents.

NARCIS (Netherlands)

Kiekens, R.M.A.; Kuijpers-Jagtman, A.M.; Hof, M.A. van 't; Hof, B.E. van 't; Maltha, J.C.

2008-01-01

INTRODUCTION: In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. METHODS: Seventy-six adult laypeople

Putative Dementia Cases Fluctuate as a Function of Mini-Mental State Examination Cut-Off Points.

Science.gov (United States)

Rosa, Ilka M; Henriques, Ana G; Wiltfang, Jens; da Cruz E Silva, Odete A B

2018-01-01

As the population ages, there is a growing need to quickly and accurately identify putative dementia cases. Many cognitive tests are available; among those commonly used are the Cognitive Dementia Rating (CDR) and the Mini-Mental Status Examination (MMSE). The aim of this work was to compare the validity and reliability of these cognitive tests in a primary care based cohort (pcb-Cohort). The MMSE and the CDR were applied to 568 volunteers in the pcb-Cohort. Distinct cut-off points for the MMSE were considered, namely MMSE 27, MMSE 24, and MMSE PT (adapted for the Portuguese population). The MMSE 27 identified the greatest number of putative dementia cases, and, as determined by the ROC curve, it was the most sensitive and specific of the MMSE cut-offs considered. Putative predictive or risk factors identified included age, literacy, depression, and diabetes mellitus (DM). DM has previously been indicated as a risk factor for dementia and Alzheimer's disease. Comparatively, the MMSE 27 cut-off has the greatest sensibility (94.9%) and specificity (66.3%) when compared to MMSE PT and MMSE 24. Upon comparing MMSE and CDR scores, the latter identified a further 146 putative dementia cases, thus permitting one to propose that in an ideal situation, both tests should be employed. This increases the likelihood of identifying putative dementia cases for subsequent follow up work, thus these cognitive tests represent important tools in patient care. Further, this is a significant study for Portuguese populations, where few of these studies have been carried out.

Novel ancestral Dysferlin splicing mutation which migrated from the Iberian peninsula to South America.

Science.gov (United States)

Vernengo, Luis; Oliveira, Jorge; Krahn, Martin; Vieira, Emilia; Santos, Rosário; Carrasco, Luisa; Negrão, Luís; Panuncio, Ana; Leturcq, France; Labelle, Veronique; Bronze-da-Rocha, Elsa; Mesa, Rosario; Pizzarossa, Carlos; Lévy, Nicolas; Rodriguez, Maria-Mirta

2011-05-01

Primary dysferlinopathies are a group of recessive heterogeneous muscular dystrophies. The most common clinical presentations are Miyoshi myopathy and LGMD2B. Additional presentations range from isolated hyperCKemia to severe functional disability. Symptomatology begins in the posterior muscle compartment of the calf and its clinical course progresses slowly in Miyoshi myopathy whereas LGMD2B involves predominantly the proximal muscles of the lower limbs. The age of onset ranges from 13 to 60years in Caucasians. We present five patients that carry a novel mutation in the exon12/intron12 boundary: c.1180_1180+7delAGTGCGTG (r.1054_1284del). We provide evidence of a founder effect due to a common ancestral origin of this mutation, detected in heterozygosity in four patients and in homozygosity in one patient. Copyright © 2011 Elsevier B.V. All rights reserved.

Effects of ancestral x irradiation followed by random mating on body weight of rats

International Nuclear Information System (INIS)

Gianola, D.; Chapman, A.B.; Rutledge, J.J.

