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Sample records for picornavirus rna replication

  1. MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

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    Qian Feng

    2012-11-01

    Full Text Available RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C, a synthetic dsRNA mimic. Here, we dissected the IFN-α/β-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/β response. IFN-α/β production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions.

  2. Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

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    Dylan eFlather

    2015-06-01

    Full Text Available The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review.

  3. Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus.

    Science.gov (United States)

    Flather, Dylan; Semler, Bert L

    2015-01-01

    The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review.

  4. PDTC inhibits picornavirus polyprotein processing and RNA replication by transporting zinc ions into cells.

    NARCIS (Netherlands)

    Lanke, K.H.W.; Krenn, B.M.; Melchers, W.J.G.; Seipelt, J.; Kuppeveld, F.J.M. van

    2007-01-01

    Previously, it was shown that pyrrolidine dithiocarbamate (PDTC) inhibits proteolytic polyprotein processing and replication of human rhinovirus by transporting metal ions into cells. Here, it is shown that PDTC also inhibits replication of two other picornaviruses: coxsackievirus B3 (CVB3), a close

  5. Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus.

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    Cristina M Dorobantu

    2015-09-01

    Full Text Available Cardioviruses, including encephalomyocarditis virus (EMCV and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+RNA] viruses. All (+RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs. For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB, while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA, to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P proved important for the recruitment of oxysterol-binding protein (OSBP, which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+RNA viruses of a host

  6. Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus.

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    Dorobantu, Cristina M; Albulescu, Lucian; Harak, Christian; Feng, Qian; van Kampen, Mirjam; Strating, Jeroen R P M; Gorbalenya, Alexander E; Lohmann, Volker; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2015-09-01

    Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid

  7. Fat(al) attraction : Picornaviruses Usurp Lipid Transfer at Membrane Contact Sites to Create Replication Organelles

    NARCIS (Netherlands)

    van der Schaar, Hilde M; Dorobantu, Cristina M; Albulescu, Lucian; Strating, Jeroen R P M; van Kuppeveld, Frank J M

    All viruses that carry a positive-sense RNA genome (+RNA), such as picornaviruses, hepatitis C virus, dengue virus, and SARS- and MERS-coronavirus, confiscate intracellular membranes of the host cell to generate new compartments (i.e., replication organelles) for amplification of their genome.

  8. Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA

    NARCIS (Netherlands)

    Langereis, Martijn A; Feng, Qian; Nelissen, Frank H T; Virgen-Slane, Richard; van der Heden van Noort, Gerbrand J; Maciejewski, Sonia; Filippov, Dmitri V; Semler, Bert L; van Delft, Floris L; van Kuppeveld, Frank J M

    2014-01-01

    Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for vir

  9. Modulation of the Host Lipid Landscape to Promote RNA Virus Replication : The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus

    NARCIS (Netherlands)

    Dorobantu, Cristina M; Albulescu, Lucian; Harak, Christian; Feng, Qian; van Kampen, Mirjam; Strating, Jeroen R P M; Gorbalenya, Alexander E; Lohmann, Volker; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2015-01-01

    Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome

  10. Diverse Strategies Used by Picornaviruses to Escape Host RNA Decay Pathways

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    Wendy Ullmer

    2016-12-01

    Full Text Available To successfully replicate, viruses protect their genomic material from degradation by the host cell. RNA viruses must contend with numerous destabilizing host cell processes including mRNA decay pathways and viral RNA (vRNA degradation resulting from the antiviral response. Members of the Picornaviridae family of small RNA viruses have evolved numerous diverse strategies to evade RNA decay, including incorporation of stabilizing elements into vRNA and re-purposing host stability factors. Viral proteins are deployed to disrupt and inhibit components of the decay machinery and to redirect decay machinery to the advantage of the virus. This review summarizes documented interactions of picornaviruses with cellular RNA decay pathways and processes.

  11. Unusual loop-sequence flexibility of the proximal RNA replication element in EMCV

    NARCIS (Netherlands)

    Zoll, J.; Hahn, M.M.; Gielen, P.R.; Heus, H.A.; Melchers, W.J.G.; Kuppeveld, F.J.M. van

    2011-01-01

    Picornaviruses contain stable RNA structures at the 5' and 3' ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5' end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of

  12. Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication

    DEFF Research Database (Denmark)

    Nayak, A.; Goodfellow, I. G.; Woolaway, K. E.;

    2006-01-01

    The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3D(pol)), the precursor 3CD, and an RNA template containing the cre/bus...

  13. Divergent picornavirus IRES elements

    DEFF Research Database (Denmark)

    Belsham, Graham

    2009-01-01

    Internal ribosome entry site (IRES) elements were first identified about 20 years ago within the 5' untranslated region of picornavirus RNAs. They direct a cap-independent mechanism of translation initiation on the viral RNA. Within the picornavirus family it is now known that there are four...... classes of IRES element which vary in size (450-270nt), they also have different, complex, secondary structures and distinct requirements for cellular proteins to allow them to function. This review describes the features of each class of picornavirus IRES element but focuses on the characteristics...... of the most recently described group, initially identified within the porcine teschovirus-1 RNA, which has strong similarities to the IRES elements from within the genomes of hepatitis C virus and the pestiviruses which are members of the flavivirus family. The selection of the initiation codon...

  14. Alphavirus polymerase and RNA replication.

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    Pietilä, Maija K; Hellström, Kirsi; Ahola, Tero

    2017-01-16

    Alphaviruses are typically arthropod-borne, and many are important pathogens such as chikungunya virus. Alphaviruses encode four nonstructural proteins (nsP1-4), initially produced as a polyprotein P1234. nsP4 is the core RNA-dependent RNA polymerase but all four nsPs are required for RNA synthesis. The early replication complex (RC) formed by the polyprotein P123 and nsP4 synthesizes minus RNA strands, and the late RC composed of fully processed nsP1-nsP4 is responsible for the production of genomic and subgenomic plus strands. Different parts of nsP4 recognize the promoters for minus and plus strands but the binding also requires the other nsPs. The alphavirus polymerase has been purified and is capable of de novo RNA synthesis only in the presence of the other nsPs. The purified nsP4 also has terminal adenylyltransferase activity, which may generate the poly(A) tail at the 3' end of the genome. Membrane association of the nsPs is vital for replication, and alphaviruses induce membrane invaginations called spherules, which form a microenvironment for RNA synthesis by concentrating replication components and protecting double-stranded RNA intermediates. The RCs isolated as crude membrane preparations are active in RNA synthesis in vitro, but high-resolution structure of the RC has not been achieved, and thus the arrangement of viral and possible host components remains unknown. For some alphaviruses, Ras-GTPase-activating protein (Src-homology 3 (SH3) domain)-binding proteins (G3BPs) and amphiphysins have been shown to be essential for RNA replication and are present in the RCs. Host factors offer an additional target for antivirals, as only few alphavirus polymerase inhibitors have been described.

  15. Unusual loop-sequence flexibility of the proximal RNA replication element in EMCV.

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    Jan Zoll

    Full Text Available Picornaviruses contain stable RNA structures at the 5' and 3' ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5' end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of the poliovirus cloverleaf depends on a specific structure in this RNA element. Little is known about the OriL of cardioviruses. Here, we investigated structural aspects and requirements of the apical loop of proximal stem-loop SL-A of mengovirus, a strain of EMCV. Using NMR spectroscopy, we showed that the mengovirus SL-A apical loop consists of an octaloop. In vivo SELEX experiments demonstrated that a large number of random sequences are tolerated in the apical octaloop that support virus replication. Mutants in which the SL-A loop size and the length of the upper part of the stem were varied showed that both stem-length and stability of the octaloop are important determinants for viral RNA replication and virus reproduction. Together, these data show that stem-loop A plays an important role in virus replication. The high degree of sequence flexibility and the lack of selective pressure on the octaloop argue against a role in sequence specific RNA-protein or RNA-RNA interactions in which octaloop nucleotides are involved.

  16. An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

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    Lötzerich Mark

    2010-10-01

    Full Text Available Abstract Background Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2 detecting double-strand RNA. Results Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs blocking enterovirus infections. Conclusions We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens

  17. An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

    Science.gov (United States)

    2010-01-01

    Background Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV) are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2) detecting double-strand RNA. Results Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV) B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs) blocking enterovirus infections. Conclusions We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens for different serotypes

  18. Viral and host proteins involved in picornavirus life cycle

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    Weng Kuo-Feng

    2009-11-01

    Full Text Available Abstract Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions.

  19. Self-replicating alphavirus RNA vaccines.

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    Ljungberg, Karl; Liljeström, Peter

    2015-02-01

    Recombinant nucleic acids are considered as promising next-generation vaccines. These vaccines express the native antigen upon delivery into tissue, thus mimicking live attenuated vaccines without having the risk of reversion to pathogenicity. They also stimulate the innate immune system, thus potentiating responses. Nucleic acid vaccines are easy to produce at reasonable cost and are stable. During the past years, focus has been on the use of plasmid DNA for vaccination. Now mRNA and replicon vaccines have come into focus as promising technology platforms for vaccine development. This review discusses self-replicating RNA vaccines developed from alphavirus expression vectors. These replicon vaccines can be delivered as RNA, DNA or as recombinant virus particles. All three platforms have been pre-clinically evaluated as vaccines against a number of infectious diseases and cancer. Results have been very encouraging and propelled the first human clinical trials, the results of which have been promising.

  20. The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

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    McKnight, K L; Lemon, S M

    1998-12-01

    Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of

  1. Picornavirus uncoating intermediate captured in atomic detail

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    Ren, Jingshan; Wang, Xiangxi; Hu, Zhongyu; Gao, Qiang; Sun, Yao; Li, Xuemei; Porta, Claudine; Walter, Thomas S.; Gilbert, Robert J.; Zhao, Yuguang; Axford, Danny; Williams, Mark; McAuley, Katherine; Rowlands, David J.; Yin, Weidong; Wang, Junzhi; Stuart, David I.; Rao, Zihe; Fry, Elizabeth E.

    2013-01-01

    It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release. PMID:23728514

  2. Phagocytosis of Picornavirus-Infected Cells Induces an RNA-Dependent Antiviral State in Human Dendritic Cells▿

    Science.gov (United States)

    Kramer, Matthijs; Schulte, Barbara M.; Toonen, Liza W. J.; Barral, Paola M.; Fisher, Paul B.; Lanke, Kjerstin H. W.; Galama, Jochem M. D.; van Kuppeveld, Frank J. M.; Adema, Gosse J.

    2008-01-01

    Dendritic cells (DCs) play a central role in instructing antiviral immune responses. DCs, however, can become targeted by different viruses themselves. We recently demonstrated that human DCs can be productively infected with echoviruses (EVs), but not coxsackie B viruses (CVBs), both of which are RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. We now show that phagocytosis of CVB-infected, type I interferon-deficient cells induces an antiviral state in human DCs. Uptake of infected cells increased the expression of the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5 as well as other interferon-stimulated genes and protected DCs against subsequent infection with EV9. These effects depended on recognition of viral RNA and could be mimicked by exposure to the synthetic double-stranded RNA analogue poly(I:C) but not other Toll-like receptor (TLR) ligands. Blocking endosomal acidification abrogated protection, suggesting a role for TLRs in the acquisition of an antiviral state in DCs. In conclusion, recognition of viral RNA rapidly induces an antiviral state in human DCs. This might provide a mechanism by which DCs protect themselves against viruses when attracted to an environment with ongoing infection. PMID:18184700

  3. Mutual Interference between Genomic RNA Replication and Subgenomic mRNA Transcription in Brome Mosaic Virus

    OpenAIRE

    Grdzelishvili, Valery Z.; Garcia-Ruiz, Hernan; Watanabe, Tokiko; Ahlquist, Paul

    2005-01-01

    Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expres...

  4. Flaviviral Replication Complex: Coordination between RNA Synthesis and 5'-RNA Capping.

    Science.gov (United States)

    Klema, Valerie J; Padmanabhan, Radhakrishnan; Choi, Kyung H

    2015-08-13

    Genome replication in flavivirus requires (-) strand RNA synthesis, (+) strand RNA synthesis, and 51-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (-) RNA synthesis, (+) RNA synthesis, and 51-RNA capping and methylation.

  5. A diarrheic chicken simultaneously co-infected with multiple picornaviruses: Complete genome analysis of avian picornaviruses representing up to six genera.

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    Boros, Ákos; Pankovics, Péter; Adonyi, Ádám; Fenyvesi, Hajnalka; Day, J Michael; Phan, Tung Gia; Delwart, Eric; Reuter, Gábor

    2016-02-01

    In this study all currently known chicken picornaviruses including a novel one (chicken phacovirus 1, KT880670) were identified by viral metagenomic and RT-PCR methods from a single specimen of a diarrheic chicken suffering from a total of eight picornavirus co-infections, in Hungary. The complete genomes of six picornaviruses were determined and their genomic and phylogenetic characteristics and UTR RNA structural models analyzed in details. Picornaviruses belonged to genera Sicinivirus (the first complete genome), Gallivirus, Tremovirus, Avisivirus and "Orivirus" (two potential genotypes). In addition, the unassigned phacoviruses were also detected in multiple samples of chickens in the USA. Multiple co-infections promote and facilitate the recombination and evolution of picornaviruses and eventually could contribute to the severity of the diarrhea in chicken, in one of the most important food sources of humans.

  6. DBR1 siRNA inhibition of HIV-1 replication

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    Naidu Yathi

    2005-10-01

    Full Text Available Abstract Background HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1 expression would specifically inhibit HIV-1 replication. Results We designed three short interfering RNA (siRNA molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. Conclusion These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

  7. Nidovirus replication structures : hijacking membranes to support viral RNA synthesis

    NARCIS (Netherlands)

    Knoops, Kèvin

    2011-01-01

    Positive-stranded RNA viruses replicate in the cytoplasm of host cells and their replication complexes are associated with modified cell membranes. We investigated the structure of the nidovirus-induced membrane modifications and found that nidoviruses transform the endoplasmic reticulum into a reti

  8. Intracellular Detection of Viral Transcription and Replication Using RNA FISH

    Science.gov (United States)

    2016-05-26

    Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses...examine the mechanisms in which viruses replicate, assemble, and traffic through the cell. An additional benefit of this method is that the robust...Visualization of single RNA transcripts in situ. Science, 1998. 280(5363): p. 585-90. 4. Jambo, K.C., et al., Small alveolar macrophages are infected

  9. Effects of DNA replication on mRNA noise.

    Science.gov (United States)

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories.

  10. RNA interference-mediated inhibition of Hepatitis B Virus replication

    Institute of Scientific and Technical Information of China (English)

    TANG Ni; ZHANG Bingqiang; YAN Ge; PU Dan; GAO Xiaolin; Tong-Chuan He; HUANG Ailong

    2004-01-01

    Persistent and recurrent infection of hepatitis B virus (HBV) represents one of the most common and severe viral infections of humans, and has caused a formidable health problem in the affected countries. Currently used antiviral drugs have a very limited success on controlling HBV replication and infection. RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of target mRNA in mammalian and plant cells, has recently been used to knockdown gene expression in various species. In this study, we sought to determine whether RNAi-mediated silencing of HBV viral gene expression could lead to the effective inhibition of HBV replication. We first developed RNAi vectors that expressed small interfering RNA (siRNA) and targeted the HBV core or surface gene sequence. Our results demonstrated that these specific siRNAs efficiently reduced the levels of corresponding viral RNAs and proteins, and thus suppressed viral replication. Treatment with siRNA gave the greatest reduction in the levels of HBsAg (92%) and in HBeAg (85%) respectively in the cultured cell medium. Our findings further demonstrated that the RNAi-mediated antiviral effect was sequence-specific and dose-dependent. Therefore, our findings strongly suggest that RNAi-mediated silencing of HBV viral genes could effectively inhibit the replication of HBV, hence RNAi-based strategy should be further explored as a more efficacious antiviral therapy of HBV infection.

  11. siRNA-mediated inhibition of HBV replication and expression

    Institute of Scientific and Technical Information of China (English)

    Xiao-Nan Zhang; Wei Xiong; Jia-Dong Wang; Yun-Wen Hu; Li Xiang; Zheng-Hong Yuan

    2004-01-01

    AIM: RNA interference (RNAi) is a newly discovered phenomenon provoked by dsRNA. The dsRNA is initially cleaved by Dicer into 21-23 nt small interfering RNA (siRNA)and can then specifically target homologous mRNA for degradation by cellular ribonucleases. RNAi has been successfully utilized to down-regulate the endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, we investigated whether vector-based siRNA promoted by U6 (pSilencer1.0-U6)could efficiently inhibit HBV replication in cell culture.METHODS: pSilencer vectors with inserts targeting on different regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 cells via lipofection and viral antigens were measured by ELISA.Viral RNA was analyzed by Northern blot. The mRNA of MxA and 2′-5′OAS was reverse transcribed and quantified by real-time PCR.RESULTS: Vector-based siRNA could potently reduce hepatitis B virus antigen expression in transient replicative cell culture. Furthermore, Northern blot analysis showed that viral RNA was effectively degraded, thus eliminating the messengers for protein expression as well as template for reverse transcription. Real-time PCR analysis of cellular MxA and 2′-5′OAS gene expression revealed that vectorbased siRNA did not provoke the interferon pathway which reassured the specificity of the vector-based RNA interference technique.CONCLUSION: Our results indicate that RNA interference may be a potential tool to control HBV infection.

  12. Reinitiated viral RNA-dependent RNA polymerase resumes replication at a reduced rate

    NARCIS (Netherlands)

    Vilfan, I.D.; Candelli, A.; Hage, S.; Aalto, A.P.; Poranen, M.M.; Bamford, D.H.; Dekker, N.H.

    2008-01-01

    RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regul

  13. Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

    Directory of Open Access Journals (Sweden)

    Kiwamu Hyodo

    2015-05-01

    Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

  14. Mutations in Encephalomyocarditis Virus 3A Protein Uncouple the Dependency of Genome Replication on Host Factors Phosphatidylinositol 4-Kinase IIIα and Oxysterol-Binding Protein

    NARCIS (Netherlands)

    Dorobantu, Cristina M; Albulescu, Lucian; Lyoo, Heyrhyoung; van Kampen, Mirjam; De Francesco, Raffaele; Lohmann, Volker; Harak, Christian; van der Schaar, Hilde M; Strating, Jeroen R P M; Gorbalenya, Alexander E; van Kuppeveld, Frank J M

    2016-01-01

    Positive-strand RNA [(+)RNA] viruses are true masters of reprogramming host lipid trafficking and synthesis to support virus genome replication. Via their membrane-associated 3A protein, picornaviruses of the genus Enterovirus (e.g., poliovirus, coxsackievirus, and rhinovirus) subvert Golgi

  15. Internal RNA Replication Elements are Prevalent in Tombusviridae

    Directory of Open Access Journals (Sweden)

    Beth L Nicholson

    2012-08-01

    Full Text Available Internal replication elements (IREs are RNA structures that are present at internal positions in the genomes of different types of plus-strand RNA viruses. Members of the genus Tombusvirus (family Tombusviridae contain an IRE within the polymerase coding region of their genomes and this RNA element participates in both genome targeting to sites of replication and replicase complex assembly. Here we propose that other members of the virus family Tombusviridae also possess comparable IREs. Through sequence and structural analyses, candidate IREs in several genera of this family were identified, including aureusviruses, necroviruses, carmoviruses and pelarspoviruses. The results from subsequent mutational analysis of selected proposed IREs were consistent with a critical role for these structures in viral genome accumulation during infections. Our study supports the existence of IREs in several genera in Tombusviridae and points to previously unappreciated similarities in genome replication strategies between members of this virus family.

  16. Inhibition of Monkeypox virus replication by RNA interference

    Directory of Open Access Journals (Sweden)

    Jahrling Peter B

    2009-11-01

    Full Text Available Abstract The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV, Vaccinia (VACV, monkeypox (MPV and Variola (VARV viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA. Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R or an important gene in viral entry (E8L, inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.

  17. COPI is required for enterovirus 71 replication.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

    Full Text Available Enterovirus 71 (EV71, a member of the Picornaviridae family, is found in Asian countries where it causes a wide range of human diseases. No effective therapy is available for the treatment of these infections. Picornaviruses undergo RNA replication in association with membranes of infected cells. COPI and COPII have been shown to be involved in the formation of picornavirus-induced vesicles. Replication of several picornaviruses, including poliovirus and Echovirus 11 (EV11, is dependent on COPI or COPII. Here, we report that COPI, but not COPII, is required for EV71 replication. Replication of EV71 was inhibited by brefeldin A and golgicide A, inhibitors of COPI activity. Furthermore, we found EV71 2C protein interacted with COPI subunits by co-immunoprecipitation and GST pull-down assay, indicating that COPI coatomer might be directed to the viral replication complex through viral 2C protein. Additionally, because the pathway is conserved among different species of enteroviruses, it may represent a novel target for antiviral therapies.

  18. Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells.

    Science.gov (United States)

    Spuul, Pirjo; Balistreri, Giuseppe; Hellström, Kirsi; Golubtsov, Andrey V; Jokitalo, Eija; Ahola, Tero

    2011-05-01

    For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in trans, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the trans-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the trans-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.

  19. Hydrogen peroxide thermochemical oscillator as driver for primordial RNA replication.

    Science.gov (United States)

    Ball, Rowena; Brindley, John

    2014-06-06

    This paper presents and tests a previously unrecognized mechanism for driving a replicating molecular system on the prebiotic earth. It is proposed that cell-free RNA replication in the primordial soup may have been driven by self-sustained oscillatory thermochemical reactions. To test this hypothesis, a well-characterized hydrogen peroxide oscillator was chosen as the driver and complementary RNA strands with known association and melting kinetics were used as the substrate. An open flow system model for the self-consistent, coupled evolution of the temperature and concentrations in a simple autocatalytic scheme is solved numerically, and it is shown that thermochemical cycling drives replication of the RNA strands. For the (justifiably realistic) values of parameters chosen for the simulated example system, the mean amount of replicant produced at steady state is 6.56 times the input amount, given a constant supply of substrate species. The spontaneous onset of sustained thermochemical oscillations via slowly drifting parameters is demonstrated, and a scheme is given for prebiotic production of complementary RNA strands on rock surfaces.

  20. DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication.

    Science.gov (United States)

    Ariumi, Yasuo; Kuroki, Misao; Abe, Ken-ichi; Dansako, Hiromichi; Ikeda, Masanori; Wakita, Takaji; Kato, Nobuyuki

    2007-12-01

    DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supernatants were suppressed in DDX3 knockdown cells after inoculation of the cell culture-generated HCVcc. Thus, DDX3 is required for HCV RNA replication.

  1. The 3A Protein from Multiple Picornaviruses Utilizes the Golgi Adaptor Protein ACBD3 To Recruit PI4KIIIβ

    OpenAIRE

    Greninger, Alexander L.; Giselle M Knudsen; Betegon, Miguel; Burlingame, Alma L.; Joseph L Derisi

    2012-01-01

    The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIβ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of Strep-Tagged transiently transfected constructs...

  2. Regulation of Human Adenovirus Replication by RNA Interference.

    Science.gov (United States)

    Nikitenko, N A; Speiseder, T; Lam, E; Rubtsov, P M; Tonaeva, Kh D; Borzenok, S A; Dobner, T; Prassolov, V S

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

  3. Replication protein of tobacco mosaic virus cotranslationally binds the 5′ untranslated region of genomic RNA to enable viral replication

    Science.gov (United States)

    Kawamura-Nagaya, Kazue; Ishibashi, Kazuhiro; Huang, Ying-Ping; Miyashita, Shuhei; Ishikawa, Masayuki

    2014-01-01

    Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5′-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5′-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided. PMID:24711385

  4. RNA interference targets arbovirus replication in Culicoides cells.

    Science.gov (United States)

    Schnettler, Esther; Ratinier, Maxime; Watson, Mick; Shaw, Andrew E; McFarlane, Melanie; Varela, Mariana; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

    2013-03-01

    Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks, and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study, we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses, including those with double-stranded RNA (dsRNA) genomes, such as BTV. Using reporter gene-based assays, we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses.

  5. Membranous Replication Factories Induced by Plus-Strand RNA Viruses

    Directory of Open Access Journals (Sweden)

    Inés Romero-Brey

    2014-07-01

    Full Text Available In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+ RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.

  6. BARE retrotransposons are translated and replicated via distinct RNA pools.

    Directory of Open Access Journals (Sweden)

    Wei Chang

    Full Text Available The replication of Long Terminal Repeat (LTR retrotransposons, which can constitute over 80% of higher plant genomes, resembles that of retroviruses. A major question for retrotransposons and retroviruses is how the two conflicting roles of their transcripts, in translation and reverse transcription, are balanced. Here, we show that the BARE retrotransposon, despite its organization into just one open reading frame, produces three distinct classes of transcripts. One is capped, polyadenylated, and translated, but cannot be copied into cDNA. The second is not capped or polyadenylated, but is destined for packaging and ultimate reverse transcription. The third class is capped, polyadenylated, and spliced to favor production of a subgenomic RNA encoding only Gag, the protein forming virus-like particles. Moreover, the BARE2 subfamily, which cannot synthesize Gag and is parasitic on BARE1, does not produce the spliced sub-genomic RNA for translation but does make the replication competent transcripts, which are packaged into BARE1 particles. To our knowledge, this is first demonstration of distinct RNA pools for translation and transcription for any retrotransposon.

  7. Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus.

    Science.gov (United States)

    Hales, Laura M; Knowles, Nick J; Reddy, P Seshidar; Xu, Ling; Hay, Carl; Hallenbeck, Paul L

    2008-05-01

    The complete genome sequence of Seneca Valley virus-001 (SVV-001), a small RNA virus, was determined and was shown to have typical picornavirus features. The 7280 nt long genome was predicted to contain a 5' untranslated region (UTR) of 666 nt, followed by a single long open reading frame consisting of 6543 nt, which encodes a 2181 aa polyprotein. This polyprotein could potentially be cleaved into 12 polypeptides in the standard picornavirus L-4-3-4 layout. A 3' UTR of 71 nt was followed by a poly(A) tail of unknown length. Comparisons with other picornaviruses showed that the P1, 2C, 3C and 3D polypeptides of SVV-001 were related most closely to those of the cardioviruses, although they were not related as closely to those of encephalomyocarditis virus and Theiler's murine encephalomyelitis virus as the latter were to each other. Most other regions of the polyprotein differed considerably from those of all other known picornaviruses. SVV-001 contains elements of an internal ribosome entry site reminiscent of that found in hepatitis C virus and a number of genetically diverse picornaviruses. SVV-001 is a novel picornavirus and it is proposed that it be classified as the prototype species in a novel genus named 'Senecavirus'.

  8. Role for RNA: DNA hybrids in origin-independent replication priming in a eukaryotic system

    OpenAIRE

    Stuckey, Ruth; García Rodriguez, Néstor; Aguilera López, Andrés; Wellinger, Ralf Erik

    2015-01-01

    DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops)...

  9. Structure-infectivity analysis of the human rhinovirus genomic RNA 3' non-coding region.

    OpenAIRE

    1996-01-01

    The specific recognition of genomic positive strand RNAS as templates for the synthesis of intermediate negative strands by the picornavirus replication machinery is presumably mediated by cis-acting sequences within the genomic RNA 3' non-coding region (NCR). A structure-infectivity analysis was conducted on the 44 nt human rhinovirus 14 (HRV14) 3' NCR to identify the primary sequence and/or secondary structure determinants required for viral replication. Using biochemical RNA secondary stru...

  10. Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

    Energy Technology Data Exchange (ETDEWEB)

    Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Hampton-Smith, Rachel J.; Aloia, Amanda L. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Eddes, James S. [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Simpson, Kaylene J. [Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville (Australia); Hoffmann, Peter [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide (Australia); Beard, Michael R. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia)

    2016-04-15

    Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.

  11. Silencing E1A mRNA by RNA interference inhibits adenovirus replication.

    Science.gov (United States)

    Chung, Y-S; Kim, M-K; Lee, W-J; Kang, C

    2007-01-01

    The adenovirus family contains 51 human serotypes, and most human adenoviruses cause widespread respiratory tract infections. Adenovirus infections can result in severe complications in some cases, such as in adenovirus type 11 infection in immunocompromised patients. However, effective treatment methods for adenovirus infections are currently unavailable. This prompted the search for antiviral agents effective against adenovirus infections. In the present study, adenovirus E1A was targeted by RNA interference (RNAi) using synthetic small interfering RNAs (siRNAs) in an attempt to inhibit viral replication, since adenovirus E1A proteins are known to be involved in the transcriptional activation of the viral and cellular genes necessary for controlling the cell cycle and viral replication. The results indicated that the siRNAs effectively reduced the amount of adenovirus E1A mRNA and the levels of replicative intermediates. Additionally, siRNA-mediated gene silencing inhibited adenovirus replication by suppressing the E1A mRNA. These results suggest that the RNAi-mediated targeting of adenovirus E1A may have a potentially therapeutic effect in controlling adenovirus infections.

  12. Picornaviruses and reoviruses of fishes

    Science.gov (United States)

    Winton, J.R.; Ahne, Winfried; Kurstak, E.

    1989-01-01

    The number of fish viruses isolated in cell culture or observed by electron microscopy continues to increase rapidly. Until recently, most viruses that were isolated from finfish and characterized were found to be members of the Rhabdoviridae, Iridoviridae, or Herpesviridae (Wolf and Mann 1980). In a comprehensive review of fish viruses published in 1984, there were no picornaviruses and only two reoviruses listed (Wolf 1984). The expansion of aquaculture into the rearing of new species at high density in different geographic areas, and the use of improved methods of detection that include newly developed cell lines and increased sampling effort, have led to the discovery of fish viruses representing nearly all families of animal viruses. Among the newest additions, are a member of the family Picornaviridae and several new viruses that belong within the Reoviridae.

  13. Picornaviruses

    DEFF Research Database (Denmark)

    Alexandersen, S.; Knowles, N.; Dekker, A.;

    2012-01-01

    Diseases of Swine, Tenth Edition is a fully revised and updated version of this indispensable reference for detailed and comprehensive information on diseases in the pig. Now published in association with the American Association of Swine Veterinarians, this new edition adds new knowledge through...

  14. Picornaviruses

    DEFF Research Database (Denmark)

    Alexandersen, S.; Knowles, N.; Dekker, A.

    2012-01-01

    Diseases of Swine, Tenth Edition is a fully revised and updated version of this indispensable reference for detailed and comprehensive information on diseases in the pig. Now published in association with the American Association of Swine Veterinarians, this new edition adds new knowledge...... for swine practitioners at all levels, including students, swine veterinarians, and researchers....

  15. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  16. Endocytosis of integrin-binding human picornaviruses.

    Science.gov (United States)

    Merilahti, Pirjo; Koskinen, Satu; Heikkilä, Outi; Karelehto, Eveliina; Susi, Petri

    2012-01-01

    Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  17. THE INHIBITORY EFFECT OF ASTRAGALUS MEMBRANACEUS ON COXSACKIE B-3 VIRUS RNA REPLICATION

    Institute of Scientific and Technical Information of China (English)

    彭天庆; 杨英珍; HelgaRiesemann; ReinhardKandolf

    1995-01-01

    Using mice infected with coxsackie B-3 virus (CVB3) as a viral myocarditis model,we observed the inhibitory effect of Astragalus membranaceus (AM) on CVB3-RNA replication in myocardial tissue of mice by RNA-RNA in situ hybridization with negative-strand RNA probes labelled with 35S and quantitative imaging analysis of positive signals.The mechanism of its effect on CVB3-RNA replication has been inves-tigated by detection of beta-interferon (β-IFN) as well.Results showed that the copy numbers of CVB3-RNA as well as the histologic scores (necrosis) in myocardial tissues of infected-AM treated mice were sig-nificantly lower than those in infected and normal saline treated mice,suggesting that AM could inhibit the replication of CVB3-RNA,but its effect on CVB3-RNA replication had no correlation with induction of β-IFN.

  18. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

    Directory of Open Access Journals (Sweden)

    Arturo Diaz

    2015-03-01

    Full Text Available Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV RNA replication occurs on perinuclear endoplasmic reticulum (ER membranes in ~70 nm vesicular invaginations (spherules. BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

  19. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

    Science.gov (United States)

    Diaz, Arturo; Zhang, Jiantao; Ollwerther, Abigail; Wang, Xiaofeng; Ahlquist, Paul

    2015-03-01

    Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

  20. RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins.

    Science.gov (United States)

    Kallio, Katri; Hellström, Kirsi; Jokitalo, Eija; Ahola, Tero

    2015-11-18

    The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules. Recruitment of RNA to membranes in the absence of spherule formation was not detected.

  1. Inhibition of RNA recruitment and replication of an RNA virus by acridine derivatives with known anti-prion activities.

    Directory of Open Access Journals (Sweden)

    Zsuzsanna Sasvari

    Full Text Available BACKGROUND: Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV, a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ and quinacrine (QC, which are active against prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii reduction of minus-strand synthesis by the tombusvirus replicase; and (iii inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication. CONCLUSION/SIGNIFICANCE: Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.

  2. Isolation and molecular characterization of a novel picornavirus from baitfish in the USA.

    Directory of Open Access Journals (Sweden)

    Nicholas B D Phelps

    Full Text Available During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20 of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas and brassy minnows (Hybognathus hankinsoni. Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (∼ 30-32 nm, with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV. The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV isolated from bluegill (Lepomis macrochirus. Based on complete polyprotein analysis, the FHMPV shared 58% (P1, 33% (P2 and 43% (P3 amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.

  3. Selective cytotoxicity and modification activity of picornaviruses on transformed cell lines

    Directory of Open Access Journals (Sweden)

    Avagyan H. R.

    2011-10-01

    Full Text Available Aim. We do analyze of the dynamics of morphometabolic changes in transformed cells (of susceptoible lines demonstrating resistance to picrnaviral infection. Methods. The study was performed by application of cell culture technology and a complex of cytochemical and cytophotometric assays. Were used picornaviruses from various genu. Results. According to the results obtained, resistant to picornavirus infection cells of different susceptible lines have similar changes in the phenotype. They have decreased number of nucleoli and increased percentage of euploidy (and near euploid. In resistant cells of all cultures the reduction in amount of DNA and RNA both in nucleus and in cytoplasm was found. All these data correlated with the increased euploidy (and near euploid of the resistant population. All picornavirus resistant cells had a less transformed phenotype, and decreased proliferative activity. Decreased nucleolar status becomes apparent by reduction of all nucleolar indices. Conclusions. Picornaviruses on the susceptible cells produce 2 types of changes – selection and modification. Whatever the mechanism, it is specific for an individual virus, since no restrictions occur in case of infection caused by another picornavirus

  4. RNA interference of influenza A virus replication by microRNA-adapted lentiviral loop short hairpin RNA.

    Science.gov (United States)

    Xu, Fang; Liu, Guanqun; Liu, Qiang; Zhou, Yan

    2015-10-01

    Limitations of the current vaccines and antivirals against influenza A virus (IAV) pandemic underscore the urgent need for developing novel anti-influenza strategies. RNA interference (RNAi) induced by small interfering RNA (siRNA) has become a powerful new means to inhibit viral infection in a gene-specific manner. However, the efficacy of the siRNA delivery platform and the relatively high cost of administration have hindered widespread application of siRNA. In this study, we developed a microRNA (miRNA)-30-based lentivirus delivery system by embedding a synthetic short hairpin RNA (shRNA) stem into the context of endogenous precursor of miRNA-30 (shRNAmir) to express a silencer of the influenza gene. We showed that the miRNA-based lentivirus vector was able to express and process a single nucleoprotein (NP)-targeting shRNAmir, which could potently inhibit IAV replication. We further showed that miRNA-based lentivirus vector carrying tandemly linked NP and polymerase PB1 shRNAmirs could express and process double shRNAmirs. Despite the relatively low levels of NP and PB1 miRNAs produced in the stably transduced cells, the combination of two miRNAs exerted a great degree of inhibition on influenza infection. Given the advantage of combinatorial RNAi in preventing emergence of mutant virus, miRNA-based lentiviral vectors are valuable tools for anitiviral activities. To the best of our knowledge, this is the first study demonstrating that a miRNA-based RNAi strategy can be applied for better control of influenza virus infection.

  5. Intersection of the multivesicular body pathway and lipid homeostasis in RNA replication by a positive-strand RNA virus.

    Science.gov (United States)

    Wang, Xiaofeng; Diaz, Arturo; Hao, Linhui; Gancarz, Brandi; den Boon, Johan A; Ahlquist, Paul

    2011-06-01

    Like many positive-strand RNA viruses, brome mosaic virus (BMV) RNA replication occurs in membrane-invaginated vesicular compartments. BMV RNA replication compartments show parallels with membrane-enveloped, budding retrovirus virions, whose release depends on the cellular multivesicular body (MVB) sorting pathway. BMV RNA replication compartments are not released from their parent membranes, but might depend on MVB functions for membrane invagination. Prior results show that BMV RNA replication is severely inhibited by deletion of the crucial MVB gene DOA4 or BRO1. We report here that involvement of DOA4 and BRO1 in BMV RNA replication is not dependent on the MVB pathway's membrane-shaping functions but rather is due to their roles in recycling ubiquitin from MVB cargos. We show that deleting DOA4 or BRO1 inhibits the ubiquitination- and proteasome-dependent activation of homologous transcription factors Mga2p and Spt23p, which regulate many lipid metabolism genes, including the fatty acid desaturase gene OLE1, which is essential for BMV RNA replication. However, Mga2p processing and BMV RNA replication are restored by supplementing free ubiquitin, which is depleted in doa4Δ and bro1Δ cells. The results identify Mga2p and Spt23p processing and lipid regulation as sensitive targets of ubiquitin depletion and correctly predict multiple effects of modulating additional host genes RFU1, UBP6, and UFD3. Our results also show that BMV RNA replication depends on additional Mga2p-regulated genes likely involved in lipid metabolism beyond OLE1. Among other points, these findings show the potential for blocking viral RNA replication by modulating lipid synthesis at multiple levels.

  6. High diversity of picornaviruses in rats from different continents revealed by deep sequencing

    DEFF Research Database (Denmark)

    Arn Hansen, Thomas; Mollerup, Sarah; Nguyen, Nam-Phuong;

    2016-01-01

    ) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus...

  7. Studies on the effects of persistent RNA priming on DNA replication and genomic stability

    OpenAIRE

    Stuckey, Ruth

    2014-01-01

    [EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, ...

  8. Studies on the effects of persistent RNA priming on DNA replication and genomic stability

    OpenAIRE

    Stuckey, Ruth

    2014-01-01

    [EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, ...

  9. Anti-HCV RNA Aptamers Targeting the Genomic cis-Acting Replication Element

    Directory of Open Access Journals (Sweden)

    Alfredo Berzal-Herranz

    2011-12-01

    Full Text Available Hepatitis C virus (HCV replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE, located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for efficient viral replication. Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80% of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents.

  10. Diversity of picornaviruses in rural Bolivia

    Science.gov (United States)

    Nix, W. Allan; Khetsuriani, Nino; Peñaranda, Silvia; Maher, Kaija; Venczel, Linda; Cselkó, Zsuzsa; Freire, Maria Cecelia; Cisterna, Daniel; Lema, Cristina L.; Rosales, Patricia; Rodriguez, Jacqueline R.; Rodriguez, Wilma; Halkyer, Percy; Ronveaux, Olivier; Pallansch, Mark A.; Oberste, M. Steven

    2015-01-01

    The family Picornaviridae is a large and diverse group of viruses that infect humans and animals. Picornaviruses are among the most common infections of humans and cause a wide spectrum of acute human disease. This study began as an investigation of acute flaccid paralysis (AFP) in a small area of eastern Bolivia, where surveillance had identified a persistently high AFP rate in children. Stools were collected and diagnostic studies ruled out poliovirus. We tested stool specimens from 51 AFP cases and 34 healthy household or community contacts collected during 2002–2003 using real-time and semi-nested RT-PCR assays for enterovirus, parechovirus, cardiovirus, kobuvirus, salivirus, and cosavirus. Anecdotal reports suggested a temporal association with neurologic disease in domestic pigs, so six porcine stools were also collected and tested with the same set of assays, with the addition of an assay for porcine teschovirus. A total of 126 picornaviruses were detected in 73 of 85 human individuals, consisting of 53 different picornavirus types encompassing five genera (all except Kobuvirus). All six porcine stools contained porcine and/or human picornaviruses. No single virus, or combination of viruses, specifically correlated with AFP; however, the study revealed a surprising complexity of enteric picornaviruses in a single community. PMID:23804569

  11. Adaptation and Diversification of an RNA Replication System under Initiation- or Termination-Impaired Translational Conditions.

    Science.gov (United States)

    Mizuuchi, Ryo; Ichihashi, Norikazu; Yomo, Tetsuya

    2016-07-01

    Adaptation to various environments is a remarkable characteristic of life. Is this limited to extant complex living organisms, or is it also possible for a simpler self-replication system to adapt? In this study, we addressed this question by using a translation-coupled RNA replication system that comprised a reconstituted translation system and an RNA "genome" that encoded a replicase gene. We performed RNA replication reactions under four conditions, under which different components of translation were partly inhibited. We found that replication efficiency increased with the number of rounds of replication under all the tested conditions. The types of dominant mutations differed depending on the condition, thus indicating that this simple system adapted to different environments in different ways. This suggests that even a primitive self-replication system composed of a small number of genes on the early earth could have had the ability to adapt to various environments.

  12. Inhibition of avian leukosis virus replication by vector-based RNA interference

    Science.gov (United States)

    RNAi has recently emerged as a promising antiviral technique in vertebrates. To date, most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral replication, though vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) are...

