Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA

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Ristic Natalia

2010-09-01

Full Text Available Abstract Background The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1 sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants. Results HIV-1 lacking SL1 (NLΔSL1 did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K on day 18 postinfection and C1907T in the SP1 domain (P10L on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1

Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.

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Rajashekara, Gireesh; Glover, David A; Banai, Menachem; O'Callaghan, David; Splitter, Gary A

2006-05-01

In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

A Selective Bottleneck Shapes the Evolutionary Mutant Spectra of Enterovirus A71 during Viral Dissemination in Humans.

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Huang, Sheng-Wen; Huang, Yi-Hui; Tsai, Huey-Pin; Kuo, Pin-Hwa; Wang, Shih-Min; Liu, Ching-Chuan; Wang, Jen-Ren

2017-12-01

RNA viruses accumulate mutations to rapidly adapt to environmental changes. Enterovirus A71 (EV-A71) causes various clinical manifestations with occasional severe neurological complications. However, the mechanism by which EV-A71 evolves within the human body is unclear. Utilizing deep sequencing and haplotype analyses of viruses from various tissues of an autopsy patient, we sought to define the evolutionary pathway by which enterovirus A71 evolves fitness for invading the central nervous system in humans. Broad mutant spectra with divergent mutations were observed at the initial infection sites in the respiratory and digestive systems. After viral invasion, we identified a haplotype switch and dominant haplotype, with glycine at VP1 residue 31 (VP1-31G) in viral particles disseminated into the integumentary and central nervous systems. In vitro viral growth and fitness analyses indicated that VP1-31G conferred growth and a fitness advantage in human neuronal cells, whereas VP1-31D conferred enhanced replication in human colorectal cells. A higher proportion of VP1-31G was also found among fatal cases, suggesting that it may facilitate central nervous system infection in humans. Our data provide the first glimpse of EV-A71 quasispecies from oral tissues to the central nervous system within humans, showing broad implications for the surveillance and pathogenesis of this reemerging viral pathogen. IMPORTANCE EV-A71 continues to be a worldwide burden to public health. Although EV-A71 is the major etiological agent of hand, foot, and mouth disease, it can also cause neurological pulmonary edema, encephalitis, and even death, especially in children. Understanding selection processes enabling dissemination and accurately estimating EV-A71 diversity during invasion in humans are critical for applications in viral pathogenesis and vaccine studies. Here, we define a selection bottleneck appearing in respiratory and digestive tissues. Glycine substitution at VP1 residue 31

Site-directed mutagenesis of HIV-1 vpu gene demonstrates two clusters of replication-defective mutants with distinct ability to down-modulate cell surface CD4 and tetherin

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Masako Nomaguchi

2010-11-01

Full Text Available HIV-1 Vpu acts positively on viral infectivity by mediating CD4 degradation in endoplasmic reticulum and enhances virion release by counteracting a virion release restriction factor, tetherin. In order to define the impact of Vpu activity on HIV-1 replication, we have generated a series of site-specific proviral vpu mutants. Of fifteen mutants examined, seven exhibited a replication-defect similar to that of a vpu-deletion mutant in a lymphocyte cell line H9. These mutations clustered in narrow regions within transmembrane domain (TMD and cytoplasmic domain (CTD. Replication-defective mutants displayed the reduced ability to enhance virion release from a monolayer cell line HEp2 without exception. Upon transfection with Vpu expression vectors, neither TMD mutants nor CTD mutants blocked CD4 expression at the cell surface in another monolayer cell line MAGI. While TMD mutants were unable to down-modulate cell surface tetherin in HEp2 cells, CTD mutants did quite efficiently. Confocal microscopy analysis revealed the difference of intracellular localization between TMD and CTD mutants. In total, replication capability of HIV-1 carrying vpu mutations correlates well with the ability of Vpu to enhance virion release and to impede the cell surface expression of CD4 but not with the ability to down-modulate cell surface tetherin. Our results here suggest that efficient viral replication requires not only down-regulation of cell surface tetherin but also its degradation.