1977-01-01

Effects of nine generations of 450R per generation of ancestral spermatogonial x irradiation of inbred rats on body weight were examined. After six generations of random mating (avoiding inbreeding) following the termination of irradiation, descendants of irradiated males (R) were significantly lighter than their controls (C) at 3 and 6 weeks, but not at 10 weeks of age. However, differences in growth between R and C populations were small. Among-litter and within-litter variance estimates were generally larger in the R lines than in the C lines, suggesting that selection responses would be greater in R than in C lines. In conjunction with previous evidence--obtained during the irradiation phase of the experiment--this suggested that more rapid response to selection for 6-week body weight, in particular, might accrue in the R lines

Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System

Science.gov (United States)

Dziewit, Lukasz; Radlinska, Monika

2016-01-01

Two novel prophages ФAH14a and ФAH14b of a psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_H14 have been characterized. They were simultaneously induced with mitomycin C and packed into capsids of the same size and protein composition. The genome sequences of ФAH14a and ФAH14b have been determined. ФAH14b, the phage with a smaller genome (16,812 bp) seems to parasitize ФAH14a (55,060 bp) and utilizes its capsids, as only the latter encodes a complete set of structural proteins. Both viruses probably constitute a phage helper-satellite system, analogous to the P2-P4 duo. This study describes the architecture and function of the ФAH14a and ФAH14b genomes. Moreover, a functional analysis of a ФAH14a-encoded lytic enzyme and a DNA methyltransferase was performed. In silico analysis revealed the presence of the homologs of ФAH14a and ФAH14b in other Pseudomonas genomes, which may suggest that helper-satellite systems related to the one described in this work are common in pseudomonads. PMID:27387973

Heterologous expression and characterization of a putative glycoside hydrolase family 43 arabinofuranosidase from Clostridium thermocellum B8

NARCIS (Netherlands)

Camargo, de Brenda R.; Claassens, Nico J.; Quirino, Betania Ferraz; Noronha, Eliane F.; Kengen, Servé W.M.

2018-01-01

An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose

Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/DNA complex following virus entry.

Science.gov (United States)

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L; Sapp, Martin

2012-09-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.

Inferred L/M cone opsin polymorphism of ancestral tarsiers sheds dim light on the origin of anthropoid primates.

Science.gov (United States)

Melin, Amanda D; Matsushita, Yuka; Moritz, Gillian L; Dominy, Nathaniel J; Kawamura, Shoji

2013-05-22

Tarsiers are small nocturnal primates with a long history of fuelling debate on the origin and evolution of anthropoid primates. Recently, the discovery of M and L opsin genes in two sister species, Tarsius bancanus (Bornean tarsier) and Tarsius syrichta (Philippine tarsier), respectively, was interpreted as evidence of an ancestral long-to-middle (L/M) opsin polymorphism, which, in turn, suggested a diurnal or cathemeral (arrhythmic) activity pattern. This view is compatible with the hypothesis that stem tarsiers were diurnal; however, a reversion to nocturnality during the Middle Eocene, as evidenced by hyper-enlarged orbits, predates the divergence of T. bancanus and T. syrichta in the Late Miocene. Taken together, these findings suggest that some nocturnal tarsiers possessed high-acuity trichromatic vision, a concept that challenges prevailing views on the adaptive origins of the anthropoid visual system. It is, therefore, important to explore the plausibility and antiquity of trichromatic vision in the genus Tarsius. Here, we show that Sulawesi tarsiers (Tarsius tarsier), a phylogenetic out-group of Philippine and Bornean tarsiers, have an L opsin gene that is more similar to the L opsin gene of T. syrichta than to the M opsin gene of T. bancanus in non-synonymous nucleotide sequence. This result suggests that an L/M opsin polymorphism is the ancestral character state of crown tarsiers and raises the possibility that many hallmarks of the anthropoid visual system evolved under dim (mesopic) light conditions. This interpretation challenges the persistent nocturnal-diurnal dichotomy that has long informed debate on the origin of anthropoid primates.

Multiple chromosomal rearrangements structured the ancestral vertebrate Hox-bearing protochromosomes.