  13. Suppression of feline calicivirus replication using small interfering RNA targeted to its polymerase gene.

    Science.gov (United States)

    Taharaguchi, Satoshi; Matsuhiro, Takahisa; Harima, Hayato; Sato, Atsuko; Ohe, Kyoko; Sakai, Sachi; Takahashi, Toshikazu; Hara, Motonobu

    2012-06-01

    Feline calicivirus (FCV) is a pathogenic microorganism that causes upper respiratory diseases in cats. Recently, an FCV infection with a high mortality rate has been confirmed, and there is need to develop a treatment for cases of acute infection. We evaluated whether the replication of FCV could be prevented by RNA interference. For this study, we designed an siRNA targeted to the polymerase region of the strain FCV-B isolated from a cat that died after exhibiting neurological symptoms. Cells transfected with siR-pol dose-dependently suppressed the replication of FCV-B. siR-pol suppressed its replication by suppressing the target viral RNA.

  14. Evolutionary dynamics of RNA-like replicator systems: A bioinformatic approach to the origin of life.

    Science.gov (United States)

    Takeuchi, Nobuto; Hogeweg, Paulien

    2012-09-01

    We review computational studies on prebiotic evolution, focusing on informatic processes in RNA-like replicator systems. In particular, we consider the following processes: the maintenance of information by replicators with and without interactions, the acquisition of information by replicators having a complex genotype-phenotype map, the generation of information by replicators having a complex genotype-phenotype-interaction map, and the storage of information by replicators serving as dedicated templates. Focusing on these informatic aspects, we review studies on quasi-species, error threshold, RNA-folding genotype-phenotype map, hypercycle, multilevel selection (including spatial self-organization, classical group selection, and compartmentalization), and the origin of DNA-like replicators. In conclusion, we pose a future question for theoretical studies on the origin of life.

  15. Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus.

    Science.gov (United States)

    Marvil, P; Knowles, N J; Mockett, A P; Britton, P; Brown, T D; Cavanagh, D

    1999-03-01

    The complete RNA genome of avian encephalomyelitis virus (AEV) has been molecularly cloned and sequenced. This revealed AEV to be a member of the Picornaviridae and consequently it is the first avian picornavirus for which the genome has been sequenced. Excluding the poly(A) tail the genome comprises 7032 nucleotides, which is shorter than that of any mammalian picornavirus sequenced to date. An open reading frame commencing at nucleotide 495 and terminating at position 6896 (6402 nucleotides) potentially encodes a polyprotein of 2134 amino acids. The polyprotein sequence has 39% overall amino acid identity with hepatitis A virus (HAV; genus Hepatovirus), compared to 19 to 21% for viruses from the other five picornavirus genera. Eleven cleavage products were predicted. The highest identity (49%) with HAV was in the P1 region, encoding the capsid proteins. The 5' and 3' untranslated regions (UTRs) comprise 494 and 136 nucleotides, respectively. The 5' UTR is the shortest of any picornavirus sequenced to date and, unlike HAV, it does not contain a long polypyrimidine tract.

  16. Ultrastructure of the replication sites of positive-strand RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

    2015-05-15

    Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.

  17. The silent defense: Micro-RNA directed defense against HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Kumar Ajit

    2007-04-01

    Full Text Available Abstract MicroRNAs play critical role in regulating gene expression. MicroRNA profile of particular cell type bears the signature of cell type specific gene expression. Given that viral pathogens replicate by evading host defenses, research is now focused on the miRNA-regulated genes that critically regulate HIV-1 propagation in human host cells.

  18. SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5'UTR RNA.

    Science.gov (United States)

    Han, Yang; Wang, Lvyin; Cui, Jin; Song, Yu; Luo, Zhen; Chen, Junbo; Xiong, Ying; Zhang, Qi; Liu, Fang; Ho, Wenzhe; Liu, Yingle; Wu, Kailang; Wu, Jianguo

    2016-12-15

    Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5' untranslated region (5'UTR) and a polyadenylated 3'UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3D(pol) protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3D(pol), resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5'UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5'UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication. © 2016. Published by The Company of Biologists Ltd.

  19. Systematic identification of novel, essential host genes affecting bromovirus RNA replication.

    Directory of Open Access Journals (Sweden)

    Brandi L Gancarz

    Full Text Available Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ∼100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ∼900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ∼81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol levels were significantly increased in strains depleted for a heat shock protein (HSF1 or proteasome components (PRE1 and RPT6, suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.

  20. Systematic identification of novel, essential host genes affecting bromovirus RNA replication.

    Science.gov (United States)

    Gancarz, Brandi L; Hao, Linhui; He, Qiuling; Newton, Michael A; Ahlquist, Paul

    2011-01-01

    Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ∼100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ∼900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ∼81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.

  1. Small noncoding RNA modulates japanese encephalitis virus replication and translation in trans

    Directory of Open Access Journals (Sweden)

    Fan Yi-Hsin

    2011-11-01

    Full Text Available Abstract Background Sequence and structural elements in the 3'-untranslated region (UTR of Japanese encephalitis virus (JEV are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA which is collinear with the highly conserved regions of the 3'-UTR in JEV-infected cells. However, function of the sfRNA in JEV life cycle remains unknown. Results Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. Trans-acting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation. Conclusions Our results indicate that sfRNA modulates viral translation and replication in trans.

  2. Emergence of a replicating species from an in vitro RNA evolution reaction

    Science.gov (United States)

    Breaker, R. R.; Joyce, G. F.

    1994-01-01

    The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

  3. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

    Directory of Open Access Journals (Sweden)

    Ying Wen Huang

    Full Text Available Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA or that of genomic RNA of other viruses, such as Potato virus X (PVX or Cucumber mosaic virus (CMV. Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

  4. Active RNA replication of hepatitis C virus downregulates CD81 expression.

    Science.gov (United States)

    Ke, Po-Yuan; Chen, Steve S-L

    2013-01-01

    So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  5. Active RNA replication of hepatitis C virus downregulates CD81 expression.

    Directory of Open Access Journals (Sweden)

    Po-Yuan Ke

    Full Text Available So far how hepatitis C virus (HCV replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp infection and downregulated cell surface level of CD81, a critical HCV entry (coreceptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  6. Adenovirus-mediated shRNA interference against HSV-1 replication in vitro.

    Science.gov (United States)

    Song, Bo; Liu, Xinjing; Wang, Qingzhi; Zhang, Rui; Yang, Ting; Han, Zhiqiang; Xu, Yuming

    2016-12-01

    The UL29 and UL28 proteins encoded by herpes simplex virus type 1 (HSV-1) are critical for its replication and packaging, respectively. Research has demonstrated that synthesized siRNA molecules targeting the UL29 gene are able to suppress HSV-2 replication and the UL28-null HSV-1 gene cannot form infectious viruses in vitro. Silencing the UL28 and UL29 genes by RNAi might lead to the development of novel antiviral agents for the treatment of HSV-1 infections. Two kinds of short hairpin RNAs (shRNAs) targeting the UL29 and UL28 genes were chemically synthesized and then delivered into cells by a replication-defective human adenovirus type 5 (Adv5) vector. (-) shRNAs targeting none of the genome of HSV-1 were used as the control. Vero cells were inoculated with Ad-UL28shRNA or Ad-UL29shRNA at a multiplicity of infection (MOI) of 100 and challenged 24 h later with HSV-1 at an MOI of 0.01 to inhibit HSV-1 replication, as measured by the level of the corresponding RNA and proteins. In addition, the amount of progeny virus was assessed at daily intervals. The antiviral effects of Ad-shRNAs at ongoing HSV-1 infection were explored at 12 h after inoculation of the HSV-1. The results showed that the shRNAs delivered by Adv5 significantly suppressed HSV-1 replication in vitro, as determined by the levels of viral RNA transcription, viral protein synthesis, and viral production. The Ad-UL28shRNA and Ad-UL29shRNA suppressed the replication of HSV-1, respectively, compared with the control group (P HSV-1 infection (P HSV-1 infection.

  7. [Determination of genomic and replicative RNA of hepatitis C virus in patients treated with interferon].

    Science.gov (United States)

    Zeilicoff, R; Ameigeiras, B; Ojeda, E; Isla Rodríguez, R; Grünbaum, S; Genero, M; Cappelletti, C; Tielli, G; Roatta, R; Koch, O

    1995-01-01

    We have investigated the presence of genomic and replicative RNA strands of hepatitis C virus in liver and serum. Eleven patients with proven chronic hepatitis C, received Interferon a2a 4,5 MU, three times a week during six months. RT-PCR was used with sense primer to detect the replicative strand and an antisense primer to identify genomic strand. Before treatment, genomic strands were present in liver and serum of all patients. Replicative strands were present in liver and serum in five and six cases, respectively. Seven out of eleven responded to treatment. In responders, genomic strands were absent in liver of 3 cases (43%) and replicative strands in liver of 4 (57%). In plasma genomic and replicative strands were absent in 5 (71%) and 7 (100%), respectively. In all non responders, genomic strands in liver and plasma remained present. Replicative strands in liver and plasma were present in 100% and 25%, respectively. Knodell score improved in 5 out of 7 responders and remained unchanged in 3 out of 4 non responders. In 2 out of 4 responders with genomic and replicative strands in liver, Knodell score remained unchanged or worse. In all non responders, genomic and replicative strands in liver were present and Knodell score remained unchanged or worse. Genomic and replicative strands in plasma tended to be negative after treatment in responders. Genomic strands in plasma remained present in non responders. Conversely, genomic and replicative strands in liver were present in all non responders. It seems to exist a relationship between genomic and replicative strands in liver and the same or worse Knodell score. After a follow up, it will be possible to determined whether responders who still present viral RNA in liver would be prone to a relapse.

  8. Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation.

    Science.gov (United States)

    Sunami, Takeshi; Ichihashi, Norikazu; Nishikawa, Takehiro; Kazuta, Yasuaki; Yomo, Tetsuya

    2016-07-01

    Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation-coupled RNA replication (TcRR) system. The RNA was replicated by Qβ replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system. A reporter RNA encoding the antisense strand of β-glucuronidase was introduced into the system to yield a TcRR read-out (green fluorescence). We demonstrate that TcRR was hardly detectable in larger liposomes (230 fL) but was more effective in smaller (7.7 fL) liposomes. Our experimental and theoretical results show that smaller microcompartments considerably enhance TcRR because the synthesized molecules, such as RNA and replicases, are more concentrated in smaller liposomes.

  9. Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation.

    Science.gov (United States)

    Ogram, Sushma A; Boone, Christopher D; McKenna, Robert; Flanegan, James B

    2014-09-01

    The mechanism of amiloride inhibition of Coxsackievirus B3 (CVB3) and poliovirus type 1 (PV1) RNA replication was investigated using membrane-associated RNA replication complexes. Amiloride was shown to inhibit viral RNA replication and VPgpUpU synthesis. However, the drug had no effect on polymerase elongation activity during either (-) strand or (+) strand synthesis. These findings indicated that amiloride inhibited the initiation of RNA synthesis by inhibiting VPg uridylylation. In addition, in silico binding studies showed that amiloride docks in the VPg binding site on the back of the viral RNA polymerase, 3D(pol). Since VPg binding at this site on PV1 3D(pol) was previously shown to be required for VPg uridylylation, our results suggest that amiloride inhibits VPg binding to 3D(pol). In summary, our findings are consistent with a model in which amiloride inhibits VPgpUpU synthesis and viral RNA replication by competing with VPg for binding to 3D(pol).

  10. Detection of Human Picornaviruses by Multiplex Reverse Transcription-PCR and Liquid Hybridization

    OpenAIRE

    Jokela, Pia; Joki-Korpela, Päivi; Maaronen, Marita; Glumoff, Virpi; Hyypiä, Timo

    2005-01-01

    A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5′ untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with g...

  11. Inhibition of IκB kinase by thalidomide increases hepatitis C virus RNA replication.

    Science.gov (United States)

    Rance, E; Tanner, J E; Alfieri, C

    2012-02-01

    Hepatic fibrosis is an integral element in the progression of chronic liver disease. Elevated hepatic interleukin (IL)-8 is an important contributor to fibrosis in patients chronically infected with the hepatitis C virus (HCV). Thalidomide has been used to reduce liver inflammation and fibrosis in HCV-infected patients, but its impact on HCV replication remains unclear. This study examined the effect of thalidomide on HCV replication in vitro. Results revealed that while thalidomide reduced IL-8 and nuclear factor kappa B (NF-κB) activity by 95% and 46% in Huh-7 cells, increasing concentrations of thalidomide correlated with a linear rise in HCV replication (17-fold at 200 μm). The NF-κB inhibitors, wedelolactone and NF-κB activation inhibitor-1, which mimic the actions of thalidomide by preventing phosphorylation and activation of IκB kinase (IKK) and hence block NF-κB activity, increased HCV RNA by 18- and 19-fold, respectively. During in vitro HCV replication in Huh-7 cells, we observed a 30% increase in IKKα protein and 55% decrease in NF-κB(p65)/RelA protein relative to cellular β-actin. Ectopic expression of IKKα to enhance the inactive form of IKK in cells undergoing virus replication led to a 13-fold increase in HCV RNA. Conversely, enhanced expression of NF-κB(p65)/RelA in infected cells resulted in a 17-fold reduction in HCV RNA. In conclusion, HCV RNA replication was significantly augmented by the inhibition of IKK activation and subsequent NF-κB signalling, whereas a restoration of NF-κB activity by the addition of NF-κB/RelA markedly reduced HCV replication. This study lends added importance to the role of the NF-κB signalling pathway in controlling HCV replication.

  12. Parasites Sustain and Enhance RNA-Like Replicators through Spatial Self-Organisation.

    Directory of Open Access Journals (Sweden)

    Enrico Sandro Colizzi

    2016-04-01

    Full Text Available In a prebiotic RNA world, parasitic behaviour may be favoured because template dependent replication happens in trans, thus being altruistic. Spatially extended systems are known to reduce harmful effects of parasites. Here we present a spatial system to show that evolution of replication is (indirectly enhanced by strong parasites, and we characterise the phase transition that leads to this mode of evolution. Building on the insights of this analysis, we identify two scenarios, namely periodic disruptions and longer replication time-span, in which speciation occurs and an evolved parasite-like lineage enables the evolutionary increase of replication rates in replicators. Finally, we show that parasites co-evolving with replicators are selected to become weaker, i.e. worse templates for replication when the duration of replication is increased. We conclude that parasites may not be considered a problem for evolution in a prebiotic system, but a degree of freedom that can be exploited by evolution to enhance the evolvability of replicators, by means of emergent levels of selection.

  13. Parasites Sustain and Enhance RNA-Like Replicators through Spatial Self-Organisation.

    Science.gov (United States)

    Colizzi, Enrico Sandro; Hogeweg, Paulien

    2016-04-01

    In a prebiotic RNA world, parasitic behaviour may be favoured because template dependent replication happens in trans, thus being altruistic. Spatially extended systems are known to reduce harmful effects of parasites. Here we present a spatial system to show that evolution of replication is (indirectly) enhanced by strong parasites, and we characterise the phase transition that leads to this mode of evolution. Building on the insights of this analysis, we identify two scenarios, namely periodic disruptions and longer replication time-span, in which speciation occurs and an evolved parasite-like lineage enables the evolutionary increase of replication rates in replicators. Finally, we show that parasites co-evolving with replicators are selected to become weaker, i.e. worse templates for replication when the duration of replication is increased. We conclude that parasites may not be considered a problem for evolution in a prebiotic system, but a degree of freedom that can be exploited by evolution to enhance the evolvability of replicators, by means of emergent levels of selection.

  14. Enhancer of Rudimentary Homolog Affects the Replication Stress Response through Regulation of RNA Processing

    Science.gov (United States)

    Kavanaugh, Gina; Zhao, Runxiang; Guo, Yan; Mohni, Kareem N.; Glick, Gloria; Lacy, Monica E.; Hutson, M. Shane; Ascano, Manuel

    2015-01-01

    Accurate replication of DNA is imperative for the maintenance of genomic integrity. We identified Enhancer of Rudimentary Homolog (ERH) using a whole-genome RNA interference (RNAi) screen to discover novel proteins that function in the replication stress response. Here we report that ERH is important for DNA replication and recovery from replication stress. ATR pathway activity is diminished in ERH-deficient cells. The reduction in ATR signaling corresponds to a decrease in the expression of multiple ATR pathway genes, including ATR itself. ERH interacts with multiple RNA processing complexes, including splicing regulators. Furthermore, splicing of ATR transcripts is deficient in ERH-depleted cells. Transcriptome-wide analysis indicates that ERH depletion affects the levels of ∼1,500 transcripts, with DNA replication and repair genes being highly enriched among those with reduced expression. Splicing defects were evident in ∼750 protein-coding genes, which again were enriched for DNA metabolism genes. Thus, ERH regulation of RNA processing is needed to ensure faithful DNA replication and repair. PMID:26100022

  15. Consensus siRNA for inhibition of HCV genotype-4 replication

    Directory of Open Access Journals (Sweden)

    El-Din Hanaa

    2009-01-01

    Full Text Available Abstract Background HCV is circulating as a heterogeneous group of quasispecies. It has been addressed that siRNA can inhibit HCV replication in-vitro using HCV clone and/or replicon which have only one genotype. The current study was conducted to assess whether siRNA can inhibit different HCV genotypes with many quasispecies and to assess whether consensus siRNA have the same effect as regular siRNA. Methods We generated two chemically synthesized consensus siRNAs (Z3 and Z5 which cover most known HCV genotype sequences and quasispecies using Ambium system. Highly positive HCV patient's serum with nine quasispecies was transfected in-vitro to Huh-7 cell line which supports HCV genotype-4 replication. siRNA (Z3&Z5 were transfected according to Qiagen Porta-lipid technique and subsequently cultured for eight days. HCV replication was monitored by RT-PCR for detection of plus and minus strands. Real-time PCR was used for quantification of HCV, whereas detection of the viral core protein was performed by western blot. Results HCV RNA levels decreased 18-fold (P = 0.001 and 25-fold (P = 0.0005 in cells transfected with Z3 and Z5, respectively, on Day 2 post transfection and continued for Day 3 by Z3 and Day 7 by Z5. Reduction of core protein expression was reported at Day 2 post Z3 siRNA transfection and at Day 1 post Z5 siRNA, which was persistent for Day 4 for the former and for Day 6 for the latter. Conclusion Consensus siRNA could be used as a new molecular target therapy to effectively inhibit HCV replication in the presence of more than one HCV quasispecies.

  16. A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

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    Hui Feng

    Full Text Available BACKGROUND: The specific interaction between hepatitis B virus (HBV polymerase (P protein and the ε RNA stem-loop on pregenomic (pg RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP, to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.

  17. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  18. Inhibition of BmNPV replication in Bombyx mori cell by dsRNA triggered RNA interference

    Institute of Scientific and Technical Information of China (English)

    XU Ying; ZHU Chenggang; JIN Yongfeng; ZHANG Yaozhou

    2004-01-01

    RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms, To investigate the effect of dsRNA on silkworm cells, we transfected three kinds of synthetic dsRNAs of 435 bp(Ap1), 300bp(Ape) and 399 bp(Au) in length against the various regions of BmNPV's DNA polymerase gene and DNA helicase gene,which are indispensable for viral replication in silkworm cells by TransMessengerTM transfection Reagent. Results indicated that in the experiment where silkworm cells were infected with wild-strain BmNPV of the three dsRNAs, Ap2 and AH can effectively suppress the replication of virus, but Ap1 had no effect on the inhibition of viral replication. Ap2 and Au can reduce the infective titer of BmNPV with a peak change of approximately 3-4 logs on day 4 post-infection.The results of reverse transcript polylnerase chain reaction (RT-PCR) and DNA dot blotting also indicated that the expression level of the two target genes and the quantity of viral DNA both distinctly decreased under the influence of Ap2 or Au. Furthermore, using fluorescence microscopy we analyzed the distribution patterns of dsRNA. Our studies revealed that a majority of dsRNA was localized in the nuclear periphery discontinuously after 24 h of transfection.

  19. Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

    OpenAIRE

    Geiss Brian J; Phillips Aaron T; Scott Jaclyn C; Cirimotich Chris M; Olson Ken E

    2009-01-01

    Abstract Background Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus), a pr...

  20. The role of water in human picornavirus transmission

    NARCIS (Netherlands)

    Lodder, W.J.

    2014-01-01

    The overall research question of the work presented in this thesis was whether the presence of human picornaviruses in the aquatic environment poses a problem to public health. A multidisciplinary approach was used to determine the extent to which human picornaviruses circulate in the general popula

  1. MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Shun; Huang, Haijiao; Li, Nanhong; Zhang, Bing; Jia, Yubin; Yang, Yukun; Yuan, Yuan; Xiong, Xing-dong; Wang, Dengchuan; Zheng, Hui-ling [Institute of Aging Research, Guangdong Medical University, Dongguan (China); Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang (China); Liu, Xinguang, E-mail: xgliu64@126.com [Institute of Aging Research, Guangdong Medical University, Dongguan (China); Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang (China)

    2016-05-13

    MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33 promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.

  2. Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra; Tao, Yizhi Jane; Vasquez-Del Carpio, Rodrigo; Nibert, Max L.; Patton, John T.; Harrison, Stephen C. (Harvard-Med); (NIH); (CH-Boston)

    2009-04-08

    Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 {angstrom} resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus {lambda}3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence. Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.

  3. Efficient inhibition of HIV-1 replication by an artificial polycistronic miRNA construct

    Directory of Open Access Journals (Sweden)

    Zhang Tao

    2012-06-01

    Full Text Available Abstract Background RNA interference (RNAi has been used as a promising approach to inhibit human immunodeficiency virus type 1 (HIV-1 replication for both in vitro and in vivo animal models. However, HIV-1 escape mutants after RNAi treatment have been reported. Expressing multiple small interfering RNAs (siRNAs against conserved viral sequences can serve as a genetic barrier for viral escape, and optimization of the efficiency of this process was the aim of this study. Results An artificial polycistronic transcript driven by a CMV promoter was designed to inhibit HIV-1 replication. The artificial polycistronic transcript contained two pre-miR-30a backbones and one pre-miR-155 backbone, which are linked by a sequence derived from antisense RNA sequence targeting the HIV-1 env gene. Our results demonstrated that this artificial polycistronic transcript simultaneously expresses three anti-HIV siRNAs and efficiently inhibits HIV-1 replication. In addition, the biosafety of MT-4 cells expressing this polycistronic miRNA transcript was evaluated, and no apparent impacts on cell proliferation rate, interferon response, and interruption of native miRNA processing were observed. Conclusions The strategy described here to generate an artificial polycistronic transcript to inhibit viral replication provided an opportunity to select and optimize many factors to yield highly efficient constructs expressing multiple siRNAs against viral infection.

  4. Oligoarginine peptides slow strand annealing and assist non-enzymatic RNA replication

    Science.gov (United States)

    Jia, Tony Z.; Fahrenbach, Albert C.; Kamat, Neha P.; Adamala, Katarzyna P.; Szostak, Jack W.

    2016-10-01

    The non-enzymatic replication of RNA is thought to have been a critical process required for the origin of life. One unsolved difficulty with non-enzymatic RNA replication is that template-directed copying of RNA results in a double-stranded product. After strand separation, rapid strand reannealing outcompetes slow non-enzymatic template copying, which renders multiple rounds of RNA replication impossible. Here we show that oligoarginine peptides slow the annealing of complementary oligoribonucleotides by up to several thousand-fold; however, short primers and activated monomers can still bind to template strands, and template-directed primer extension can still occur, all within a phase-separated condensed state, or coacervate. Furthermore, we show that within this phase, partial template copying occurs even in the presence of full-length complementary strands. This method to enable further rounds of replication suggests one mechanism by which short non-coded peptides could have enhanced early cellular fitness, and potentially explains how longer coded peptides, that is, proteins, came to prominence in modern biology.

  5. Cyclophilin A binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly.

    Science.gov (United States)

    Kovalev, Nikolay; Nagy, Peter D

    2013-12-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.

  6. Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons.

    Science.gov (United States)

    Hamazaki, Hiroyuki; Ujino, Saneyuki; Miyano-Kurosaki, Naoko; Shimotohno, Kunitada; Takaku, Hiroshi

    2006-05-12

    RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.

  7. CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

    Directory of Open Access Journals (Sweden)

    Yu-Chen Lin

    2017-05-01

    Full Text Available Influenza A virus (IAV RNA segments are individually packaged with viral nucleoprotein (NP and RNA polymerases to form a viral ribonucleoprotein (vRNP complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4, which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication.

  8. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication

    Science.gov (United States)

    Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia; Li, Yize; Wilhelm, Jochen; Rabouw, Huib H.; Züst, Roland; Marti, Sabrina; Habjan, Matthias; Cervantes-Barragan, Luisa; Elliot, Ruth; Karl, Nadja; Gaughan, Christina; Silverman, Robert H.; Keller, Markus; Ludewig, Burkhard; Bergmann, Cornelia C.; Ziebuhr, John; Kalinke, Ulrich

    2017-01-01

    Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis–within the replicase complex—suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses. PMID:28158275

  9. Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses

    Directory of Open Access Journals (Sweden)

    Vladimir A. Gushchin

    2013-03-01

    Full Text Available In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER, Golgi, endo/lysosomes, mitochondria, chloroplasts and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV and other closteroviruses, the region between the methyltransferase (MTR and helicase (HEL domains (1a central region, 1a CR is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2 of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-m mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of virus factories in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses and negative-strand RNA viruses (bunyaviruses.

  10. Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses.

    Science.gov (United States)

    Gushchin, Vladimir A; Solovyev, Andrey G; Erokhina, Tatyana N; Morozov, Sergey Y; Agranovsky, Alexey A

    2013-01-01

    In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-μm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of "virus factories" in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).

  11. Inhibition of hepatitis B virus gene expression and replication by artificial microRNA

    Institute of Scientific and Technical Information of China (English)

    Yu-Feng Gao; Li Yu; Wei Wei; Jia-Bin Li; Qing-Li Luo; Ji-Long Shen

    2008-01-01

    AIM: To investigate the inhibitory effects of hepatitis B virus (HBV) replication and expression by transfecting artificial microRNA (amiRNA) into HepG2.2.15 cells.METHODS: Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells.HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays (TRFIA). HBV DNA replication was examined by fluorescence quantitative PCR, and the level of HBV S mRNA was measured by semi-quantitative RT-PCR.RESULTS: The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection (P< 0.01 for all). The secretion of HBsAg and HBeAginto the supernatant was in hibited by 49.8% + 4.7%and 39.9% ± 6.7%, respectively, at 72 h in amiRNA-HBV-S608 plasmid transfection group. The copy of HBVDNA within culture supernatant was also significantlydecreased at 72 h and 96 h after transfection (P <0.01 for all). In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups (P < 0.01 for all, vs negative control). The copies of HBV DNA were inhibited by 33.4% ± 3.0%,60.8% ± 2.3% and 70.1% ± 3.3%, respectively.CONCLUSION: In HepG2.2.15 cells, HBV replication and expression could be inhibited by artificial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.

  12. Naturally acquired picornavirus infections in primates at the Dhaka zoo.

    Science.gov (United States)

    Oberste, M Steven; Feeroz, Mohammed M; Maher, Kaija; Nix, W Allan; Engel, Gregory A; Begum, Sajeda; Hasan, Kamrul M; Oh, Gunwha; Pallansch, Mark A; Jones-Engel, Lisa

    2013-01-01

    The conditions in densely populated Bangladesh favor picornavirus transmission, resulting in a high rate of infection in the human population. Data suggest that nonhuman primates (NHP) may play a role in the maintenance and transmission of diverse picornaviruses in Bangladesh. At the Dhaka Zoo, multiple NHP species are caged in close proximity. Their proximity to other species and to humans, both zoo workers and visitors, provides the potential for cross-species transmission. To investigate possible interspecies and intraspecies transmission of picornaviruses among NHP, we collected fecal specimens from nine NHP taxa at the Dhaka Zoo at three time points, August 2007, January 2008, and June 2008. Specimens were screened using real-time PCR for the genera Enterovirus, Parechovirus, and Sapelovirus, and positive samples were typed by VP1 sequencing. Fifty-two picornaviruses comprising 10 distinct serotypes were detected in 83 fecal samples. Four of these serotypes, simian virus 19 (SV19), baboon enterovirus (BaEV), enterovirus 112 (EV112), and EV115, have been solely associated with infection in NHP. EV112, EV115, and SV19 accounted for 88% of all picornaviruses detected. Over 80% of samples from cages housing rhesus macaques, olive baboons, or hamadryas baboons were positive for a picornavirus, while no picornaviruses were detected in samples from capped langurs or vervet monkeys. In contrast to our findings among synanthropic NHP in Bangladesh where 100% of the picornaviruses detected were of human serotypes, in the zoo population, only 15% of picornaviruses detected in NHP were of human origin. Specific serotypes tended to persist over time, suggesting either persistent infection of individuals or cycles of reinfection.

  13. Multiplexing seven miRNA-Based shRNAs to suppress HIV replication.

    Science.gov (United States)

    Choi, Jang-Gi; Bharaj, Preeti; Abraham, Sojan; Ma, Hongming; Yi, Guohua; Ye, Chunting; Dang, Ying; Manjunath, N; Wu, Haoquan; Shankar, Premlata

    2015-02-01

    Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc(-/-) mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.

  14. Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication.

    Science.gov (United States)

    Wang, Chunfu; Pflugheber, Jill; Sumpter, Rhea; Sodora, Donald L; Hui, Daniel; Sen, Ganes C; Gale, Michael

    2003-04-01

    Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2alpha) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2alpha phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively

  15. Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses...... to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA...... mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning...

  16. RNA-dependent RNA polymerase 6 delays accumulation and precludes meristem invasion of a viroid that replicates in the nucleus.

    Science.gov (United States)

    Di Serio, Francesco; Martínez de Alba, Angel-Emilio; Navarro, Beatriz; Gisel, Andreas; Flores, Ricardo

    2010-03-01

    The detection of viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs (siRNAs, 21 to 24 nucleotides [nt]) in plants infected by nuclear-replicating members of the family Pospiviroidae (type species, Potato spindle tuber viroid [PSTVd]) indicates that they are inducers and targets of the RNA-silencing machinery of their hosts. RNA-dependent RNA polymerase 6 (RDR6) catalyzes an amplification circuit producing the double-stranded precursors of secondary siRNAs. Recently, the role of RDR6 in restricting systemic spread of certain RNA viruses and precluding their invasion of the apical growing tip has been documented using RDR6-silenced Nicotiana benthamiana (NbRDR6i) plants. Here we show that RDR6 is also engaged in regulating PSTVd levels: accumulation of PSTVd genomic RNA was increased in NbRDR6i plants with respect to the wild-type controls (Nbwt) early in infection, whereas this difference decreased or disappeared in later infection stages. Moreover, in situ hybridization revealed that RDR6 is involved in restricting PSTVd access in floral and vegetative meristems, thus providing firm genetic evidence for an antiviroid RNA silencing mechanism. RNA gel blot hybridization and deep sequencing showed in wt and RDR6i backgrounds that PSTVd sRNAs (i) accumulate to levels paralleling their genomic RNA, (ii) display similar patterns with prevailing 22- or 21-nt plus-strand species, and (iii) adopt strand-specific hot spot profiles along the genomic RNA. Therefore, the surveillance mechanism restraining entry of some RNA viruses into meristems likely also controls PSTVd access in N. benthamiana. Unexpectedly, deep sequencing also disclosed in NbRDR6i plants a profile of RDR6-derived siRNA dominated by 21-nt plus-strand species mapping within a narrow window of the hairpin RNA stem expressed transgenically for silencing RDR6, indicating that minus-strand siRNAs silencing the NbRDR6 mRNA represent a minor fraction of the total siRNA population.

  17. Inhibition of hepatitis C virus replication by single-stranded RNA structural mimics

    Institute of Scientific and Technical Information of China (English)

    Robert; Smolic; Martina; Smolic; John; H; Andorfer; Catherine; H; Wu; Robert; M; Smith; George; Y; Wu

    2010-01-01

    AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were trans...

  18. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

    Science.gov (United States)

    Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

    2012-11-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.

  19. Adenovirus vector-mediated RNA interference for the inhibition of human parvovirus B19 replication.

    Science.gov (United States)

    Brandt, Marius R G; Kirste, Ariane G; Pozzuto, Tanja; Schubert, Steffen; Kandolf, Reinhard; Fechner, Henry; Bock, C-Thomas; Kurreck, Jens

    2013-09-01

    Human parvovirus B19 (B19V) has been considered to cause acute and chronic myocarditis, which is accompanied by endothelial dysfunction. Currently, no causative treatment option for B19V-infections is available. Since RNA interference (RNAi) has proven to be a highly potent antiviral approach, the aim of the current study was to develop an RNAi-based strategy to inhibit B19V replication. Three B19V-VP2-specific short hairpin RNAs (shRNAs) were designed and tested for their silencing activity in reporter assays and the expression cassette of the best one was introduced into an adenoviral shuttle vector (Ad5). B19V-permissive UT7/Epo-S1 cells were infected with B19V and the RNAi triggers were delivered by the adenoviral vector (Ad5shVP2) 24h thereafter. The shRNA targeting the B19V-VP2 gene significantly suppressed VP2 mRNA levels as determined by quantitative RT-PCR. Additionally, also the expression levels of the non-targeted non-structural B19V-NS1 mRNA were strongly reduced. Our results demonstrate that vector-mediated delivery of shRNA expression cassettes targeting the structural B19-VP2 gene is a suitable approach to inhibit B19V replication.

  20. A critical role of a cellular membrane traffic protein in poliovirus RNA replication.

    Directory of Open Access Journals (Sweden)

    George A Belov

    2008-11-01

    Full Text Available Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA, implicating some components(s of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA.

  1. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    Energy Technology Data Exchange (ETDEWEB)

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J., E-mail: nmoorman@med.unc.edu

    2016-02-15

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

  2. A Novel Mechanism Underlying the Innate Immune Response Induction upon Viral-Dependent Replication of Host Cell mRNA: A Mistake of +sRNA Viruses' Replicases

    Science.gov (United States)

    Delgui, Laura R.; Colombo, María I.

    2017-01-01

    Viruses are lifeless particles designed for setting virus-host interactome assuring a new generation of virions for dissemination. This interactome generates a pressure on host organisms evolving mechanisms to neutralize viral infection, which places the pressure back onto virus, a process known as virus-host cell co-evolution. Positive-single stranded RNA (+sRNA) viruses are an important group of viral agents illustrating this interesting phenomenon. During replication, their genomic +sRNA is employed as template for translation of viral proteins; among them the RNA-dependent RNA polymerase (RdRp) is responsible of viral genome replication originating double-strand RNA molecules (dsRNA) as intermediates, which accumulate representing a potent threat for cellular dsRNA receptors to initiate an antiviral response. A common feature shared by these viruses is their ability to rearrange cellular membranes to serve as platforms for genome replication and assembly of new virions, supporting replication efficiency increase by concentrating critical factors and protecting the viral genome from host anti-viral systems. This review summarizes current knowledge regarding cellular dsRNA receptors and describes prototype viruses developing replication niches inside rearranged membranes. However, for several viral agents it's been observed both, a complex rearrangement of cellular membranes and a strong innate immune antiviral response induction. So, we have included recent data explaining the mechanism by, even though viruses have evolved elegant hideouts, host cells are still able to develop dsRNA receptors-dependent antiviral response. PMID:28164038

  3. Role of RNA Structures in Genome Terminal Sequences of the Hepatitis C Virus for Replication and Assembly▿ †

    Science.gov (United States)

    Friebe, Peter; Bartenschlager, Ralf

    2009-01-01

    Hepatitis C virus (HCV) is a positive-strand RNA virus replicating its genome via a negative-strand [(−)] intermediate. Little is known about replication signals residing in the 3′ end of HCV (−) RNA. Recent studies identified seven stem-loop structures (SL-I′, -IIz′, -IIy′, -IIIa′, -IIIb′, -IIIcdef′, and -IV′) in this region. In the present study, we mapped the minimal region required for RNA replication to SL-I′ and -IIz′, functionally confirmed the SL-IIz′ structure, and identified SL-IIIa′ to -IV′ as auxiliary replication elements. In addition, we show that the 5′ nontranslated region of the genome most likely does not contain cis-acting RNA structures required for RNA packaging into infectious virions. PMID:19740989

  4. Role of RNA structures in genome terminal sequences of the hepatitis C virus for replication and assembly.

    Science.gov (United States)

    Friebe, Peter; Bartenschlager, Ralf

    2009-11-01

    Hepatitis C virus (HCV) is a positive-strand RNA virus replicating its genome via a negative-strand [(-)] intermediate. Little is known about replication signals residing in the 3' end of HCV (-) RNA. Recent studies identified seven stem-loop structures (SL-I', -IIz', -IIy', -IIIa', -IIIb', -IIIcdef', and -IV') in this region. In the present study, we mapped the minimal region required for RNA replication to SL-I' and -IIz', functionally confirmed the SL-IIz' structure, and identified SL-IIIa' to -IV' as auxiliary replication elements. In addition, we show that the 5' nontranslated region of the genome most likely does not contain cis-acting RNA structures required for RNA packaging into infectious virions.

  5. Discovery of replicating circular RNAs by RNA-seq and computational algorithms.

    Directory of Open Access Journals (Sweden)

    Zhixiang Zhang

    2014-12-01

    Full Text Available Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR. However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd and Apple hammerhead viroid-like RNA (AHVd-like RNA, respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small

  6. Discovery of replicating circular RNAs by RNA-seq and computational algorithms.

    Science.gov (United States)

    Zhang, Zhixiang; Qi, Shuishui; Tang, Nan; Zhang, Xinxin; Chen, Shanshan; Zhu, Pengfei; Ma, Lin; Cheng, Jinping; Xu, Yun; Lu, Meiguang; Wang, Hongqing; Ding, Shou-Wei; Li, Shifang; Wu, Qingfa

    2014-12-01

    Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.

  7. Discovery of replicating circular RNAs by RNA-seq and computational algorithms.

    Directory of Open Access Journals (Sweden)

    Zhixiang Zhang

    2014-12-01

    Full Text Available Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR. However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd and Apple hammerhead viroid-like RNA (AHVd-like RNA, respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small

  8. PEGylated poly(ethylene imine) copolymer-delivered siRNA inhibits HIV replication in vitro.

    Science.gov (United States)

    Weber, Nick D; Merkel, Olivia M; Kissel, Thomas; Muñoz-Fernández, María Ángeles

    2012-01-10

    RNA interference is increasingly being utilized for the specific targeting and down-regulation of disease-causing genes, including targeting viral infections such as HIV. T lymphocytes, the primary target for HIV, are very difficult to treat with gene therapy applications such as RNA interference because of issues with drug delivery. To circumvent these problems, we investigated poly(ethylene imine) (PEI) as a method of improving transfection efficiency of siRNA to T lymphocytes. Additionally, polyethylene glycol (PEG) moieties were engrafted to the PEI polymers with the goals of improving stability and reducing cytotoxicity. Initial studies on PEG-PEI/siRNA polyplex formation, size and their interaction with cell membranes demonstrated their feasibility as drug delivery agents. Assays with lymphocytes revealed low cytotoxicity profiles of the polyplexes at pharmacologically relevant concentrations with PEGylated copolymers obtaining the best results. Successful transfection of a T cell line or primary T cells with siRNA was observed via flow cytometry and confocal microscopy. Finally, the biological effect of copolymer-delivered siRNA was measured. Of particular significance, siRNA targeted to the HIV gene nef and delivered by one of the PEG-PEI copolymers in repetitive treatments every 2-3 days was observed to inhibit HIV replication to the same extent as azidothymidine over the course of 15 days. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. IFNγ inhibits the cytosolic replication of Shigella flexneri via the cytoplasmic RNA sensor RIG-I.

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    Stephanie P Jehl

    Full Text Available The activation of host cells by interferon gamma (IFNγ is essential for inhibiting the intracellular replication of most microbial pathogens. Although significant advances have been made in identifying IFNγ-dependent host factors that suppress intracellular bacteria, little is known about how IFNγ enables cells to recognize, or restrict, the growth of pathogens that replicate in the host cytoplasm. The replication of the cytosolic bacterial pathogen Shigella flexneri is significantly inhibited in IFNγ-stimulated cells, however the specific mechanisms that mediate this inhibition have remained elusive. We found that S. flexneri efficiently invades IFNγ-activated mouse embryonic fibroblasts (MEFs and escapes from the vacuole, suggesting that IFNγ acts by blocking S. flexneri replication in the cytosol. This restriction on cytosolic growth was dependent on interferon regulatory factor 1 (IRF1, an IFNγ-inducible transcription factor capable of inducing IFNγ-mediated cell-autonomous immunity. To identify host factors that restrict S. flexneri growth, we used whole genome microarrays to identify mammalian genes whose expression in S. flexneri-infected cells is controlled by IFNγ and IRF1. Among the genes we identified was the pattern recognition receptor (PRR retanoic acid-inducible gene I (RIG-I, a cytoplasmic sensor of foreign RNA that had not been previously known to play a role in S. flexneri infection. We found that RIG-I and its downstream signaling adaptor mitochondrial antiviral signaling protein (MAVS--but not cytosolic Nod-like receptors (NLRs--are critically important for IFNγ-mediated S. flexneri growth restriction. The recently described RNA polymerase III pathway, which transcribes foreign cytosolic DNA into the RIG-I ligand 5'-triphosphate RNA, appeared to be involved in this restriction. The finding that RIG-I responds to S. flexneri infection during the IFNγ response extends the range of PRRs that are capable of recognizing

  10. Post-transcriptional inhibition of hepatitis C virus replication through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-03-01

    Full Text Available Abstract Background Hepatitis C Virus (HCV infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN α and ribavirin. This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV. Results This study was design to examine the ability of exogenous small interfering RNAs (siRNAs to block the replication of HCV in human liver cells. In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase. siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064. We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858 showed 58% and 88% reduction in viral titer respectively. Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 μM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.