Nonsense mutants in the bacteriophage T4D v gene

Energy Technology Data Exchange (ETDEWEB)

Minderhout, L van; Grimbergen, J; Groot, B de [Rijksuniversiteit Leiden (Netherlands). Lab. voor Stralengenetica en Chemische Mutagenese; Cohen (J.A.) Instituut voor Radiopathologie en Stralenbescherming, Leiden (Netherlands))

1975-09-01

Ten UV-sensitive mutants of T4D with the v phenotype were isolated. Of these ten mutants, two are amber and two opal. In UV curves and in photoreactivation and multiplicity reactivation experiments the nonsense mutants show the v phenotype in su/sup -/ hosts and almost the T4/sup +/ phenotype in su/sup +/ hosts. The mutations are located between rl and e and are alleles of v/sub 1/. In crosses with irradiated and non-irradiated phages the recombinant frequency is not reduced by uvs5. Amber uvs5 propagated in CR63 su/sup +/ is with B su/sup -/ just as sensitive to UV as uvs5 propagated in B su/sup -/, which permits the conclusion that the capsid of T4 phage particles does not contain the v gene product.

The same allele of translation initiation factor 4E mediates resistance against two Potyvirus spp. in Pisum sativum

DEFF Research Database (Denmark)

Bruun-Rasmussen, M.; Møller, I.S.; Tulinius, G.

2007-01-01

to linkage group VI together with other Potyvirus resistances. One of these, sbm1, confers resistance to strains of Pea seedborne mosaic virus and previously has been identified as a mutant allele of the eukaryotic translation initiation factor 4E gene (eIF4E). Sequence comparison of eIF4E from BYMV...... was overcome, and virus from these plants had a codon change causing an Arg to His change at position 116 of the predicted viral genome-linked protein (VPg). Accordingly, plants carrying the wlv resistance gene were infected upon inoculation with BYMV-W derived from cDNA with a His codon at position 116...

Progress in diagnosis of viral hepatitis A, B, C, D and E

International Nuclear Information System (INIS)

Kurstak, E.; Hossain, A.; Kurstak, C.

1996-01-01

The effective use of new molecular biological techniques towards the reliable diagnosis of HCV and other viral liver infections has been updated in this review. The applications PCR techniques with amplification of reverse transcribed cDNA seems to prove an effective means for assaying HCV infections. A very recent one-stage PCR assay of HCV RNA combined with either liquid hybridization or Southern blot analysis, equal in sensitivity to the nested PCR assay but with sharply reduced potential for contamination appears to be promising. Future further development of reliable and automated RT-PCR assay would be particularly interesting for diagnosis of HCV infections. PCR apparently remains the most useful test for the appraisal of HBV infection in sera-negative patients with liver disease. It has now made possible the confirmation of observations made with the spot blot or Southern blot test and provided access to the nucleotide sequence analysis of these viral mutant forms. The rapidity and simplicity of these viral forms. The rapidity and simplicity of the the newly developed latex agglutination method using ISTA also makes it a viable alternative for the determination of HBsAg. Cloning of HEV, sequencing of the viral genome and expression of recombinant HEV proteins has undoubtedly facilitated significant progress in the development of methods for identification of HEV infection in patients. Recently the availability of specific, more sensitive assays as recombinant-based EIAs has made the diagnosis of hepatitis E very much practicable. (author)

The prevalence of the pre-existing hepatitis C viral variants and the evolution of drug resistance in patients treated with the NS3-4a serine protease inhibitor telaprevir

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Rong, Libin [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory

2008-01-01

Telaprevir (VX-950), a novel hepatitis C virus (HCV) NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients infected with HCV genotype 1. Some patients experience viral breakthrough, which has been shown to be associated with emergence of telaprevir-resistant HCV variants during treatment. The exact mechanisms underlying the rapid selection of drug resistant viral variants during dosing are not fully understood. In this paper, we develop a two-strain model to study the pre-treatment prevalence of the mutant virus and derive an analytical solution of the mutant frequency after administration of the protease inhibitor. Our analysis suggests that the rapid increase of the mutant frequency during therapy is not due to mutant growth but rather due to the rapid and profound loss of wild-type virus, which uncovers the pre-existing mutant variants. We examine the effects of backward mutation and hepatocyte proliferation on the pre-existence of the mutant virus and the competition between wild-type and drug resistant virus during therapy. We then extend the simple model to a general model with multiple viral strains. Mutations during therapy do not play a significant role in the dynamics of various viral strains, although they are capable of generating low levels of HCV variants that would otherwise be completely suppressed because of fitness disadvantages. Hepatocyte proliferation may not affect the pretreatment frequency of mutant variants, but is able to influence the quasispecies dynamics during therapy. It is the relative fitness of each mutant strain compared with wild-type that determines which strain(s) will dominate the virus population. The study provides a theoretical framework for exploring the prevalence of pre-existing mutant variants and the evolution of drug resistance during treatment with other protease inhibitors or HCV polymerase inhibitors.

Programmed cell death in the leaves of the Arabidopsis spontaneous necrotic spots (sns-D mutant correlates with increased expression of the eukaryotic translation initiation factor eIF4B2

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Gwenael M.D.J.-M. Gaussand

2011-04-01

Full Text Available From a pool of transgenic Arabidopsis (Arabidopsis thaliana plants harboring an activator T-DNA construct, one mutant was identified that developed spontaneous necrotic spots (sns-D on the rosette leaves under aseptic conditions. The sns-D mutation is dominant and homozygous plants are embryo lethal. The mutant produced smaller rosettes with a different number of stomata than the wild-type. DNA fragmentation in the nuclei of cells in the necrotic spots and a significant increase of caspase-3 and caspase-6 like activities in sns-D leaf extracts indicated that the sns-D mutation caused programmed cell death (PCD. The integration of the activator T-DNA caused an increase of the expression level of At1g13020, which encodes the eukaryotic translation initiation factor eIF4B2. The expression level of eIF4B2 was positively correlated with the severity of sns-D mutant phenotype. Overexpression of the eIF4B2 cDNA mimicked phenotypic traits of the sns-D mutant indicating that the sns-D mutant phenotype is indeed caused by activation tagging of eIF4B2. Thus, incorrect regulation of translation initiation may result in PCD.

BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression

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Shwu-Yuan Wu

2016-08-01

Full Text Available Post-translational modification can modulate protein conformation and alter binding partner recruitment within gene regulatory regions. Here, we report that bromodomain-containing protein 4 (BRD4, a transcription co-factor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV to viral early gene and cellular matrix metalloproteinase-9 (MMP-9 promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NF-κB to their target sequences. This is triggered by replacement of AP-1 family members JunB and JunD by c-Jun and by re-localization of NF-κB from the cytoplasm to the nucleus. In addition, BRD4 phosphorylation is critical for E2- and origin-dependent HPV DNA replication. A class of phospho-BRD4-targeting compounds, distinct from the BET bromodomain inhibitors, effectively blocks BRD4 phosphorylation-specific functions in transcription and factor recruitment.

Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

International Nuclear Information System (INIS)

Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.; Dunn, J.J.; Semler, B.L.; Wimmer, E.

1988-01-01

By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s)

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

International Nuclear Information System (INIS)

Iourin, Oleg; Harlos, Karl; El Omari, Kamel; Lu, Weixian; Kadlec, Jan; Iqbal, Munir; Meier, Christoph; Palmer, Andrew; Jones, Ian; Thomas, Carole; Brownlie, Joe; Grimes, Jonathan M.; Stuart, David I.

2012-01-01

The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the ectodomain of BVDV E2 are described. Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress

Translation Initiation Factor eIF4E and eIFiso4E Are Both Required for Peanut stripe virus Infection in Peanut (Arachis hypogaea L.).