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Vincent J Lynch

2009-01-01

Full Text Available While the proposal that large-scale genome expansions occurred early in vertebrate evolution is widely accepted, the exact mechanisms of the expansion--such as a single or multiple rounds of whole genome duplication, bloc chromosome duplications, large-scale individual gene duplications, or some combination of these--is unclear. Gene families with a single invertebrate member but four vertebrate members, such as the Hox clusters, provided early support for Ohno's hypothesis that two rounds of genome duplication (the 2R-model occurred in the stem lineage of extant vertebrates. However, despite extensive study, the duplication history of the Hox clusters has remained unclear, calling into question its usefulness in resolving the role of large-scale gene or genome duplications in early vertebrates. Here, we present a phylogenetic analysis of the vertebrate Hox clusters and several linked genes (the Hox "paralogon" and show that different phylogenies are obtained for Dlx and Col genes than for Hox and ErbB genes. We show that these results are robust to errors in phylogenetic inference and suggest that these competing phylogenies can be resolved if two chromosomal crossover events occurred in the ancestral vertebrate. These results resolve conflicting data on the order of Hox gene duplications and the role of genome duplication in vertebrate evolution and suggest that a period of genome reorganization occurred after genome duplications in early vertebrates.

PRÁCTICAS SOSTENIBLES BASADAS EN EL CONOCIMIENTO ANCESTRAL Y LA TECNOLOGÍA. APLICACIÓN EN EL CULTIVO DE MAÍZ

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Tony JinsopYangüés Pappa

2012-11-01

Full Text Available Este trabajo fue desarrollado en la microcuenca Membrillo del cantón Bolívar, provincia de Manabí, su objetivo fue incrementar la productividad del cultivo de maíz aplicando prácticas sostenibles basadas en el conocimiento ancestral y la tecnología. Para lograr el objetivo propuesto se realizó un Diagnóstico Rural Participativo (DRP y se aplicaron las Metodologías de Escuelas de Aprendizaje (ERAs. Entre los principales resultados obtenidos se destacan el incremento de la productividad promedio del cultivo del maíz a 120 qq/ha con prácticas alternativas sostenibles basadas en el conocimiento ancestral y la tecnología en las comunidades donde se aplicaron las experiencias, alcanzando estas mayores ingresos que satisfacen las necesidades de los productores y consecuentemente se logró reducir la presión hacia los bosques por la producción intensiva alcanzada. Se capacitaron a los agricultores involucrados en técnicas de elaboración de bioinsumos que se utilizaron para la producción de maíz, alternativas para el manejo técnico sostenible y amigable con el ambiente en este cultivo. También se demostró que las prácticas implementadas en el desarrollo de este trabajo contribuyen al desarrollo sostenible de la Microcuenca Membrillo.

Synthesis of dispersive iron or iron–silver nanoparticles on engineered capsid pVIII of M13 virus with electronegative terminal peptides

Energy Technology Data Exchange (ETDEWEB)

Zhang, Shuai; Nakano, Kazuhiko; Zhang, Shu-liang; Yu, Hui-min, E-mail: yuhm@tsinghua.edu.cn [Tsinghua University, Key Laboratory of Industrial Biocatalysis of the Ministry of Education, Department of Chemical Engineering (China)

2015-10-15

M13 is a filamentous Escherichia coli virus covered with five types of capsid proteins, in which pVIII with ∼2700 copies was around the cylindered surface and pIII with five copies located at one end of the phage particle. The pIII-engineered M13 phages with enhanced binding specificity toward Fe were screened after five rounds of biopanning, and the one containing ATPTVAMSLSPL peptide at pIII-terminus was selected for mediated synthesis of zero valent (ZV) Fe nanoparticles (NPs) with the wild M13 as control. Under a reducing environment, uniformly dispersed ZVFeNPs with diameter of 5–10 nm were both synthesized and the morphologies after annealing were confirmed to be face-centered cubic type. The synthesized FeNPs mediated by the two phages showed no significant difference, revealing that the pVIII capsid did dominant contribution to metal binding in comparison with the pIII. A novel pVIII-engineered M13 containing AAEEEDPAK at terminus, named as 4ED-pVIII-M13, was constructed and it carried one more negatively charged residue than the wild one (AEGDDPAK). Metal adsorption quantification showed that the binding affinity of the 4ED-pVIII-M13 toward Ag and Ni ions improved to 62 and 18 % from original 21 and 6 %, respectively. The binding affinity toward Fe remained constant (∼85 %). ZVFe–Ag bi-NPs were successfully synthesized through mediation of 4ED-pVIII-M13. Particularly, the Fe:Ag ratio in the bi-NPs was conveniently controlled through changing the molar concentration of FeCl{sub 2} and AgNO{sub 3} solution before reduction.

Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV for downstream use in virus characterization

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Molly E. Church

2016-06-01

Full Text Available Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA, and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus.

Supplementary data: Variation in the PTEN-induced putative kinase ...

Indian Academy of Sciences (India)

Variation in the PTEN-induced putative kinase 1 gene associated with the increase risk of type 2 diabetes in northern Chinese. Yanchun Qu, Liang Sun, Ze Yang and Ruifa Han. J. Genet. 90, 125–128. Table 1. Clinical characteristics of cases and controls. Phenotype. T2DM. Controls. P value. Age (years). 49.5 ± 11.1. 50.4 ± ...

Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

Energy Technology Data Exchange (ETDEWEB)

Sharma, Rohit B. [Department of Microbiology and Molecular Genetics, University of Pittsburgh, PA 15261 (United States); Wang, Qingde [Department of Surgery, University of Pittsburgh, PA 15261 (United States); Khillan, Jaspal S., E-mail: khillan@pitt.edu [Department of Microbiology and Molecular Genetics, University of Pittsburgh, PA 15261 (United States)

2013-07-12

Highlights: •Vitamin A supports self renewal of putative CSCs from mammary tumors. •These cells exhibit impaired retinol metabolism into retinoic acid. •CSCs from mammary tumors differentiate into mammary specific cell lineages. •The cells express mammary stem cell specific CD29 and CD49f markers. •Putative CSCs form highly metastatic tumors in NOD SCID mouse. -- Abstract: Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibit mammary stem cell specific CD29{sup hi}/CD49f{sup hi}/CD24{sup hi} markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.

Alkenenitrile Transmissive Olefination: Synthesis of the Putative Lignan "Morinol I"

Science.gov (United States)

Fleming, Fraser F.; Liu, Wang; Yao, Lihua; Pitta, Bhaskar; Purzycki, Matthew; Ravikumar, P. C.

2012-01-01

Grignard reagents trigger an addition-elimination with α'-hydroxy acrylonitriles to selectively generate Z-alkenenitriles. The modular assembly of Z-alkenenitriles from a Grignard reagent, acrylonitrile, and an aldehyde is ideal for stereoselectively synthesizing alkenes as illustrated in the synthesis of the putative lignan "morinol I." PMID:22545004

Analysis on the reconstruction accuracy of the Fitch method for inferring ancestral states

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Grünewald Stefan

2011-01-01

Full Text Available Abstract Background As one of the most widely used parsimony methods for ancestral reconstruction, the Fitch method minimizes the total number of hypothetical substitutions along all branches of a tree to explain the evolution of a character. Due to the extensive usage of this method, it has become a scientific endeavor in recent years to study the reconstruction accuracies of the Fitch method. However, most studies are restricted to 2-state evolutionary models and a study for higher-state models is needed since DNA sequences take the format of 4-state series and protein sequences even have 20 states. Results In this paper, the ambiguous and unambiguous reconstruction accuracy of the Fitch method are studied for N-state evolutionary models. Given an arbitrary phylogenetic tree, a recurrence system is first presented to calculate iteratively the two accuracies. As complete binary tree and comb-shaped tree are the two extremal evolutionary tree topologies according to balance, we focus on the reconstruction accuracies on these two topologies and analyze their asymptotic properties. Then, 1000 Yule trees with 1024 leaves are generated and analyzed to simulate real evolutionary scenarios. It is known that more taxa not necessarily increase the reconstruction accuracies under 2-state models. The result under N-state models is also tested. Conclusions In a large tree with many leaves, the reconstruction accuracies of using all taxa are sometimes less than those of using a leaf subset under N-state models. For complete binary trees, there always exists an equilibrium interval [a, b] of conservation probability, in which the limiting ambiguous reconstruction accuracy equals to the probability of randomly picking a state. The value b decreases with the increase of the number of states, and it seems to converge. When the conservation probability is greater than b, the reconstruction accuracies of the Fitch method increase rapidly. The reconstruction

Adenoviruses using the cancer marker EphA2 as a receptor in vitro and in vivo by genetic ligand insertion into different capsid scaffolds.