  11. MicroRNA Regulation of Human Genes Essential for Influenza A (H7N9) Replication

    Science.gov (United States)

    Wolf, Stefan; Wu, Weilin; Jones, Cheryl; Perwitasari, Olivia; Mahalingam, Suresh; Tripp, Ralph A.

    2016-01-01

    Influenza A viruses are important pathogens of humans and animals. While seasonal influenza viruses infect humans every year, occasionally animal-origin viruses emerge to cause pandemics with significantly higher morbidity and mortality rates. In March 2013, the public health authorities of China reported three cases of laboratory confirmed human infection with avian influenza A (H7N9) virus, and subsequently there have been many cases reported across South East Asia and recently in North America. Most patients experience severe respiratory illness, and morbidity with mortality rates near 40%. No vaccine is currently available and the use of antivirals is complicated due the frequent emergence of drug resistant strains. Thus, there is an imminent need to identify new drug targets for therapeutic intervention. In the current study, a high-throughput screening (HTS) assay was performed using microRNA (miRNA) inhibitors to identify new host miRNA targets that reduce influenza H7N9 replication in human respiratory (A549) cells. Validation studies lead to a top hit, hsa-miR-664a-3p, that had potent antiviral effects in reducing H7N9 replication (TCID50 titers) by two logs. In silico pathway analysis revealed that this microRNA targeted the LIF and NEK7 genes with effects on pro-inflammatory factors. In follow up studies using siRNAs, anti-viral properties were shown for LIF. Furthermore, inhibition of hsa-miR-664a-3p also reduced virus replication of pandemic influenza A strains H1N1 and H3N2. PMID:27166678

  12. Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis

    Science.gov (United States)

    Shakeel, Shabih; Westerhuis, Brenda M.; Domanska, Ausra; Koning, Roman I.; Matadeen, Rishi; Koster, Abraham J.; Bakker, Arjen Q.; Beaumont, Tim; Wolthers, Katja C.; Butcher, Sarah J.

    2016-07-01

    The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in complex with human monoclonal antibody Fabs demonstrates the expected picornavirus capsid structure with three distinct features. First, 25% of the HPeV3 RNA genome in 60 sites is highly ordered as confirmed by asymmetric reconstruction, and interacts with conserved regions of the capsid proteins VP1 and VP3. Second, the VP0 N terminus stabilizes the capsid inner surface, in contrast to other picornaviruses where on expulsion as VP4, it forms an RNA translocation channel. Last, VP1's hydrophobic pocket, the binding site for the antipicornaviral drug, pleconaril, is blocked and thus inappropriate for antiviral development. Together, these results suggest a direction for development of neutralizing antibodies, antiviral drugs based on targeting the RNA-protein interactions and dissection of virus assembly on the basis of RNA nucleation.

  13. Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells

    OpenAIRE

    Liu, Yu-Tsueng; Brazas, Rob; Ganem, Don

    2001-01-01

    Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian...

  14. High diversity of picornaviruses in rats from different continents revealed by deep sequencing

    DEFF Research Database (Denmark)

    Arn Hansen, Thomas; Mollerup, Sarah; Nguyen, Nam-Phuong

    2016-01-01

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus......) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus...

  15. RNA helicase DDX3: at the crossroad of viral replication and antiviral immunity.

    Science.gov (United States)

    Valiente-Echeverría, Fernando; Hermoso, Marcela A; Soto-Rifo, Ricardo

    2015-09-01

    Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3, or DDX3, belongs to the DEAD-box family of ATP-dependent RNA helicases and is known to play different roles in RNA metabolism ranging from transcription to nuclear export, translation, and assembly of stress granules. In addition, there is growing evidence that DDX3 is a component of the innate immune response against viral infections. As such, DDX3 has been shown to play roles both upstream and downstream of I-kappa beta kinase ε (IKKε)/TANK-binding kinase 1, leading to IFN-β production. Interestingly, several RNA viruses, including human threats such as HIV-1 and hepatitis C virus, hijack DDX3 to accomplish various steps of their replication cycles. Thus, it seems that viruses have evolved to exploit DDX3's functions while threatening the innate immune response. Understanding this interesting dichotomy in DDX3 function will help us not only to improve our knowledge of virus-host interactions but also to develop novel antiviral drugs targeting the multifaceted roles of DDX3 in viral replication.

  16. Phosphorylation of hepatitis C virus RNA polymerases ser29 and ser42 by protein kinase C-related kinase 2 regulates viral RNA replication.

    Science.gov (United States)

    Han, Song-Hee; Kim, Seong-Jun; Kim, Eun-Jung; Kim, Tae-Eun; Moon, Jae-Su; Kim, Geon-Woo; Lee, Seung-Hoon; Cho, Kun; Yoo, Jong Shin; Son, Woo Sung; Rhee, Jin-Kyu; Han, Seung Hyun; Oh, Jong-Won

    2014-10-01

    Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified

  17. Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Takegami, T.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.

  18. Structural Features of the Seneca Valley Virus Internal Ribosome Entry Site (IRES) Element: a Picornavirus with a Pestivirus-Like IRES

    DEFF Research Database (Denmark)

    Willcocks, Margaret M.; Locker, Nicolas; Gomwalk, Zarmwa

    2011-01-01

    The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the FLAVIVIRIDAE: The SVV IRES ...

  19. Structural Features of the Seneca Valley Virus Internal Ribosome Entry Site (IRES) Element: a Picornavirus with a Pestivirus-Like IRES

    DEFF Research Database (Denmark)

    Willcocks, Margaret M.; Locker, Nicolas; Gomwalk, Zarmwa

    2011-01-01

    The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the FLAVIVIRIDAE: The SVV IRES...

  20. Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14.

    Science.gov (United States)

    Yang, Yan; Rijnbrand, Rene; McKnight, Kevin L; Wimmer, Eckard; Paul, Aniko; Martin, Annette; Lemon, Stanley M

    2002-08-01

    Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA

  1. In vitro and in vivo characterization of microRNA-targeted alphavirus replicon and helper RNAs.

    Science.gov (United States)

    Kamrud, Kurt I; Coffield, V McNeil; Owens, Gary; Goodman, Christin; Alterson, Kim; Custer, Max; Murphy, Michael A; Lewis, Whitney; Timberlake, Sarah; Wansley, Elizabeth K; Berglund, Peter; Smith, Jonathan

    2010-08-01

    Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.

  2. Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes

    Directory of Open Access Journals (Sweden)

    Tiley Laurence S

    2006-08-01

    Full Text Available Abstract Background The RNA-dependent RNA polymerase of Influenza A virus is a determinant of viral pathogenicity and host range that is responsible for transcribing and replicating the negative sense segmented viral genome (vRNA. Transcription produces capped and polyadenylated mRNAs whereas genome replication involves the synthesis of an alternative plus-sense transcript (cRNA with unmodified termini that is copied back to vRNA. Viral mRNA transcription predominates at early stages of viral infection, while later, negative sense genome replication is favoured. However, the "switch" that regulates the transition from transcription to replication is poorly understood. Results We show that temperature strongly affects the balance between plus and minus-sense RNA synthesis with high temperature causing a large decrease in vRNA accumulation, a moderate decrease in cRNA levels but (depending on genome segment either increased or unchanged levels of mRNA. We found no evidence implicating cellular heat shock protein activity in this effect despite the known association of hsp70 and hsp90 with viral polymerase components. Temperature-shift experiments indicated that polymerase synthesised at 41°C maintained transcriptional activity even though genome replication failed. Reduced polymerase association with viral RNA was seen in vivo and in confirmation of this, in vitro binding assays showed that temperature increased the rate of dissociation of polymerase from both positive and negative sense promoters. However, the interaction of polymerase with the cRNA promoter was particularly heat labile, showing rapid dissociation even at 37°C. This suggested that vRNA synthesis fails at elevated temperatures because the polymerase does not bind the promoter. In support of this hypothesis, a mutant cRNA promoter with vRNA-like sequence elements supported vRNA synthesis at higher temperatures than the wild-type promoter. Conclusion The differential stability of

  3. Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.

    Directory of Open Access Journals (Sweden)

    Benjamin G Kopek

    2007-09-01

    Full Text Available Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV-infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside approximately 50-nm vesicles (spherules, which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of approximately 100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

  4. Zika Virus RNA Replication and Persistence in Brain and Placental Tissue

    Science.gov (United States)

    Rabeneck, Demi B.; Martines, Roosecelis B.; Reagan-Steiner, Sarah; Ermias, Yokabed; Estetter, Lindsey B.C.; Suzuki, Tadaki; Ritter, Jana; Keating, M. Kelly; Hale, Gillian; Gary, Joy; Muehlenbachs, Atis; Lambert, Amy; Lanciotti, Robert; Oduyebo, Titilope; Meaney-Delman, Dana; Bolaños, Fernando; Saad, Edgar Alberto Parra; Shieh, Wun-Ju; Zaki, Sherif R.

    2017-01-01

    Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients (brains from 8 infants with microcephaly and placental/fetal tissues from 24 women) were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections. PMID:27959260

  5. Adenosine deaminase acting on RNA-1 (ADAR1 inhibits HIV-1 replication in human alveolar macrophages.

    Directory of Open Access Journals (Sweden)

    Michael D Weiden

    Full Text Available While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1 in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages.

  6. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Science.gov (United States)

    Ylösmäki, Erkko; Lavilla-Alonso, Sergio; Jäämaa, Sari; Vähä-Koskela, Markus; af Hällström, Taija; Hemminki, Akseli; Arola, Johanna; Mäkisalo, Heikki; Saksela, Kalle

    2013-01-01

    MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  7. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Directory of Open Access Journals (Sweden)

    Erkko Ylösmäki

    Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  8. MicroRNA-141 Targets Sirt1 and Inhibits Autophagy to Reduce HBV Replication.

    Science.gov (United States)

    Yang, Ying; Liu, Yanning; Xue, Jihua; Yang, Zhenggang; Shi, Yu; Shi, Yixian; Lou, Guohua; Wu, Shanshan; Qi, Jinjin; Liu, Weixia; Chen, Zhi; Wang, Jing

    2017-01-01

    About 400 million individuals are chronically infected with hepatitis B virus, at high risk of developing liver cirrhosis and hepatocellular carcinoma. Recent studies have demonstrated an interaction between hepatitis B virus replication and autophagy activity of hepatocytes. In the present study, we aimed to investigate the role of miR-141 in regulating autophagy and hepatitis B virus replication. The expression of HBV-DNA, miR-141 and Sirt1 mRNA was determined by quantitative real-time PCR analysis. The expression of HBsAg and HBeAg was determined by ELISA. Western blotting was performed to detect protein expression. The LC3 puncta was determined by immunofluorescence. To test whether miR-141 directly regulate the expression level of Sirt1 mRNA, dual-luciferase reporter gene assay was performed. In vitro studies showed that miR-141 mimic inhibited the autophagic response, hepatitis B virus and the expression of Sirt1 in hepatocytes. And transfection with miR-141 inhibitor enhanced autophagic response and Sirt1 expression. The autophagy induced by overexpression of Sirt1 was inhibited by miR-141 mimic. In addition, miR-141 mimic also decreased the expression of Sirt1 mRNA. Sirt1 was predicted as a potential miR-141 target by bioinformatic analysis of its 3'-UTR, and confirmed by luciferase reporter assays which analyzing the interaction of miR-141 with the wild- type or the mutated Sirt1 3'-UTR. We have therefore demonstrated a role of miR-141 in regulating autophagy-mediated hepatitis B virus inhibition by targeting Sirt1, and may provide potential targets for drug development. © 2017 The Author(s) Published by S. Karger AG, Basel.

  9. MicroRNA-141 Targets Sirt1 and Inhibits Autophagy to Reduce HBV Replication

    Directory of Open Access Journals (Sweden)

    Ying Yang

    2017-01-01

    Full Text Available Background/Aims: About 400 million individuals are chronically infected with hepatitis B virus, at high risk of developing liver cirrhosis and hepatocellular carcinoma. Recent studies have demonstrated an interaction between hepatitis B virus replication and autophagy activity of hepatocytes. In the present study, we aimed to investigate the role of miR-141 in regulating autophagy and hepatitis B virus replication. Methods: The expression of HBV-DNA, miR-141 and Sirt1 mRNA was determined by quantitative real-time PCR analysis. The expression of HBsAg and HBeAg was determined by ELISA. Western blotting was performed to detect protein expression. The LC3 puncta was determined by immunofluorescence. To test whether miR-141 directly regulate the expression level of Sirt1 mRNA, dual-luciferase reporter gene assay was performed. Results: In vitro studies showed that miR-141 mimic inhibited the autophagic response, hepatitis B virus and the expression of Sirt1 in hepatocytes. And transfection with miR-141 inhibitor enhanced autophagic response and Sirt1 expression. The autophagy induced by overexpression of Sirt1 was inhibited by miR-141 mimic. In addition, miR-141 mimic also decreased the expression of Sirt1 mRNA. Sirt1 was predicted as a potential miR-141 target by bioinformatic analysis of its 3'-UTR, and confirmed by luciferase reporter assays which analyzing the interaction of miR-141 with the wild- type or the mutated Sirt1 3’-UTR. Conclusion: We have therefore demonstrated a role of miR-141 in regulating autophagy-mediated hepatitis B virus inhibition by targeting Sirt1, and may provide potential targets for drug development.

  10. Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

    Directory of Open Access Journals (Sweden)

    Geiss Brian J

    2009-03-01

    Full Text Available Abstract Background Arthropod-borne viruses (arboviruses can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus, a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition. Results B2 protein expression from SINV (TE/3'2J inhibited the accumulation of non-specific small RNAs in Aedes aegypti mosquito cell culture and virus-specific small RNAs both in infected cell culture and Ae. aegypti mosquitoes. More viral genomic and subgenomic RNA accumulated in cells and mosquitoes infected with TE/3'2J virus expressing B2 (TE/3'2J/B2 compared to TE/3'2J and TE/3'2J virus expressing GFP. TE/3'2J/B2 exhibited increased infection rates, dissemination rates, and infectious virus titers in mosquitoes following oral bloodmeal. Following infectious oral bloodmeal, significantly more mosquitoes died when TE/3'2J/B2 was ingested. The virus was 100% lethal following intrathoracic inoculation of multiple mosquito species and lethality was dose-dependent in Ae. aegypti. Conclusion We show that RNAi is active in Ae. aegypti cell culture and that B2 protein inhibits RNAi in mosquito cells when expressed by a recombinant SINV. Also, SINV more efficiently replicates in mosquito cells when RNAi is inhibited. Finally, TE/3'2J/B2 kills mosquitoes in a dose-dependent manner independent of infection route and mosquito species.

  11. Quantitative and integrated proteome and microRNA analysis of endothelial replicative senescence.

    Science.gov (United States)

    Yentrapalli, Ramesh; Azimzadeh, Omid; Kraemer, Anne; Malinowsky, Katharina; Sarioglu, Hakan; Becker, Karl-Friedrich; Atkinson, Michael J; Moertl, Simone; Tapio, Soile

    2015-08-01

    Age-related changes in vascular functioning are a harbinger of cardiovascular disease but the biological mechanisms during the progression of endothelial senescence have not been studied. We investigated alterations in the proteome and miRNA profiles in the course of replicative senescence using primary human umbilical vein endothelial cells as an in vitro vascular model. Quantitative proteomic profiling from early growth stage to senescence was performed by isotope-coded protein label coupled to LC-ESI-MS/MS analysis. Some proteins consistently changed their expression during the senescence whereas others appeared as deregulated only during the late senescence. The latter was accompanied by alterations in morphology of senescent endothelial cells. MicroRNA expression profiling revealed transient changes in the level of miR-16-5p, miR-28-3p and miR-886-5p in the early senescence, decrease in the level of miR-106b-3p at the late stage, and continuous changes in the expression of miR-181a-5p and miR-376a-3p during the whole senescence process. Integrating data on proteomic and microRNA changes indicated potential crosstalk between specific proteins and non-coding RNAs in the regulation of metabolism, cell cycle progression and cytoskeletal organization in the endothelial senescence. The knowledge of molecular targets that change during the senescence can ultimately contribute to a better understanding and prevention of age-related vascular diseases.

  12. shRNA-armed conditionally replicative adenoviruses: a promising approach for cancer therapy

    Science.gov (United States)

    Zhang, Jie; Ding, Meng; Xu, Kai; Mao, Lijun; Zheng, Junian

    2016-01-01

    The small-interfering RNAs (siRNAs) have been employed to knockdown the expression of cancer-associated genes and shown some promise in cancer therapy. However, synthetic siRNA duplexes or plasmid mediated delivery of siRNAs have several problems, such as short half-life, low transfection efficiency and cytotoxicity associated with transfection. Conditionally replicating adenovirus (CRAds) as the delivery vector for short hairpin RNAs (shRNAs) could overcome these limitations and have shown augmented anti-tumor effects in experimental studies and preclinical trials. In this review, we summarize recent progress in the development of CRAds-shRNA for cancer treatment. Combination of CRAds-shRNA with chemotherapeutics, radiation, dendritic cells, monoclonal antibodies and small-molecule inhibitors will be necessary to eradicate cancer cells and cancer stem cells and achieve superior outcomes. The use of CRAd platform for efficient delivery of shRNAs and foreign genes will open a new avenue for cancer therapy. PMID:26980708

  13. The DNA/RNA-dependent RNA polymerase QDE-1 generates aberrant RNA and dsRNA for RNAi in a process requiring replication protein A and a DNA helicase.

    Directory of Open Access Journals (Sweden)

    Heng-Chi Lee

    Full Text Available The production of aberrant RNA (aRNA is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs to generate double-stranded RNA (dsRNA is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP. Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA, a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.

  14. The evolution of strand preference in simulated RNA replicators with strand displacement: implications for the origin of transcription.

    Science.gov (United States)

    Takeuchi, Nobuto; Salazar, Laura; Poole, Anthony M; Hogeweg, Paulien

    2008-08-11

    The simplest conceivable example of evolving systems is RNA molecules that can replicate themselves. Since replication produces a new RNA strand complementary to a template, all templates would eventually become double-stranded and, hence, become unavailable for replication. Thus the problem of how to separate the two strands is considered a major issue for the early evolution of self-replicating RNA. One biologically plausible way to copy a double-stranded RNA is to displace a preexisting strand by a newly synthesized strand. Such copying can in principle be initiated from either the (+) or (-) strand of a double-stranded RNA. Assuming that only one of them, say (+), can act as replicase when single-stranded, strand displacement produces a new replicase if the (-) strand is the template. If, however, the (+) strand is the template, it produces a new template (but no replicase). Modern transcription exhibits extreme strand preference wherein anti-sense strands are always the template. Likewise, replication by strand displacement seems optimal if it also exhibits extreme strand preference wherein (-) strands are always the template, favoring replicase production. Here we investigate whether such strand preference can evolve in a simple RNA replicator system with strand displacement. We first studied a simple mathematical model of the replicator dynamics. Our results indicated that if the system is well-mixed, there is no selective force acting upon strand preference per se. Next, we studied an individual-based simulation model to investigate the evolution of strand preference under finite diffusion. Interestingly, the results showed that selective forces "emerge" because of finite diffusion. Strikingly, the direction of the strand preference that evolves [i.e. (+) or (-) strand excess] is a complex non-monotonic function of the diffusion intensity. The mechanism underlying this behavior is elucidated. Furthermore, a speciation-like phenomenon is observed under

  15. The evolution of strand preference in simulated RNA replicators with strand displacement: Implications for the origin of transcription

    Directory of Open Access Journals (Sweden)

    Poole Anthony M

    2008-08-01

    Full Text Available Abstract Background The simplest conceivable example of evolving systems is RNA molecules that can replicate themselves. Since replication produces a new RNA strand complementary to a template, all templates would eventually become double-stranded and, hence, become unavailable for replication. Thus the problem of how to separate the two strands is considered a major issue for the early evolution of self-replicating RNA. One biologically plausible way to copy a double-stranded RNA is to displace a preexisting strand by a newly synthesized strand. Such copying can in principle be initiated from either the (+ or (- strand of a double-stranded RNA. Assuming that only one of them, say (+, can act as replicase when single-stranded, strand displacement produces a new replicase if the (- strand is the template. If, however, the (+ strand is the template, it produces a new template (but no replicase. Modern transcription exhibits extreme strand preference wherein anti-sense strands are always the template. Likewise, replication by strand displacement seems optimal if it also exhibits extreme strand preference wherein (- strands are always the template, favoring replicase production. Here we investigate whether such strand preference can evolve in a simple RNA replicator system with strand displacement. Results We first studied a simple mathematical model of the replicator dynamics. Our results indicated that if the system is well-mixed, there is no selective force acting upon strand preference per se. Next, we studied an individual-based simulation model to investigate the evolution of strand preference under finite diffusion. Interestingly, the results showed that selective forces "emerge" because of finite diffusion. Strikingly, the direction of the strand preference that evolves [i.e. (+ or (- strand excess] is a complex non-monotonic function of the diffusion intensity. The mechanism underlying this behavior is elucidated. Furthermore, a speciation

  16. Alterations in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Zeichner Steven L

    2004-12-01

    Full Text Available Abstract Recent results showed that certain DEAD box protein RNA helicases, DDX3 and DDX1, play an important role in the HIV infection cycle by facilitating the export of long, singly spliced or unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Close examination of an extensive microarray expression profiling dataset obtained from cells latently infected with HIV induced to undergo lytic viral replication indicated that additional DEAD box proteins, beyond DDX3 and DDX1, exhibit differential expression during lytic HIV replication, and in latently infected cells prior to induction into active replication. This finding provides additional evidence that the involvement of DEAD box proteins and other RNA-binding proteins may play roles in active HIV replication and in the control of viral latency. Agents targeting these functions may offer new approaches to antiretroviral therapy and the therapeutic manipulation of HIV latency.

  17. SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

    Science.gov (United States)

    Han, Yang; Wang, Lvyin; Cui, Jin; Song, Yu; Luo, Zhen; Chen, Junbo; Xiong, Ying; Zhang, Qi; Liu, Fang; Ho, Wenzhe; Liu, Yingle; Wu, Jianguo

    2016-01-01

    ABSTRACT Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5′ untranslated region (5′UTR) and a polyadenylated 3′UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3Dpol protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3Dpol, resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5′UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5′UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication. PMID:27875274

  18. Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells

    Science.gov (United States)

    Dixit, Updesh; Pandey, Ashutosh K.; Mishra, Priya; Sengupta, Amitabha; Pandey, Virendra N.

    2016-01-01

    Persistent hepatitis C virus (HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish CHC and cause its subsequent development into LC and HCC are poorly understood. We have identified a cytoplasmic double-stranded RNA binding protein, Stau1, which is crucial for HCV replication. In this study, Stau1 specifically interacted with the variable-stem-loop region in the 3′ NTR and domain IIId of the HCV-IRES in the 5′ NTR, and promoted HCV replication and translation. Stau1 coimmunoprecipitates HCV NS5B and a cell factor, protein kinase R (PKR), which is critical for interferon-induced cellular antiviral and antiproliferative responses. Like Stau1, PKR displayed binding specificity to domain IIId of HCV-IRES. Stau1 binds to PKR and strongly inhibits PKR-autophosphorylation. We demonstrated that the transport of HCV RNA on the polysomes is Stau1-dependent, being mainly localized in the monosome fractions when Stau1 is downregulated and exclusively localized in the polysomes when Stau1 is overexpressed. Our findings suggest that HCV may appropriate Stau1 to its advantage to prevent PKR-mediated inhibition of eIF2α, which is required for the synthesis of HCV proteins for translocation of viral RNA genome to the polysomes for efficient translation and replication. PMID:27106056

  19. A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo

    Institute of Scientific and Technical Information of China (English)

    Xue-Song Liang; Jian-Qi Lian; Yong-Xing Zhou; Qing-He Nie; Chun-Qiu Hao

    2003-01-01

    AIM: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA).METHODS: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was tansfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccinas line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay.RESULTS: Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated capindependent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus.CONCLUSION: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication.

  20. Rescue of foot-and-mouth disease viruses that are pathogenic for cattle from preserved viral RNA samples

    DEFF Research Database (Denmark)

    Belsham, Graham; Jamal, Syed Muhammad; Tjørnehøj, Kirsten;

    2011-01-01

    Background: Foot and mouth disease is an economically important disease of cloven-hoofed animals including cattle, sheep and pigs. It is caused by a picornavirus, foot-and-mouth disease virus (FMDV), which has a positive sense RNA genome which, when introduced into cells, can initiate virus...... replication. Principal Findings: A system has been developed to rescue infectious FMDV from RNA preparations generated from clinical samples obtained under experimental conditions and then applied to samples collected in the ‘‘field’’. Clinical samples from suspect cases of foot-and-mouth disease (FMD) were...

  1. Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, "UCAA-motif" RNA Aptamers.

    Science.gov (United States)

    Whatley, Angela S; Ditzler, Mark A; Lange, Margaret J; Biondi, Elisa; Sawyer, Andrew W; Chang, Jonathan L; Franken, Joshua D; Burke, Donald H

    2013-02-05

    RNA aptamers that bind the reverse transcriptase (RT) of human immunodeficiency virus (HIV) compete with nucleic acid primer/template for access to RT, inhibit RT enzymatic activity in vitro, and suppress viral replication when expressed in human cells. Numerous pseudoknot aptamers have been identified by sequence analysis, but relatively few have been confirmed experimentally. In this work, a screen of nearly 100 full-length and >60 truncated aptamer transcripts established the predictive value of the F1Pk and F2Pk pseudoknot signature motifs. The screen also identified a new, nonpseudoknot motif with a conserved unpaired UCAA element. High-throughput sequence (HTS) analysis identified 181 clusters capable of forming this novel element. Comparative sequence analysis, enzymatic probing and RT inhibition by aptamer variants established the essential requirements of the motif, which include two conserved base pairs (AC/GU) on the 5' side of the unpaired UCAA. Aptamers in this family inhibit RT in primer extension assays with IC(50) values in the low nmol/l range, and they suppress viral replication with a potency that is comparable with that of previously studied aptamers. All three known anti-RT aptamer families (pseudoknots, the UCAA element, and the recently described "(6/5)AL" motif) are therefore suitable for developing aptamer-based antiviral gene therapies.Molecular Therapy - Nucleic Acids (2013) 2, e71; doi:10.1038/mtna.2012.62; published online 5 February 2013.

  2. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening.

    Science.gov (United States)

    Trotard, Maud; Lepère-Douard, Charlotte; Régeard, Morgane; Piquet-Pellorce, Claire; Lavillette, Dimitri; Cosset, François-Loic; Gripon, Philippe; Le Seyec, Jacques

    2009-11-01

    The entry pathway of the hepatitis C virus (HCV), a major human pathogen, into the cell is incompletely defined. To better characterize this viral life cycle stage, we screened a small interfering RNA library dedicated to the membrane trafficking and remodeling with the infection model of Huh-7.5.1 cells by HCV pseudoparticles (HCVpp). Results showed that the down-regulation of different factors implied in clathrin-mediated endocytosis (CME) inhibits HCVpp cell infection. In addition, knockdown of the phosphatidylinositol 4-kinase type III-alpha (PI4KIIIalpha) prevented infection by HCVpp or by cell-culture grown JFH-1-based HCV. Moreover, the replication activity of an HCV replicon was also affected by the PI4KIIIalpha knockdown. Additional investigations on the different members of the PI4K family revealed that the presence of PI4KIIIbeta in the host cells influenced their susceptibility to HCVpp infection and their capacity to sustain the HCV replication. The PI4KIII involvement during the HCV life cycle seemed to occur by other ways than the control of the CME or of the membranous expression of HCV receptors. Finally, our library screening completed data on the CME-dependant entry route of HCV and identified 2 kinases, PI4KIIIalpha and beta, as relevant potential therapeutic targets.

  3. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

    Energy Technology Data Exchange (ETDEWEB)

    Bienz, K.; Egger, D.; Troxler, M.; Pasamontes, L. (Univ. of Basel (Switzerland))

    1990-03-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.

  4. The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms.

    Science.gov (United States)

    Jungfleisch, Jennifer; Chowdhury, Ashis; Alves-Rodrigues, Isabel; Tharun, Sundaresan; Díez, Juana

    2015-08-01

    The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.

  5. Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

    Directory of Open Access Journals (Sweden)

    Stephanie M Rainey

    2016-04-01

    Full Text Available The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus. Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to

  6. Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

    Science.gov (United States)

    Rainey, Stephanie M; Martinez, Julien; McFarlane, Melanie; Juneja, Punita; Sarkies, Peter; Lulla, Aleksei; Schnettler, Esther; Varjak, Margus; Merits, Andres; Miska, Eric A; Jiggins, Francis M; Kohl, Alain

    2016-04-01

    The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced

  7. Hepatitis A virus and the origins of picornaviruses

    Science.gov (United States)

    Hu, Zhongyu; Sun, Yao; Li, Xuemei; Rowlands, David J.; Yin, Weidong; Wang, Junzhi; Stuart, David I.; Rao, Zihe; Fry, Elizabeth E.

    2016-01-01

    Hepatitis A virus (HAV) remains enigmatic, despite some 1.4 million cases worldwide annually1. It differs radically from other picornaviruses, existing in an enveloped form2 and being unusually stable, both genetically and physically3, but has proved difficult to study. We report high-resolution X-ray structures for the mature virus and empty particles. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, while the empty particle harbors only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions which might correspond to receptor binding sites. Peptide scanning data extends the previously reported VP3 antigenic site4, while structure-based predictions5 suggest further epitopes. HAV contains no pocket factor, can withstand remarkably high temperature and low pH, with empty particles being even more robust than full particles. The virus probably uncoats via a novel mechanism, being built differently to other picornaviruses. It utilizes a VP2 ‘domain swap’ characteristic of insect picorna-like viruses6,7 and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between ‘modern’ picornaviruses and the more ‘primitive’ precursor insect viruses, for instance HAV retains the ability to move from cell-to-cell by transcytosis8,9. PMID:25327248

  8. Traffic jam on the cellular secretory pathway generated by a replication protein from a plant RNA virus.

    Science.gov (United States)

    Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

    2014-01-01

    Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a plant RNA virus, the Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on plant-virus interaction are discussed.

  9. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S. [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa); Arbuthnot, Patrick, E-mail: Patrick.Arbuthnot@wits.ac.za [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)

    2009-11-20

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  10. High diversity of picornaviruses in rats from different continents revealed by deep sequencing.

    Science.gov (United States)

    Hansen, Thomas Arn; Mollerup, Sarah; Nguyen, Nam-Phuong; White, Nicole E; Coghlan, Megan; Alquezar-Planas, David E; Joshi, Tejal; Jensen, Randi Holm; Fridholm, Helena; Kjartansdóttir, Kristín Rós; Mourier, Tobias; Warnow, Tandy; Belsham, Graham J; Bunce, Michael; Willerslev, Eske; Nielsen, Lars Peter; Vinner, Lasse; Hansen, Anders Johannes

    2016-08-17

    Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler's encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission.

  11. RNAi suppressor P19 can be broadly exploited for enhanced adenovirus replication and microRNA knockdown experiments.

    Science.gov (United States)

    Rauschhuber, Christina; Mueck-Haeusl, Martin; Zhang, Wenli; Nettelbeck, Dirk M; Ehrhardt, Anja

    2013-01-01

    RNA interference (RNAi) is a key regulator of various biological systems including viral infection. Within a virus life cycle gene products can be modulated by the RNA interference (RNAi) pathway which can crucially impact productive virus replication. Herein we explored the RNA interference suppressor protein P19 derived from a plant virus and we found that P19 enhanced adenovirus replication up to 100-fold. Critical factors responsible for this observation were overexpression of adenovirus encoded genes on mRNA and protein levels. To investigate the impact of this phenomenon on recombinant viruses, we exploited its feasibility for therapeutic and genomic applications. We found that P19 significantly increased recombinant adenovirus yields enabling up-scaling for preclinical and clinical studies. Moreover, adenoviruses possessed significantly higher oncolytic activity by expression of P19. Finally, we show that introducing a p19 expression cassette into high-capacity adenovirus provides a strategy to analyze RNAi knockdown in a tissue-specific manner.

  12. Control of the rescue and replication of Semliki Forest virus recombinants by the insertion of miRNA target sequences.

    Directory of Open Access Journals (Sweden)

    Kaspar Ratnik

    Full Text Available Due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. These properties may, however, be potentially hazardous unless the virus infection is controlled. While the RNA genome of alphaviruses precludes the standard control techniques, host miRNAs can be used to down-regulate viral replication. In this study, target sites from ubiquitous miRNAs and those of miRNAs under-represented in cervical cancer cells were inserted into replication-competent DNA/RNA layered vectors of Semliki Forest virus. It was found that in order to achieve the most efficient suppression of recombinant virus rescue, the introduced target sequences must be fully complementary to those of the corresponding miRNAs. Target sites of ubiquitous miRNAs, introduced into the 3' untranslated region of the viral vector, profoundly reduced the rescue of recombinant viruses. Insertion of the same miRNA targets into coding region of the viral vector was approximately 300-fold less effective. Viruses carrying these miRNAs were genetically unstable and rapidly lost the target sequences. This process was delayed, but not completely prevented, by miRNA inhibitors. Target sites of miRNA under-represented in cervical cancer cells had much smaller but still significant effects on recombinant virus rescue in cervical cancer-derived HeLa cells. Over-expression of miR-214, one of these miRNAs, reduced replication of the targeted virus. Though the majority of rescued viruses maintained the introduced miRNA target sequences, genomes with deletions of these sequences were also detected. Thus, the low-level repression of rescue and replication of targeted virus in HeLa cells was still sufficient to cause genetic instability.

  13. Control of the rescue and replication of Semliki Forest virus recombinants by the insertion of miRNA target sequences.

    Science.gov (United States)

    Ratnik, Kaspar; Viru, Liane; Merits, Andres

    2013-01-01

    Due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. These properties may, however, be potentially hazardous unless the virus infection is controlled. While the RNA genome of alphaviruses precludes the standard control techniques, host miRNAs can be used to down-regulate viral replication. In this study, target sites from ubiquitous miRNAs and those of miRNAs under-represented in cervical cancer cells were inserted into replication-competent DNA/RNA layered vectors of Semliki Forest virus. It was found that in order to achieve the most efficient suppression of recombinant virus rescue, the introduced target sequences must be fully complementary to those of the corresponding miRNAs. Target sites of ubiquitous miRNAs, introduced into the 3' untranslated region of the viral vector, profoundly reduced the rescue of recombinant viruses. Insertion of the same miRNA targets into coding region of the viral vector was approximately 300-fold less effective. Viruses carrying these miRNAs were genetically unstable and rapidly lost the target sequences. This process was delayed, but not completely prevented, by miRNA inhibitors. Target sites of miRNA under-represented in cervical cancer cells had much smaller but still significant effects on recombinant virus rescue in cervical cancer-derived HeLa cells. Over-expression of miR-214, one of these miRNAs, reduced replication of the targeted virus. Though the majority of rescued viruses maintained the introduced miRNA target sequences, genomes with deletions of these sequences were also detected. Thus, the low-level repression of rescue and replication of targeted virus in HeLa cells was still sufficient to cause genetic instability.

  14. Evaluation of canonical siRNA and Dicer substrate RNA for inhibition of hepatitis C virus genome replication--a comparative study.

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    Bruno Carneiro

    Full Text Available Hepatitis C virus (HCV frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis and, potentially, to liver cirrhosis and hepatocellular carcinoma at later stages. The objective of this study was to test the ability of five Dicer substrate siRNAs (DsiRNA to inhibit HCV replication and to compare these molecules to canonical 21 nt siRNA. DsiRNA molecules were designed to target five distinct regions of the HCV genome - the 5' UTR and the coding regions for NS3, NS4B, NS5A or NS5B. These molecules were transfected into Huh7.5 cells that stably harboured an HCV subgenomic replicon expressing a firefly luciferase/neoR reporter (SGR-Feo-JFH-1 and were also tested on HCVcc-infected cells. All of the DsiRNAs inhibited HCV replication in both the subgenomic system and HCVcc-infected cells. When DsiRNAs were transfected prior to infection with HCVcc, the inhibition levels reached 99.5%. When directly compared, canonical siRNA and DsiRNA exhibited similar potency of virus inhibition. Furthermore, both types of molecules exhibited similar dynamics of inhibition and frequencies of resistant mutants after 21 days of treatment. Thus, DsiRNA molecules are as potent as 21 nt siRNAs for the inhibition of HCV replication and may provide future approaches for HCV therapy if the emergence of resistant mutants can be addressed.

  15. MicroRNA-221 modulates RSV replication in human bronchial epithelium by targeting NGF expression.

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    Sreekumar Othumpangat

    Full Text Available BACKGROUND: Early-life infection by respiratory syncytial virus (RSV is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs, and that this effect favors viral growth by interfering with programmed death of infected cells. METHODOLOGY: Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into non-infected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication. PRINCIPAL FINDINGS: RSV caused downregulation of 24 miRNAs and upregulation of 2 (p<0.01. Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75(NTR. Among the selected miRNAs, miR-221 was significantly downregulated by RSV and its transfection in bronchial epithelial cells maximally inhibited gene and protein expression of NGF and TrKA, increased apoptotic cell death, and reduced viral replication and infectivity. CONCLUSIONS/SIGNIFICANCE: Our data suggest that RSV upregulates the NGF-TrKA axis in human airways by silencing miR-221 expression, and this favors viral replication by interfering with the apoptotic death of infected cells. Consequently, the targeted delivery of

  16. Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

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    Gregory Upert

    2012-01-01

    Full Text Available Human immunodeficiency virus-1 (HIV-1 replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR, to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP cation-based vectors that efficiently deliver nucleotide analogs (PNAs into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

  17. Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication.

    Science.gov (United States)

    Nasar, Farooq; Palacios, Gustavo; Gorchakov, Rodion V; Guzman, Hilda; Da Rosa, Amelia P Travassos; Savji, Nazir; Popov, Vsevolod L; Sherman, Michael B; Lipkin, W Ian; Tesh, Robert B; Weaver, Scott C

    2012-09-04

    Most alphaviruses and many other arboviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. Consequently, they can be propagated in most vertebrate and insect cell cultures. This ability of arboviruses to infect arthropods and vertebrates is usually essential for their maintenance in nature. However, several flaviviruses have recently been described that infect mosquitoes but not vertebrates, although the mechanism of their host restriction has not been determined. Here we describe a unique alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani mosquitoes from the Negev desert of Israel. Phylogenetic analyses placed EILV as a sister to the Western equine encephalitis antigenic complex within the main clade of mosquito-borne alphaviruses. Electron microscopy revealed that, like other alphaviruses, EILV virions were spherical, 70 nm in diameter, and budded from the plasma membrane of mosquito cells in culture. EILV readily infected a variety of insect cells with little overt cytopathic effect. However, in contrast to typical mosquito-borne alphaviruses, EILV could not infect mammalian or avian cell lines, and viral as well as RNA replication could not be detected at 37 °C or 28 °C. Evolutionarily, these findings suggest that EILV lost its ability to infect vertebrate cells. Thus, EILV seems to be mosquito-specific and represents a previously undescribed complex within the genus Alphavirus. Reverse genetic studies of EILV may facilitate the discovery of determinants of alphavirus host range that mediate disease emergence.

  18. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

    Science.gov (United States)

    Owen, Carolyn A.; Moukarzel, Romy; Huang, Xiao; Kassem, Mona A.; Eliasco, Eleonora; Aranda, Miguel A.; Coutts, Robert H. A.; Livieratos, Ioannis C.

    2016-01-01

    Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants. PMID:27314380

  19. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

    Directory of Open Access Journals (Sweden)

    Carolyn A. Owen

    2016-06-01

    Full Text Available Cucurbit yellow stunting disorder virus (CYSDV, a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

  20. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts.

    Science.gov (United States)

    Owen, Carolyn A; Moukarzel, Romy; Huang, Xiao; Kassem, Mona A; Eliasco, Eleonora; Aranda, Miguel A; Coutts, Robert H A; Livieratos, Ioannis C

    2016-06-14

    Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5'- and 3'-UTRs plus the 3'-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

  1. Metabolically Coupled Replicator Systems: Overview of an RNA-world model concept of prebiotic evolution on mineral surfaces.

    Science.gov (United States)

    Czárán, Tamás; Könnyű, Balázs; Szathmáry, Eörs

    2015-09-21

    Metabolically Coupled Replicator Systems (MCRS) are a family of models implementing a simple, physico-chemically and ecologically feasible scenario for the first steps of chemical evolution towards life. Evolution in an abiotically produced RNA-population sets in as soon as any one of the RNA molecules become autocatalytic by engaging in template directed self-replication from activated monomers, and starts increasing exponentially. Competition for the finite external supply of monomers ignites selection favouring RNA molecules with catalytic activity helping self-replication by any possible means. One way of providing such autocatalytic help is to become a replicase ribozyme. An additional way is through increasing monomer supply by contributing to monomer synthesis from external resources, i.e., by evolving metabolic enzyme activity. Retroevolution may build up an increasingly autotrophic, cooperating community of metabolic ribozymes running an increasingly complicated and ever more efficient metabolism. Maintaining such a cooperating community of metabolic replicators raises two serious ecological problems: one is keeping the system coexistent in spite of the different replicabilities of the cooperating replicators; the other is constraining parasitism, i.e., keeping "cheaters" in check. Surface-bound MCRS provide an automatic solution to both problems: coexistence and parasite resistance are the consequences of assuming the local nature of metabolic interactions. In this review we present an overview of results published in previous articles, showing that these effects are, indeed, robust in different MCRS implementations, by considering different environmental setups and realistic chemical details in a few different models. We argue that the MCRS model framework naturally offers a suitable starting point for the future modelling of membrane evolution and extending the theory to cover the emergence of the first protocell in a self-consistent manner. The

  2. Structure of Seneca Valley Virus-001: an oncolytic picornavirus representing a new genus.

    Science.gov (United States)

    Venkataraman, Sangita; Reddy, Seshidhar P; Loo, Jackie; Idamakanti, Neeraja; Hallenbeck, Paul L; Reddy, Vijay S

    2008-10-01

    The crystal structure of Seneca Valley Virus-001 (SVV-001), the representative member of a new genus, Senecavirus, is reported at 2.3A resolution. SVV-001 is the first naturally occurring nonpathogenic picornavirus shown to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features. The nonsegmented (+) ssRNA genome of SVV-001 shares closest sequence similarity with the genomes of the members of Cardiovirus. The overall tertiary structure of VP1-VP4 subunits is conserved with the exception of loops, especially those of VP1 that show large deviations relative to the members of the cardioviruses. The surface loops of VP1 and VP2 are predicted to mediate cell tropism of SVV-001. In addition, the organization of the packaged nucleic acid density indicates that certain regions of VP2 and VP4 interact closely with the packaged nucleic acid.

  3. Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology.

    Science.gov (United States)

    Negro, F; Pacchioni, D; Shimizu, Y; Miller, R H; Bussolati, G; Purcell, R H; Bonino, F

    1992-01-01

    A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or delta, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver

  4. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

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    Stacia L. Phillips

    2016-01-01

    Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

  5. An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

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    Ling Liu

    2009-03-01

    Full Text Available Brome mosaic virus (BMV protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a(Pol and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a(Pol accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a(Pol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

  6. Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method

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    Dan Dou

    2017-07-01

    Full Text Available Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

  7. Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

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    Wimmer Eckard

    2005-11-01

    Full Text Available Abstract Poliovirus protein 3CDpro possesses both proteinase and RNA binding activities, which are located in the 3Cpro domain of the protein. The RNA polymerase (3Dpol domain of 3CDpro modulates these activities of the protein. We have recently shown that the level of 3CDpro in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. However, the addition of either 3CDpro mRNA or of purified 3CDpro protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CDpro indicated that RNA binding by the 3Cpro domain and the integrity of interface I in the 3Dpol domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CDpro enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CDpro to in vitro translation RNA-replication reactions results in a mild enhancement of both minus and plus strand RNA synthesis. By examining the viral particles formed in the in vitro reactions on sucrose gradients we determined that 3CDpro has only a slight stimulating effect on the synthesis of capsid precursors but it strikingly enhances the maturation of virus particles. Both the stimulation of RNA synthesis and the maturation of the virus particles are dependent on the presence of an intact RNA binding site within the 3Cpro domain of 3CDpro. In addition, the integrity of interface I in the 3Dpol domain of 3CDpro is required for efficient production of mature virus. Surprisingly, plus strand RNA synthesis and virus production in in vitro reactions, programmed with full-length transcript RNA, are not enhanced by the addition of extra 3CDpro. Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CDpro in vitro is dependent on the presence of a VPg-linked RNA template.

  8. Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

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    Deepika Bhullar

    Full Text Available Japanese encephalitis virus (JEV has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs. The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+, and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-. The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(- RNA with viral RNA-dependent RNA polymerase (NS5 protein, an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

  9. MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts.

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    Laura N Bonifacio

    Full Text Available Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

  10. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis

    Science.gov (United States)

    Rey, Félix A.

    2017-01-01

    The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. PMID:28151973

  11. Quasispecies spatial models for RNA viruses with different replication modes and infection strategies.

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    Josep Sardanyés

    Full Text Available Empirical observations and theoretical studies suggest that viruses may use different replication strategies to amplify their genomes, which impact the dynamics of mutation accumulation in viral populations and therefore, their fitness and virulence. Similarly, during natural infections, viruses replicate and infect cells that are rarely in suspension but spatially organized. Surprisingly, most quasispecies models of virus replication have ignored these two phenomena. In order to study these two viral characteristics, we have developed stochastic cellular automata models that simulate two different modes of replication (geometric vs stamping machine for quasispecies replicating and spreading on a two-dimensional space. Furthermore, we explored these two replication models considering epistatic fitness landscapes (antagonistic vs synergistic and different scenarios for cell-to-cell spread, one with free superinfection and another with superinfection inhibition. We found that the master sequences for populations replicating geometrically and with antagonistic fitness effects vanished at low critical mutation rates. By contrast, the highest critical mutation rate was observed for populations replicating geometrically but with a synergistic fitness landscape. Our simulations also showed that for stamping machine replication and antagonistic epistasis, a combination that appears to be common among plant viruses, populations further increased their robustness by inhibiting superinfection. We have also shown that the mode of replication strongly influenced the linkage between viral loci, which rapidly reached linkage equilibrium at increasing mutations for geometric replication. We also found that the strategy that minimized the time required to spread over the whole space was the stamping machine with antagonistic epistasis among mutations. Finally, our simulations revealed that the multiplicity of infection fluctuated but generically increased along

  12. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex.

    Science.gov (United States)

    Spear, Allyn; Ogram, Sushma A; Morasco, B Joan; Smerage, Lucia Eisner; Flanegan, James B

    2015-11-01

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showed that 3D entered the replication complex in the form of its precursor, P3 (or 3CD), and was cleaved to release active 3D polymerase. Furthermore, our results showed that P3 is the preferred precursor that binds to the 5'CL. Using reciprocal complementation assays, we showed that one molecule of P3 binds the 5'CL and that a second molecule of P3 provides 3D. In addition, we showed that a second molecule of P3 served as the VPg provider. These results support a model in which P3 binds to the 5'CL and recruits additional molecules of P3, which are cleaved to release either 3D or VPg to initiate RNA replication.

  13. Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

    NARCIS (Netherlands)

    Vliet, P.C. van der; Dam, D. van; Kwant, M.M.

    1984-01-01

    Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, r

  14. Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication.

    Science.gov (United States)

    Muylaert, Isabella; Elias, Per

    2007-04-13

    Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.

  15. The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling.

    Science.gov (United States)

    Fros, Jelke J; van der Maten, Erika; Vlak, Just M; Pijlman, Gorben P

    2013-09-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-induced JAK-STAT signaling, and (iii) nsP2 independently affects RNA replication, cytopathicity, and JAK-STAT signaling.

  16. Small interfering RNA targeting the nonstructural gene 1 transcript inhibits influenza A virus replication in experimental mice.

    Science.gov (United States)

    Rajput, Roopali; Khanna, Madhu; Kumar, Prashant; Kumar, Binod; Sharma, Sonal; Gupta, Neha; Saxena, Latika

    2012-12-01

    Nonstructural protein 1 (NS1) of influenza A viruses counteracts the host immune response against the influenza viruses by not only inhibiting the nuclear export and maturation of host cell messenger RNA (mRNA), but by also blocking the double-stranded RNA-activated protein kinase-mediated inhibition of viral RNA translation. Reduction of NS1 gene product in the host cell may be a potent antiviral strategy to provide protection against the influenza virus infection. We used small interfering RNAs (siRNAs) synthesized against the viral mRNA to down regulate the NS1 gene and observed its effect on inhibition of virus replication. When NS1 gene-specific siRNA were transfected in Madin Darby canine kidney (MDCK) cells followed by influenza A virus infection, approximately 60% inhibition in intracellular levels of NS1 RNA was observed. When siRNA was administered in BALB/c mice, 92% reduction in the levels of NS1 gene expression in mice lungs was observed. A significant reduction in the lung virus titers and cytokine levels was also detected in the presence of siRNAs as compared with the untreated control. The study was validated by the use of selectively disabled mutants of each set of siRNA. Our findings suggest that siRNA targeted against NS1 gene of influenza A virus can provide considerable protection to the virus-infected host cells and may be used as potential candidates for nucleic acid-based antiviral therapy for prevention of influenza A virus infection.

  17. MicroRNA-23b Promotes Avian Leukosis Virus Subgroup J (ALV-J) Replication by Targeting IRF1.

    Science.gov (United States)

    Li, Zhenhui; Chen, Biao; Feng, Min; Ouyang, Hongjia; Zheng, Ming; Ye, Qiao; Nie, Qinghua; Zhang, Xiquan

    2015-05-18

    Avian leukosis virus subgroup J (ALV-J) can cause several different leukemia-like proliferative diseases in the hemopoietic system of chickens. Here, we investigated the transcriptome profiles and miRNA expression profiles of ALV-J-infected and uninfected chicken spleens to identify the genes and miRNAs related to ALV-J invasion. In total, 252 genes and 167 miRNAs were differentially expressed in ALV-J-infected spleens compared to control uninfected spleens. miR-23b expression was up-regulated in ALV-J-infected spleens compared with the control spleens, and transcriptome analysis revealed that the expression of interferon regulatory factor 1 (IRF1) was down-regulated in ALV-J-infected spleens compared to uninfected spleens. A dual-luciferase reporter assay showed that IRF1 was a direct target of miR-23b. miR-23b overexpression significantly (P = 0.0022) decreased IRF1 mRNA levels and repressed IRF1-3'-UTR reporter activity. In vitro experiments revealed that miR-23b overexpression strengthened ALV-J replication, whereas miR-23b loss of function inhibited ALV-J replication. IRF1 overexpression inhibited ALV-J replication, and IRF1 knockdown enhanced ALV-J replication. Moreover, IRF1 overexpression significantly (P = 0.0014) increased IFN-β expression. In conclusion, these results suggested that miR-23b may play an important role in ALV-J replication by targeting IRF1.

  18. Evolutionary dynamics of RNA-like replicators : A bioinformatic approach to the origin of life

    NARCIS (Netherlands)

    Takeuchi, N.

    2010-01-01

    The origin of life has always attracted scientific inquiries. The RNA world hypothesis suggests that, before the evolution of DNAs and proteins, primordial life was based on RNAs both for information storage and chemical catalysis. In its simplest form, an RNA world consists of RNA molecules that ca

  19. Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

    DEFF Research Database (Denmark)

    Jacobsen, Kari Stougaard; Nielsen, Kirstine Overgaard; Nordmann Winther, Thilde;

    2016-01-01

    BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially...... of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes...... identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model....

  20. Addition of polyadenylate sequences to virus-specific RNA during adenovirus replication.

    Science.gov (United States)

    Philipson, L; Wall, R; Glickman, G; Darnell, J E

    1971-11-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA.

  1. Identification of a novel human tRNA(Ser(CGA)) functional in murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Schmitz, A; Pedersen, F S

    2000-01-01

    (CGA)) was detected in cell lines of human, monkey and mouse origin. The UCG codon is the most rarely used codon in human genes. The cloned human tRNA(Ser(CGA)) gene encodes an 85 nucleotide, intron-less tRNA, contains a consensus split intragenic promoter and is located at region p21.3-22.2 on chromosome 6......We have identified a human tRNA(Ser) isoacceptor matching the UCG codon. The tRNA was discovered via its ability to act in reverse transcription of a murine leukemia virus vector containing a complementary tRNA primer binding site (Lund et al., Nucleic Acids Res., 28 (2000) 791-799). The tRNA(Ser....... The integrity and functionality of the cloned tRNA(Ser(CGA)) gene was verified by in vitro transcription analysis in HeLa nuclear extracts....

  2. SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

    Science.gov (United States)

    Neuvonen, Maarit; Kazlauskas, Arunas; Martikainen, Miika; Hinkkanen, Ari; Ahola, Tero; Saksela, Kalle

    2011-11-01

    Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

  3. SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

    Directory of Open Access Journals (Sweden)

    Maarit Neuvonen

    2011-11-01

    Full Text Available Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3 domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV, Sindbis (SINV, and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

  4. Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus

    Science.gov (United States)

    Xu, Kai; Nagy, Peter D.

    2016-01-01

    Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment. PMID:27760128

  5. Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus.

    Science.gov (United States)

    Sehgal, O P; Sinha, R C; Gellatly, D L; Ivanov, I; AbouHaidar, M G

    1993-04-01

    Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species.

  6. The C-Terminal Domain of Chikungunya Virus nsP2 Independently Governs Viral RNA Replication, Cytopathicity, and Inhibition of Interferon Signaling

    OpenAIRE

    Fros, J. J.; van der Maten, E.; Vlak, J. M.; Pijlman, G.P.

    2013-01-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-...

  7. Persistent hepatitis C virus RNA replication in haemophiliacs: role of co-infection with human immunodeficiency virus.

    Science.gov (United States)

    Chambost, H; Gerolami, V; Halfon, P; Thuret, I; Michel, G; Sicardi, F; Rousseau, S; Perrimond, H; Cartouzou, G

    1995-11-01

    In order to evaluate the evolution of transfusional hepatitis C in haemophiliacs, we performed a retrospective study of ALT levels and HCV viraemia with a RNA PCR assay in 57 patients. We found that the vast majority of HCV-infected patients remained viraemic (43/57 = 75%) and higher ALT levels correlated with HCV viraemia. Although indicators of the transfusional viral load (age, severity of haemophilia) and HBV co-infection did not correlate with HCV RNA replication, HIV seropositivity was strongly associated with persistence of HCV viraemia (23/25 = 92% in HIV-positive versus 20/32 = 62% in HIV-negative patients), without any correlation with CD4 counts. Genotyping of HCV in the 43 viraemic patients shows more frequent genotype 1 in the HIV-seropositive group (14/23) than in the seronegative group (6/20). Our data emphasize that besides the role of the immunodeficiency status, the genotypes of HCV might be involved in the differences observed in terms of HCV RNA replication between the HIV-seropositive and seronegative haemophiliacs.

  8. A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral

    Directory of Open Access Journals (Sweden)

    Claire M. Smith

    2016-08-01

    Full Text Available Defective interfering (DI viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8 was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1; it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral.

  9. The expanding functions of cellular helicases: the tombusvirus RNA replication enhancer co-opts the plant eIF4AIII-like AtRH2 and the DDX5-like AtRH5 DEAD-box RNA helicases to promote viral asymmetric RNA replication.

    Directory of Open Access Journals (Sweden)

    Nikolay Kovalev

    2014-04-01

    Full Text Available Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. Several of the co-opted host factors bind to the viral RNA, which plays multiple roles, including mRNA function, as an assembly platform for the viral replicase (VRC, template for RNA synthesis, and encapsidation during infection. It is likely that remodeling of the viral RNAs and RNA-protein complexes during the switch from one step to another requires RNA helicases. In this paper, we have discovered a second group of cellular RNA helicases, including the eIF4AIII-like yeast Fal1p and the DDX5-like Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD box helicases, which are co-opted by tombusviruses. Unlike the previously characterized DDX3-like AtRH20/Ded1p helicases that bind to the 3' terminal promoter region in the viral minus-strand (-RNA, the other class of eIF4AIII-like RNA helicases bind to a different cis-acting element, namely the 5' proximal RIII(- replication enhancer (REN element in the TBSV (-RNA. We show that the binding of AtRH2 and AtRH5 helicases to the TBSV (-RNA could unwind the dsRNA structure within the RIII(- REN. This unique characteristic allows the eIF4AIII-like helicases to perform novel pro-viral functions involving the RIII(- REN in stimulation of plus-strand (+RNA synthesis. We also show that AtRH2 and AtRH5 helicases are components of the tombusvirus VRCs based on co-purification experiments. We propose that eIF4AIII-like helicases destabilize dsRNA replication intermediate within the RIII(- REN that promotes bringing the 5' and 3' terminal (-RNA sequences in close vicinity via long-range RNA-RNA base pairing. This newly formed RNA structure promoted by eIF4AIII helicase together with AtRH20 helicase might facilitate the recycling of the viral replicases for multiple rounds of (+-strand synthesis, thus resulting in asymmetrical viral replication.

  10. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    Science.gov (United States)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  11. Ascorbic acid inhibits replication and infectivity of avian RNA tumor virus

    Energy Technology Data Exchange (ETDEWEB)

    BISSELL, MINA J; HATIE, CARROLL; FARSON, DEBORAH A.; SCHWARZ, RICHARD I.; SOO, WHAI-JEN

    1980-04-01

    Ascorbic acid, at nontoxic concentrations, causes a substantial reduction in the ability of avian tumor viruses to replicate in both primary avian tendon cells and chicken embryo fibroblasts. The virus-infected cultures appear to be less transformed in the presence of ascorbic acid by the criteria of morphology, reduced glucose uptake, and increased collagen synthesis. The vitamin does not act by altering the susceptibility of the cells to initial infection and transformation, but instead appears to interfere with the spread of infection through a reduction in virus replication and virus infectivity. The effect is reversible and requires the continuous presence of the vitamin in the culture medium.

  12. Multiple functions of the 32K and 60K proteins in cowpea mosaic virus RNA replication.

    NARCIS (Netherlands)

    Peters, S.A.

    1994-01-01

    Cowpea mosaic virus (CPMV) is the type member of the comoviridae , a group of 14 different plant viruses that have a divided genome consisting of two plus-strand RNAs. These RNAs, designated B-RNA and M-RNA, have a small protein, VPg, attached to the 5'-end and a poly(A) tail at the 3'-end and are s

  13. A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.

    Directory of Open Access Journals (Sweden)

    Hao Gong

    2011-09-01

    Full Text Available Small non-coding RNAs (sRNAs that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA and small interfering RNA (siRNA in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.

  14. Epithelial Distribution and Replication of Foot-and-Mouth Disease Virus RNA in Infected Pigs

    DEFF Research Database (Denmark)

    Durand, S.; Murphy, C.; Zhang, Z.

    2008-01-01

    Although the pathogenesis of foot-and-mouth disease (FMD) has been extensively investigated relatively few studies have addressed the localization of FMD virus (FMDV) and in particular its replication in relation to the typical in-vivo sites of FMD lesions. In the present study, pigs were infecte...

  15. Inhibition of polyprotein processing and RNA replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions.

    NARCIS (Netherlands)

    Krenn, B.M.; Holzer, B.; Gaudernak, E.; Triendl, A.; Kuppeveld, F.J.M. van; Seipelt, J.

    2005-01-01

    Pyrrolidine dithiocarbamate (PDTC) is an antiviral compound that was shown to inhibit the replication of human rhinoviruses (HRVs), poliovirus, and influenza virus. To elucidate the mechanism of PDTC, the effects on the individual steps of the infection cycle of HRV were investigated. PDTC did not i

  16. Analysis of classical swine fever virus RNA replication determinants using replicons

    DEFF Research Database (Denmark)

    Risager, Peter Christian; Fahnøe, Ulrik; Gullberg, Maria;

    2013-01-01

    Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome (BAC), was modified by targeted...

  17. Kissing interaction between 3' noncoding and coding sequences is essential for porcine arterivirus RNA replication

    NARCIS (Netherlands)

    Verheije, M.H.; Olsthoorn, R.C.L.; Kroese, M.V.; Rottier, P.J.M.; Meulenberg, J.J.M.

    2002-01-01

    We used an infectious cDNA clone of Porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686)

  18. SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

    OpenAIRE

    Maarit Neuvonen; Arunas Kazlauskas; Miika Martikainen; Ari Hinkkanen; Tero Ahola; Kalle Saksela

    2011-01-01

    Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homolo...

  19. Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase*

    Science.gov (United States)

    Moustafa, Ibrahim M.; Korboukh, Victoria K.; Arnold, Jamie J.; Smidansky, Eric D.; Marcotte, Laura L.; Gohara, David W.; Yang, Xiaorong; Sánchez-Farrán, María Antonieta; Filman, David; Maranas, Janna K.; Boehr, David D.; Hogle, James M.; Colina, Coray M.; Cameron, Craig E.

    2014-01-01

    RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity. PMID:25378410

  20. Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Pedersen, F S

    2000-01-01

    . While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown......Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian...... retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites...

  1. MicroRNA-649 promotes HSV-1 replication by directly targeting MALT1.

    Science.gov (United States)

    Zhang, Yi; Dai, Jun; Tang, Jinfeng; Zhou, Li; Zhou, Mengzhou

    2016-11-03

    Herpes simplex virus type 1 (HSV-1), a member of the Herpes viridae, is associated with a wide variety of nervous system diseases including meningitis and encephalitis. The data presented here demonstrate that miR-649 promotes the replication of HSV-1 without affecting cell viability. Further mechanistic studies revealed that MALT1 (mucosa associated lymphoid tissue lymphoma translocation gene 1) is directly targeted by miR-649. We then found that MALT1 and the downstream NF-κB signaling pathway, are involved in miR-649-induced HSV-1 replication. Interestingly, miR-649 levels were downregulated after HSV-1 infection, and miR-649 expression was negatively associated with MALT1 expression in HSV-1-infected HeLa cells. Taken together, this present study indicates that miR-649 promotes HSV-1 replication through regulation of the MALT1-mediated antiviral signaling pathway and suggests a promising target for antiviral therapies. J. Med. Virol. © 2016 Wiley Periodicals, Inc.

  2. RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies.

    Science.gov (United States)

    Tulloch, Fiona; Atkinson, Nicky J; Evans, David J; Ryan, Martin D; Simmonds, Peter

    2014-12-09

    Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective.

  3. Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice

    Institute of Scientific and Technical Information of China (English)

    WU Ying; HUANG Ai-long; TANG Ni; ZHANG Bing-qiang; LU Nian-fang

    2005-01-01

    Background RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs (siRNAs) have sufficiently inhibited hepatitis B virus (HBV) replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects.Methods A mutant RNAi vector (pSI-C mut) with two base pairs different from the original target gene sequence at the RNAi vector (pSI-C) was constructed according to the method described in this study. A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload. pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein (GFP) gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay (ELISA) assay was used to measure the concentration of HBV surface antigen (HBsAg) in mouse serum, immunohistochemical straining method was used to visualize the expressin of HBV core protein (HBcAg) in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detectedd by reverse transcriptase PCR (RT-PCR) analysis.Results Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection (p.i.), the OD values were shown to be 5.07±1.07 in infecting group and 0.62±0.59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group (P<0.01). Liver intracellular synthesis of viral core protein was sharply reduced to 0.9%±0.1%, compared with 5.4%±1.2% of positive hepatocytes in infecting group (P<0.01), and the transcriptional level of HBV C mRNA was greatly reduced by 84.7%. However, the irrelevant RNAi control plasmid

  4. Long non-coding RNA GAS5 inhibited hepatitis C virus replication by binding viral NS3 protein.

    Science.gov (United States)

    Qian, Xijing; Xu, Chen; Zhao, Ping; Qi, Zhongtian

    2016-05-01

    HCV infection has a complex and dynamic process which involves a large number of viral and host factors. Long non-coding RNA GAS5 inhibits liver fibrosis and liver tumor migration and invasion. However, the contribution of GAS5 on HCV infection remains unknown. In this study, GAS5 was gradually upregulated during HCV infection in Huh7 cells. In addition, GAS5 attenuated virus replication with its 5' end sequences, as confirmed by different GAS5 truncations. Moreover, this 5' end sequences showed RNA-protein interaction with HCV NS3 protein that could act as a decoy to inhibit its functions, which contributed to the suppression of HCV replication. Finally, the innate immune responses remained low in HCV infected Huh7 cells, ruling out the possibility of GAS5 to modulate innate immunity. Thus, HCV stimulated endogenous GAS5 can suppress HCV infection by acting as HCV NS3 protein decoy, providing a potential role of GAS5 as a diagnostic or therapeutic target.

  5. p53 induces formation of NEAT1 lncRNA-containing paraspeckles that modulate replication stress response and chemosensitivity.

    Science.gov (United States)

    Adriaens, Carmen; Standaert, Laura; Barra, Jasmine; Latil, Mathilde; Verfaillie, Annelien; Kalev, Peter; Boeckx, Bram; Wijnhoven, Paul W G; Radaelli, Enrico; Vermi, William; Leucci, Eleonora; Lapouge, Gaëlle; Beck, Benjamin; van den Oord, Joost; Nakagawa, Shinichi; Hirose, Tetsuro; Sablina, Anna A; Lambrechts, Diether; Aerts, Stein; Blanpain, Cédric; Marine, Jean-Christophe

    2016-08-01

    In a search for mediators of the p53 tumor suppressor pathway, which induces pleiotropic and often antagonistic cellular responses, we identified the long noncoding RNA (lncRNA) NEAT1. NEAT1 is an essential architectural component of paraspeckle nuclear bodies, whose pathophysiological relevance remains unclear. Activation of p53, pharmacologically or by oncogene-induced replication stress, stimulated the formation of paraspeckles in mouse and human cells. Silencing Neat1 expression in mice, which prevents paraspeckle formation, sensitized preneoplastic cells to DNA-damage-induced cell death and impaired skin tumorigenesis. We provide mechanistic evidence that NEAT1 promotes ATR signaling in response to replication stress and is thereby engaged in a negative feedback loop that attenuates oncogene-dependent activation of p53. NEAT1 targeting in established human cancer cell lines induced synthetic lethality with genotoxic chemotherapeutics, including PARP inhibitors, and nongenotoxic activation of p53. This study establishes a key genetic link between NEAT1 paraspeckles, p53 biology and tumorigenesis and identifies NEAT1 as a promising target to enhance sensitivity of cancer cells to both chemotherapy and p53 reactivation therapy.

  6. Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements

    Science.gov (United States)

    Fernandez-Chamorro, Javier; Lozano, Gloria; Garcia-Martin, Juan Antonio; Ramajo, Jorge; Dotu, Ivan; Clote, Peter; Martinez-Salas, Encarnacion

    2016-04-01

    The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both in vitro and in vivo systems. Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.

  7. The Role of RNA Polymerase II Elongation Control in HIV-1 Gene Expression, Replication, and Latency

    Directory of Open Access Journals (Sweden)

    Kyle A. Nilson

    2011-01-01

    Full Text Available HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II, leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure.

  8. Replicates, Read Numbers, and Other Important Experimental Design Considerations for Microbial RNA-seq Identified Using Bacillus thuringiensis Datasets.

    Science.gov (United States)

    Manga, Punita; Klingeman, Dawn M; Lu, Tse-Yuan S; Mehlhorn, Tonia L; Pelletier, Dale A; Hauser, Loren J; Wilson, Charlotte M; Brown, Steven D

    2016-01-01

    RNA-seq is being used increasingly for gene expression studies and it is revolutionizing the fields of genomics and transcriptomics. However, the field of RNA-seq analysis is still evolving. Therefore, we specifically designed this study to contain large numbers of reads and four biological replicates per condition so we could alter these parameters and assess their impact on differential expression results. Bacillus thuringiensis strains ATCC10792 and CT43 were grown in two Luria broth medium lots on four dates and transcriptomics data were generated using one lane of sequence output from an Illumina HiSeq2000 instrument for each of the 32 samples, which were then analyzed using DESeq2. Genome coverages across samples ranged from 87 to 465X with medium lots and culture dates identified as major variation sources. Significantly differentially expressed genes (5% FDR, two-fold change) were detected for cultures grown using different medium lots and between different dates. The highly differentially expressed iron acquisition and metabolism genes, were a likely consequence of differing amounts of iron in the two media lots. Indeed, in this study RNA-seq was a tool for predictive biology since we hypothesized and confirmed the two LB medium lots had different iron contents (~two-fold difference). This study shows that the noise in data can be controlled and minimized with appropriate experimental design and by having the appropriate number of replicates and reads for the system being studied. We outline parameters for an efficient and cost effective microbial transcriptomics study.

  9. Inhibition of avian leukosis virus subgroup J replication by miRNA targeted against env.

    Science.gov (United States)

    Wang, Wei; Zhang, Zai-Ping; Tian, Jin; Xiao, Zhi-Guang; Meng, Qing-Wen

    2013-08-01

    No effective vaccine has been developed against the subgroup J avian leukosis virus (ALV-J). The genetic diversity of ALV-J might be related to the env gene, therefore, we selected conserved sequences of the env gene and designed interference sequence. In this study, microRNAs (miRNAs) were designed and synthesized, corresponding to conserved regions of the env gene. These miRNAs were cloned into the linearized eukaryotic expression vector. The recombinant plasmids were transfected into DF-1 cells. After transfection, the cells were inoculated with ALV-J. In reporter assays, the transfection efficiency is 80% by indirect immunofluorescence (IFA). Expression of the virus envelope glycoprotein was measured by IFA and western blotting assays. The relative expression of env gene was determined using quantitative PCR. Our results show that the mi-env 231 and mi-env 1384 could effectively suppress the replication of ALV-J with an efficiency of 68.7-75.2%. These data suggest that the miRNAs targeting the env can inhibit replication of ALV-J efficiently. This finding provides evidence that miRNAs could be used as a potential tool against ALV infection.

  10. A whole-genome RNA interference screen for human cell factors affecting myxoma virus replication.

    Science.gov (United States)

    Teferi, Wondimagegnehu M; Dodd, Kristopher; Maranchuk, Rob; Favis, Nicole; Evans, David H

    2013-04-01

    Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes ("hits") and nonsignificant genes ("nonhits") of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G(1), or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G(1)/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-D-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.

  11. Replication of honey bee-associated RNA viruses across multiple bee species in apple orchards of Georgia, Germany and Kyrgyzstan.

    Science.gov (United States)

    Radzevičiūtė, Rita; Theodorou, Panagiotis; Husemann, Martin; Japoshvili, George; Kirkitadze, Giorgi; Zhusupbaeva, Aigul; Paxton, Robert J

    2017-06-01

    The essential ecosystem service of pollination is provided largely by insects, which are considered threatened by diverse biotic and abiotic global change pressures. RNA viruses are one such pressure, and have risen in prominence as a major threat for honey bees (Apis mellifera) and global apiculture, as well as a risk factor for other bee species through pathogen spill-over between managed honey bees and sympatric wild pollinator communities. Yet despite their potential role in global bee decline, the prevalence of honey bee-associated RNA viruses in wild bees is poorly known from both geographic and taxonomic perspectives. We screened members of pollinator communities (honey bees, bumble bees and other wild bees belonging to four families) collected from apple orchards in Georgia, Germany and Kyrgyzstan for six common honey bee-associated RNA virus complexes encompassing nine virus targets. The Deformed wing virus complex (DWV genotypes A and B) had the highest prevalence across all localities and host species and was the only virus complex found in wild bee species belonging to all four studied families. Based on amplification of negative-strand viral RNA, we found evidence for viral replication in wild bee species of DWV-A/DWV-B (hosts: Andrena haemorrhoa and several Bombus spp.) and Black queen cell virus (hosts: Anthophora plumipes, several Bombus spp., Osmia bicornis and Xylocopa spp.). Viral amplicon sequences revealed that DWV-A and DWV-B are regionally distinct but identical in two or more bee species at any one site, suggesting virus is shared amongst sympatric bee taxa. This study demonstrates that honey bee associated RNA viruses are geographically and taxonomically widespread, likely infective in wild bee species, and shared across bee taxa. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Gastroenteritis and the novel picornaviruses aichi virus, cosavirus, saffold virus, and salivirus in young children

    DEFF Research Database (Denmark)

    Nielsen, Alex Christian Yde; Gyhrs, Mette Louise; Nielsen, Lars Peter

    2013-01-01

    During the last few years many new human picornaviruses have been discovered due to advances in metagenomics and other molecular biological approaches. The clinical significance and the occurrence are only sparsely described....

  13. SiRNA Inhibits Replication of Langat Virus, a Member of the Tick-Borne Encephalitis Virus Complex in Organotypic Rat Brain Slices

    Science.gov (United States)

    Maffioli, Carola; Grandgirard, Denis; Leib, Stephen L.; Engler, Olivier

    2012-01-01

    Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro. PMID:22984545

  14. Functional significance of a hepta nucleotide motif present at the junction of Cucumber mosaic virus satellite RNA multimers in helper-virus dependent replication.

    Science.gov (United States)

    Seo, Jang-Kyun; Kwon, Sun-Jung; Chaturvedi, Sonali; Choi, Soon Ho; Rao, A L N

    2013-01-20

    Satellite RNAs (satRNA) associated with Cucumber mosaic virus (CMV) have been shown to generate multimers during replication. We have discovered that multimers of a CMV satRNA generated in the absence of its helper virus (HV) are characterized by the addition of a hepta nucleotide motif (HNM) at the monomer junctions. Here, we evaluated the functional significance of HNM in HV-dependent replication by ectopically expressing wild type and mutant forms of satRNA multimers in planta either in (+) or (-)-strand polarity. Comparative replication profiles revealed that (-)-strand multimers with complementary HNM (cHNM) are the preferred initial templates for HV-dependent replication than (-)-strand monomers and multimers lacking the cHNM. Further mutational analyses of the HNM accentuate that preservation of the sequence and native length of HNM is obligatory for efficient replication of satRNA. A model implicating the significance of HNM in HV-dependent production of monomeric and multimeric forms of satRNA is presented.

  15. Quinacrine impairs enterovirus 71 RNA replication by preventing binding of polypyrimidine-tract binding protein with internal ribosome entry sites.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

    Full Text Available Since the 1980s, epidemics of enterovirus 71 (EV71 and other enteroviruses have occurred in Asian countries and regions, causing a wide range of human diseases. No effective therapy is available for the treatment of these infections. Internal ribosome entry sites (IRESs are indispensable for the initiation of translation in enteroviruses. Several cellular factors, as well as the ribosome, are recruited to the conserved IRES during this process. Quinacrine intercalates into the RNA architecture and inhibits RNA transcription and protein synthesis, and a recent study showed that quinacrine inhibited encephalomyocarditis virus and poliovirus IRES-mediated translation in vitro without disrupting internal cellular IRES. Here, we report that quinacrine was highly active against EV71, protecting cells from EV71 infection. Replication of viral RNA, expression of viral capsid protein, and production of virus were all strongly inhibited by quinacrine. Interaction of the polypyrimidine tract-binding protein (PTB with the conserved IRES was prevented by quinacrine. Coxsackieviruses and echovirus were also inhibited by quinacrine in cultured cells. These results indicate that quinacrine may serve as a potential protective agent for use in the treatment of patients with chronic enterovirus infection.

  16. Identification and complete genome characterization of a novel picornavirus in turkey (Meleagris gallopavo).

    Science.gov (United States)

    Boros, Ákos; Nemes, Csaba; Pankovics, Péter; Kapusinszky, Beatrix; Delwart, Eric; Reuter, Gábor

    2012-10-01

    Members of the family Picornaviridae are important pathogens of humans and animals, although compared with the thousands of known bird species (>10 000), only a few (n = 11) picornaviruses have been identified from avian sources. This study reports the metagenomic detection and complete genome characterization of a novel turkey picornavirus from faecal samples collected from eight turkey farms in Hungary. Using RT-PCR, both healthy (two of three) and affected (seven of eight) commercial turkeys with enteric and/or stunting syndrome were shown to be shedding viruses in seven (88 %) of the eight farms. The viral genome sequence (turkey/M176/2011/HUN; GenBank accession no. JQ691613) shows a high degree of amino acid sequence identity (96 %) to the partial P3 genome region of a picornavirus reported recently in turkey and chickens from the USA and probably belongs to the same species. In the P1 and P2 regions, turkey/M176/2011/HUN is related most closely to, but distinct from, the kobuviruses and turdivirus 1. Complete genome analysis revealed the presence of characteristic picornaviral amino acid motifs, a potential type II-like 5' UTR internal ribosome entry site (first identified among avian-origin picornaviruses) and a conserved, 48 nt long 'barbell-like' structure found at the 3' UTR of turkey/M176/2011/HUN and members of the picornavirus genera Avihepatovirus and Kobuvirus. The general presence of turkey picornavirus - a novel picornavirus species - in faecal samples from healthy and affected turkeys in Hungary and in the USA suggests the worldwide occurrence and endemic circulation of this virus in turkey farms. Further studies are needed to investigate the aetiological role and pathogenic potential of this picornavirus in food animals.

  17. Identification and complete genome analysis of novel picornavirus in bovine in Japan

    DEFF Research Database (Denmark)

    Nagai, Makoto; Omatsu, Tsutomu; Aoki, Hiroshi

    2015-01-01

    We identified novel viruses in feces from cattle with diarrhea collected in 2009 in Hokkaido Prefecture, Japan, by using a metagenomics approach and determined the (near) complete sequences of the virus. Sequence analyses revealed that they had a standard picornavirus genome organization, i.e. 5'...... with diarrhea were positive, indicating the prevalence of these picornavirus in the Japanese cattle population in Hokkaido Prefecture. However, further studies are needed to investigate the pathogenic potential and etiological role of these viruses in cattle....

  18. In Vitro and In Vivo Characterization of MicroRNA-Targeted Alphavirus Replicon and Helper RNAs ▿ ‡

    Science.gov (United States)

    Kamrud, Kurt I.; Coffield, V. McNeil; Owens, Gary; Goodman, Christin; Alterson, Kim; Custer, Max; Murphy, Michael A.; Lewis, Whitney; Timberlake, Sarah; Wansley, Elizabeth K.; Berglund, Peter; Smith, Jonathan

    2010-01-01

    Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans. PMID:20504925

  19. Expression of plasmid-based shRNA against the E1 and nsP1 genes effectively silenced Chikungunya virus replication.

    Directory of Open Access Journals (Sweden)

    Shirley Lam

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. METHODS: Plasmid-based small hairpin RNA (shRNA was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: Taken together, these

  20. Cytonuclear conflict in interpopulation hybrids: the role of RNA polymerase in mtDNA transcription and replication.

    Science.gov (United States)

    Ellison, C K; Burton, R S

    2010-03-01

    Organismal fitness requires functional integration of nuclear and mitochondrial genomes. Structural and regulatory elements coevolve within lineages and several studies have found that interpopulation hybridization disrupts mitonuclear interactions. Because mitochondrial RNA polymerase (mtRPOL) plays key roles in both mitochondrial DNA (mtDNA) replication and transcription, the interaction between mtRPOL and coevolved regulatory sites in the mtDNA may be central to mitonuclear integration. Here, we generate interpopulation hybrids between divergent populations of the copepod Tigriopus californicus to obtain lines having different combinations of mtRPOL and mtDNA. Lines were scored for mtDNA copy number and ATP6 (mtDNA) gene expression. We find that there is a genotype-dependent negative association between mitochondrial transcriptional response and mtDNA copy number. We argue that an observed increase in mtDNA copy number and reduced mtDNA transcription in hybrids reflects the regulatory role of mtRPOL; depending on the mitonuclear genotype, hybridization may disrupt the normal balance between transcription and replication of the mitochondrial genome.

  1. Use of microRNA Let-7 to control the replication specificity of oncolytic adenovirus in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Huajun Jin

    Full Text Available Highly selective therapy for hepatocellular carcinoma (HCC remains an unmet medical need. In present study, we found that the tumor suppressor microRNA, let-7 was significantly downregulated in a proportion of primary HCC tissues (12 of 33, 36.4% and HCC cell lines. In line with this finding, we have engineered a chimeric Ad5/11 fiber oncolytic adenovirus, SG7011(let7T, by introducing eight copies of let-7 target sites (let7T into the 3' untranslated region of E1A, a key gene associated with adenoviral replication. The results showed that the E1A expression (both RNA and protein levels of the SG7011(let7T was tightly regulated according to the endogenous expression level of the let-7. As contrasted with the wild-type adenovirus and the control virus, the replication of SG7011(let7T was distinctly inhibited in normal liver cells lines (i.e. L-02 and WRL-68 expressing high level of let-7 (>300 folds, whereas was almost not impaired in HCC cells (i.e. Hep3B and PLC/PRF/5 with low level of let-7. Consequently, the cytotoxicity of SG7011(let7T to normal liver cells was successfully decreased while was almost not attenuated in HCC cells in vitro. The antitumor ability of SG7011(let7Tin vivo was maintained in mice with Hep3B xenograft tumor, whereas was greatly decreased against the SMMC-7721 xenograft tumor expressing a high level of let-7 similar with L-02 when compared to the wild-type adenovirus. These results suggested that SG7011(let7T may be a promising anticancer agent or vector to mediate the expression of therapeutic gene, broadly applicable in the treatment for HCC and other cancers where the let-7 gene is downregulated.