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Michael Behr

Full Text Available Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK. This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.

Adenoviruses Using the Cancer Marker EphA2 as a Receptor In Vitro and In Vivo by Genetic Ligand Insertion into Different Capsid Scaffolds

Science.gov (United States)

Behr, Michael; Kaufmann, Johanna K.; Ketzer, Patrick; Engelhardt, Sarah; Mück-Häusl, Martin; Okun, Pamela M.; Petersen, Gabriele; Neipel, Frank; Hassel, Jessica C.; Ehrhardt, Anja; Enk, Alexander H.; Nettelbeck, Dirk M.

2014-01-01

Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis. PMID:24760010

Evolution of sweet taste perception in hummingbirds by transformation of the ancestral umami receptor

Science.gov (United States)

Baldwin, Maude W.; Toda, Yasuka; Nakagita, Tomoya; O'Connell, Mary J.; Klasing, Kirk C.; Misaka, Takumi; Edwards, Scott V.; Liberles, Stephen D.

2015-01-01

Sensory systems define an animal's capacity for perception and can evolve to promote survival in new environmental niches. We have uncovered a noncanonical mechanism for sweet taste perception that evolved in hummingbirds since their divergence from insectivorous swifts, their closest relatives. We observed the widespread absence in birds of an essential subunit (T1R2) of the only known vertebrate sweet receptor, raising questions about how specialized nectar feeders such as hummingbirds sense sugars. Receptor expression studies revealed that the ancestral umami receptor (the T1R1-T1R3 heterodimer) was repurposed in hummingbirds to function as a carbohydrate receptor. Furthermore, the molecular recognition properties of T1R1-T1R3 guided taste behavior in captive and wild hummingbirds. We propose that changing taste receptor function enabled hummingbirds to perceive and use nectar, facilitating the massive radiation of hummingbird species. PMID:25146290

Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle.

Science.gov (United States)

Hartman, Emily C; Jakobson, Christopher M; Favor, Andrew H; Lobba, Marco J; Álvarez-Benedicto, Ester; Francis, Matthew B; Tullman-Ercek, Danielle

2018-04-11

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

Energy Technology Data Exchange (ETDEWEB)

Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino (Rutgers); (LBNL); (Connecticut); (TJU); (MSU)

2017-01-30

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

Science.gov (United States)

Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino

2017-01-01

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging' is a DNA-dependent symmetrization of portal protein. PMID:28134243

Identification of a putative nuclear export signal motif in human NANOG homeobox domain

International Nuclear Information System (INIS)

Park, Sung-Won; Do, Hyun-Jin; Huh, Sun-Hyung; Sung, Boreum; Uhm, Sang-Jun; Song, Hyuk; Kim, Nam-Hyung; Kim, Jae-Hwan

2012-01-01

Highlights: ► We found the putative nuclear export signal motif within human NANOG homeodomain. ► Leucine-rich residues are important for human NANOG homeodomain nuclear export. ► CRM1-specific inhibitor LMB blocked the potent human NANOG NES-mediated nuclear export. -- Abstract: NANOG is a homeobox-containing transcription factor that plays an important role in pluripotent stem cells and tumorigenic cells. To understand how nuclear localization of human NANOG is regulated, the NANOG sequence was examined and a leucine-rich nuclear export signal (NES) motif ( 125 MQELSNILNL 134 ) was found in the homeodomain (HD). To functionally validate the putative NES motif, deletion and site-directed mutants were fused to an EGFP expression vector and transfected into COS-7 cells, and the localization of the proteins was examined. While hNANOG HD exclusively localized to the nucleus, a mutant with both NLSs deleted and only the putative NES motif contained (hNANOG HD-ΔNLSs) was predominantly cytoplasmic, as observed by nucleo/cytoplasmic fractionation and Western blot analysis as well as confocal microscopy. Furthermore, site-directed mutagenesis of the putative NES motif in a partial hNANOG HD only containing either one of the two NLS motifs led to localization in the nucleus, suggesting that the NES motif may play a functional role in nuclear export. Furthermore, CRM1-specific nuclear export inhibitor LMB blocked the hNANOG potent NES-mediated export, suggesting that the leucine-rich motif may function in CRM1-mediated nuclear export of hNANOG. Collectively, a NES motif is present in the hNANOG HD and may be functionally involved in CRM1-mediated nuclear export pathway.