  2. Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroid-binding protein

    Science.gov (United States)

    Lv, Dian-Qiu; Liu, Shang-Wu; Zhao, Jian-Hua; Zhou, Bang-Jun; Wang, Shao-Peng; Guo, Hui-Shan; Fang, Yuan-Yuan

    2016-01-01

    Viroids are plant-pathogenic molecules made up of single-stranded circular non-coding RNAs. How replicating viroids interfere with host silencing remains largely unknown. In this study, we investigated the effects of a nuclear-replicating Potato spindle tuber viroid (PSTVd) on interference with plant RNA silencing. Using transient induction of silencing in GFP transgenic Nicotiana benthamiana plants (line 16c), we found that PSTVd replication accelerated GFP silencing and increased Virp1 mRNA, which encodes bromodomain-containing viroid-binding protein 1 and is required for PSTVd replication. DNA methylation was increased in the GFP transgene promoter of PSTVd-replicating plants, indicating involvement of transcriptional gene silencing. Consistently, accelerated GFP silencing and increased DNA methylation in the of GFP transgene promoter were detected in plants transiently expressing Virp1. Virp1 mRNA was also increased upon PSTVd infection in natural host potato plants. Reduced transcript levels of certain endogenous genes were also consistent with increases in DNA methylation in related gene promoters in PSTVd-infected potato plants. Together, our data demonstrate that PSTVd replication interferes with the nuclear silencing pathway in that host plant, and this is at least partially attributable to Virp1. This study provides new insights into the plant-viroid interaction on viroid pathogenicity by subverting the plant cell silencing machinery. PMID:27767195

  3. The Multifaceted Poliovirus 2A Protease: Regulation of Gene Expression by Picornavirus Proteases

    Directory of Open Access Journals (Sweden)

    Alfredo Castelló

    2011-01-01

    Full Text Available After entry into animal cells, most viruses hijack essential components involved in gene expression. This is the case of poliovirus, which abrogates cellular translation soon after virus internalization. Abrogation is achieved by cleavage of both eIF4GI and eIF4GII by the viral protease 2A. Apart from the interference of poliovirus with cellular protein synthesis, other gene expression steps such as RNA and protein trafficking between nucleus and cytoplasm are also altered. Poliovirus 2Apro is capable of hydrolyzing components of the nuclear pore, thus preventing an efficient antiviral response by the host cell. Here, we compare in detail poliovirus 2Apro with other viral proteins (from picornaviruses and unrelated families as regard to their activity on key host factors that control gene expression. It is possible that future analyses to determine the cellular proteins targeted by 2Apro will uncover other cellular functions ablated by poliovirus infection. Further understanding of the cellular proteins hydrolyzed by 2Apro will add further insight into the molecular mechanism by which poliovirus and other viruses interact with the host cell.

  4. Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein.

    Directory of Open Access Journals (Sweden)

    Dorothee A Vogt

    Full Text Available The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web and assists viral assembly in the close vicinity of lipid droplets (LDs. To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1-31, a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47 as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon, indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47--via its interaction with NS5A--serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.

  5. Knockdown of cellular RNA helicase DDX3 by short hairpin RNAs suppresses HIV-1 viral replication without inducing apoptosis.

    Science.gov (United States)

    Ishaq, Musarat; Hu, Jiajie; Wu, Xiaoyun; Fu, Qiong; Yang, Yalin; Liu, Qingzhen; Guo, Deyin

    2008-07-01

    The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.

  6. Investigating the role of viral integral membrane proteins in promoting the assembly of nepovirus and comovirus replication factories

    Directory of Open Access Journals (Sweden)

    Helene eSanfacon

    2013-01-01

    Full Text Available Formation of plant virus membrane-associated replication factories requires the association of viral replication proteins and viral RNA with intracellular membranes, the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex. Many viruses encode integral membrane proteins that act as anchors for the replication complex. These hydrophobic proteins contain trans-membrane domains and/or amphipathic helices that associate with the membrane and modify its structure. The comovirus Co-Pro and NTP-binding (NTB, putative helicase proteins and the cognate nepovirus X2 and NTB proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2B, 2C and 3A membrane proteins. The identification of membrane-association domains and analysis of the membrane topology of these proteins is discussed. The evidence suggesting that these proteins have the ability to induce membrane proliferation, alter the structure and integrity of intracellular membranes and modulate the induction of symptoms in infected plants is also reviewed. Finally, areas of research that need further investigation are highlighted.

  7. Non coding extremities of the seven influenza virus type C vRNA segments: effect on transcription and replication by the type C and type A polymerase complexes

    Directory of Open Access Journals (Sweden)

    van der Werf Sylvie

    2008-10-01

    Full Text Available Abstract Background The transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC regions. The plasmid-based system for the in vivo reconstitution of functional ribonucleoproteins, upon expression of viral-like RNAs together with the nucleoprotein and polymerase proteins has been widely used to analyze transcription/replication of influenza viruses. It was thus shown that the type A polymerase could transcribe and replicate type A, B, or C vRNA templates whereas neither type B nor type C polymerases were able to transcribe and replicate type A templates efficiently. Here we studied the importance of the NC regions from the seven segments of type C influenza virus for efficient transcription/replication by the type A and C polymerases. Results The NC sequences of the seven genomic segments of the type C influenza virus C/Johannesburg/1/66 strain were found to be more variable in length than those of the type A and B viruses. The levels of transcription/replication of viral-like vRNAs harboring the NC sequences of the respective type C virus segments flanking the CAT reporter gene were comparable in the presence of either type C or type A polymerase complexes except for the NS and PB2-like vRNAs. For the NS-like vRNA, the transcription/replication level was higher after introduction of a U residue at position 6 in the 5' NC region as for all other segments. For the PB2-like vRNA the CAT expression level was particularly reduced with the type C polymerase. Analysis of mutants of the 5' NC sequence in the PB2-like vRNA, the shortest 5' NC sequence among the seven segments, showed that additional sequences within the PB2 ORF were essential for the efficiency of transcription but not replication by the type C polymerase complex. Conclusion In the context of a PB2-like reporter

  8. Identification and complete genome analysis of a novel bovine picornavirus in Japan.

    Science.gov (United States)

    Nagai, Makoto; Omatsu, Tsutomu; Aoki, Hiroshi; Kaku, Yoshihiro; Belsham, Graham J; Haga, Kei; Naoi, Yuki; Sano, Kaori; Umetsu, Moeko; Shiokawa, Mai; Tsuchiaka, Shinobu; Furuya, Tetsuya; Okazaki, Sachiko; Katayama, Yukie; Oba, Mami; Shirai, Junsuke; Katayama, Kazuhiko; Mizutani, Tetsuya

    2015-12-02

    We identified novel viruses in feces from cattle with diarrhea collected in 2009 in Hokkaido Prefecture, Japan, by using a metagenomics approach and determined the (near) complete sequences of the virus. Sequence analyses revealed that they had a standard picornavirus genome organization, i.e. 5' untranslated region (UTR) - L- P1 (VP4- VP3- VP2- VP1) - P2 (2A- 2B- 2C) - P3 (3A- 3B- 3C-3D) - 3'UTR- poly(A). They are closely related to other unclassified Chinese picornaviruses; bat picornaviruses group 1-3, feline picornavirus, and canine picornavirus, sharing 45.4-51.4% (P1), 38.0-44.9% (P2), and 49.6-53.3% (P3) amino acid identities, respectively. The phylogenetic analyses and detailed genome characterization showed that they, together with the unclassified Chinese picornaviruses, grouped as a cluster for the P1, 2C, 3CD and VP1 coding regions. These viruses had conserved features (e.g. predicted protein cleavage sites, presence of a leader protein, 2A, 2C, 3C, and 3D functional domains), suggesting they have a common ancestor. Reverse-transcription-PCR assays, using specific primers designed from the 5'UTR sequence of these viruses, showed that 23.0% (20/87) of fecal samples from cattle with diarrhea were positive, indicating the prevalence of these picornavirus in the Japanese cattle population in Hokkaido Prefecture. However, further studies are needed to investigate the pathogenic potential and etiological role of these viruses in cattle.

  9. TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase

    OpenAIRE

    Taylor, R. Travis; Lubick, Kirk J.; Robertson, Shelly J.; Broughton, James P.; Bloom, Marshall E.; Bresnahan, Wade A.; Best, Sonja M.

    2011-01-01

    In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential f...

  10. Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly.

    Science.gov (United States)

    Mine, Akira; Hyodo, Kiwamu; Takeda, Atsushi; Kaido, Masanori; Mise, Kazuyuki; Okuno, Tetsuro

    2010-11-25

    Red clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we identified domains in p27 and p88 responsible for their protein-protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27-p27 and p27-p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly.

  11. Chronic DNA Replication Stress Reduces Replicative Lifespan of Cells by TRP53-Dependent, microRNA-Assisted MCM2-7 Downregulation.

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    Gongshi Bai

    2016-01-01

    Full Text Available Circumstances that compromise efficient DNA replication, such as disruptions to replication fork progression, cause a state known as DNA replication stress (RS. Whereas normally proliferating cells experience low levels of RS, excessive RS from intrinsic or extrinsic sources can trigger cell cycle arrest and senescence. Here, we report that a key driver of RS-induced senescence is active downregulation of the Minichromosome Maintenance 2-7 (MCM2-7 factors that are essential for replication origin licensing and which constitute the replicative helicase core. Proliferating cells produce high levels of MCM2-7 that enable formation of dormant origins that can be activated in response to acute, experimentally-induced RS. However, little is known about how physiological RS levels impact MCM2-7 regulation. We found that chronic exposure of primary mouse embryonic fibroblasts (MEFs to either genetically-encoded or environmentally-induced RS triggered gradual MCM2-7 repression, followed by inhibition of replication and senescence that could be accelerated by MCM hemizygosity. The MCM2-7 reduction in response to RS is TRP53-dependent, and involves a group of Trp53-dependent miRNAs, including the miR-34 family, that repress MCM expression in replication-stressed cells before they undergo terminal cell cycle arrest. miR-34 ablation partially rescued MCM2-7 downregulation and genomic instability in mice with endogenous RS. Together, these data demonstrate that active MCM2-7 repression is a physiologically important mechanism for RS-induced cell cycle arrest and genome maintenance on an organismal level.

  12. 5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication

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    García-Sastre Adolfo

    2010-05-01

    Full Text Available Abstract Background Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I has recently been shown to induce antiviral state. Results In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA, a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication in vivo in mice. Conclusions Our findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.

  13. Avian picornaviruses: molecular evolution, genome diversity and unusual genome features of a rapidly expanding group of viruses in birds.

    Science.gov (United States)

    Boros, Ákos; Pankovics, Péter; Reuter, Gábor

    2014-12-01

    Picornaviridae is one of the most diverse families of viruses infecting vertebrate species. In contrast to the relative small number of mammal species compared to other vertebrates, the abundance of mammal-infecting picornaviruses was significantly overrepresented among the presently known picornaviruses. Therefore most of the current knowledge about the genome diversity/organization patterns and common genome features were based on the analysis of mammal-infecting picornaviruses. Beside the well known reservoir role of birds in case of several emerging viral pathogens, little is known about the diversity of picornaviruses circulating among birds, although in the last decade the number of known avian picornavirus species with complete genome was increased from one to at least 15. However, little is known about the geographic distribution, host spectrum or pathogenic potential of the recently described picornaviruses of birds. Despite the low number of known avian picornaviruses, the phylogenetic and genome organization diversity of these viruses were remarkable. Beside the common L-4-3-4 and 4-3-4 genome layouts unusual genome patterns (3-4-4; 3-5-4, 3-6-4; 3-8-4) with variable, multicistronic 2A genome regions were found among avian picornaviruses. The phylogenetic and genomic analysis revealed the presence of several conserved structures at the untranslated regions among phylogenetically distant avian and non-avian picornaviruses as well as at least five different avian picornavirus phylogenetic clusters located in every main picornavirus lineage with characteristic genome layouts which suggests the complex evolution history of these viruses. Based on the remarkable genetic diversity of the few known avian picornaviruses, the emergence of further divergent picornaviruses causing challenges in the current taxonomy and also in the understanding of the evolution and genome organization of picornaviruses will be strongly expected. In this review we would like to

  14. The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling

    NARCIS (Netherlands)

    Fros, J.J.; Maten, van der E.; Vlak, J.M.; Pijlman, G.P.

    2013-01-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S

  15. The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling

    NARCIS (Netherlands)

    Fros, J.J.; Maten, van der E.; Vlak, J.M.; Pijlman, G.P.

    2013-01-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S

  16. Novel Picornavirus Associated with Avian Keratin Disorder in Alaskan Birds

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    Maxine Zylberberg

    2016-07-01

    Full Text Available Avian keratin disorder (AKD, characterized by debilitating overgrowth of the avian beak, was first documented in black-capped chickadees (Poecile atricapillus in Alaska. Subsequently, similar deformities have appeared in numerous species across continents. Despite the widespread distribution of this emerging pathology, the cause of AKD remains elusive. As a result, it is unknown whether suspected cases of AKD in the afflicted species are causally linked, and the impacts of this pathology at the population and community levels are difficult to evaluate. We applied unbiased, metagenomic next-generation sequencing to search for candidate pathogens in birds affected with AKD. We identified and sequenced the complete coding region of a novel picornavirus, which we are calling poecivirus. Subsequent screening of 19 AKD-affected black-capped chickadees and 9 control individuals for the presence of poecivirus revealed that 19/19 (100% AKD-affected individuals were positive, while only 2/9 (22% control individuals were infected with poecivirus. Two northwestern crows (Corvus caurinus and two red-breasted nuthatches (Sitta canadensis with AKD-consistent pathology also tested positive for poecivirus. We suggest that poecivirus is a candidate etiological agent of AKD.

  17. Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

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    Clerzius Guerline

    2005-10-01

    Full Text Available Abstract Increasing evidence indicates that RNA interference (RNAi may be used to provide antiviral immunity in mammalian cells. Human micro (miRNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN-induced protein kinase R (PKR but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si and micro (miRNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.

  18. The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

    Science.gov (United States)

    Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  19. HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV-infection: does it really mean viral replication?

    Institute of Scientific and Technical Information of China (English)

    Volker Meier; Sabine Mihm; Perdita Wietzke-Braun; Guliano Ramadori

    2001-01-01

    AIM To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC)and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication,METHODS HCV-RNA was monitored in serumand PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-α therapy using a nested RT/ PCRtechnique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma.RESULTS In the IFN-α responding patients,HCV-RNA disappeared first from total RNApreparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNAreappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNAconcentration in serum was performed before and after transition from detectable to nondetectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMCpreparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMCfrom healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration-dependent PCR-positivity for HCV-RNA in reisolated PBMC.CONCLUSION The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMCpreparations of chronically infected patients.

  20. In vitro antiviral activity of circular triple helix forming oligonucleotide RNA towards Feline Infectious Peritonitis virus replication.

    Science.gov (United States)

    Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman

    2014-01-01

    Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

  1. In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication

    Directory of Open Access Journals (Sweden)

    Oi Kuan Choong

    2014-01-01

    Full Text Available Feline Infectious Peritonitis (FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV, a virulent mutant strain of Feline Enteric Coronavirus (FECV. Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO RNAs (TFO1 to TFO5, which target the different regions of virulent feline coronavirus (FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 1014 in the virus-inoculated cells to 109 in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

  2. Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells

    Institute of Scientific and Technical Information of China (English)

    Gui-Qiu Li; Wei-Zhen Xu; Jing-Xia Wang; Wen-Wei Deng; Di Li; Hong-Xi Gu

    2007-01-01

    AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression by combination of siRNA and lamivudine in HepG2.2.15 cells.METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. The transfected cells were cultured in lamivudine-containing medium (0.05 μmol/L) and harvested at 48, 72 and 96 h. The concentration of HBeAg and HBsAg was determined using ELISA. HBV DMA replication was examined by realtime PCR and the level of HBV mRNA was measured by RT-PCR.RESULTS: In HepG2.2.15 cells treated with combination of siRNA and lamivudine, the secretion of HBeAg and HBsAg into the supernatant was found to be inhibited by 91.80% and 82.40% (2.89 ± 0.48 vs 11.73±0.38,P<0.05;4.59±0.57 vs 16.25±O.48,P<0.05) at 96 h,respectively;the number of HBV DNA copies within culture medium was also significantly decreased at 96 h (1.04±0.26 vs 8.35±0.33,P<0.05).Moreover,mRNA concentration in HepG2.2.15 cells treated with combination of siRNA and lamivudine was obviously lower compared to those treated either with siRNA or lamivudine (19.44±0.17 vs 33.27±0.21 or 79.9±0.13,P<O.05).CONCLUSION:Combination of siRNA and lamivudine is more effective in inhibiting HBV replication as compared to the single use of siRNA or lamivudine in HepG2.2.15 cells.

  3. Zn(2+ inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture.

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    Aartjan J W te Velthuis

    Full Text Available Increasing the intracellular Zn(2+ concentration with zinc-ionophores like pyrithione (PT can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn(2+ and PT at low concentrations (2 µM Zn(2+ and 2 µM PT inhibits the replication of SARS-coronavirus (SARS-CoV and equine arteritis virus (EAV in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp, which is the core enzyme of their multiprotein replication and transcription complex (RTC. Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV--thus eliminating the need for PT to transport Zn(2+ across the plasma membrane--we show that Zn(2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9 purified from E. coli subsequently revealed that Zn(2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn(2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn(2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.

  4. The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast

    Science.gov (United States)

    Nagy, Peter D.

    2017-01-01

    Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (endosomal sorting complexes required for transport I) or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs) used for in vitro assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in pex3Δ yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases. PMID:28759634

  5. The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast.

    Directory of Open Access Journals (Sweden)

    Nikolay Kovalev

    2017-07-01

    Full Text Available Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (endosomal sorting complexes required for transport I or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs used for in vitro assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in pex3Δ yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases.

  6. Transient replication of Hepatitis C Virus sub-genomic RNA in murine cell lines is enabled by miR-122 and varies with cell passage.

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    Patricia A Thibault

    Full Text Available Hepatitis C Virus (HCV is a serious global health problem, infecting almost 3% of the world's population. The lack of model systems for studying this virus limit research options in vaccine and therapeutic development, as well as for studying the pathogenesis of chronic HCV infection. Herein we make use of the liver-specific microRNA miR-122 to render mouse cell lines permissive to HCV replication in an attempt to develop additional model systems for the identification of new features of the virus and its life cycle. We have determined that some wild-type and knockout mouse cell lines--NCoA6 and PKR knockout embryonic fibroblasts--can be rendered permissive to transient HCV sub-genomic RNA replication upon addition of miR-122, but we did not observe replication of full-length HCV RNA in these cells. However, other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122, and in fact, different NCoA6 and PKR knockout cell line passages and isolates from the same mice demonstrated varying permissiveness phenotypes and eventually complete loss of permissiveness. When we tested knockdown of NCoA6 and PKR in Huh7.5 cells, we saw no substantial impact in sub-genomic HCV replication, which we would expect if these genes were inhibitory to the virus' life cycle. This leads us to conclude that along with the influence of specific gene knockouts there are additional factors within the cell lines that affect their permissiveness for HCV replication; we suggest that these may be epigenetically regulated, or modulated by cell line immortalization and transformation.

  7. RNA interference-mediated targeting of human cytomegalovirus immediate-early or early gene products inhibits viral replication with differential effects on cellular functions.

    Science.gov (United States)

    Xiaofei, E; Stadler, Bradford M; Debatis, Michelle; Wang, Shixia; Lu, Shan; Kowalik, Timothy F

    2012-05-01

    Viral drug toxicity, resistance, and an increasing immunosuppressed population warrant continued research into new avenues for limiting diseases associated with human cytomegalovirus (HCMV). In this study, a small interfering RNA (siRNA), siX3, was designed to target coding sequences within shared exon 3 of UL123 and UL122 transcripts encoding IE1 and IE2 immediate-early proteins of HCMV. Pretreatment of cells with siX3 reduced the levels of viral protein expression, DNA replication, and progeny virus production compared to control siRNA. Two siRNAs against UL54 and overlapping transcripts (UL55-57) were compared to siX3 in HCMV infection and were also found to be effective at inhibiting HCMV replication. Further investigation into the effects of the siRNAs on viral replication showed that pretreatment with each of the siRNAs resulted in an inhibition in the formation of mature replication compartments. The ability of these siRNAs to prevent or reduce certain cytopathic effects associated with HCMV infection was also examined. Infected cells pretreated with siX3, but not siUL54, retained promyelocytic leukemia (PML) protein in cellular PML bodies, an essential component of this host intrinsic antiviral defense. DNA damage response proteins, which are localized in nuclear viral replication compartments, were reduced in the siX3- and siUL54-treated cells. siX3, but not siUL54, prevented DNA damage response signaling early after infection. Therapeutic efficacy was demonstrated by treating cells with siRNAs after HCMV replication had commenced. Together, these findings suggest that siRNAs targeting exon 3 of the major IE genes or the UL54-57 transcripts be further studied for their potential development into anti-HCMV therapeutics.

  8. 人工microRNA抗乙型肝炎病毒效果初探%Artificial microRNA Can Suppress Hepatitis B Virus Replication Efficiently In Vitro

    Institute of Scientific and Technical Information of China (English)

    赵海峰; 李丹丹; 李英; 胡伟; 王学军; 王升启

    2011-01-01

    Objective: To screen artificial microRNA (amiRNA) which can inhibit hepatitis B virus (HBV) replication in vitro. Methods: amiRNA targeted HBV conserved regions were designed and cloned into the vector pcDNA6.2-GW/EmGFP-miR. The inhibition rate of different amiRNA were compared by cotransfection amiRNA vectors and the HBV replicon vector pHBV1.31 into HepG2 cells. After 72 h, HBsAg, HBeAg and HBV DNA were assayed and analyzed. Results: amiRNA can suppress HBV replication efficiently in vitro. Conclusion: amiRNA as a potential anti-HBV technology shound be studied deeply in future.%目的:设计靶向乙型肝炎病毒(HBV)基因保守区的人工microRNA (amiRNA),考察其对HBV基因表达的抑制作用.方法:比对HBV全基因组现有序列,选择保守区设计amiRNA,定向克隆到pcDNA6.2-GW/EmGFP-miR载体,将amiRNA载体与HBV复制载体pHBV1.31共转染HepG2细胞,72 h后收取细胞上清,ELISA检测HBV表面抗原(HBsAg)及e抗原(HBeAg)的含量,荧光定量PCR检测HBV DNA含量.结果:amiRNA可显著抑制细胞上清HBsAg、HBeAg和HBV DNA的水平.结论:amiRNA作为防治HBV感染的潜在有效手段之一值得进一步深入研究.

  9. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    Science.gov (United States)

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.

  10. Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

    Science.gov (United States)

    Wang, H-M; Xia, X-Z; Hu, G-X; Yu, L; He, H-B

    2016-03-01

    Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6 hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA.

  11. A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.

    Science.gov (United States)

    Hatakeyama, Dai; Shoji, Masaki; Yamayoshi, Seiya; Hirota, Takenori; Nagae, Monami; Yanagisawa, Shin; Nakano, Masahiro; Ohmi, Naho; Noda, Takeshi; Kawaoka, Yoshihiro; Kuzuhara, Takashi

    2014-09-05

    The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

  13. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export.

    Science.gov (United States)

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation.

  14. The host-dependent interaction of alpha-importins with influenza PB2 polymerase subunit is required for virus RNA replication.

    Directory of Open Access Journals (Sweden)

    Patricia Resa-Infante

    Full Text Available The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with alpha-importins is required for virus RNA replication. To ascertain whether PB2-alpha-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host.

  15. Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus

    Science.gov (United States)

    Fukuhara, Takasuke; Kambara, Hiroto; Shiokawa, Mai; Ono, Chikako; Katoh, Hiroshi; Morita, Eiji; Okuzaki, Daisuke; Maehara, Yoshihiko; Koike, Kazuhiko

    2012-01-01

    Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly. PMID:22593164

  16. Origin of life. Primordial genetics: Information transfer in a pre-RNA world based on self-replicating beta-sheet amyloid conformers.

    Science.gov (United States)

    Maury, Carl Peter J

    2015-10-07

    The question of the origin of life on Earth can largely be reduced to the question of what was the first molecular replicator system that was able to replicate and evolve under the presumably very harsh conditions on the early Earth. It is unlikely that a functional RNA could have existed under such conditions and it is generally assumed that some other kind of information system preceded the RNA world. Here, I present an informational molecular system that is stable, self-replicative, environmentally responsive, and evolvable under conditions characterized by high temperatures, ultraviolet and cosmic radiation. This postulated pregenetic system is based on the amyloid fold, a functionally unique polypeptide fold characterized by a cross beta-sheet structure in which the beta strands are arranged perpendicular to the fiber axis. Beside an extraordinary structural robustness, the amyloid fold possesses a unique ability to transmit information by a three-dimensional templating mechanism. In amyloidogenesis short peptide monomers are added one by one to the growing end of the fiber. From the same monomeric subunits several structural variants of amyloid may be formed. Then, in a self-replicative mode, a specific amyloid conformer can act as a template and confer its spatially encoded information to daughter molecular entities in a repetitive way. In this process, the specific conformational information, the spatially changed organization, is transmitted; the coding element is the steric zipper structure, and recognition occurs by amino acid side chain complementarity. The amyloid information system fulfills several basic requirements of a primordial evolvable replicator system: (i) it is stable under the presumed primitive Earth conditions, (ii) the monomeric building blocks of the informational polymer can be formed from available prebiotic compounds, (iii) the system is self-assembling and self-replicative and (iv) it is adaptive to changes in the environment and

  17. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Anna Garbelli

    Full Text Available DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  18. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    Science.gov (United States)

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  19. Mouse hepatitis coronavirus replication induces host translational shutoff and mRNA decay, with concomitant formation of stress granules and processing bodies.

    Science.gov (United States)

    Raaben, Matthijs; Groot Koerkamp, Marian J A; Rottier, Peter J M; de Haan, Cornelis A M

    2007-09-01

    Many viruses, including coronaviruses, induce host translational shutoff, while maintaining synthesis of their own gene products. In this study we performed genome-wide microarray analyses of the expression patterns of mouse hepatitis coronavirus (MHV)-infected cells. At the time of MHV-induced host translational shutoff, downregulation of numerous mRNAs, many of which encode protein translation-related factors, was observed. This downregulation, which is reminiscent of a cellular stress response, was dependent on viral replication and caused by mRNA decay. Concomitantly, phosphorylation of the eukaryotic translation initiation factor 2alpha was increased in MHV-infected cells. In addition, stress granules and processing bodies appeared, which are sites for mRNA stalling and degradation respectively. We propose that MHV replication induces host translational shutoff by triggering an integrated stress response. However, MHV replication per se does not appear to benefit from the inhibition of host protein synthesis, at least in vitro, since viral replication was not negatively affected but rather enhanced in cells with impaired translational shutoff.

  20. A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication

    Directory of Open Access Journals (Sweden)

    Stoltzfus C Martin

    2006-02-01

    Full Text Available Abstract Background Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1 primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss, which are positively regulated by exonic splicing enhancers (ESE and negatively regulated by exonic splicing silencers (ESS and intronic splicing silencers (ISS. We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. Results Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. Conclusion These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non

  1. Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation.

    Science.gov (United States)

    Kim, Mi-Gyeong; Moon, Jae-Su; Kim, Eun-Jung; Lee, Seung-Hoon; Oh, Jong-Won

    2012-04-27

    Heat shock protein 90 (Hsp90), which chaperones multiple client proteins, has been shown to be implicated in HCV replication. Pharmacological inhibitors of Hsp90 display an anti-HCV activity. However, little is known about the mechanisms of regulation of HCV replication by Hsp90. Here, we show that Hsp90 inhibition by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) destabilizes phosphoinositide-dependent kinase-1 (PDK1), an upstream kinase of the protein kinase C-related kinase 2 (PRK2) responsible for phosphorylation of HCV RNA polymerase, through the proteosome pathway. Destabilization of PDK1 led to inhibition of phosphorylation of the viral RNA polymerase through a decrease in the abundance of active form PRK2 level. Consequently, Hsp90 inhibition resulted in suppression of HCV replication both in human hepatoma Huh7 cells harboring an HCV subgenomic replicon and in HCV-infected cells. 17-DMAG treatment did not interfere with HCV internal ribosome entry site-mediated translation and the cell cycle in Huh7 cells. Co-treatment of 17-DMAG with interferon-α or HA1077, an inhibitor of PRK2, enhanced the anti-HCV activity of 17-DMAG. Taken together, these findings suggest that Hsp90 plays a critical role in the regulation of HCV RNA polymerase phosphorylation via the PDK1-PRK2 signaling pathway.

  2. Species-Specific Transmission of Novel Picornaviruses in Lemurs

    Science.gov (United States)

    Lim, Efrem S.; Deem, Sharon L.; Porton, Ingrid J.; Cao, Song

    2015-01-01

    ABSTRACT The roles of host genetics versus exposure and contact frequency in driving cross-species transmission remain the subject of debate. Here, we used a multitaxon lemur collection at the Saint Louis Zoo in the United States as a model to gain insight into viral transmission in a setting of high interspecies contact. Lemurs are a diverse and understudied group of primates that are highly endangered. The speciation of lemurs, which are endemic to the island of Madagascar, occurred in geographic isolation apart from that of continental African primates. Although evidence of endogenized viruses in lemur genomes exists, no exogenous viruses of lemurs have been described to date. Here we identified two novel picornaviruses in fecal specimens of ring-tailed lemurs (Lemur catta) and black-and-white ruffed lemurs (Varecia variegata). We found that the viruses were transmitted in a species-specific manner (lesavirus 1 was detected only in ring-tailed lemurs, while lesavirus 2 was detected only in black-and-white ruffed lemurs). Longitudinal sampling over a 1-year interval demonstrated ongoing infection in the collection. This was supported by evidence of viral clearance in some animals and new infections in previously uninfected animals, including a set of newly born triplets that acquired the infection. While the two virus strains were found to be cocirculating in a mixed-species exhibit of ring-tailed lemurs, black-and-white ruffed lemurs, and black lemurs, there was no evidence of cross-species transmission. This suggests that despite high-intensity contact, host species barriers can prevent cross-species transmissions of these viruses. IMPORTANCE Up to 75% of emerging infectious diseases in humans today are the result of zoonotic transmission. However, a challenge in understanding transmission dynamics has been the limited models of cross-species transmission. Zoos provide a unique opportunity to explore parameters defining viral transmission. We demonstrated that

  3. Structural features of the Seneca Valley virus internal ribosome entry site (IRES) element: a picornavirus with a pestivirus-like IRES.

    Science.gov (United States)

    Willcocks, Margaret M; Locker, Nicolas; Gomwalk, Zarmwa; Royall, Elizabeth; Bakhshesh, Mehran; Belsham, Graham J; Idamakanti, Neeraja; Burroughs, Kevin D; Reddy, P Seshidhar; Hallenbeck, Paul L; Roberts, Lisa O

    2011-05-01

    The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId(2) domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.

  4. mRNA decay factor AUF1 binds the internal ribosomal entry site of enterovirus 71 and inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Jing-Yi Lin

    Full Text Available AU-rich element binding factor 1 (AUF1 has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES of enterovirus 71 (EV71 and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.

  5. A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching

    Science.gov (United States)

    Hopkins, Kaycie C.; McLane, Laura M.; Maqbool, Tariq; Panda, Debasis; Gordesky-Gold, Beth; Cherry, Sara

    2013-01-01

    Bunyaviruses are an emerging group of medically important viruses, many of which are transmitted from insects to mammals. To identify host factors that impact infection, we performed a genome-wide RNAi screen in Drosophila and identified 131 genes that impacted infection of the mosquito-transmitted bunyavirus Rift Valley fever virus (RVFV). Dcp2, the catalytic component of the mRNA decapping machinery, and two decapping activators, DDX6 and LSM7, were antiviral against disparate bunyaviruses in both insect cells and adult flies. Bunyaviruses 5′ cap their mRNAs by “cap-snatching” the 5′ ends of poorly defined host mRNAs. We found that RVFV cap-snatches the 5′ ends of Dcp2 targeted mRNAs, including cell cycle-related genes. Loss of Dcp2 allows increased viral transcription without impacting viral mRNA stability, while ectopic expression of Dcp2 impedes viral transcription. Furthermore, arresting cells in late S/early G2 led to increased Dcp2 mRNA targets and increased RVFV replication. Therefore, RVFV competes for the Dcp2-accessible mRNA pool, which is dynamically regulated and can present a bottleneck for viral replication. PMID:23824541

  6. RNA genetics

    Energy Technology Data Exchange (ETDEWEB)

    Domingo, E. (Instituto de Biologia Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, Canto Blanco, Madrid (ES)); Holland, J.J. (California Univ., San Diego, La Jolla, CA (USA). Dept. of Biology); Ahlquist, P. (Wisconsin Univ., Madison, WI (USA). Dept. of Plant Pathology)

    1988-01-01

    This book contains the proceedings on RNA genetics: RNA-directed virus replication Volume 1. Topics covered include: Replication of the poliovirus genome; Influenza viral RNA transcription and replication; and Relication of the reoviridal: Information derived from gene cloning and expression.

  7. TGF-β and iron differently alter HBV replication in human hepatocytes through TGF-β/BMP signaling and cellular microRNA expression.

    Directory of Open Access Journals (Sweden)

    Sun O Park

    Full Text Available The nature of host-virus interactions in hepatitis B virus infection is incompletely understood. Since soluble factors, e.g., cytokines and metals, may exacerbate liver injury in chronic hepatitis, we considered that defining the effects of receptor-mediated signaling upon viral replication will be significant. Consequently, we studied effects of iron or TGF-β-induced TGF-β/BMP signaling in the HepG2 2.2.15 cell model of hepatitis B virus replication. We found iron and TGF-β increased hepcidin mRNA expression or TGF-β receptor kinase activity, respectively, which indicated that 2.2.15 cells responded appropriately to these substances. However, iron increased but TGF-β decreased hepatitis B virus mRNA and DNA expression. TGF-β induced expression at the mRNA level of multiple TGF-β/BMP pathway genes. This change was not observed in iron-treated cells. On the other hand, presence of SMAD proteins in iron or TGF-β-treated cells, including of SMAD4, did confirm convergence of TGF-β/BMP signaling pathways under these conditions. Since transcription factors in TGF-β/BMP signaling pathways could not have directly targeted hepatitis B virus itself, we studied whether iron or TGF-β exerted their effects through alternative mechanisms, such as by involvement of antiviral cellular microRNAs. We discovered cellular microRNA expression profiles were significantly different in iron or TGF-β-treated cells compared with untreated control cells. In many cases, exposure to iron or TGF-β changed microRNA expression in opposite directions. Introduction in cells of sequences representing such differentially expressed microRNAs, e.g., hsa-miR-125a-5p and -151-5p, even reproduced effects on virus replication of iron- or TGF-β. We surmised that TGF-β/BMP pathway members, i.e., SMADs, likely governed iron or TGF-β-induced microRNA expression. Iron may have mediated Drosha/DGCR8/heme-mediated processing of microRNAs. In turn, cellular microRNAs regulated

  8. Cellular microRNA miR-181b inhibits replication of mink enteritis virus by repression of non-structural protein 1 translation.

    Science.gov (United States)

    Sun, Jia-zeng; Wang, Jigui; Yuan, Daoli; Wang, Shuang; Li, Zhili; Yi, Bao; Mao, Yaping; Hou, Qiang; Liu, Weiquan

    2013-01-01

    Mink enteritis virus (MEV) is one of the most important viral pathogens in the mink industry. Recent studies have showed that microRNAs (miRNAs), small noncoding RNAs of length ranging from 18-23 nucleotides (nt) participate in host-pathogen interaction networks; however, whether or not miRNAs are involved in MEV infection has not been reported. Our study revealed that miRNA miR-181b inhibited replication of MEV in the feline kidney (F81) cell line by targeting the MEV non-structural protein 1 (NS1) messenger RNA (mRNA) coding region, resulting in NS1 translational repression, while MEV infection reduced miR-181b expression. This is the first description of cellular miRNAs modulating MEV infection in F81 cells, providing further insight into the mechanisms of viral infection, and may be useful in development of naturally-occurring miRNAs antiviral strategies.

  9. RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications.

    Science.gov (United States)

    Mutso, Margit; Nikonov, Andrei; Pihlak, Arno; Žusinaite, Eva; Viru, Liane; Selyutina, Anastasia; Reintamm, Tõnu; Kelve, Merike; Saarma, Mart; Karelson, Mati; Merits, Andres

    2015-01-01

    The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.

  10. RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications.

    Directory of Open Access Journals (Sweden)

    Margit Mutso

    Full Text Available The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA residues at its termini (LNA/DNA gapmer by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50: 0.13 nM, whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively. However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.

  11. RNA interference against Enterovirus 71 replication%RNA干扰技术抗肠道病毒71型复制

    Institute of Scientific and Technical Information of China (English)

    李国栋; 杨倬; 张景海

    2013-01-01

    Objective To provide certain scientific basis to suppress Enterovirus 71 (EV 71) replication by RNA interference (RNAi). Methods RNA interference (RNAi) was used as a therapeutic approach to inhibit EV 71 replication in rhabdomyosarcoma(RD) cells. The inhibition effects were confirmed by a corresponding decrease in viral RNA of the infected cells,the expression level of viral protein and the number of progeny virus in the cultured supernatants. The inhibitory effect was validated through Western-blot, Real-time PCR and virus titer. Results The siRNAs targeting viral genome 5 'UTR and VP2 could effectively block viral replications. Conclusions This study confirms that RNAi method possesses potential and feasibility in the specific viral inhibition.%目的 肠道病毒71型(Enterovirus 71,EV 71)是手足口病(hand,foot and mouth disease,HFMD)主要的病原体之一.研究RNA干扰技术抑制肠道病毒71型复制.方法 以RNA干扰技术(RNAi)为干预手段,抑制病毒EV 71在人横纹肌肉瘤(rhabdomyosarcoma,RD)细胞中的复制.通过Western blot、Real-time PCR和病毒滴度3种方法验证,其抑制效果体现在感染细胞内部病毒RNA,病毒蛋白质的表达水平和培养基上清中子代病毒颗粒的数量上.结果 靶向病毒基因组5'UTR和VP2的siRNAs具有明显的病毒抑制效应.结论 此研究证实RNAi方法具有特异性抑制病毒复制的潜能和可行性.

  12. Antiviral Innate Immune Response Interferes with the Formation of Replication-Associated Membrane Structures Induced by a Positive-Strand RNA Virus

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    Diede Oudshoorn

    2016-12-01

    Full Text Available Infection with nidoviruses like corona- and arteriviruses induces a reticulovesicular network of interconnected endoplasmic reticulum (ER-derived double-membrane vesicles (DMVs and other membrane structures. This network is thought to accommodate the viral replication machinery and protect it from innate immune detection. We hypothesized that the innate immune response has tools to counteract the formation of these virus-induced replication organelles in order to inhibit virus replication. Here we have investigated the effect of type I interferon (IFN treatment on the formation of arterivirus-induced membrane structures. Our approach involved ectopic expression of arterivirus nonstructural proteins nsp2 and nsp3, which induce DMV formation in the absence of other viral triggers of the interferon response, such as replicating viral RNA. Thus, this setup can be used to identify immune effectors that specifically target the (formation of virus-induced membrane structures. Using large-scale electron microscopy mosaic maps, we found that IFN-β treatment significantly reduced the formation of the membrane structures. Strikingly, we also observed abundant stretches of double-membrane sheets (a proposed intermediate of DMV formation in IFN-β-treated samples, suggesting the disruption of DMV biogenesis. Three interferon-stimulated gene products, two of which have been reported to target the hepatitis C virus replication structures, were tested for their possible involvement, but none of them affected membrane structure formation. Our study reveals the existence of a previously unknown innate immune mechanism that antagonizes the viral hijacking of host membranes. It also provides a solid basis for further research into the poorly understood interactions between the innate immune system and virus-induced replication structures.

  13. Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution.

    Science.gov (United States)

    Gritsun, Dmitri J; Jones, Ian M; Gould, Ernest A; Gritsun, Tamara S

    2014-01-01

    RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.

  14. Crystal structure of complete rhinovirus RNA polymerase suggests front loading of protein primer.

    Science.gov (United States)

    Appleby, Todd C; Luecke, Hartmut; Shim, Jae Hoon; Wu, Jim Z; Cheney, I Wayne; Zhong, Weidong; Vogeley, Lutz; Hong, Zhi; Yao, Nanhua

    2005-01-01

    Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3Dpol) at a 2.4-A resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3Dpol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3Dpo1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.

  15. Molecular epidemiology of canine picornavirus in Hong Kong and Dubai and proposal of a novel genus in Picornaviridae.

    Science.gov (United States)

    Woo, Patrick C Y; Lau, Susanna K P; Choi, Garnet K Y; Huang, Yi; Sivakumar, Saritha; Tsoi, Hoi-Wah; Yip, Cyril C Y; Jose, Shanty V; Bai, Ru; Wong, Emily Y M; Joseph, Marina; Li, Tong; Wernery, Ulrich; Yuen, Kwok-Yung

    2016-07-01

    Previously, we reported the discovery of a novel canine picornavirus (CanPV) in the fecal sample of a dog. In this molecular epidemiology study, CanPV was detected in 15 (1.11%) of 1347 canine fecal samples from Hong Kong and one (0.76%) of 131 canine fecal samples from Dubai, with viral loads 1.06×10(3) to 6.64×10(6) copies/ml. Complete genome sequencing and phylogenetic analysis showed that CanPV was clustered with feline picornavirus (FePV), bat picornavirus (BatPV) 1 to 3, Ia io picornavirus 1 (IaioPV1) and bovine picornavirus (BoPV), and this cluster was most closely related to the genera Enterovirus and Sapelovirus. The Ka/Ks ratios of all the coding regions were <0.1. According to the definition of the Picornavirus Study Group of ICTV, CanPV, FePV, BatPV 1 to 3, IaioPV1 and BoPV should constitute a novel genus in Picornaviridae. BEAST analysis showed that this genus diverged from its most closely related genus, Sapelovirus, about 49 years ago.

  16. Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease's active site cysteine residue.

    Science.gov (United States)

    Rausalu, Kai; Utt, Age; Quirin, Tania; Varghese, Finny S; Žusinaite, Eva; Das, Pratyush Kumar; Ahola, Tero; Merits, Andres

    2016-11-15

    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.

  17. Respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children.

    Science.gov (United States)

    Jartti, Tuomas; Lehtinen, Pasi; Vuorinen, Tytti; Osterback, Riika; van den Hoogen, Bernadette; Osterhaus, Albert D M E; Ruuskanen, Olli

    2004-06-01

    We studied the viral etiology of acute expiratory wheezing (bronchiolitis, acute asthma) in 293 hospitalized children in a 2-year prospective study in Finland. A potential causative viral agent was detected in 88% of the cases. Eleven different viruses were represented. Respiratory syncytial virus (RSV) (27%), enteroviruses (25%), rhinovirus (24%), and nontypable rhino/enterovirus (16%) were found most frequently. In infants, RSV was found in 54% and respiratory picornaviruses (rhinovirus and enteroviruses) in 42% of the cases. In older children, respiratory picornaviruses dominated (65% of children ages 1-2 years and 82% of children ages > or =3 years). Human metapneumovirus was detected in 4% of all children and in 11% of infants. To prevent and treat acute expiratory wheezing illnesses in children, efforts should be focused on RSV, enterovirus, and rhinovirus infections.

  18. A divergent picornavirus from a turkey with gastro-intestinal disease

    Science.gov (United States)

    A novel picornavirus, turkey avisivirus (TuASV), was identified from the feces of turkeys (Meleagris gallopavo) with gastro-intestinal disease from a farm in Indiana, USA. Its genome organization is 5’UTR**IRES-II[VP0,VP3,VP1,2A,2B,2C,3A,3B,3Cpro,3Dpol]3’UTR-poly(A). TuASV only shares 34% (P1), 36% ...

  19. Discovery of SCH446211 (SCH6): A New Ketoamide Inhibitor of the HCV NS3 Serine Protease and HCV Subgenomic RNA Replication

    Energy Technology Data Exchange (ETDEWEB)

    Bogen, Stephane L.; Arasappan, Ashok; Bennett, Frank; Chen, Kevin; Jao, Edwin; Liu, Yi-Tsung; Lovey, Raymond G.; Venkatraman, Srikanth; Pan, Weidong; Parekh, Tajel; Pike, Russel E.; Ruan, Sumei; Liu, Rong; Baroudy, Bahige; Agrawal, Sony; Chase, Robert; Ingravallo, Paul; Pichardo, John; Prongay, Andrew; Brisson, Jean-Marc; Hsieh, Tony Y.; Cheng, Kuo-Chi; Kemp, Scott J.; Levy, Odile E.; Lim-Wilby, Marguerita; Tamura, Susan Y.; Saksena, Anil K.; Girijavallabhan, Viyyoor; Njoroge, F. George (SPRI)

    2008-06-30

    Introduction of various modified prolines at P{sub 2} and optimization of the P{sub 1} side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K*{sub i} = 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC{sub 50} and IC{sub 90} of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.

  20. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    Science.gov (United States)

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-07-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  1. Gastroenteritis and the novel picornaviruses aichi virus, cosavirus, saffold virus, and salivirus in young children.

    Science.gov (United States)

    Nielsen, Alex Christian Yde; Gyhrs, Mette Louise; Nielsen, Lars Peter; Pedersen, Court; Böttiger, Blenda

    2013-07-01

    During the last few years many new human picornaviruses have been discovered due to advances in metagenomics and other molecular biological approaches. The clinical significance and the occurrence are only sparsely described. To determine the epidemiology and clinical significance of infections with the novel human picornaviruses, aichi virus, cosavirus, salivirus, and saffold virus in infants in Denmark. We tested 1393 stool samples from a birth cohort of 454 children for these viruses. Samples were collected at ages 6, 10 and 15 months, and at episodes of gastroenteritis. Samples were tested by real-time reverse-transcriptase polymerase chain reaction assays. Each study participant had a diary, where the parents reported episodes of disease, including gastroenteritis. Aichi virus, salivirus and saffold virus were detected in 6, 9 and 38 of the children, respectively, but cosavirus was not detected in any of the children. There was a clear seasonal variation with most infections occurring in autumn and winter. A statistically significant association between the findings of salivirus and gastrointestinal disease was demonstrated. There was no association between gastrointestinal disease and the presence of aichi virus or saffold virus. The newly discovered human picornaviruses aichi virus, saffold virus, and salivirus are circulating in Danish children, with the most common being saffold virus. Saffold virus was seen almost exclusively in the autumn and winter period. Salivirus was the only virus, which was significantly associated with gastroenteritis, although the number of positive samples was rather low. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Picornaviruses in cerebrospinal fluid of children with meningitis in Luanda, Angola.

    Science.gov (United States)

    Pelkonen, Tuula; Roine, Irmeli; Anjos, Elizabete; Kaijalainen, Svetlana; Roivainen, Merja; Peltola, Heikki; Pitkäranta, Anne

    2012-07-01

    Human enteroviruses are the most common cause of viral meningitis. Viral-bacterial interaction may affect the clinical course and outcome of bacterial meningitis. In Africa, viruses might be responsible for 14-25% of all meningitis cases. However, only few studies from Africa have reported detection of viruses in the cerebrospinal fluid (CSF) or mixed viral-bacterial infections of the central nervous system (CNS). The aim of the present study was to investigate the presence of picornaviruses in the CSF of children suffering from meningitis in Luanda, Angola. The study included 142 consecutive children enrolled in a prospective study of bacterial meningitis in Luanda between 2005 and 2006, from whom a CSF sample was available. CSF samples were obtained at hospital admission, stored in a deep-freeze, and transported to Finland for testing by real-time PCR for picornaviruses. Enteroviruses were detected in 4 (3%) of 142 children with presumed bacterial meningitis. A 5-month-old girl with rhinovirus and Haemophilus influenzae meningitis recovered uneventfully. An 8-year-old girl with human enterovirus and pneumococcal meningitis developed no sequelae. A 2-month-old girl with human enterovirus and malaria recovered quickly. A 7-month-old girl with human enterovirus was treated for presumed tuberculous meningitis and survived with severe sequelae. Mixed infections of the CNS with picornaviruses and bacteria are rare. Detection of an enterovirus does not affect the clinical picture and outcome of bacterial meningitis.

  3. The complete genome of klassevirus – a novel picornavirus in pediatric stool

    Directory of Open Access Journals (Sweden)

    Ganem Donald

    2009-06-01

    Full Text Available Abstract Background Diarrhea kills 2 million children worldwide each year, yet an etiological agent is not found in approximately 30–50% of cases. Picornaviral genera such as enterovirus, kobuvirus, cosavirus, parechovirus, hepatovirus, teschovirus, and cardiovirus have all been found in human and animal diarrhea. Modern technologies, especially deep sequencing, allow rapid, high-throughput screening of clinical samples such as stool for new infectious agents associated with human disease. Results A pool of 141 pediatric gastroenteritis samples that were previously found to be negative for known diarrheal viruses was subjected to pyrosequencing. From a total of 937,935 sequence reads, a collection of 849 reads distantly related to Aichi virus were assembled and found to comprise 75% of a novel picornavirus genome. The complete genome was subsequently cloned and found to share 52.3% nucleotide pairwise identity and 38.9% amino acid identity to Aichi virus. The low level of sequence identity suggests a novel picornavirus genus which we have designated klassevirus. Blinded screening of 751 stool specimens from both symptomatic and asymptomatic individuals revealed a second positive case of klassevirus infection, which was subsequently found to be from the index case's 11-month old twin. Conclusion We report the discovery of human klassevirus 1, a member of a novel picornavirus genus, in stool from two infants from Northern California. Further characterization and epidemiological studies will be required to establish whether klasseviruses are significant causes of human infection.

  4. Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts.

    Science.gov (United States)

    Kim, Kyongmin Hwang; Rümenapf, Tillmann; Strauss, Ellen G; Strauss, James H

    2004-05-20

    Alphavirus nonstructural proteins are translated as a polyprotein that is ultimately cleaved into four mature proteins called nsP1, nsP2, nsP3, and nsP4 from their order in the polyprotein. The role of this nonstructural polyprotein, of cleavage intermediates, and of mature proteins in synthesis of Semliki Forest virus (SFV) RNA has been studied using mutants unable to cleave one or more of the sites in the nonstructural polyprotein or that had the arginine sense codon between nsP3 and nsP4 changed to an opal termination codon. The results were compared with those obtained for Sindbis virus (SINV), which has a naturally occurring opal codon between nsP2 and nsP3. We found that (1) an active nonstructural protease in nsP2 is required for RNA synthesis. This protease is responsible for all three cleavages in the nonstructural polyprotein. (2) Cleavage between nsP3 and nsP4 (the viral RNA polymerase) is required for RNA synthesis by SFV. (3) SFV mutants that are able to produce only polyprotein P123 and nsP4 synthesize minus-strand RNA early after infection as efficiently as SF wild type but are defective in the synthesis of plus-strand RNA. The presence of sense or opal following nsP3 did not affect this result. At 30 degrees C, they give rise to low yields of virus after a delay, but at 39 degrees C, they are nonviable. (4) SFV mutants that produce nsP1, P23, nsP4, as well as the precursor P123 are viable but produce an order of magnitude less virus than wild type at 30 degrees C and two orders of magnitude less virus at 39 degrees C. The ratio of subgenomic mRNA to genomic RNA is much reduced in these mutants relative to the parental viruses. (5) At 30 degrees C, the variants containing an opal codon grow as well as or slightly better than the corresponding virus with a sense codon. At 39 degrees C, however, the opal variants produce significantly more virus. These results support the conclusion that SFV and SINV, and by extension all alphaviruses, regulate their

  5. APOBEC3G induces a hypermutation gradient: purifying selection at multiple steps during HIV-1 replication results in levels of G-to-A mutations that are high in DNA, intermediate in cellular viral RNA, and low in virion RNA

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    Pathak Vinay K

    2009-02-01

    Full Text Available Abstract Background Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown. Results To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y40RHHY44, were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA, and the levels of G-to-A mutations in virion RNA (vRNA were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA. Conclusion These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual

  6. Identification and characterization of a cis-encoded antisense RNA associated with the replication process of Salmonella enterica serovar Typhi.

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    Isaac Dadzie

    Full Text Available Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise while others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned.

  7. Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

    Science.gov (United States)

    Jiwaji, Meesbah; Short, James Roswell; Dorrington, Rosemary Ann

    2016-10-01

    Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

  8. Targeting the human DEAD-box polypeptide 3 (DDX3) RNA helicase as a novel strategy to inhibit viral replication.

    Science.gov (United States)

    Garbelli, A; Radi, M; Falchi, F; Beermann, S; Zanoli, S; Manetti, F; Dietrich, U; Botta, M; Maga, G

    2011-01-01

    Compounds currently used for the treatment of HIV-1 Infections are targeted to viral proteins. However, the high intrinsic mutation and replication rates of HIV-1 often lead to the emergence of drug resistant strains and consequent therapeutic failure. On this basis, cellular cofactors represent attractive new targets for HIV-1 chemotherapy, since targeting a cellular factor that is required for viral replication should help to overcome the problem of viral resistance. We and others have recently reported the identification of compounds suppressing HIV-1 replication by targeting the cellular DEAD-box helicase DDX3. These results provide a proof-of-principle for the feasibility of blocking HIV-1 infection by rendering the host cell environment less favorable for the virus. The rationale for such an approach and its implications in potentially overcoming the problem of drug resistance related to drugs targeting viral proteins will be discussed in the context of the known cellular functions of the DEAD-box helicase DDX3.

  9. Small interfering RNAs targeting peste des petits ruminants virus M mRNA increase virus-mediated fusogenicity and inhibit viral replication in vitro.

    Science.gov (United States)

    Liu, Fuxiao; Wu, Xiaodong; Zou, Yanli; Li, Lin; Liu, Shan; Chi, Tianying; Wang, Zhiliang

    2015-11-01

    Peste des petits ruminants (PPR), caused by peste des petits ruminants virus (PPRV), is an acute or subacute, highly contagious and economically important disease of small ruminants. The PPRV is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) protein possesses an intrinsic ability to bind to lipid membranes, and plays a crucial role in viral assembly and further budding. In this study, three different small interfering RNAs (siRNA) were designed on the basis of translated region for PPRV Nigeria 75/1M mRNA, and were subsequently synthesized for their transfection into Vero-SLAM cells, followed by infection with PPRVs. The results showed that two out of three siRNAs robustly induced cell-to-cell fusion as early as 36h post-infection with PPRVs, effectively suppressed expression of the M protein by interference for the M mRNA, and eventually inhibited viral replication in vitro. These findings led us to speculate that siRNA-mediated knockdown of the M protein would alter its interaction with viral glycoproteins, thus exacerbating intercellular fusion but hampering virus release. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Tomato bushy stunt virus and DI RNAs as a model for studying mechanisms of RNA virus replication, pathogenicity and recombination. Final technical report for 1994--1997

    Energy Technology Data Exchange (ETDEWEB)

    Morris, T.J. [Univ. of Nebraska, Lincoln, NE (United States). School of Biological Sciences; Jackson, A.O. [Univ. of California, Berkeley, CA (United States). Dept. of Plant Biology

    1997-12-31

    Tomato bushy stunt virus (TBSV) is a small icosahedral virus with a very broad host-range. The symptoms of systemic infection range from mild mosaic to severe necrosis that often results in death. The genome of TBSV is composed of a single plus stranded RNA molecule with five genes. Two 5 inch genes are translated from the viral RNA, and the remaining three are translated from two subgenomic RNAs. Prior to the DOE supported studies, TBSV gene function had been assigned solely on the basis of sequence similarity with other virus genes of known function. The two 5 inch proximal genes (p33 and p92) were thought to be involved in viral replication, the middle gene encoded the capsid protein (p41), but no clear function was assigned to two nested 3 inch genes (p19 and p22), although it was suggested that at least one could be involved in movement. This research has determined the roles of each of the viral genes in the infection process, and the authors have obtained considerable genetic information pertinent to the contributions of the coat protein and the nested genes to the disease phenotypes observed in several host plants. They have also identified another genetic element with a short open reading frame in the 3 inch-noncoding region of the genome that provides a host-dependent replication function.

  11. Induction and suppression of innate antiviral responses by picornaviruses

    NARCIS (Netherlands)

    Feng, Qian; Langereis, Martijn A; van Kuppeveld, Frank J M

    2014-01-01

    The family Picornaviridae comprises of small, non-enveloped, positive-strand RNA viruses and contains many human and animal pathogens including enteroviruses (e.g. poliovirus, coxsackievirus, enterovirus 71 and rhinovirus), cardioviruses (e.g. encephalomyocarditis virus), hepatitis A virus and foot-

  12. Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

    Directory of Open Access Journals (Sweden)

    Sallie Richard

    2005-08-01

    Full Text Available Abstract Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNApol generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNApol causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNApol infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations.

  13. Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue

    Science.gov (United States)

    Rausalu, Kai; Utt, Age; Quirin, Tania; Varghese, Finny S.; Žusinaite, Eva; Das, Pratyush Kumar; Ahola, Tero; Merits, Andres

    2016-01-01

    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease. PMID:27845418

  14. The NS3 and NS4A genes as the targets of RNA interference inhibit replication of Japanese encephalitis virus in vitro and in vivo.

    Science.gov (United States)

    Yuan, Lei; Wu, Rui; Liu, Hanyang; Wen, Xintian; Huang, Xiaobo; Wen, Yiping; Ma, Xiaoping; Yan, Qigui; Huang, Yong; Zhao, Qin; Cao, Sanjie

    2016-12-15

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that can cause acute encephalitis with a high fatality rate. RNA interference (RNAi) is a powerful tool to silence gene expression and a potential therapy for virus infection. In this study, the antiviral ability of eight shRNA expression plasmids targeting different sites of the NS3 and NS4A genes of JEV was determined in BHK21 cells and mice. The pGP-NS3-3 and pGP-NS4A-4 suppressed 93.9% and 82.0% of JEV mRNA in cells, respectively. The virus titer in cells was reduced approximately 950-fold by pretreating with pGP-NS3-4, and 640-fold by pretreating with pGP-NS4A-4. The results of western blot and immunofluorescence analysis showed JEV E protein and viral load in cells were remarkably inhibited by shRNA expression plasmids. The viral load in brains of mice pretreated with pGP-NS3-4 or pGP-NS4A-4 were reduced approximately 2400-fold and 800-fold, respectively, and the survival rate of mice challenged with JEV were 70% and 50%, respectively. However, the antiviral ability of shRNA expression plasmids was decreased over time. This study indicates that RNAi targeting of the NS3 and NS4A genes of JEV can sufficiently inhibit the replication of JEV in vitro and in vivo, and NS3 and NS4A genes might be potential targets of molecular therapy for JEV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Interaction of Sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) with the p10 domain of polyprotein 2a and its implications in SeMV replication.

    Science.gov (United States)

    Govind, Kunduri; Bakshi, Arindam; Savithri, Handanahal S

    2014-01-01

    Identification of viral encoded proteins that interact with RNA-dependent RNA polymerase (RdRp) is an important step towards unraveling the mechanism of replication. Sesbania mosaic virus (SeMV) RdRp was shown to interact strongly with p10 domain of polyprotein 2a and moderately with the protease domain. Mutational analysis suggested that the C-terminal disordered domain of RdRp is involved in the interaction with p10. Coexpression of full length RdRp and p10 resulted in formation of RdRp-p10 complex which showed significantly higher polymerase activity than RdRp alone. Interestingly, CΔ43 RdRp also showed a similar increase in activity. Thus, p10 acts as a positive regulator of RdRp by interacting with the C-terminal disordered domain of RdRp.

  16. Efficient inhibition of human immunodeficiency virus replication using novel modified microRNA-30a targeting 3′-untranslated region transcripts

    Science.gov (United States)

    NEJATI, AHMAD; SHAHMAHMOODI, SHOHREH; AREFIAN, EHSAN; SHOJA, ZABIHOLLAH; MARASHI, SAYED-MAHDI; TABATABAIE, HAMIDEH; MOLLAEI-KANDELOUS, YAGHOUB; SOLEIMANI, MASOUD; NATEGH, RAKHSHANDEH

    2016-01-01

    RNA interference (RNAi)-based gene therapy is currently considered to be a combinatorial anti-human immunodeficiency virus-1 (HIV-1) therapy. Although artificial polycistronic microRNAs (miRs) can reduce HIV-1 escape mutant variants, this approach may increase the risk of side effects. The present study aimed to optimize the efficiency of anti-HIV RNAi gene therapy in order to reduce the cell toxicity induced by multi-short hairpin RNA expression. An artificial miR-30a-3′-untranslated region (miR-3-UTR) obtained from a single RNA polymerase II was used to simultaneously target all viral transcripts. The results of the present study demonstrated that HIV-1 replication was significantly inhibited in the cells with the miR-3-UTR construct, suggesting that miR-3′-UTR may serve as a promising tool for RNAi-based gene therapy in the treatment of HIV-1. PMID:27168813

  17. Cellular microRNA miR-181b inhibits replication of mink enteritis virus by repression of non-structural protein 1 translation.

    Directory of Open Access Journals (Sweden)

    Jia-zeng Sun

    Full Text Available Mink enteritis virus (MEV is one of the most important viral pathogens in the mink industry. Recent studies have showed that microRNAs (miRNAs, small noncoding RNAs of length ranging from 18-23 nucleotides (nt participate in host-pathogen interaction networks; however, whether or not miRNAs are involved in MEV infection has not been reported. Our study revealed that miRNA miR-181b inhibited replication of MEV in the feline kidney (F81 cell line by targeting the MEV non-structural protein 1 (NS1 messenger RNA (mRNA coding region, resulting in NS1 translational repression, while MEV infection reduced miR-181b expression. This is the first description of cellular miRNAs modulating MEV infection in F81 cells, providing further insight into the mechanisms of viral infection, and may be useful in development of naturally-occurring miRNAs antiviral strategies.

  18. Analysis of protein-protein interactions in the feline calicivirus replication complex.

    Science.gov (United States)

    Kaiser, William J; Chaudhry, Yasmin; Sosnovtsev, Stanislav V; Goodfellow, Ian G

    2006-02-01

    Caliciviruses are a major cause of gastroenteritis in humans and cause a wide variety of other diseases in animals. Here, the characterization of protein-protein interactions between the individual proteins of Feline calicivirus (FCV), a model system for other members of the family Caliciviridae, is reported. Using the yeast two-hybrid system combined with a number of other approaches, it is demonstrated that the p32 protein (the picornavirus 2B analogue) of FCV interacts with p39 (2C), p30 (3A) and p76 (3CD). The FCV protease/RNA polymerase (ProPol) p76 was found to form homo-oligomers, as well as to interact with VPg and ORF2, the region encoding the major capsid protein VP1. A weak interaction was also observed between p76 and the minor capsid protein encoded by ORF3 (VP2). ORF2 protein was found to interact with VPg, p76 and VP2. The potential roles of the interactions in calicivirus replication are discussed.

  19. Hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA,ε, as template, and depends on cellular chaperones;moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids.This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV),now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cellfree systems. At this time, they can, unfortunately,not be complemented by three-dimensional structural information on the involved components. However, at least for the s RNA element such information is emerging,raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal,will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.

  20. Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ishida, Hisashi; Tatsumi, Tomohide; Hosui, Atsushi; Nawa, Takatoshi; Kodama, Takahiro; Shimizu, Satoshi; Hikita, Hayato; Hiramatsu, Naoki; Kanto, Tatsuya [Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita 565-0871 (Japan); Hayashi, Norio [Kansai Rosai Hospital, 3-1-69, Inabaso, Amagasaki 660-8511 (Japan); Takehara, Tetsuo, E-mail: takehara@gh.med.osaka-u.ac.jp [Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita 565-0871 (Japan)

    2011-08-19

    Highlights: {yields} HCV infection upregulated miR-192, -194, -215, downregulated miR-320, -491. {yields} Transfection of miR-192, -215, and -491 enhanced HCV replication. {yields} Transfection of miR-491 inhibited Akt phosphorylation. {yields} Akt inhibition could be responsible for augmentation of HCV replication by miR-491. -- Abstract: The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

  1. Hibiscus chlorotic ringspot virus coat protein is essential for cell-to-cell and long-distance movement but not for viral RNA replication.

    Directory of Open Access Journals (Sweden)

    Shengniao Niu

    Full Text Available Hibiscus chlorotic ringspot virus (HCRSV is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP functions on virus replication and movement in kenaf (Hibiscus cannabinus L., two HCRSV mutants, designated as p2590 (A to G in which the first start codon ATG was replaced with GTG and p2776 (C to G in which proline 63 was replaced with alanine, were constructed. In vitro transcripts of p2590 (A to G were able to replicate to a similar level as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for β-annulus formation during virion assembly. Progeny of transcripts derived from p2776 (C to G was able to move from cell-to-cell in inoculated cotyledons but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples isolated from 2776 (C to G inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including the β-annulus domain is required in T = 3 assembly in vitro.

  2. Replications of Two Closely Related Groups of Jumbo Phages Show Different Level of Dependence on Host-encoded RNA Polymerase

    Science.gov (United States)

    Matsui, Takeru; Yoshikawa, Genki; Mihara, Tomoko; Chatchawankanphanich, Orawan; Kawasaki, Takeru; Nakano, Miyako; Fujie, Makoto; Ogata, Hiroyuki; Yamada, Takashi

    2017-01-01

    Ralstonia solanacearum phages ΦRP12 and ΦRP31 are jumbo phages isolated in Thailand. Here we show that they exhibit similar virion morphology, genome organization and host range. Genome comparisons as well as phylogenetic and proteomic tree analyses support that they belong to the group of ΦKZ-related phages, with their closest relatives being R. solanacearum phages ΦRSL2 and ΦRSF1. Compared with ΦRSL2 and ΦRSF1, ΦRP12 and ΦRP31 possess larger genomes (ca. 280 kbp, 25% larger). The replication of ΦRP12 and ΦRP31 was not affected by rifampicin treatment (20 μg/ml), suggesting that phage-encoded RNAPs function to start and complete the infection cycle of these phages without the need of host-encoded RNAPs. In contrast, ΦRSL2 and ΦRSF1, encoding the same set of RNAPs, did not produce progeny phages in the presence of rifampicin (5 μg/ml). This observation opens the possibility that some ΦRP12/ΦRP31 factors that are absent in ΦRSL2 and ΦRSF1 are involved in their host-independent transcription. PMID:28659872

  3. Cold-Inducible RNA-Binding Protein Bypasses Replicative Senescence in Primary Cells through Extracellular Signal-Regulated Kinase 1 and 2 Activation▿ †

    Science.gov (United States)

    Artero-Castro, Ana; Callejas, Francisco B.; Castellvi, Josep; Kondoh, Hiroshi; Carnero, Amancio; Fernández-Marcos, Pablo J.; Serrano, Manuel; Ramón y Cajal, Santiago; Lleonart, Matilde E.

    2009-01-01

    Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16INK4a, p21WAF1, and p19ARF upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research. PMID:19158277

  4. MicroRNA-146a represses mycobacteria-induced inflammatory response and facilitates bacterial replication via targeting IRAK-1 and TRAF-6.

    Directory of Open Access Journals (Sweden)

    Shuo Li

    Full Text Available BACKGROUND: Apart from triggering host immune responses, macrophages also act as a major reservoir for mycobacteria. For better survival, mycobacteria have evolved various mechanisms to modulate the production of proinflammatory cytokines in macrophages, and manipulation of micro-RNA (miRNA expression has been considered as an important one. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that miR-146a expression was significantly increased in a time- and dose-dependent manner in mycobacteria-infected macrophages. It could obviously reduce the induction of proinflammatory cytokines TNF-α, IL-1β, IL-6 and chemokine MCP-1 by targeting interleukin-1 receptor-associated kinase-1 (IRAK-1 and TNF receptor-associated factor-6 (TRAF-6, two key elements involved in the TLR/NF-κB signaling pathway cascades. Consistent with the anti-inflammation effect, a higher bacterial burden was seen in miR-146a mimics-treated macrophages. CONCLUSION/SIGNIFICANCE: Here, we demonstrated that mycobacteria-induced miR-146a could modulate inflammatory response by targeting IRAK1 and TRAF6 and facilitate mycobacteria replication in macrophages.

  5. Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

    Directory of Open Access Journals (Sweden)

    Balart Luis A

    2010-10-01

    Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

  6. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle.

    Directory of Open Access Journals (Sweden)

    Seyed Hanif Mahboobi

    Full Text Available Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC, which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV's reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA, computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

  7. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle.

    Science.gov (United States)

    Mahboobi, Seyed Hanif; Javanpour, Alex A; Mofrad, Mohammad R K

    2015-01-01

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV's reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

  8. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  9. The picornavirus avian encephalomyelitis virus possesses a hepatitis C virus-like internal ribosome entry site element

    DEFF Research Database (Denmark)

    Bakhshesh, M.; Groppelli, E.; Willcocks, M.A.

    2008-01-01

    in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3' end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-Iike IRES element within its...

  10. Transgene-mediated suppression of the RNA interference pathway in Aedes aegypti interferes with gene silencing and enhances Sindbis virus and dengue virus type 2 replication.

    Science.gov (United States)

    Khoo, C C H; Doty, J B; Heersink, M S; Olson, K E; Franz, A W E

    2013-02-01

    RNA interference (RNAi) is the major innate antiviral pathway in Aedes aegypti that responds to replicating arboviruses such as dengue virus (DENV) and Sindbis virus (SINV). On the one hand, the mosquito's RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. On the other, transient silencing of key genes of the RNAi pathway increases replication of SINV and DENV2, allowing the viruses to temporally overcome dose-dependent midgut infection and midgut escape barriers (MEB) more efficiently. Here we expressed Flock house virus B2 (FHV-B2) from the poly-ubiquitin (PUb) promoter in Ae. aegypti using the ΦC31 site-directed recombination system to investigate the impact of transgene-mediated RNAi pathway suppression on infections with SINV-TR339eGFP and DENV2-QR94, the latter of which has been shown to be confronted with a strong MEB in Ae. aegypti. FHV-B2 was constitutively expressed in midguts of sugar- and blood-fed mosquitoes of transgenic line PUbB2 P61. B2 over-expression suppressed RNA silencing of carboxypeptidase A-1 (AeCPA-1) in midgut tissue of PUbB2 P61 mosquitoes. Following oral challenge with SINV-TR339eGFP or DENV2-QR94, mean titres in midguts of PUbB2 P61 females were significantly higher at 7 days post-bloodmeal (pbm) than in those of nontransgenic control mosquitoes. At 14 days pbm, infection rates of carcasses were significantly increased in PubB2 P61 mosquitoes infected with SINV-TR339eGFP. Following infection with DENV2-QR94, midgut infection rates were significantly increased in the B2-expressing mosquitoes at 14 days pbm. However, B2 expression in PUbB2 P61 did not increase the DENV2-QR94 dissemination rate, indicating that the infection phenotype was not primarily controlled by RNAi. © 2013 Royal Entomological Society.

  11. Replication and Inhibitors of Enteroviruses and Parechoviruses

    Directory of Open Access Journals (Sweden)

    Lonneke van der Linden

    2015-08-01

    Full Text Available The Enterovirus (EV and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV. They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.

  12. Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome

    Directory of Open Access Journals (Sweden)

    Kahori Shimizu

    2014-01-01

    Full Text Available Leaky expression of adenovirus (Ad genes occurs following transduction with a conventional replication-incompetent Ad vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3′-untranslated region (UTR of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a–targeted sequences into the 3′-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a–mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

  13. miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication.

    Science.gov (United States)

    Amaral, Andreia J; Andrade, Jorge; Foxall, Russell B; Matoso, Paula; Matos, Ana M; Soares, Rui S; Rocha, Cheila; Ramos, Christian G; Tendeiro, Rita; Serra-Caetano, Ana; Guerra-Assunção, José A; Santa-Marta, Mariana; Gonçalves, João; Gama-Carvalho, Margarida; Sousa, Ana E

    2017-02-01

    Cell activation is a vital step for T-cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next-generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up-regulation of miR-34c-5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV-1 and HIV-2 infections. Overexpression of miR-34c-5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T-cell activation. We additionally show that miR-34c-5p promotes HIV-1 replication, suggesting that its down-regulation during HIV infection may be part of an anti-viral host response. © 2016 The Authors.

  14. Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

    Directory of Open Access Journals (Sweden)

    Jun Takezawa

    2010-01-01

    Full Text Available When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s, which insert nucleotide(s opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.

  15. Coronavirus Attachment and Replication

    Science.gov (United States)

    1988-03-28

    synthesis during RNA replication of vesicular stomatitis virus. J. Virol. 49:303-309. Pedersen, N.C. 1976a. Feline infectious peritonitis: Something old...receptors on intestinal brush border membranes from normal host species were developed for canine (CCV), feline (FIPV), porcine (TGEV), human (HCV...gastroenteritis receptor on pig BBMs ...... ................. ... 114 Feline infectious peritonitis virus receptor on cat BBMs ... .............. 117 Human

  16. Abiotic self-replication.

    Science.gov (United States)

    Meyer, Adam J; Ellefson, Jared W; Ellington, Andrew D

    2012-12-18

    The key to the origins of life is the replication of information. Linear polymers such as nucleic acids that both carry information and can be replicated are currently what we consider to be the basis of living systems. However, these two properties are not necessarily coupled. The ability to mutate in a discrete or quantized way, without frequent reversion, may be an additional requirement for Darwinian evolution, in which case the notion that Darwinian evolution defines life may be less of a tautology than previously thought. In this Account, we examine a variety of in vitro systems of increasing complexity, from simple chemical replicators up to complex systems based on in vitro transcription and translation. Comparing and contrasting these systems provides an interesting window onto the molecular origins of life. For nucleic acids, the story likely begins with simple chemical replication, perhaps of the form A + B → T, in which T serves as a template for the joining of A and B. Molecular variants capable of faster replication would come to dominate a population, and the development of cycles in which templates could foster one another's replication would have led to increasingly complex replicators and from thence to the initial genomes. The initial genomes may have been propagated by RNA replicases, ribozymes capable of joining oligonucleotides and eventually polymerizing mononucleotide substrates. As ribozymes were added to the genome to fill gaps in the chemistry necessary for replication, the backbone of a putative RNA world would have emerged. It is likely that such replicators would have been plagued by molecular parasites, which would have been passively replicated by the RNA world machinery without contributing to it. These molecular parasites would have been a major driver for the development of compartmentalization/cellularization, as more robust compartments could have outcompeted parasite-ridden compartments. The eventual outsourcing of metabolic

  17. The enterovirus 71 A-particle forms a gateway to allow genome release: a cryoEM study of picornavirus uncoating.

    Directory of Open Access Journals (Sweden)

    Kristin L Shingler

    2013-03-01

    Full Text Available Since its discovery in 1969, enterovirus 71 (EV71 has emerged as a serious worldwide health threat. This human pathogen of the picornavirus family causes hand, foot, and mouth disease, and also has the capacity to invade the central nervous system to cause severe disease and death. Upon binding to a host receptor on the cell surface, the virus begins a two-step uncoating process, first forming an expanded, altered "A-particle", which is primed for genome release. In a second step after endocytosis, an unknown trigger leads to RNA expulsion, generating an intact, empty capsid. Cryo-electron microscopy reconstructions of these two capsid states provide insight into the mechanics of genome release. The EV71 A-particle capsid interacts with the genome near the icosahedral two-fold axis of symmetry, which opens to the external environment via a channel ∼10 Å in diameter that is lined with patches of negatively charged residues. After the EV71 genome has been released, the two-fold channel shrinks, though the overall capsid dimensions are conserved. These structural characteristics identify the two-fold channel as the site where a gateway forms and regulates the process of genome release.

  18. Sequence-specific cleavage of BM2 gene transcript of influenza B virus by 10-23 catalytic motif containing DNA enzymes significantly inhibits viral RNA translation and replication.

    Science.gov (United States)

    Kumar, Binod; Kumar, Prashant; Rajput, Roopali; Saxena, Latika; Daga, Mradul K; Khanna, Madhu

    2013-10-01

    One of the hallmarks of progression of influenza virus replication is the step involving the virus uncoating that occurs in the host cytoplasm. The BM2 ion channel protein of influenza B virus is highly conserved and is essentially required during the uncoating processes of virus, thus an attractive target for designing antiviral drugs. We screened several DNA enzymes (Dzs) containing the 10-23 catalytic motif against the influenza B virus BM2 RNA. Dzs directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. The Dz209 not only showed significant intracellular reduction of BM2 gene expression in transient-expression system but also provided considerable protection against influenza B virus challenge in MDCK cells. Our findings suggest that the Dz molecule can be used as selective and effective inhibitor of viral RNA replication, and can be explored further for development of a potent therapeutic agent against influenza B virus infection.

  19. Co-opted oxysterol-binding ORP and VAP proteins channel sterols to RNA virus replication sites via membrane contact sites.

    Science.gov (United States)

    Barajas, Daniel; Xu, Kai; de Castro Martín, Isabel Fernández; Sasvari, Zsuzsanna; Brandizzi, Federica; Risco, Cristina; Nagy, Peter D

    2014-10-01

    Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication.

  20. Database Replication

    CERN Document Server

    Kemme, Bettina

    2010-01-01

    Database replication is widely used for fault-tolerance, scalability and performance. The failure of one database replica does not stop the system from working as available replicas can take over the tasks of the failed replica. Scalability can be achieved by distributing the load across all replicas, and adding new replicas should the load increase. Finally, database replication can provide fast local access, even if clients are geographically distributed clients, if data copies are located close to clients. Despite its advantages, replication is not a straightforward technique to apply, and

  1. Structure-function relationship of viral cis-acting RNA elements : the role of the OriI and OriR in enterovirus replication

    NARCIS (Netherlands)

    Ooij, Martinus Johannes Maria van

    2007-01-01

    The genus Enterovirus belongs to Picornaviridae, a family of small, non-enveloped, lytic RNA viruses. They contain a single-stranded RNA genome of positive polarity of approximately 7,500 nucleotides. A viral protein VPg is specifically linked to the 5'terminus of the viral RNA. IRES-mediated initi

  2. Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Kenneth C McCullough

    2014-01-01

    Full Text Available Self-amplifying replicon RNA (RepRNA possesses high potential for increasing antigen load within dendritic cells (DCs. The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs, there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements—slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.

  3. Inhibition of porcine reproductive and respiratory syndrome virus replication with exosome-transferred artificial microRNA targeting the 3' untranslated region.

    Science.gov (United States)

    Zhu, Li; Bao, Liping; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2015-10-01

    Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease. As part of the development of RNA interference (RNAi) strategy against the disease, in this study a recombinant adenovirus (rAd) expressing the artificial microRNA (amiRNA) targeting the 3' untranslated region (UTR) was used to investigate the exosome-mediated amiRNA transfer from different pig cell types to porcine alveolar macrophages (PAMs). Quantitative RT-PCR showed that the sequence-specific amiRNA was expressed in and secreted via exosomes from the rAd-transduced pig kidney cell line PK-15, PAM cell line 3D4/163, kidney fibroblast cells (PFCs) and endometrial endothelial cells (PEECs) with different secretion efficiencies. Fluorescent microscopy revealed that the dye-labeled amiRNA-containing exosomes of different cell origins were efficiently taken up by all of the five types of pig cells tested, including primary PAMs. Quantitative RT-PCR showed that the amiRNA-containing exosomes of different cell origins were taken up by primary PAMs in both time- and dose-dependent manners. Both quantitative RT-PCR and viral titration assays showed that the exosome-delivered amiRNA had potent anti-viral effects against three different PRRSV strains. These data suggest that the exosomes derived from pig cells could serve as an efficient miRNA transfer vehicle, and that the exosome-delivered amiRNA had potent anti-viral effects against different PRRSV strains.

  4. Crinivirus replication and host interactions

    Directory of Open Access Journals (Sweden)

    Zsofia A Kiss

    2013-05-01

    Full Text Available Criniviruses comprise one of the genera within the family Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense ssRNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was available. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as BYV-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP, CPm, Hsp70h, and p59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5’ end of RNA 2 as ORF1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the

  5. Toward genetics-based virus taxonomy: comparative analysis of a genetics-based classification and the taxonomy of picornaviruses.

    Science.gov (United States)

    Lauber, Chris; Gorbalenya, Alexander E

    2012-04-01

    Virus taxonomy has received little attention from the research community despite its broad relevance. In an accompanying paper (C. Lauber and A. E. Gorbalenya, J. Virol. 86:3890-3904, 2012), we have introduced a quantitative approach to hierarchically classify viruses of a family using pairwise evolutionary distances (PEDs) as a measure of genetic divergence. When applied to the six most conserved proteins of the Picornaviridae, it clustered 1,234 genome sequences in groups at three hierarchical levels (to which we refer as the "GENETIC classification"). In this study, we compare the GENETIC classification with the expert-based picornavirus taxonomy and outline differences in the underlying frameworks regarding the relation of virus groups and genetic diversity that represent, respectively, the structure and content of a classification. To facilitate the analysis, we introduce two novel diagrams. The first connects the genetic diversity of taxa to both the PED distribution and the phylogeny of picornaviruses. The second depicts a classification and the accommodated genetic diversity in a standardized manner. Generally, we found striking agreement between the two classifications on species and genus taxa. A few disagreements concern the species Human rhinovirus A and Human rhinovirus C and the genus Aphthovirus, which were split in the GENETIC classification. Furthermore, we propose a new supergenus level and universal, level-specific PED thresholds, not reached yet by many taxa. Since the species threshold is approached mostly by taxa with large sampling sizes and those infecting multiple hosts, it may represent an upper limit on divergence, beyond which homologous recombination in the six most conserved genes between two picornaviruses might not give viable progeny.

  6. Regulation of arginine methyltransferase 3 by a Wolbachia-induced microRNA in Aedes aegypti and its effect on Wolbachia and dengue virus replication.

    Science.gov (United States)

    Zhang, Guangmei; Hussain, Mazhar; Asgari, Sassan

    2014-10-01

    The gram-negative endosymbiotic bacteria, Wolbachia, have been found to colonize a wide range of invertebrates, including over 40% of insect species. Best known for host reproductive manipulations, some strains of Wolbachia have been shown to reduce the host life span by about 50% and inhibit replication and transmission of dengue virus (DENV) in the mosquito vector, Aedes aegypti. The molecular mechanisms underlying these effects still are not well understood. Our previous studies showed that Wolbachia uses host microRNAs (miRNAs) to manipulate host gene expression for its efficient maintenance and limiting replication of DENV in Ae. aegypti. Protein arginine methyltransferases are structurally and functionally conserved proteins from yeast to human. In mammals, it has been reported that protein arginine methyltransferases such as PRMT1, 5 and 6 could regulate replication of different viruses. Ae. aegypti contains eight members of protein arginine methyltransferases (AaArgM1-8). Here, we show that the wMelPop strain of Wolbachia introduced into Ae. aegypti significantly induces the expression of AaArgM3. Interestingly, we found that Wolbachia uses aae-miR-2940, which is highly upregulated in Wolbachia-infected mosquitoes, to upregulate the expression of AaArgM3. Silencing of AaArgM3 in a mosquito cell line led to a significant reduction in Wolbachia replication, but had no effect on the replication of DENV. These results provide further evidence that Wolbachia uses the host miRNAs to manipulate host gene expression and facilitate colonization in Ae. aegypti mosquito.

  7. Detection of foot-and-mouth disease virus RNA in pharyngeal epithelium biopsy samples obtained from infected cattle: Investigation of possible sites of virus replication and persistence

    DEFF Research Database (Denmark)

    Stenfeldt, Anna Carolina; Belsham, Graham

    2012-01-01

    was used to investigate the level of FMDV RNA present at this site at sequential time points during the infection. Results were compared to measurements of virus excretion in samples of oropharyngeal fluid collected at corresponding time points. Possible sites of virus persistence were investigated through...... measurements of the levels of FMDV RNA in the DSP as well as mandibular and retropharyngeal lymph nodes beyond 28 days after infection. Results indicated only low levels of FMDV RNA present in samples of pharyngeal epithelia during both early and persistent phases of infection with significantly higher levels...