Centromere Destiny in Dicentric Chromosomes: New Insights from the Evolution of Human Chromosome 2 Ancestral Centromeric Region.

Science.gov (United States)

Chiatante, Giorgia; Giannuzzi, Giuliana; Calabrese, Francesco Maria; Eichler, Evan E; Ventura, Mario

2017-07-01

Dicentric chromosomes are products of genomic rearrangements that place two centromeres on the same chromosome. Due to the presence of two primary constrictions, they are inherently unstable and overcome their instability by epigenetically inactivating and/or deleting one of the two centromeres, thus resulting in functionally monocentric chromosomes that segregate normally during cell division. Our understanding to date of dicentric chromosome formation, behavior and fate has been largely inferred from observational studies in plants and humans as well as artificially produced de novo dicentrics in yeast and in human cells. We investigate the most recent product of a chromosome fusion event fixed in the human lineage, human chromosome 2, whose stability was acquired by the suppression of one centromere, resulting in a unique difference in chromosome number between humans (46 chromosomes) and our most closely related ape relatives (48 chromosomes). Using molecular cytogenetics, sequencing, and comparative sequence data, we deeply characterize the relicts of the chromosome 2q ancestral centromere and its flanking regions, gaining insight into the ancestral organization that can be easily broadened to all acrocentric chromosome centromeres. Moreover, our analyses offered the opportunity to trace the evolutionary history of rDNA and satellite III sequences among great apes, thus suggesting a new hypothesis for the preferential inactivation of some human centromeres, including IIq. Our results suggest two possible centromere inactivation models to explain the evolutionarily stabilization of human chromosome 2 over the last 5-6 million years. Our results strongly favor centromere excision through a one-step process. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Search strings for the study of putative occupational determinants of disease

NARCIS (Netherlands)

Mattioli, Stefano; Zanardi, Francesca; Baldasseroni, Alberto; Schaafsma, Frederieke; Cooke, Robin M. T.; Mancini, Gianpiero; Fierro, Mauro; Santangelo, Chiara; Farioli, Andrea; Fucksia, Serenella; Curti, Stefania; Violante, Francesco S.; Verbeek, Jos

2010-01-01

To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms describing

E Pluribus Unum: 50 Years of Research, Millions of Viruses, and One Goal--Tailored Acceleration of AAV Evolution.

Science.gov (United States)

Grimm, Dirk; Zolotukhin, Sergei

2015-12-01

Fifty years ago, a Science paper by Atchison et al. reported a newly discovered virus that would soon become known as adeno-associated virus (AAV) and that would subsequently emerge as one of the most versatile and most auspicious vectors for human gene therapy. A large part of its attraction stems from the ease with which the viral capsid can be engineered for particle retargeting to cell types of choice, evasion from neutralizing antibodies or other desirable properties. Particularly powerful and in the focus of the current review are high-throughput methods aimed at expanding the repertoire of AAV vectors by means of directed molecular evolution, such as random mutagenesis, DNA family shuffling, in silico reconstruction of ancestral capsids, or peptide display. Here, unlike the wealth of prior reviews on this topic, we especially emphasize and critically discuss the practical aspects of the different procedures that affect the ultimate outcome, including diversification protocols, combinatorial library complexity, and selection strategies. Our overall aim is to provide general guidance that should help users at any level, from novice to expert, to safely navigate through the rugged space of directed AAV evolution while avoiding the pitfalls that are associated with these challenging but promising technologies.