  8. Vitamin D Potentiates the Inhibitory Effect of MicroRNA-130a in Hepatitis C Virus Replication Independent of Type I Interferon Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xiaoqiong Duan

    2015-01-01

    Full Text Available Calcitriol, the bioactive metabolite of vitamin D, was reported to inhibit HCV production in a synergistic fashion with interferon, a treatment in vitro. Our previous study established that miR-130a inhibits HCV replication by restoring the host innate immune response. We aimed to determine whether there is additive inhibitory effect of calcitriol and miR-130a on HCV replication. Here we showed that calcitriol potentiates the anti-HCV effect of miR-130a in both Con1b replicon and J6/JFH1 culture systems. Intriguingly, this potentiating effect of calcitriol on miR-130a was not through upregulating the expression of cellular miR-130a or through increasing the miR-130a-mediated IFNα/β production. All these findings may contribute to the development of novel anti-HCV therapeutic strategies although the antiviral mechanism needs to be further investigated.

  9. Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES).

    Science.gov (United States)

    Torrecilla, Josune; Del Pozo-Rodríguez, Ana; Solinís, María Ángeles; Apaolaza, Paola S; Berzal-Herranz, Beatriz; Romero-López, Cristina; Berzal-Herranz, Alfredo; Rodríguez-Gascón, Alicia

    2016-10-01

    Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3'). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon.

  10. The DNA replication licensing factor miniature chromosome maintenance 7 is essential for RNA splicing of epidermal growth factor receptor, c-Met, and platelet-derived growth factor receptor.

    Science.gov (United States)

    Chen, Zhang-Hui; Yu, Yan P; Michalopoulos, George; Nelson, Joel; Luo, Jian-Hua

    2015-01-16

    Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221-248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein.

  11. MKT1, a nonessential Saccharomyces cerevisiae gene with a temperature-dependent effect on replication of M2 double-stranded RNA.

    OpenAIRE

    Wickner, R B

    1987-01-01

    The MKT1 gene was defined by recessive alleles present in many laboratory strains of Saccharomyces cerevisiae that result in loss of M2 double-stranded RNA at temperatures above 30 degrees C if L-A-HN double-stranded RNA is present but not if L-A-H is present. I mapped MKT1 near TOP2 and isolated the gene by chromosome walking from TOP2. The gene location was defined by deletions, and a 2.8-kilobase transcript corresponding to the gene was detected. The recessive natural-variant mutations are...

  12. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

    DEFF Research Database (Denmark)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

    2016-01-01

    Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depl...

  13. Neither the RNA nor the Proteins of Open Reading Frames 3a and 3b of the Coronavirus Infectious Bronchitis Virus Are Essential for Replication

    Science.gov (United States)

    Hodgson, Teri; Britton, Paul; Cavanagh, Dave

    2006-01-01

    Gene 3 of infectious bronchitis virus is tricistronic; open reading frames (ORFs) 3a and 3b encode two small nonstructural (ns) proteins, 3a and 3b, of unknown function, and a third, structural protein E, is encoded by ORF 3c. To determine if either the 3a or the 3b protein is required for replication, we first modified their translation initiation codons to prevent translation of the 3a and 3b proteins from recombinant infectious bronchitis viruses (rIBVs). Replication in primary chick kidney (CK) cells and in chicken embryos was not affected. In chicken tracheal organ cultures (TOCs), the recombinant rIBVs reached titers similar to those of the wild-type virus, but in the case of viruses lacking the 3a protein, the titer declined reproducibly earlier. Translation of the IBV E protein is believed to be initiated by internal entry of ribosomes at a structure formed by the sequences corresponding to ORFs 3a and 3b. To assess the necessity of this mechanism, we deleted most of the sequence representing 3a and 3b to produce a gene in which ORF 3c (E) was adjacent to the gene 3 transcription-associated sequence. Western blot analysis revealed that the recombinant IBV produced fivefold less E protein. Nevertheless, titers produced in CK cells, embryos, and TOCs were similar to those of the wild-type virus, although they declined earlier in TOCs, probably due to the absence of the 3a protein. Thus, neither the tricistronic arrangement of gene 3, the internal initiation of translation of E protein, nor the 3a and 3b proteins are essential for replication per se, suggesting that these proteins are accessory proteins that may have roles in vivo. PMID:16352554

  14. Role of picornaviruses in flu-like illnesses of adults enrolled in an oseltamivir treatment study who had no evidence of influenza virus infection

    NARCIS (Netherlands)

    G. Boivin (Guy); A. Gaudreau (Annie); H.C. Jackson (Helen); J. Groen (Jan); P. Ward (Penelope); A.D.M.E. Osterhaus (Albert)

    2002-01-01

    textabstractThe primary objective of this study was to determine the role of picornavirus in flu-like episodes (temperature of > or =38.0 degrees C plus one respiratory and one constitutional symptom) among otherwise healthy adults enrolled in a placebo-controlled, double-blind, randomized

  15. Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain

    Directory of Open Access Journals (Sweden)

    Howe Charles L

    2012-03-01

    hippocampal neuroprotection and preservation of cognitive function. Specific depletion of neutrophils with the 1A8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain. These effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection.

  16. An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication

    NARCIS (Netherlands)

    Mauthe, Mario; Langereis, Martijn; Jung, Jennifer; Zhou, Xingdong; Jones, Alex; Omta, Wienand; Tooze, Sharon A.; Stork, Bjoern; Paludan, Soren Riis; Ahola, Tero; Egan, Dave; Behrends, Christian; Mokry, Michal; de Haan, Cornelis; van Kuppeveld, Frank; Reggiori, Fulvio

    2016-01-01

    Autophagy is a catabolic process regulated by the orchestrated action of the autophagy-related (ATG) proteins. Recent work indicates that some of the ATG proteins also have autophagy-independent roles. Using an unbiased siRNA screen approach, we explored the extent of these unconventional functions

  17. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    NARCIS (Netherlands)

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M

    2014-01-01

    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  18. A whole genome RNAi screen identifies replication stress response genes.

    Science.gov (United States)

    Kavanaugh, Gina; Ye, Fei; Mohni, Kareem N; Luzwick, Jessica W; Glick, Gloria; Cortez, David

    2015-11-01

    Proper DNA replication is critical to maintain genome stability. When the DNA replication machinery encounters obstacles to replication, replication forks stall and the replication stress response is activated. This response includes activation of cell cycle checkpoints, stabilization of the replication fork, and DNA damage repair and tolerance mechanisms. Defects in the replication stress response can result in alterations to the DNA sequence causing changes in protein function and expression, ultimately leading to disease states such as cancer. To identify additional genes that control the replication stress response, we performed a three-parameter, high content, whole genome siRNA screen measuring DNA replication before and after a challenge with replication stress as well as a marker of checkpoint kinase signalling. We identified over 200 replication stress response genes and subsequently analyzed how they influence cellular viability in response to replication stress. These data will serve as a useful resource for understanding the replication stress response.

  19. Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue

    OpenAIRE

    Kai Rausalu; Age Utt; Tania Quirin; Varghese, Finny S.; Eva Žusinaite; Pratyush Kumar Das; Tero Ahola; Andres Merits

    2016-01-01

    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensa...

  20. Cross-talk between human dendritic cell subsets influences expression of RNA sensors and inhibits picornavirus infection.

    NARCIS (Netherlands)

    Kramer, M.; Schulte, B.M.; Eleveld-Trancikova, D.; Hout-Kuijer, M.A. van; Toonen, L.W.J.; Tel, J.; Vries, I.J.M. de; Kuppeveld, F.J.M. van; Jansen, B.J.H.; Adema, G.J.

    2010-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells that provide a link between innate and adaptive immunity. Multiple DC subsets exist and their activation by microorganisms occurs through binding of conserved pathogen-derived structures to so-called pattern recognition receptors (PRRs)

  1. Cost-effective method of siRNA preparation and its application to inhibit hepatitis B virus replication in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Zhi-Kang Qian; Bao-Qin Xuan; Tai-Shan Min; Jian-Feng Xu; Lin Li; Wei-Da Huang

    2005-01-01

    AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation,digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture.METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation.cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase Ⅲ in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2000 to test their inhibition efficiency.RESULTS: siRNAs corresponding to part of the hepatitis3 virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP.CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.

  2. Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication.

    Science.gov (United States)

    Kundu, Pallob; Raychaudhuri, Santanu; Tsai, Weimin; Dasgupta, Asim

    2005-08-01

    The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

  3. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2014-03-15

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers.

  4. RIG-I and dsRNA-induced IFNbeta activation.

    Directory of Open Access Journals (Sweden)

    Stéphane Hausmann

    Full Text Available Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses, most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral (pppRNAs remain unblocked during the infection. Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5. We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5' tri-phosphate ends, and found that this infection as well requires RIG-I and not mda-5. We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.

  5. Replication of Avocado Sunblotch Viroid in the Yeast Saccharomyces cerevisiae▿

    Science.gov (United States)

    Delan-Forino, Clémentine; Maurel, Marie-Christine; Torchet, Claire

    2011-01-01

    Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, “naked” RNAs, which replicate through RNA-RNA transcription. Viroids of the Avsunviroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, viroids have only been detected in plant cells. Here, we investigate the replication of Avocado sunblotch viroid (ASBVd) of the Avsunviroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrate that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We show that the viroid monomeric RNA is destabilized by the nuclear 3′ and the cytoplasmic 5′ RNA degradation pathways. For the first time, our results provide evidence that viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd. PMID:21270165

  6. Genetic characterization of a novel picornavirus in turkeys (Meleagris gallopavo) distinct from turkey galliviruses and megriviruses and distantly related to the members of the genus Avihepatovirus.

    Science.gov (United States)

    Boros, Akos; Nemes, Csaba; Pankovics, Péter; Kapusinszky, Beatrix; Delwart, Eric; Reuter, Gábor

    2013-07-01

    This study reports the metagenomic detection and complete genome characterization of a novel turkey picornavirus from faecal samples of healthy (1/3) and affected (6/8) commercial turkeys with enteric and/or stunting syndrome in Hungary. The virus was detected at seven of the eight farms examined. The turkey/M176-TuASV/2011/HUN genome (KC465954) was genetically different from the currently known picornaviruses of turkey origin (megriviruses and galliviruses), and showed distant phylogenetic relationship and common genomic features (e.g. uncleaved VP0 and three predicted and unrelated 2A polypeptides) to duck hepatitis A virus (DHAV) of the genus Avihepatovirus. The complete genome analysis revealed multiple distinct genome features like the presence of two in-tandem aphthovirus 2A-like sequence repeats with DxExNPG/P 'ribosome-skipping' sites (76 %, 23/30 amino acids identical), with the first aphthovirus 2A-like sequence being located at the end of the VP1 capsid protein (VP1/2A1 'ribosome-skipping' site). The phylogenetic analyses, low sequence identity (33, 32 and 36 % amino acid identity in P1, P2 and P3 regions) to DHAV, and the type II-like internal ribosome entry site suggests that this turkey picornavirus is related to, but distinct from the genus Avihepatovirus and it could be the founding member of a novel Avihepatovirus sister-clade genus. This is the third, taxonomically highly distinct picornavirus clade identified from turkeys exhibiting varied symptoms.

  7. Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Danielle N Renner

    Full Text Available Glioblastoma (GBM is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA257-264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific Kb:OVA257-264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA257-264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.

  8. Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses.

    Science.gov (United States)

    Renner, Danielle N; Jin, Fang; Litterman, Adam J; Balgeman, Alexis J; Hanson, Lisa M; Gamez, Jeffrey D; Chae, Michael; Carlson, Brett L; Sarkaria, Jann N; Parney, Ian F; Ohlfest, John R; Pirko, Istvan; Pavelko, Kevin D; Johnson, Aaron J

    2015-01-01

    Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA257-264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific Kb:OVA257-264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA257-264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.

  9. Archaeal DNA replication.

    Science.gov (United States)

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed.

  10. Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells

    NARCIS (Netherlands)

    Deddouche, Safia; Goubau, Delphine; Rehwinkel, Jan; Chakravarty, Probir; Begum, Sharmin; Maillard, Pierre V; Borg, Annabel; Matthews, Nik; Feng, Qian; van Kuppeveld, Frank J M|info:eu-repo/dai/nl/156614723; Reis e Sousa, Caetano

    2014-01-01

    The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with

  11. A transcription and translation-coupled DNA replication system using rolling-circle replication.

    Science.gov (United States)

    Sakatani, Yoshihiro; Ichihashi, Norikazu; Kazuta, Yasuaki; Yomo, Tetsuya

    2015-05-27

    All living organisms have a genome replication system in which genomic DNA is replicated by a DNA polymerase translated from mRNA transcribed from the genome. The artificial reconstitution of this genome replication system is a great challenge in in vitro synthetic biology. In this study, we attempted to construct a transcription- and translation-coupled DNA replication (TTcDR) system using circular genomic DNA encoding phi29 DNA polymerase and a reconstituted transcription and translation system. In this system, phi29 DNA polymerase was translated from the genome and replicated the genome in a rolling-circle manner. When using a traditional translation system composition, almost no DNA replication was observed, because the tRNA and nucleoside triphosphates included in the translation system significantly inhibited DNA replication. To minimize these inhibitory effects, we optimized the composition of the TTcDR system and improved replication by approximately 100-fold. Using our system, genomic DNA was replicated up to 10 times in 12 hours at 30 °C. This system provides a step toward the in vitro construction of an artificial genome replication system, which is a prerequisite for the construction of an artificial cell.

  12. Plant RNA binding proteins for control of RNA virus infection

    OpenAIRE

    Huh, Sung Un; Paek, Kyung-Hee

    2013-01-01

    Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific b...

  13. The role of host factor LSm1-7 complex in dengue virus RNA replication%宿主蛋白LSm1-7复合体在登革病毒RNA复制中的作用研究

    Institute of Scientific and Technical Information of China (English)

    董阳超; 雷迎峰; 丁天兵; 时莹; 潘鹭翔; 王晓霞; 张泽信; 徐志凯

    2013-01-01

    Objective To investigate the roles of host protein LSml-7 complex in DENV RNA replication. Methods mRNA was isolated from DENV infected HepG2 cells at different time point(2,24,36,48 h) of post-infection, and the level of DENV RNA and LSml RNA was analysed by RT-qPCR. HepG2 cells were transfected with specific siRNA for LSml and nontargeting siRNA and siNC respectively, followed by infection with DENV.The mRNA was isolated at 24 h p. i. and analyzed by RT-qPCR to determine the RNA level of DENV and LSml. Laser scanning confocal microscopy was performed to observe the localization of LSml protein and DENV dsRNA in DENV infected HepG2 cells. Results The levels of DENV RNA and LSml RNA were increased compared to 2 h p.i. during the course of DENV infection. siRNA-mediated silencing resulted in a specific 78.2% reduction of LSml RNA when using the nontargeting siNC as a negative control, and down-regulation of LSml resulted in a marked reduction of the DENV RNA level by 35.6%. LSml-7 complex distributed and colocalized with DENV dsRNA around nucleolous in the DENV infected HepG2 cells. Conclusion LSml-7 complex may act as the component of DENV replication machinery and is one of the positive regulators for DENV RNA replication.%目的 探讨LSm1-7复合体在登革病毒(DENV)RNA复制中的作用.方法 DENV感染HepG2细胞后,在不同时间点(2、24、36、48 h)收集细胞和提取总mRNA,实时定量PCR (RT-qPCR)分析LSm1表达水平;将LSm1的siRNA和非特异对照siRNA两次转染HepG2细胞后感染DENV-2,24h后收集细胞,提取总mRNA,RT-qPCR分析LSm1表达水平与病毒RNA表达水平;利用LSm1抗体与dsRNA抗体对DENV感染细胞进行免疫荧光染色,激光共聚焦分析LSm1与dsRNA共定位情况.结果 与2h比较,随着时间的推移,DENV-2 RNA和LSm1 RNA水平逐渐升高;与对照组siNC比较,siLSm1组LSm1 RNA水平明显降低,沉默效率达78.2%;与对照组siNC比较,siLSm1组DENV RNA含量降低35.6%;LSm1-7

  14. Involvement of Autophagy in Coronavirus Replication

    Directory of Open Access Journals (Sweden)

    Paul Britton

    2012-11-01

    Full Text Available Coronaviruses are single stranded, positive sense RNA viruses, which induce the rearrangement of cellular membranes upon infection of a host cell. This provides the virus with a platform for the assembly of viral replication complexes, improving efficiency of RNA synthesis. The membranes observed in coronavirus infected cells include double membrane vesicles. By nature of their double membrane, these vesicles resemble cellular autophagosomes, generated during the cellular autophagy pathway. In addition, coronavirus infection has been demonstrated to induce autophagy. Here we review current knowledge of coronavirus induced membrane rearrangements and the involvement of autophagy or autophagy protein microtubule associated protein 1B light chain 3 (LC3 in coronavirus replication.

  15. Replication Restart in Bacteria.

    Science.gov (United States)

    Michel, Bénédicte; Sandler, Steven J

    2017-07-01

    In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability. Copyright © 2017 American Society for Microbiology.

  16. The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

    Directory of Open Access Journals (Sweden)

    Lonneke van der Linden

    2015-03-01

    Full Text Available The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71 for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylenebis(oxy]bis(5-nitro-benzonitrile with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family. Surprisingly, coxsackievirus B3 (CVB3 and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight

  17. Suppression of Adenovirus Replication by Cardiotonic Steroids.

    Science.gov (United States)

    Grosso, Filomena; Stoilov, Peter; Lingwood, Clifford; Brown, Martha; Cochrane, Alan

    2017-02-01

    The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

  18. A Self-Replicating Ligase Ribozyme

    Science.gov (United States)

    Paul, Natasha; Joyce, Gerald F.

    2002-01-01

    A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of Itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 per min, whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(exp -11) M per min. Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

  19. Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5 ' untranslated regions are efficiently translated in cells by a cap-dependent mechanism

    DEFF Research Database (Denmark)

    Belsham, Graham; Nielsen, Inge; Normann, Preben;

    2008-01-01

    secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which...

  20. Structure and replication of hepatitis delta virus

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-29

    Dec 29, 2008 ... Unidade de Biologia Molecular, Centro de Malária e outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, ... molecules of both delta antigens (Ryu et al., 1993). This ..... Glenn JS, Watson JA, Havel CM, White JO (1992). ... HDV RNA encoding the large delta antigen cannot replicate. J. Gen.

  1. Control of chromosome replication in caulobacter crescentus.

    Science.gov (United States)

    Marczynski, Gregory T; Shapiro, Lucy

    2002-01-01

    Caulobacter crescentus permits detailed analysis of chromosome replication control during a developmental cell cycle. Its chromosome replication origin (Cori) may be prototypical of the large and diverse class of alpha-proteobacteria. Cori has features that both affiliate and distinguish it from the Escherichia coli chromosome replication origin. For example, requirements for DnaA protein and RNA transcription affiliate both origins. However, Cori is distinguished by several features, and especially by five binding sites for the CtrA response regulator protein. To selectively repress and limit chromosome replication, CtrA receives both protein degradation and protein phosphorylation signals. The signal mediators, proteases, response regulators, and kinases, as well as Cori DNA and the replisome, all show distinct patterns of temporal and spatial organization during cell cycle progression. Future studies should integrate our knowledge of biochemical activities at Cori with our emerging understanding of cytological dynamics in C. crescentus and other bacteria.

  2. Receptor tyrosine kinase signaling regulates replication of the peste des petits ruminants virus.

    Science.gov (United States)

    Chaudhary, K; Chaubey, K K; Singh, S V; Kumar, N

    2015-03-01

    In this study, we found out that blocking the receptor tyrosine kinase (RTK) signaling in Vero cells by tryphostin AG879 impairs the in vitro replication of the peste des petits ruminants virus (PPRV). A reduced virus replication in Trk1-knockdown (siRNA) Vero cells confirmed the essential role of RTK in the virus replication, in particular a specific regulation of viral RNA synthesis. These data represent the first evidence that the RTK signaling regulates replication of a morbillivirus.

  3. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  4. Replicative Homeostasis: A fundamental mechanism mediating selective viral replication and escape mutation

    Directory of Open Access Journals (Sweden)

    Sallie Richard

    2005-02-01

    Full Text Available Abstract Hepatitis C (HCV, hepatitis B (HBV, the human immunodeficiency viruses (HIV, and other viruses that replicate via RNA intermediaries, cause an enormous burden of disease and premature death worldwide. These viruses circulate within infected hosts as vast populations of closely related, but genetically diverse, molecules known as "quasispecies". The mechanism(s by which this extreme genetic and antigenic diversity is stably maintained are unclear, but are fundamental to understanding viral persistence and pathobiology. The persistence of HCV, an RNA virus, is especially problematic and HCV stability, maintained despite rapid genomic mutation, is highly paradoxical. This paper presents the hypothesis, and evidence, that viruses capable of persistent infection autoregulate replication and the likely mechanism mediating autoregulation – Replicative Homeostasis – is described. Replicative homeostasis causes formation of stable, but highly reactive, equilibria that drive quasispecies expansion and generates escape mutation. Replicative homeostasis explains both viral kinetics and the enigma of RNA quasispecies stability and provides a rational, mechanistic basis for all observed viral behaviours and host responses. More importantly, this paradigm has specific therapeutic implication and defines, precisely, new approaches to antiviral therapy. Replicative homeostasis may also modulate cellular gene expression.

  5. Regulation of replication fork progression through histone supply and demand

    DEFF Research Database (Denmark)

    Groth, Anja; Corpet, Armelle; Cook, Adam J L

    2007-01-01

    DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone...... chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf......1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork...

  6. The Replication Recipe: What makes for a convincing replication?

    NARCIS (Netherlands)

    Brandt, M.J.; IJzerman, H.; Dijksterhuis, A.J.; Farach, F.J.; Geller, J.; Giner-Sorolla, R.; Grange, J.A.; Perugini, M.; Spies, J.R.; Veer, A. van 't

    2014-01-01

    Psychological scientists have recently started to reconsider the importance of close replications in building a cumulative knowledge base; however, there is no consensus about what constitutes a convincing close replication study. To facilitate convincing close replication attempts we have developed

  7. Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

    Directory of Open Access Journals (Sweden)

    David Piñeiro

    2015-04-01

    Full Text Available Gemin5 is a RNA-binding protein (RBP that was first identified as a peripheral component of the survival of motor neurons (SMN complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs. Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E. Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

  8. RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

    NARCIS (Netherlands)

    Dorssers, L.C.J.

    1983-01-01

    The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.Previously, an RNA-dependent RNA polymerase produced upon infection of Vigna unguiculata

  9. Modeling DNA Replication.

    Science.gov (United States)

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  10. Eukaryotic DNA Replication Fork.

    Science.gov (United States)

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  11. An antioxidant resveratrol significantly enhanced replication of hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Mitsuyasu; Nakamura; Masanori; Ikeda; Ryota; Hokari; Nobuyuki; Kato; Toshifumi; Hibi; Soichiro; Miura

    2010-01-01

    AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of the HCV replicon system OR6.RVT is a strong antioxidant and a kind of polyphenol that inhibits replication of various viruses.AXN is also a strong antioxidant.The replication of HCV RNA was assessed by the luciferase reporter assay.An additive effect of antioxidants on antiviral effects of inter...

  12. Adenovirus DNA Replication

    OpenAIRE

    Hoeben, Rob C.; Uil, Taco G.

    2013-01-01

    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new deve...

  13. Nidovirus ribonucleases: Structures and functions in viral replication.

    NARCIS (Netherlands)

    Ulferts, R.; Ziebuhr, J.

    2011-01-01

    Nidoviruses employ unique strategies to replicate and express their exceptionally large RNA genomes. The viruses use a variety of enzymes to synthesize, modify and process an extensive set of viral RNAs of both genome and subgenome length, including RNA polymerase, primase, helicase, ribose 2'-O and

  14. GFLV replication in electroporated grapevine protoplasts.

    Science.gov (United States)

    Valat; Toutain; Courtois; Gaire; Decout; Pinck; Mauro; Burrus

    2000-06-29

    Grapevine fanleaf virus (GFLV), responsible for the economically important court-noué disease, is exclusively transmitted to its natural host in the vineyards through Xiphinema nematodes. We have developed direct inoculation of GFLV into grapevine through protoplast electroporation. Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions. Permeation conditions were determined by monitoring calcein uptake. Low salt poration medium was selected. Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation (isolate F13). GFLV replication was detected with either virus particles (2 µg) or viral RNA (10 ng) in both protoplast populations, as shown by anti-P38 Western blotting. Direct inoculation and replication were also observed with Arabis mosaic virus (ArMV), a closely related nepovirus, as well as with another GFLV isolate. These results will be valuable in grapevine biotechnology, for GFLV replication studies, transgenic plant screening for GFLV resistance, and biorisk evaluation.

  15. Minichromosome replication in vitro: inhibition of re-replication by replicatively assembled nucleosomes.

    Science.gov (United States)

    Krude, T; Knippers, R

    1994-08-19

    Single-stranded circular DNA, containing the SV40 origin sequence, was used as a template for complementary DNA strand synthesis in cytosolic extracts from HeLa cells. In the presence of the replication-dependent chromatin assembly factor CAF-1, defined numbers of nucleosomes were assembled during complementary DNA strand synthesis. These minichromosomes were then induced to semiconservatively replicate by the addition of the SV40 initiator protein T antigen (re-replication). The results indicate that re-replication of minichromosomes appears to be inhibited by two independent mechanisms. One acts at the initiation of minichromosome re-replication, and the other affects replicative chain elongation. To directly demonstrate the inhibitory effect of replicatively assembled nucleosomes, two types of minichromosomes were prepared: (i) post-replicative minichromosomes were assembled in a reaction coupled to replication as above; (ii) pre-replicative minichromosomes were assembled independently of replication on double-stranded DNA. Both types of minichromosomes were used as templates for DNA replication under identical conditions. Replicative fork movement was found to be impeded only on post-replicative minichromosome templates. In contrast, pre-replicative minichromosomes allowed one unconstrained replication cycle, but re-replication was inhibited due to a block in fork movement. Thus, replicatively assembled chromatin may have a profound influence on the re-replication of DNA.

  16. Identification of the determinants of efficient Pestivirus replication

    DEFF Research Database (Denmark)

    Risager, Peter Christian

    The key for the survival of a virus is to copy its own genome into progeny genomes that allows continued reproduction. The mechanism behind this "copy function" or "replication" is a wellorganized process that involves the formation of a replication complex in the cell and interactions between...... the viral proteins. The replication process in single-stranded RNA viruses of positive polarity requires a particular enzyme, an RNA dependent RNA polymerase, that has no direct counterpart elsewhere in nature. The variable nature of rapidly evolving viral genomes, pose a constant challenge to the host......, gives a general introduction to RNA viruses, with the focus on viruses classified within the Flaviviridae. Next, pestiviruses are described with special attention to classical swine fever virus and the disease it is responsible for. A brief history of types of viral vaccines is provided, finishing...

  17. Investigating variation in replicability: A "Many Labs" replication project

    NARCIS (Netherlands)

    Klein, R.A.; Ratliff, K.A.; Vianello, M.; Adams, R.B.; Bahnik, S.; Bernstein, M.J.; Bocian, K.; Brandt, M.J.; Brooks, B.; Brumbaugh, C.C.; Cemalcilar, Z.; Chandler, J.; Cheong, W.; Davis, W.E.; Devos, T.; Eisner, M.; Frankowska, N.; Furrow, D.; Galliani, E.M.; Hasselman, F.W.; Hicks, J.A.; Hovermale, J.F.; Hunt, S.J.; Huntsinger, J.R.; IJzerman, H.; John, M.S.; Joy-Gaba, J.A.; Kappes, H.B.; Krueger, L.E.; Kurtz, J.; Levitan, C.A.; Mallett, R.K.; Morris, W.L.; Nelson, A.J.; Nier, J.A.; Packard, G.; Pilati, R.; Rutchick, A.M.; Schmidt, K.; Skorinko, J.L.M.; Smith, R.; Steiner, T.G.; Storbeck, J.; Van Swol, L.M.; Thompson, D.; Veer, A.E. van 't; Vaughn, L.A.; Vranka, M.; Wichman, A.L.; Woodzicka, J.A.; Nosek, B.A.

    2014-01-01

    Although replication is a central tenet of science, direct replications are rare in psychology. This research tested variation in the replicability of 13 classic and contemporary effects across 36 independent samples totaling 6,344 participants. In the aggregate, 10 effects replicated consistently.

  18. Genes and sequences involved in the replication of cowpea mosaic virus RNAs

    NARCIS (Netherlands)

    Eggen, R.

    1989-01-01

    The aim of the studies described in this thesis was to gain more insight in the complex molecular mechanisms underlying the RNA replication of the cowpea mosaic virus genome. Previously the replication of CPMV RNA has been examined extensively with crude membrane fractions prepared from CP

  19. Genes and sequences involved in the replication of cowpea mosaic virus RNAs.

    NARCIS (Netherlands)

    Eggen, R.

    1989-01-01

    The aim of the studies described in this thesis was to gain more insight in the complex molecular mechanisms underlying the RNA replication of the cowpea mosaic virus genome. Previously the replication of CPMV RNA has been examined extensively with crude membrane fractions prepared from CPMV inf

  20. Psychology, replication & beyond.

    Science.gov (United States)

    Laws, Keith R

    2016-06-01

    Modern psychology is apparently in crisis and the prevailing view is that this partly reflects an inability to replicate past findings. If a crisis does exists, then it is some kind of 'chronic' crisis, as psychologists have been censuring themselves over replicability for decades. While the debate in psychology is not new, the lack of progress across the decades is disappointing. Recently though, we have seen a veritable surfeit of debate alongside multiple orchestrated and well-publicised replication initiatives. The spotlight is being shone on certain areas and although not everyone agrees on how we should interpret the outcomes, the debate is happening and impassioned. The issue of reproducibility occupies a central place in our whig history of psychology.

  1. Hepatitis B virus-3p-siRNA inhibits hepatitis B virus replication and activates interferon-βexpression in mice%乙型肝炎病毒-三磷酸-小干扰核糖核酸抑制小鼠乙型肝炎病毒复制并激活β干扰素的表达

    Institute of Scientific and Technical Information of China (English)

    邢雅玲; 陈晓娟; 闫飞; 杜鹃; 周勇; 王学军; 陈忠斌

    2014-01-01

    Objective To observe the activation of anti-viral innate immune response of type Ⅰinterferon and inhibition of hepatitis B virus (HBV)genome replication in mice by HBV-3p-siRNA. Methods HBV-3p-siRNA was designed by targeting specific sequence of HBV S/P mRNA and was generated by in vitro transcription.Negative control siRNA (NC-siRNA)and non-modified HBV-siRNA were used as control groups.Blood samples were collected from tail vein of mice and the model of HBV-infected mice were established by hydrodynamic injection.Forty mice were divided into 4 groups with 10 in each group.The model group was only injected with pGL3.0-HBV1 .2 copy plasmid.The negative control group received peritoneal injection of NC-siRNA.HBV-siRNA group received peritoneal injection of HBV-siRNA and HBV-3p-siRNA group received peritoneal injection of HBV-3p-siRNA.The interferon-β(IFN-β)and hepatitis B surface antigen (HBsAg)in serum were detected by enzyme linked immunosorbent assay (ELISA).The copies of HBV DNA were assessed by fluore scence quantitative polymerase chain reaction (PCR ).The statistical difference between groups was determined using One way-ANOVA analysis by LSD or Dunnett T3.Results Serum level of IFN-β was (12.37±5 .32)pg/mL in model group,(22.61 ±6.29 )pg/mL in negative control group,(26.40±5 .39)pg/mL in HBV-siRNA group and (68.37± 21 .00 ) pg/mL in HBV-3p-siRNA group.The secretions of IFN-β into serum were significantly enhanced by HBV-siRNA and HBV-3p-siRNA compared with model group (F =23.988 and 46.523,respectively,both P <0.01).Serum level of HBsAg was (2 864.86±907.11 )ng/mL in model group,(2 198.86±456.89 )ng/mL in negative control group,(1 049.71 ± 396.28 )ng/mL in HBV-siRNA group and (640.86±383.08)ng/mL in HBV-3p-siRNA group.The expressions of HBsAg were inhibited by HBV-3p-siRNA and HBV-siRNA compared with model group (F = 23.537 and 39.144, respectively;P =0.025 and 0.010,respectively).Serum level of HBV DNA was (2.54 ×104 ±1 .46 × 104 )copy

  2. Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication

    OpenAIRE

    Kristi L. Berger; Cooper, Jacob D.; Nicholas S. Heaton; Yoon, Rosa; Oakland, Todd E.; Jordan, Tristan X.; Mateu, Guaniri; Grakoui, Arash; Randall, Glenn

    2009-01-01

    Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock...

  3. Electron microscopic analysis of rotavirus assembly-replication intermediates

    Energy Technology Data Exchange (ETDEWEB)

    Boudreaux, Crystal E.; Kelly, Deborah F. [Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA (United States); McDonald, Sarah M., E-mail: mcdonaldsa@vtc.vt.edu [Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA (United States); Department of Biomedical Sciences and Pathobiology, Virginia—Maryland Regional College of Veterinary Medicine, Blacksburg, VA (United States)

    2015-03-15

    Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30–70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly–replicase process. - Highlights: • Rotaviruses replicate their genomes in tandem with early virion assembly. • Little is known about rotavirus assembly-replication intermediates. • Assembly-replication intermediates were imaged using electron microscopy.

  4. Unveiling the mystery of mitochondrial DNA replication in yeasts.

    Science.gov (United States)

    Chen, Xin Jie; Clark-Walker, George Desmond

    2017-08-01

    Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  5. In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803.

    Science.gov (United States)

    Yang, X; Daniell, H; McFadden, B

    1994-09-01

    Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.

  6. DNA replication origins in archaea

    OpenAIRE

    Zhenfang eWu; Jingfang eLiu; Haibo eYang; Hua eXiang

    2014-01-01

    DNA replication initiation, which starts at specific chromosomal site (known as replication origins), is the key regulatory stage of chromosome replication. Archaea, the third domain of life, use a single or multiple origin(s) to initiate replication of their circular chromosomes. The basic structure of replication origins is conserved among archaea, typically including an AT-rich unwinding region flanked by several conserved repeats (origin recognition box, ORB) that are located adjacent to ...

  7. Replication studies in longevity

    DEFF Research Database (Denmark)

    Varcasia, O; Garasto, S; Rizza, T

    2001-01-01

    In Danes we replicated the 3'APOB-VNTR gene/longevity association study previously carried out in Italians, by which the Small alleles (less than 35 repeats) had been identified as frailty alleles for longevity. In Danes, neither genotype nor allele frequencies differed between centenarians and 20...

  8. Replication-Fork Dynamics

    NARCIS (Netherlands)

    Duderstadt, Karl E.; Reyes-Lamothe, Rodrigo; van Oijen, Antoine M.; Sherratt, David J.

    2014-01-01

    The proliferation of all organisms depends on the coordination of enzymatic events within large multiprotein replisomes that duplicate chromosomes. Whereas the structure and function of many core replisome components have been clarified, the timing and order of molecular events during replication re

  9. Inhibition of Avian Influenza Virus Replication via Short Interfering RNA Expressed by Plasmid%通过载体表达siRNAs抑制禽流感病毒复制的研究

    Institute of Scientific and Technical Information of China (English)

    操胜; 刘光亮; 孟庆文; 杨增岐; 亓文宝; 于康震; 吴东来

    2006-01-01

    禽流感病毒是养禽业危害最严重的病原微生物之一.为探讨小干涉RNA(siRNA)对A型禽流感病毒复制的干扰作用,以H5亚型AIV PB2基因为靶序列,设计合成了4对编码siRNAs的DNA序列,将其克隆到psiRNA-hH1neo载体中,构建siRNAs表达载体,鉴定正确后将重组质粒转染MDCK细胞,采用G41 8筛选建立抗性细胞系,用血凝(HA)试验和real time RT-PCR试验检测抑制效果,在细胞水平筛选出具有高效抑制AIV复制的2个靶位点PB2-1154、PB2-342、为AIV的基因功能研究、抗病毒药物的开发和转基因动物的研究奠定了基础.

  10. Construction and identification of replication recombinent adenovirus vector of mice LINGO-1 shRNA%含鼠LINGO-1 shRNA重组腺病毒载体的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    高峰; 孙红辉; 杨有庚

    2009-01-01

    目的 构建含鼠LINGO-1短发夹RNA(short hairpin RNA,shRNA)的复制缺陷型腺病毒载体.方法 通过PCR法将带有LINGO-1 shRNA序列构建至U6启动子(U6sh LINGO-1)下游,与T载体连接,将质粒转化入大肠杆菌.将U6sh LINGO-1连到穿梭质粒pDC316上,与腺病毒基因组质粒pBHGlox△E1、3Cre共转染293T细胞,得到重组腺病毒载体(rAd LINGO-1).经PCR鉴定正确后,进行扩增、纯化、滴度测定及感染性鉴定.结果 PCR法得到U6sh LINGO-1,rAd LINGO-1具有良好的感染性,腺病毒滴度可达109TCID50/ml.双引物PCR法鉴定Ad LINGO-1可扩增出Ad及特异性片段U6sh LINGO-1.结论 成功构建了能表达LINGO-1 shRNA的重组腺病毒载体rAd LINGO-1,可能为临床应用RNA干涉技术促进脊髓损伤的恢复提供新的方法.

  11. Reversible Switching of Cooperating Replicators

    Science.gov (United States)

    Urtel, Georg C.; Rind, Thomas; Braun, Dieter

    2017-02-01

    How can molecules with short lifetimes preserve their information over millions of years? For evolution to occur, information-carrying molecules have to replicate before they degrade. Our experiments reveal a robust, reversible cooperation mechanism in oligonucleotide replication. Two inherently slow replicating hairpin molecules can transfer their information to fast crossbreed replicators that outgrow the hairpins. The reverse is also possible. When one replication initiation site is missing, single hairpins reemerge from the crossbreed. With this mechanism, interacting replicators can switch between the hairpin and crossbreed mode, revealing a flexible adaptation to different boundary conditions.

  12. Ribavirin Inhibits Parrot Bornavirus 4 Replication in Cell Culture.

    Science.gov (United States)

    Musser, Jeffrey M B; Heatley, J Jill; Koinis, Anastasia V; Suchodolski, Paulette F; Guo, Jianhua; Escandon, Paulina; Tizard, Ian R

    2015-01-01

    Parrot bornavirus 4 is an etiological agent of proventricular dilatation disease, a fatal neurologic and gastrointestinal disease of psittacines and other birds. We tested the ability of ribavirin, an antiviral nucleoside analog with antiviral activity against a range of RNA and DNA viruses, to inhibit parrot bornavirus 4 replication in duck embryonic fibroblast cells. Two analytical methods that evaluate different products of viral replication, indirect immunocytochemistry for viral specific nucleoprotein and qRT-PCR for viral specific phosphoprotein gene mRNA, were used. Ribavirin at concentrations between 2.5 and 25 μg/mL inhibited parrot bornavirus 4 replication, decreasing viral mRNA and viral protein load, in infected duck embryonic fibroblast cells. The addition of guanosine diminished the antiviral activity of ribavirin suggesting that one possible mechanism of action against parrot bornavirus 4 may likely be through inosine monophosphate dehydrogenase inhibition. This study demonstrates parrot bornavirus 4 susceptibility to ribavirin in cell culture.

  13. Ribavirin Inhibits Parrot Bornavirus 4 Replication in Cell Culture.

    Directory of Open Access Journals (Sweden)

    Jeffrey M B Musser

    Full Text Available Parrot bornavirus 4 is an etiological agent of proventricular dilatation disease, a fatal neurologic and gastrointestinal disease of psittacines and other birds. We tested the ability of ribavirin, an antiviral nucleoside analog with antiviral activity against a range of RNA and DNA viruses, to inhibit parrot bornavirus 4 replication in duck embryonic fibroblast cells. Two analytical methods that evaluate different products of viral replication, indirect immunocytochemistry for viral specific nucleoprotein and qRT-PCR for viral specific phosphoprotein gene mRNA, were used. Ribavirin at concentrations between 2.5 and 25 μg/mL inhibited parrot bornavirus 4 replication, decreasing viral mRNA and viral protein load, in infected duck embryonic fibroblast cells. The addition of guanosine diminished the antiviral activity of ribavirin suggesting that one possible mechanism of action against parrot bornavirus 4 may likely be through inosine monophosphate dehydrogenase inhibition. This study demonstrates parrot bornavirus 4 susceptibility to ribavirin in cell culture.

  14. Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

    Science.gov (United States)

    Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

    2017-09-15

    Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Initiation and regulation of paramyxovirus transcription and replication.

    Science.gov (United States)

    Noton, Sarah L; Fearns, Rachel

    2015-05-01

    The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle.

  16. Mitochondrial biology. Replication-transcription switch in human mitochondria.

    Science.gov (United States)

    Agaronyan, Karen; Morozov, Yaroslav I; Anikin, Michael; Temiakov, Dmitry

    2015-01-30

    Coordinated replication and expression of the mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mitochondrial DNA copy number can be regulated by the transcription machinery. We found that interaction of human transcription elongation factor TEFM with mitochondrial RNA polymerase and nascent transcript prevents the generation of replication primers and increases transcription processivity and thereby serves as a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may allow mitochondria to increase transcription rates and, as a consequence, respiration and adenosine triphosphate production without the need to replicate mitochondrial DNA, as has been observed during spermatogenesis and the early stages of embryogenesis.