Mechanical and assembly units of viral capsids identified via quasi-rigid domain decomposition.

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Guido Polles

Full Text Available Key steps in a viral life-cycle, such as self-assembly of a protective protein container or in some cases also subsequent maturation events, are governed by the interplay of physico-chemical mechanisms involving various spatial and temporal scales. These salient aspects of a viral life cycle are hence well described and rationalised from a mesoscopic perspective. Accordingly, various experimental and computational efforts have been directed towards identifying the fundamental building blocks that are instrumental for the mechanical response, or constitute the assembly units, of a few specific viral shells. Motivated by these earlier studies we introduce and apply a general and efficient computational scheme for identifying the stable domains of a given viral capsid. The method is based on elastic network models and quasi-rigid domain decomposition. It is first applied to a heterogeneous set of well-characterized viruses (CCMV, MS2, STNV, STMV for which the known mechanical or assembly domains are correctly identified. The validated method is next applied to other viral particles such as L-A, Pariacoto and polyoma viruses, whose fundamental functional domains are still unknown or debated and for which we formulate verifiable predictions. The numerical code implementing the domain decomposition strategy is made freely available.

Putative Biomarkers and Targets of Estrogen Receptor Negative Human Breast Cancer

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Stephen W. Byers

2011-07-01

Full Text Available Breast cancer is a progressive and potentially fatal disease that affects women of all ages. Like all progressive diseases, early and reliable diagnosis is the key for successful treatment and annihilation. Biomarkers serve as indicators of pathological, physiological, or pharmacological processes. Her2/neu, CA15.3, estrogen receptor (ER, progesterone receptor (PR, and cytokeratins are biomarkers that have been approved by the Food and Drug Administration for disease diagnosis, prognosis, and therapy selection. The structural and functional complexity of protein biomarkers and the heterogeneity of the breast cancer pathology present challenges to the scientific community. Here we review estrogen receptor-related putative breast cancer biomarkers, including those of putative breast cancer stem cells, a minor population of estrogen receptor negative tumor cells that retain the stem cell property of self renewal. We also review a few promising cytoskeleton targets for ER alpha negative breast cancer.

Plum pox virus capsid protein suppresses plant pathogen-associated molecular pattern (PAMP)-triggered immunity.

Science.gov (United States)

Nicaise, Valerie; Candresse, Thierry

2017-08-01

The perception of pathogen-associated molecular patterns (PAMPs) by immune receptors launches defence mechanisms referred to as PAMP-triggered immunity (PTI). Successful pathogens must suppress PTI pathways via the action of effectors to efficiently colonize their hosts. So far, plant PTI has been reported to be active against most classes of pathogens, except viruses, although this defence layer has been hypothesized recently as an active part of antiviral immunity which needs to be suppressed by viruses for infection success. Here, we report that Arabidopsis PTI genes are regulated upon infection by viruses and contribute to plant resistance to Plum pox virus (PPV). Our experiments further show that PPV suppresses two early PTI responses, the oxidative burst and marker gene expression, during Arabidopsis infection. In planta expression of PPV capsid protein (CP) was found to strongly impair these responses in Nicotiana benthamiana and Arabidopsis, revealing its PTI suppressor activity. In summary, we provide the first clear evidence that plant viruses acquired the ability to suppress PTI mechanisms via the action of effectors, highlighting a novel strategy employed by viruses to escape plant defences. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Sequence analysis of putative swrW gene required for surfactant ...

African Journals Online (AJOL)

owner

2012-07-17

Jul 17, 2012 ... These nucleotide and protein sequence analysis of the putative swrW gene provides vital information on the versatility .... chain reaction (PCR) products were stored at 4°C. Presence of ... identical to the same gene with an E-value of 0.0. .... The Prokaryotes-A Handbook on the Biol. of Bacteria:Ecophysiol.