  17. A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

    Directory of Open Access Journals (Sweden)

    Zhengyang Zeng

    2016-01-01

    Full Text Available Hepatitis B virus (HBV infection is a major cause of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma, all of which are severe threats to human health. However, current clinical therapies for HBV are limited by potential side effects, toxicity, and drug-resistance. In this study, a cell-penetrating peptide-conjugated peptide nucleic acid (PNA, Tat-PNA-DR, was designed to target the direct repeat (DR sequences of HBV. Tat-PNA-DR effectively inhibited HBV replication in HepG2.2.15 cells. Its anti-HBV effect relied on the binding of Tat-PNA-DR to the DR, whereby it suppressed the translation of hepatitis B e antigen (HBeAg, HBsAg, HBV core, hepatitis B virus x protein, and HBV reverse transcriptase (RT and the reverse transcription of the HBV genome. Furthermore, Tat-PNA-DR administered by intravenous injection efficiently cleared HBeAg and HBsAg in an acute hepatitis B mouse model. Importantly, it induced an 80% decline in HBV DNA in mouse serum, which was similar to the effect of the widely used clinical drug Lamivudine (3TC. Additionally, a long-term hydrodynamics HBV mouse model also demonstrated Tat-PNA-DR's antiviral effect. Interestingly, Tat-PNA-DR displayed low cytotoxicity, low mouse acute toxicity, low immunogenicity, and high serum stability. These data indicate that Tat-PNA-DR is a unique PNA and a promising drug candidate against HBV.

  18. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  19. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  20. Optical tweezers reveal how proteins alter replication

    Science.gov (United States)

    Chaurasiya, Kathy

    Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

  1. Initiation of adenovirus DNA replication.

    OpenAIRE

    Reiter, T; Fütterer, J; Weingärtner, B; Winnacker, E L

    1980-01-01

    In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared ...

  2. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication...... initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  3. Replication Research and Special Education

    Science.gov (United States)

    Travers, Jason C.; Cook, Bryan G.; Therrien, William J.; Coyne, Michael D.

    2016-01-01

    Replicating previously reported empirical research is a necessary aspect of an evidence-based field of special education, but little formal investigation into the prevalence of replication research in the special education research literature has been conducted. Various factors may explain the lack of attention to replication of special education…

  4. Replication Research and Special Education

    Science.gov (United States)

    Travers, Jason C.; Cook, Bryan G.; Therrien, William J.; Coyne, Michael D.

    2016-01-01

    Replicating previously reported empirical research is a necessary aspect of an evidence-based field of special education, but little formal investigation into the prevalence of replication research in the special education research literature has been conducted. Various factors may explain the lack of attention to replication of special education…

  5. Replication data collection highlights value in diversity of replication attempts

    Science.gov (United States)

    DeSoto, K. Andrew; Schweinsberg, Martin

    2017-01-01

    Researchers agree that replicability and reproducibility are key aspects of science. A collection of Data Descriptors published in Scientific Data presents data obtained in the process of attempting to replicate previously published research. These new replication data describe published and unpublished projects. The different papers in this collection highlight the many ways that scientific replications can be conducted, and they reveal the benefits and challenges of crucial replication research. The organizers of this collection encourage scientists to reuse the data contained in the collection for their own work, and also believe that these replication examples can serve as educational resources for students, early-career researchers, and experienced scientists alike who are interested in learning more about the process of replication. PMID:28291224

  6. Stearoyl coenzyme A desaturase 1 is associated with hepatitis C virus replication complex and regulates viral replication

    DEFF Research Database (Denmark)

    Nguyen, LN; Lim, YS; Pham, Long

    2014-01-01

    exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1...... is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support the idea that SCD1...

  7. Cis-acting RNA elements at the 5' end of Sindbis virus genome RNA regulate minus- and plus-strand RNA synthesis.

    OpenAIRE

    Frolov, I; Hardy, R; Rice, C M

    2001-01-01

    Alphavirus genome replication is a multistep asymmetric process. Several lines of evidence suggest that the template preference of the RNA replicase is regulated by proteolytic cleavage of the viral nonstructural polyprotein. Cis-acting RNA elements in the viral genome also play crucial roles in regulating genome replication and subgenomic RNA transcription. In this report, a series of RNA templates were analyzed in vitro and in vivo to define functional elements in the 5' end of the genome. ...

  8. Anatomy of Mammalian Replication Domains

    Science.gov (United States)

    Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

    2017-01-01

    Genetic information is faithfully copied by DNA replication through many rounds of cell division. In mammals, DNA is replicated in Mb-sized chromosomal units called “replication domains.” While genome-wide maps in multiple cell types and disease states have uncovered both dynamic and static properties of replication domains, we are still in the process of understanding the mechanisms that give rise to these properties. A better understanding of the molecular basis of replication domain regulation will bring new insights into chromosome structure and function. PMID:28350365

  9. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  10. Modeling inhomogeneous DNA replication kinetics.

    Directory of Open Access Journals (Sweden)

    Michel G Gauthier

    Full Text Available In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.

  11. Efficient Interaction between Arenavirus Nucleoprotein (NP) and RNA-Dependent RNA Polymerase (L) Is Mediated by the Virus Nucleocapsid (NP-RNA) Template.

    Science.gov (United States)

    Iwasaki, Masaharu; Ngo, Nhi; Cubitt, Beatrice; de la Torre, Juan C

    2015-05-01

    In this study, we document that efficient interaction between arenavirus nucleoprotein (NP) and RNA-dependent RNA polymerase (L protein), the two trans-acting viral factors required for both virus RNA replication and gene transcription, requires the presence of virus-specific RNA sequences located within the untranslated 5' and 3' termini of the viral genome.

  12. Replicated Spectrographs in Astronomy

    CERN Document Server

    Hill, Gary J

    2014-01-01

    As telescope apertures increase, the challenge of scaling spectrographic astronomical instruments becomes acute. The next generation of extremely large telescopes (ELTs) strain the availability of glass blanks for optics and engineering to provide sufficient mechanical stability. While breaking the relationship between telescope diameter and instrument pupil size by adaptive optics is a clear path for small fields of view, survey instruments exploiting multiplex advantages will be pressed to find cost-effective solutions. In this review we argue that exploiting the full potential of ELTs will require the barrier of the cost and engineering difficulty of monolithic instruments to be broken by the use of large-scale replication of spectrographs. The first steps in this direction have already been taken with the soon to be commissioned MUSE and VIRUS instruments for the Very Large Telescope and the Hobby-Eberly Telescope, respectively. MUSE employs 24 spectrograph channels, while VIRUS has 150 channels. We compa...

  13. SUMO and KSHV Replication

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Pei-Ching [Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan (China); Kung, Hsing-Jien, E-mail: hkung@nhri.org.tw [Institute for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan (China); Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616 (United States); UC Davis Cancer Center, University of California, Davis, CA 95616 (United States); Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan (China)

    2014-09-29

    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.

  14. Origin of the membrane compartment for cowpea mosaic virus replication

    NARCIS (Netherlands)

    Carette, J.E.

    2002-01-01

    Replication of many positive-stranded RNA viruses takes place in association with intracellular membranes. Often these membranes are induced upon infection by vesiculation or rearrangement of membranes from different organelles including the early and late endomembrane system. Upon infection of cowp

  15. DNA replication at the single-molecule level

    NARCIS (Netherlands)

    Stratmann, S.A.; Oijen, A.M. van

    2014-01-01

    A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules – metabolites, structural proteins, enzymes, oligonucleotides – multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or i

  16. DNA replication at the single-molecule level

    NARCIS (Netherlands)

    Stratmann, S A; van Oijen, A M

    2014-01-01

    A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules - metabolites, structural proteins, enzymes, oligonucleotides - multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or i

  17. RNA helicases

    OpenAIRE

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In...

  18. The RNA synthesis machinery of negative-stranded RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

    2015-05-15

    The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

  19. DDX3 DEAD-Box RNA Helicase Is Required for Hepatitis C Virus RNA Replication▿

    OpenAIRE

    2007-01-01

    DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supern...

  20. RNA topology

    OpenAIRE

    Frank-Kamenetskii, Maxim D.

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases.

  1. Errors and alternatives in prebiotic replication and catalysis.

    Science.gov (United States)

    Ninio, Jacques

    2007-04-01

    The work on nonenzymatic nucleic acid replication performed by Leslie Orgel and co-workers over the last four decades, now extended by work on artificial selection of RNA aptamers and ribozymes, is generating some pessimism concerning the 'naked gene' theories of the origin of life. It is suggested here that the low probability of finding RNA aptamers and ribozymes within pools of random sequences is not as disquieting as the poor gain in efficiency obtained with increases in information content. As acknowledged by Orgel and many other authors, primitive RNA replication and catalysis must have occurred within already complex and dynamic environments. I, thus, propose to pay attention to a number of possibilities that bridge the gap between 'naked gene' theories, on one side, and metabolic theories in which complex systems self-propagate by growth and fragmentation, on the other side. For instance, one can de-emphasize nucleotide-by-nucleotide replication leading to long informational polymers, and view instead long random polymers as storage devices, from which shorter oligomers are excised. Catalytic tasks would be mainly performed by complexes associating two or more oligomers belonging to the same or to different chemical families. It is proposed that the problems of stability, binding affinity, reactivity, and specificity could be easier to handle by heterogeneous complexes of short oligomers than by long, single-stranded polymers. Finally, I point out that replication errors in a primitive replication context should include incorporations of alternative nucleotides with interesting, chemically reactive groups. In this way, an RNA sequence could be at the same time an inert sequence when copied without error, and a ribozyme, when a chemically reactive nucleotide is inadvertently introduced during replication.

  2. Ultrastructural Characterization of Zika Virus Replication Factories

    Directory of Open Access Journals (Sweden)

    Mirko Cortese

    2017-02-01

    Full Text Available A global concern has emerged with the pandemic spread of Zika virus (ZIKV infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs. Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.

  3. The molecular biology of Bluetongue virus replication.

    Science.gov (United States)

    Patel, Avnish; Roy, Polly

    2014-03-01

    The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention.

  4. Early steps of retrovirus replicative cycle

    Directory of Open Access Journals (Sweden)

    Saïb Ali

    2004-05-01

    Full Text Available Abstract During the last two decades, the profusion of HIV research due to the urge to identify new therapeutic targets has led to a wealth of information on the retroviral replication cycle. However, while the late stages of the retrovirus life cycle, consisting of virus replication and egress, have been partly unraveled, the early steps remain largely enigmatic. These early steps consist of a long and perilous journey from the cell surface to the nucleus where the proviral DNA integrates into the host genome. Retroviral particles must bind specifically to their target cells, cross the plasma membrane, reverse-transcribe their RNA genome, while uncoating the cores, find their way to the nuclear membrane and penetrate into the nucleus to finally dock and integrate into the cellular genome. Along this journey, retroviruses hijack the cellular machinery, while at the same time counteracting cellular defenses. Elucidating these mechanisms and identifying which cellular factors are exploited by the retroviruses and which hinder their life cycle, will certainly lead to the discovery of new ways to inhibit viral replication and to improve retroviral vectors for gene transfer. Finally, as proven by many examples in the past, progresses in retrovirology will undoubtedly also provide some priceless insights into cell biology.

  5. Differential cleavage of IRES trans-acting factors (ITAFs) in cells infected by human rhinovirus.

    Science.gov (United States)

    Chase, Amanda J; Semler, Bert L

    2014-01-20

    Human rhinovirus (HRV) is a major causative agent of the common cold, and thus has several important health implications. As a member of the picornavirus family, HRV has a small genomic RNA that utilizes several host cell proteins for RNA replication. Host proteins poly(rC) binding protein 2 (PCBP2) and polypyrimidine tract binding protein (PTB) are cleaved by a viral proteinase during the course of infection by the related picornavirus, poliovirus. The cleavage of PCBP2 and PTB inhibits poliovirus translation and has been proposed to mediate a switch in poliovirus template usage from translation to RNA replication. HRV RNA replication also requires a switch in template usage from translation to RNA replication; however, the mechanism is not yet known. We demonstrate that PCBP2 and PTB are differentially cleaved during HRV infection in different cell lines, suggesting that HRV utilizes a mechanism distinct from PCBP2 or PTB cleavage to mediate a switch in template usage.

  6. Efficient usage of Adabas replication

    CERN Document Server

    Storr, Dieter W

    2011-01-01

    In today's IT organization replication becomes more and more an essential technology. This makes Software AG's Event Replicator for Adabas an important part of your data processing. Setting the right parameters and establishing the best network communication, as well as selecting efficient target components, is essential for successfully implementing replication. This book provides comprehensive information and unique best-practice experience in the field of Event Replicator for Adabas. It also includes sample codes and configurations making your start very easy. It describes all components ne

  7. Solving the Telomere Replication Problem

    Science.gov (United States)

    Maestroni, Laetitia; Matmati, Samah; Coulon, Stéphane

    2017-01-01

    Telomeres are complex nucleoprotein structures that protect the extremities of linear chromosomes. Telomere replication is a major challenge because many obstacles to the progression of the replication fork are concentrated at the ends of the chromosomes. This is known as the telomere replication problem. In this article, different and new aspects of telomere replication, that can threaten the integrity of telomeres, will be reviewed. In particular, we will focus on the functions of shelterin and the replisome for the preservation of telomere integrity. PMID:28146113

  8. DNA intercalator stimulates influenza transcription and virus replication

    Directory of Open Access Journals (Sweden)

    Poon Leo LM

    2011-03-01

    Full Text Available Abstract Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII. In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD, was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPIIa in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPIIa to hyperphosphorylated RNAPII (RNAPIIo.

  9. DNA intercalator stimulates influenza transcription and virus replication.

    Science.gov (United States)

    Li, Olive T W; Poon, Leo L M

    2011-03-15

    Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII). In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD), was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPII(a) in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPII(a)) to hyperphosphorylated RNAPII (RNAPII(o)).

  10. Non‐Canonical Replication Initiation: You’re Fired!

    Directory of Open Access Journals (Sweden)

    Bazilė Ravoitytė

    2017-01-01

    Full Text Available The division of prokaryotic and eukaryotic cells produces two cells that inherit a perfect copy of the genetic material originally derived from the mother cell. The initiation of canonical DNA replication must be coordinated to the cell cycle to ensure the accuracy of genome duplication. Controlled replication initiation depends on a complex interplay of cis‐acting DNA sequences, the so‐called origins of replication (ori, with trans‐acting factors involved in the onset of DNA synthesis. The interplay of cis‐acting elements and trans‐acting factors ensures that cells initiate replication at sequence‐specific sites only once, and in a timely order, to avoid chromosomal endoreplication. However, chromosome breakage and excessive RNA:DNA hybrid formation can cause breakinduced (BIR or transcription‐initiated replication (TIR, respectively. These non‐canonical replication events are expected to affect eukaryotic genome function and maintenance, and could be important for genome evolution and disease development. In this review, we describe the difference between canonical and non‐canonical DNA replication, and focus on mechanistic differences and common features between BIR and TIR. Finally, we discuss open issues on the factors and molecular mechanisms involved in TIR.

  11. Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Barajas, Daniel; Xu, Kai; Sharma, Monika; Wu, Cheng-Yu; Nagy, Peter D., E-mail: pdnagy2@uky.edu

    2014-12-15

    Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells. - Highlights: • Tombusvirus p33 replication protein interacts with FFAT-domain host protein. • Tombusvirus replication leads to upregulation of phospholipids. • Tombusvirus replication depends on de novo lipid synthesis. • Deletion of FFAT-domain host protein enhances TBSV replication. • TBSV rewires host phospholipid synthesis.

  12. Construction and identification of replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and its inhibition of liver cancer cell growth%survivin 启动子调控肿瘤干细胞标记 CD133基因 siRNA增殖型溶瘤腺病毒的构建及对肝癌细胞生长的抑制作用

    Institute of Scientific and Technical Information of China (English)

    牛坚; 王月; 刘斌; 王人颢; 朱志军; 申海莲

    2016-01-01

    目的:构建 survivin 启动子调控的靶向 CD133基因的 siRNA 增殖型溶瘤腺病毒,研究其对肝癌细胞生长的影响。方法RT-PCR 法扩增 survivin 启动子,测序鉴定,双酶切连接,获得 pH-XC2-survivin。酶切 pH-XC2-survivin、pZD55-CD133-siRNA 获得 survivin 启动子表达框的亚克隆和CD133-siRNA 基因表达框的亚克隆,连接获得 survivin 启动子调控的 siRNA 增殖型溶瘤腺病毒表达载体质粒 pT-ZD55-CD133-siRNA。增殖型溶瘤腺病毒 survivin-T-ZD55-CD133-siRNA 经 PCR 和测序鉴定。 qRT-PCR 法检测 CD133表达, Western blot 法检测 E1A,CCK-8法检测细胞生长,流式细胞术检测细胞凋亡。结果成功构建增殖型溶瘤腺病毒 sur-vivin-T-ZD55-CD133-siRNA。 qRT-PCR 法检测 CD133 mRNA明显下降, Western blot 证实 survivin-T-ZD55-CD133-siRNA在肿瘤细胞中表达 E1A 能抑制肝癌细胞 CD133表达及生长。结论构建的增殖型溶瘤腺病毒可有效降低肝癌细胞CD133的表达,用于肝癌基因治疗的进一步研究。%Objective To construct a replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and investigate its inhibitory effect on Hep 3B cells.Methods The fragment of the survivin promoter was amplified by PCR and inserted into pH -XC2 to reconstruct a recombinant plasmid pH -XC2-survivin.Complete digestion pH-XC2-survivin and pZD55-CD133-siRNA, combinational joining the subclones, then getting replication-competent adenovirus expressing short interference RNA targeting CD 133 gene regulated by survivin promoter, replication-competent adenovirus was constructed .The recombined adenoviruses ( T-ZD55-CD133-siRNA) were verified by PCR and sequencing .The effect of T-ZD55-CD133-siRNA on CD133 expression in Hep3B cells was detected by qRT-PCR.The expression of E1A was detected by Western blot.The antitumor po-tential of replication

  13. Charter School Replication. Policy Guide

    Science.gov (United States)

    Rhim, Lauren Morando

    2009-01-01

    "Replication" is the practice of a single charter school board or management organization opening several more schools that are each based on the same school model. The most rapid strategy to increase the number of new high-quality charter schools available to children is to encourage the replication of existing quality schools. This policy guide…

  14. LHCb experience with LFC replication

    CERN Document Server

    Bonifazi, F; Perez, E D; D'Apice, A; dell'Agnello, L; Düllmann, D; Girone, M; Re, G L; Martelli, B; Peco, G; Ricci, P P; Sapunenko, V; Vagnoni, V; Vitlacil, D

    2008-01-01

    Database replication is a key topic in the framework of the LHC Computing Grid to allow processing of data in a distributed environment. In particular, the LHCb computing model relies on the LHC File Catalog, i.e. a database which stores information about files spread across the GRID, their logical names and the physical locations of all the replicas. The LHCb computing model requires the LFC to be replicated at Tier-1s. The LCG 3D project deals with the database replication issue and provides a replication service based on Oracle Streams technology. This paper describes the deployment of the LHC File Catalog replication to the INFN National Center for Telematics and Informatics (CNAF) and to other LHCb Tier-1 sites. We performed stress tests designed to evaluate any delay in the propagation of the streams and the scalability of the system. The tests show the robustness of the replica implementation with performance going much beyond the LHCb requirements.

  15. DATABASE REPLICATION IN HETEROGENOUS PLATFORM

    Directory of Open Access Journals (Sweden)

    Hendro Nindito

    2014-01-01

    Full Text Available The application of diverse database technologies in enterprises today is increasingly a common practice. To provide high availability and survavibality of real-time information, a database replication technology that has capability to replicate databases under heterogenous platforms is required. The purpose of this research is to find the technology with such capability. In this research, the data source is stored in MSSQL database server running on Windows. The data will be replicated to MySQL running on Linux as the destination. The method applied in this research is prototyping in which the processes of development and testing can be done interactively and repeatedly. The key result of this research is that the replication technology applied, which is called Oracle GoldenGate, can successfully manage to do its task in replicating data in real-time and heterogeneous platforms.

  16. LHCb experience with LFC replication

    CERN Document Server

    Carbone, Angelo; Dafonte Perez, Eva; D'Apice, Antimo; dell'Agnello, Luca; Duellmann, Dirk; Girone, Maria; Lo Re, Giuseppe; Martelli, Barbara; Peco, Gianluca; Ricci, Pier Paolo; Sapunenko, Vladimir; Vagnoni, Vincenzo; Vitlacil, Dejan

    2007-01-01

    Database replication is a key topic in the framework of the LHC Computing Grid to allow processing of data in a distributed environment. In particular, the LHCb computing model relies on the LHC File Catalog, i.e. database which stores information about files spread across the GRID, their logical names and the physical locations of all the replicas. The LHCb computing model requires the LFC to be replicated at Tier-1s. The LCG 3D project deals with the database replication issue and provides a replication service based on Oracle Streams technology. This paper describes the deployment of the LHC File Catalog replication to the INFN National Center for Telematics and Informations (CNAF) and to other LHCb Tier-1 sites. We performed stress tests designed to evaluate any delay in the propagation of the streams and the scalability of the system. The tests show the robustness of the replica implementation with performance going much beyond the LHCb requirements.

  17. Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase.

    Science.gov (United States)

    Nohales, María-Ángeles; Flores, Ricardo; Daròs, José-Antonio

    2012-08-21

    Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95-G96 and containing 5'-phosphomonoester and 3'-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA ligase to act as RNA-dependent RNA polymerase and RNA ligase, respectively.

  18. [Physicochemical properties of Teschen disease virus RNA].

    Science.gov (United States)

    Tsybanov, S Zh; Sergeev, V A; Balysheva, V I

    1982-01-01

    The specific infectivity of virion RNA of teschen disease virus in a sensitive PP cell culture was 4-5 lg TCD50/ml per 1 microgram RNA. When virion RNA was inoculated into cell cultures insusceptible to the native virus, the virus replicated to a titre of 2.0-3.5 lg TCD50/ml. The molecular weight of virion RNA determined by two independent methods was 2.7 x 10(6) daltons. Tm calculated from the curve of virion RNA melting temperature was 57 degrees C. The double-stranded replicative form of RNA recovered from virus-infected PP cells was shown to have sucrose gradient sedimentation coefficient of 20 S. The specific infectivity was 2-3 lg TCD50/ml per 1 microgram of RNA.

  19. Inhibitory effects of conditionally replicative adenovirus armed with short hairpin RNA against GAPDH genes with hTERT promoter regulation and control on human renal carcinoma OSRC cells%荷载管家基因shRNA的双调控溶瘤腺病毒抑制肾癌细胞增殖的研究

    Institute of Scientific and Technical Information of China (English)

    李连涛; 张宝福; 李慧忠; 郑骏年

    2012-01-01

    Objective To study the effects of suppressing cell proliferation and GAPDH gene expression on human renal carcinoma OSRC cells with TD - GAPDH, a conditionally replicative adenovirus ( CRAds ) armed with short hairpin RNA against GAPDH genes with hTERT promoter regulation and control . Methods Renal carcinoma OSRC cells and human proximal tubular epithelial cells ( HK - 2 ) were infected with TD - GAPDH, TD55, ZD55 - EGFP. The protein expressions of E1A were detected by Western blot. GAPDH expressing levels of OSRC cells were detected by RT -PCR and Western blot. Cell apoptosis was measured by TUNEL assay. Cell proliferation was assayed by MTT method. OSRC cells were stained with crystal violet to detect cytotoxici effect . Results Western blot assay of E1A expression indicated OSRC cells infected with TD - GAPDH, TD55, ZD55 - EGFP expressed E1A, not detected E1A protein expression in HK - 2 cells. Significant reduction of GAPDH mRNA and protein content was observed in the lysates from OSRC cells in -fected with TD - GAPDH. TD - GAPDH treatment of OSRC cells resulted in increased apoptotie cell death than TD 55 and ZD55 - EGFP treatment. Results of crystal violet stain and MTT assay demonstrated that the antitumor effect of TD-GAPDH was more potent than both TD55 and ZD55 - EGFP. Conclusion CRAds armed with shRNA against GAPDH gene could duplicate in tumor cells , which has both the lytic ability of oncolytic adenovirus and the capacity to deliver shR -NA. TD - GAPDH armed with shRNA gene can significantly inhibit tumor cell growth , which may provide a new platform for cancer gene therapy with shRNA and mayprove to be a useful novel tool for renal cancer therapy .%目的 研究荷载管家基因GAPDH shRNA的溶瘤腺病毒(TD-GAPDH)对人肾癌细胞OSRC增殖抑制的作用.方法 病毒TD-GAPDH、TD55、ZD55-EGFP感染OSRC细胞、人肾小管上皮细胞HK-2.Western blot法检测E1A表达;逆转录-聚合酶链反应(RT-PCR)、Western blot法分别检测GAPDH

  20. NACSA Charter School Replication Guide: The Spectrum of Replication Options. Authorizing Matters. Replication Brief 1

    Science.gov (United States)

    O'Neill, Paul

    2010-01-01

    One of the most important and high-profile issues in public education reform today is the replication of successful public charter school programs. With more than 5,000 failing public schools in the United States, there is a tremendous need for strong alternatives for parents and students. Replicating successful charter school models is an…

  1. Low oxygen tension enhances hepatitis C virus replication.

    Science.gov (United States)

    Vassilaki, N; Kalliampakou, K I; Kotta-Loizou, I; Befani, C; Liakos, P; Simos, G; Mentis, A F; Kalliaropoulos, A; Doumba, P P; Smirlis, D; Foka, P; Bauhofer, O; Poenisch, M; Windisch, M P; Lee, M E; Koskinas, J; Bartenschlager, R; Mavromara, P

    2013-03-01

    Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction.

  2. International Expansion through Flexible Replication

    DEFF Research Database (Denmark)

    Jonsson, Anna; Foss, Nicolai Juul

    2011-01-01

    to local environments and under the impact of new learning. To illuminate these issues, we draw on a longitudinal in-depth study of Swedish home furnishing giant IKEA, involving more than 70 interviews. We find that IKEA has developed organizational mechanisms that support an ongoing learning process aimed......, etc.) are replicated in a uniform manner across stores, and change only very slowly (if at all) in response to learning (“flexible replication”). We conclude by discussing the factors that influence the approach to replication adopted by an international replicator....

  3. Regulation of human adenovirus replication by RNA interference

    OpenAIRE

    Nikitenko, N. A.; SPEISEDER T.; Lam, E; Rubtsov, P. M.; TONAEVA KH. D.; S. A. Borzenok; Dobner, T; Prassolov, V.S.

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to...

  4. The Psychology of Replication and Replication in Psychology.

    Science.gov (United States)

    Francis, Gregory

    2012-11-01

    Like other scientists, psychologists believe experimental replication to be the final arbiter for determining the validity of an empirical finding. Reports in psychology journals often attempt to prove the validity of a hypothesis or theory with multiple experiments that replicate a finding. Unfortunately, these efforts are sometimes misguided because in a field like experimental psychology, ever more successful replication does not necessarily ensure the validity of an empirical finding. When psychological experiments are analyzed with statistics, the rules of probability dictate that random samples should sometimes be selected that do not reject the null hypothesis, even if an effect is real. As a result, it is possible for a set of experiments to have too many successful replications. When there are too many successful replications for a given set of experiments, a skeptical scientist should be suspicious that null or negative findings have been suppressed, the experiments were run improperly, or the experiments were analyzed improperly. This article describes the implications of this observation and demonstrates how to test for too much successful replication by using a set of experiments from a recent research paper.

  5. Regulation of Replication Recovery and Genome Integrity

    DEFF Research Database (Denmark)

    Colding, Camilla Skettrup

    Preserving genome integrity is essential for cell survival. To this end, mechanisms that supervise DNA replication and respond to replication perturbations have evolved. One such mechanism is the replication checkpoint, which responds to DNA replication stress and acts to ensure replication pausing...

  6. A replicator was not involved in the origin of life.

    Science.gov (United States)

    Shapiro, R

    2000-03-01

    Many scientific theories of the origin of life suggest that life began with the spontaneous formation of a replicator (a self-copying organic polymer) within an unorganized chemical mixture, or "soup." A profound difficulty exists, however, with the idea of RNA, or any other replicator, at the start of life. Existing replicators can serve as templates for the synthesis of additional copies of themselves, but this device cannot be used for the preparation of the very first such molecule, which must arise spontaneously from an unorganized mixture. The formation of an information-bearing homopolymer through undirected chemical synthesis appears very improbable. The difficulties involved in such a synthesis are illustrated by considering the prospects for the assembly of a polypeptide of L-alpha-amino acids, based on the contents of the Murchison meteorite as an example of a mixture of abiotic origin. In that mixture, potential replicator components would be accompanied by a host of interfering substances, which include chain terminators (simple carboxylic acids and amines), branch-formers, D-amino acids, and many classes of substances for which incorporation would disrupt the necessary structural regularity of the replicator. Laboratory experiments dealing with the nonenzymatic synthesis of biopolymers have not addressed the specificity problem. The possibility that formation of the first replicator took place through a very improbable event cannot be excluded, but greater attention should be given to metabolism-first theories, which avoid this difficulty.

  7. Noncoding flavivirus RNA displays RNA interference suppressor activity in insect and Mammalian cells.

    Science.gov (United States)

    Schnettler, Esther; Sterken, Mark G; Leung, Jason Y; Metz, Stefan W; Geertsema, Corinne; Goldbach, Rob W; Vlak, Just M; Kohl, Alain; Khromykh, Alexander A; Pijlman, Gorben P

    2012-12-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3'-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.

  8. RECOVIR: An application package to automatically identify some single stranded RNA viruses using capsid protein residues that uniquely distinguish among these viruses

    Directory of Open Access Journals (Sweden)

    Fox George E

    2007-10-01

    Full Text Available Abstract Background Most single stranded RNA (ssRNA viruses mutate rapidly to generate large number of strains having highly divergent capsid sequences. Accurate strain recognition in uncharacterized target capsid sequences is essential for epidemiology, diagnostics, and vaccine development. Strain recognition based on similarity scores between target sequences and sequences of homology matched reference strains is often time consuming and ambiguous. This is especially true if only partial target sequences are available or if different ssRNA virus families are jointly analyzed. In such cases, knowledge of residues that uniquely distinguish among known reference strains is critical for rapid and unambiguous strain identification. Conventional sequence comparisons are unable to identify such capsid residues due to high sequence divergence among the ssRNA virus reference strains. Consequently, automated general methods to reliably identify strains using strain distinguishing residues are not currently available. Results We present here RECOVIR ("recognize viruses", a software tool to automatically detect strains of caliciviruses and picornaviruses by comparing their capsid residues with built-in databases of residues that uniquely distinguish among known reference strains of these viruses. The databases were created by constructing partitioned phylogenetic trees of complete capsid sequences of these viruses. Strains were correctly identified for more than 300 complete and partial target sequences by comparing the database residues with the aligned residues of these sequences. It required about 5 seconds of real time to process each sequence. A Java-based user interface coupled with Perl-coded computational modules ensures high portability of the software. RECOVIR currently runs on Windows XP and Linux platforms. The software generalizes a manual method briefly outlined earlier for human caliciviruses. Conclusion This study shows implementation of

  9. Low doses of ultraviolet radiation and oxidative damage induce dramatic accumulation of mitochondrial DNA replication intermediates, fork regression, and replication initiation shift.

    Science.gov (United States)

    Torregrosa-Muñumer, Rubén; Goffart, Steffi; Haikonen, Juha A; Pohjoismäki, Jaakko L O

    2015-11-15

    Mitochondrial DNA is prone to damage by various intrinsic as well as environmental stressors. DNA damage can in turn cause problems for replication, resulting in replication stalling and double-strand breaks, which are suspected to be the leading cause of pathological mtDNA rearrangements. In this study, we exposed cells to subtle levels of oxidative stress or UV radiation and followed their effects on mtDNA maintenance. Although the damage did not influence mtDNA copy number, we detected a massive accumulation of RNA:DNA hybrid-containing replication intermediates, followed by an increase in cruciform DNA molecules, as well as in bidirectional replication initiation outside of the main replication origin, OH. Our results suggest that mitochondria maintain two different types of replication as an adaptation to different cellular environments; the RNA:DNA hybrid-involving replication mode maintains mtDNA integrity in tissues with low oxidative stress, and the potentially more error tolerant conventional strand-coupled replication operates when stress is high.

  10. A cypovirus VP5 displays the RNA chaperone-like activity that destabilizes RNA helices and accelerates strand annealing.

    Science.gov (United States)

    Yang, Jie; Cheng, Zhenyun; Zhang, Songliu; Xiong, Wei; Xia, Hongjie; Qiu, Yang; Wang, Zhaowei; Wu, Feige; Qin, Cheng-Feng; Yin, Lei; Hu, Yuanyang; Zhou, Xi

    2014-02-01

    For double-stranded RNA (dsRNA) viruses in the family Reoviridae, their inner capsids function as the machinery for viral RNA (vRNA) replication. Unlike other multishelled reoviruses, cypovirus has a single-layered capsid, thereby representing a simplified model for studying vRNA replication of reoviruses. VP5 is one of the three major cypovirus capsid proteins and functions as a clamp protein to stabilize cypovirus capsid. Here, we expressed VP5 from type 5 Helicoverpa armigera cypovirus (HaCPV-5) in a eukaryotic system and determined that this VP5 possesses RNA chaperone-like activity, which destabilizes RNA helices and accelerates strand annealing independent of ATP. Our further characterization of VP5 revealed that its helix-destabilizing activity is RNA specific, lacks directionality and could be inhibited by divalent ions, such as Mg(2+), Mn(2+), Ca(2+) or Zn(2+), to varying degrees. Furthermore, we found that HaCPV-5 VP5 facilitates the replication initiation of an alternative polymerase (i.e. reverse transcriptase) through a panhandle-structured RNA template, which mimics the 5'-3' cyclization of cypoviral positive-stranded RNA. Given that the replication of negative-stranded vRNA on the positive-stranded vRNA template necessitates the dissociation of the 5'-3' panhandle, the RNA chaperone activity of VP5 may play a direct role in the initiation of reoviral dsRNA synthesis.

  11. Biomarkers of replicative senescence revisited

    DEFF Research Database (Denmark)

    Nehlin, Jan

    2016-01-01

    Biomarkers of replicative senescence can be defined as those ultrastructural and physiological variations as well as molecules whose changes in expression, activity or function correlate with aging, as a result of the gradual exhaustion of replicative potential and a state of permanent cell cycle...... with their chronological age and present health status, help define their current rate of aging and contribute to establish personalized therapy plans to reduce, counteract or even avoid the appearance of aging biomarkers....

  12. Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

    Science.gov (United States)

    Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R; Sakhuja, Kiran; Cerritelli, Susana M; Moss, Chloe; Bowmaker, Mark R; Jacobs, Howard T; Crouch, Robert J; Holt, Ian J

    2015-07-28

    Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

  13. Endogenous MOV10 inhibits the retrotransposition of endogenous retroelements but not the replication of exogenous retroviruses

    Science.gov (United States)

    2012-01-01

    Background The identification of cellular factors that regulate the replication of exogenous viruses and endogenous mobile elements provides fundamental understanding of host-pathogen relationships. MOV10 is a superfamily 1 putative RNA helicase that controls the replication of several RNA viruses and whose homologs are necessary for the repression of endogenous mobile elements. Here, we employ both ectopic expression and gene knockdown approaches to analyse the role of human MOV10 in the replication of a panel of exogenous retroviruses and endogenous retroelements. Results MOV10 overexpression substantially decreased the production of infectious retrovirus particles, as well the propagation of LTR and non-LTR endogenous retroelements. Most significantly, RNAi-mediated silencing of endogenous MOV10 enhanced the replication of both LTR and non-LTR endogenous retroelements, but not the production of infectious retrovirus particles demonstrating that natural levels of MOV10 suppress retrotransposition, but have no impact on infection by exogenous retroviruses. Furthermore, functional studies showed that MOV10 is not necessary for miRNA or siRNA-mediated mRNA silencing. Conclusions We have identified novel specificity for human MOV10 in the control of retroelement replication and hypothesise that MOV10 may be a component of a cellular pathway or process that selectively regulates the replication of endogenous retroelements in somatic cells. PMID:22727223

  14. Nucleotide Metabolism and DNA Replication.

    Science.gov (United States)

    Warner, Digby F; Evans, Joanna C; Mizrahi, Valerie

    2014-10-01

    The development and application of a highly versatile suite of tools for mycobacterial genetics, coupled with widespread use of "omics" approaches to elucidate the structure, function, and regulation of mycobacterial proteins, has led to spectacular advances in our understanding of the metabolism and physiology of mycobacteria. In this article, we provide an update on nucleotide metabolism and DNA replication in mycobacteria, highlighting key findings from the past 10 to 15 years. In the first section, we focus on nucleotide metabolism, ranging from the biosynthesis, salvage, and interconversion of purine and pyrimidine ribonucleotides to the formation of deoxyribonucleotides. The second part of the article is devoted to DNA replication, with a focus on replication initiation and elongation, as well as DNA unwinding. We provide an overview of replication fidelity and mutation rates in mycobacteria and summarize evidence suggesting that DNA replication occurs during states of low metabolic activity, and conclude by suggesting directions for future research to address key outstanding questions. Although this article focuses primarily on observations from Mycobacterium tuberculosis, it is interspersed, where appropriate, with insights from, and comparisons with, other mycobacterial species as well as better characterized bacterial models such as Escherichia coli. Finally, a common theme underlying almost all studies of mycobacterial metabolism is the potential to identify and validate functions or pathways that can be exploited for tuberculosis drug discovery. In this context, we have specifically highlighted those processes in mycobacterial DNA replication that might satisfy this critical requirement.

  15. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  16. Monitoring the determinants of efficient viral replication using Classical Swine Fever Virus-reporter replicons

    DEFF Research Database (Denmark)

    Risager, Peter Christian; Everett, Helen; Crooke, Helen

    2012-01-01

    proteins considered non-essential for RNA replication were constructed and these deletions were replaced with an in-frame insertion of the Renilla luciferase (Rluc) sequence. RNA transcripts from these replicons should be translated as a single functional open reading frame. Full-genome cDNAs (~10-12,3 kb...

  17. Regulation of DNA replication and chromosomal polyploidy by the MLL-WDR5-RBBP5 methyltransferases

    Science.gov (United States)

    Lu, Fei; Wu, Xiaojun; Yin, Feng; Chia-Fang Lee, Christina; Yu, Min; Mihaylov, Ivailo S.; Yu, Jiekai; Sun, Hong

    2016-01-01

    ABSTRACT DNA replication licensing occurs on chromatin, but how the chromatin template is regulated for replication remains mostly unclear. Here, we have analyzed the requirement of histone methyltransferases for a specific type of replication: the DNA re-replication induced by the downregulation of either Geminin, an inhibitor of replication licensing protein CDT1, or the CRL4CDT2 ubiquitin E3 ligase. We found that siRNA-mediated reduction of essential components of the MLL-WDR5-RBBP5 methyltransferase complexes including WDR5 or RBBP5, which transfer methyl groups to histone H3 at K4 (H3K4), suppressed DNA re-replication and chromosomal polyploidy. Reduction of WDR5/RBBP5 also prevented the activation of H2AX checkpoint caused by re-replication, but not by ultraviolet or X-ray irradiation; and the components of MLL complexes co-localized with the origin recognition complex (ORC) and MCM2-7 replicative helicase complexes at replication origins to control the levels of methylated H3K4. Downregulation of WDR5 or RBBP5 reduced the methylated H3K4 and suppressed the recruitment of MCM2-7 complexes onto replication origins. Our studies indicate that the MLL complexes and H3K4 methylation are required for DNA replication but not for DNA damage repair. PMID:27744293

  18. Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells.

    Science.gov (United States)

    Pombo, A; Ferreira, J; Bridge, E; Carmo-Fonseca, M

    1994-11-01

    We have visualized the intranuclear topography of adenovirus replication and transcription in infected HeLa cells. The results show that viral DNA replication occurs in multiple foci that are highly organized in the nucleoplasm. Pulse-chase experiments indicate that newly synthesized viral double-stranded DNA molecules are displaced from the replication foci and spread throughout the nucleoplasm, while the single-stranded DNA replication intermediates accumulate in adjacent sites. Double-labelling experiments and confocal microscopy show that replication occurs in foci localized at the periphery of the sites where single-stranded DNA accumulates. The simultaneous visualization of viral replication and transcription reveals that the sites of transcription are predominantly separated from the sites of replication. Transcription is detected adjacent to the replication foci and extends around the sites of single-stranded DNA accumulation. These data indicate that newly synthesized double-stranded DNA molecules are displaced from the replication foci and spread in the surrounding nucleoplasm, where they are used as templates for transcription. Splicing snRNPs are shown to co-localize with the sites of transcription and to be excluded from the sites of replication. This provides evidence that splicing of viral RNAs occurs co-transcriptionally and that the sites of viral DNA replication are spatially distinct from the sites of RNA transcription and processing.

  19. Dengue virus binding and replication by platelets.

    Science.gov (United States)

    Simon, Ayo Y; Sutherland, Michael R; Pryzdial, Edward L G

    2015-07-16

    Dengue virus (DENV) infection causes ∼200 million cases of severe flulike illness annually, escalating to life-threatening he