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Xu, Manlin; Xie, Hongfeng; Wu, Juxiang; Xie, Lianhui; Yang, Jinguang; Chi, Yucheng

2017-01-01

Peanut stripe virus (PStV) belongs to the genus Potyvirus and is the most important viral pathogen of cultivated peanut ( Arachis hypogaea L.). The eukaryotic translation initiation factor, eIF4E, and its isoform, eIF(iso)4E, play key roles during virus infection in plants, particularly Potyvirus . In the present study, we cloned the eIF4E and eIF(iso)4E homologs in peanut and named these as PeaeIF4E and PeaeIF(iso)4E , respectively. Quantitative real-time PCR (qRT-PCR) analysis showed that these two genes were expressed during all growth periods and in all peanut organs, but were especially abundant in young leaves and roots. These also had similar expression levels. Yeast two-hybrid analysis showed that PStV multifunctional helper component proteinase (HC-Pro) and viral protein genome-linked (VPg) both interacted with PeaeIF4E and PeaeIF(iso)4E. Bimolecular fluorescence complementation assay showed that there was an interaction between HC-Pro and PeaeIF4E/PeaeIF(iso)4E in the cytoplasm and between VPg and PeaeIF4E/PeaeIF(iso)4E in the nucleus. Silencing either PeaeIF4E or PeaeIF(iso)4E using a virus-induced gene silencing system did not significantly affect PStV accumulation. However, silencing both PeaeIF4E and PeaeIF(iso)4E genes significantly weakened PStV accumulation. The findings of the present study suggest that PeaeIF4E and PeaeIF(iso)4E play important roles in the PStV infection cycle and may potentially contribute to PStV resistance.

Adenovirus E4 open reading frame 4-induced dephosphorylation inhibits E1A activation of the E2 promoter and E2F-1-mediated transactivation independently of the retinoblastoma tumor suppressor protein

DEFF Research Database (Denmark)

Mannervik, M; Fan, S; Ström, A C

1999-01-01

of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation......Previous studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression...... of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the E2 promoter. In agreement with this, we find that E4-ORF4 inhibits E2F-1/DP-1-mediated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expression...

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts

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Hannah L. Fox

2017-06-01

Full Text Available Herpes simplex virus 1 (HSV-1 genes are transcribed by cellular RNA polymerase II (RNA Pol II. While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22 function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16 was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq. The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq, we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production.

Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

Science.gov (United States)

Hosmillo, Myra; Chaudhry, Yasmin; Kim, Deok-Song; Goodfellow, Ian; Cho, Kyoung-Oh

2014-11-01

Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5' end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and

Viral phenotype, antiretroviral resistance and clinical evolution in human immunodeficiency virus-infected children.

Science.gov (United States)

Mellado, M J; Cilleruelo, M J; Ortiz, M; Villota, J; García, M; Perez-Jurado, M L; Barreiro, G; Martín-Fontelos, P; Bernal, A

1997-11-01

The syncytium-inducing (SI) viral phenotype and the emergence of viral strains resistant to zidovudine have been described in persons infected with HIV, and in some cases they have been associated with poor prognosis. HIV isolates obtained from 37 HIV-infected children were analyzed to determine whether the SI viral phenotype and the mutation on the 215 position of the reverse transcriptase (M215) could be used as markers of disease progression. We performed peripheral blood coculture mononuclear cells, and we analyzed the induction of syncytia using the MT-2 cell line. The emergence of mutations on the 215 position was determined by PCR. We found a statistically significant association (P < 0.05) between SI viral phenotype and (1) recurrent serious bacterial infections, (2) absolute CD4+ cell counts <2 SD, (3) progression to AIDS and (4) death. Sixty percent of the children treated with zidovudine developed 215 mutant viral strains without statistically significant association with clinical or immunologic findings. The SI viral phenotype was statistically associated with the presence of the 215 mutation (P < 0.05). SI viral phenotype is a marker associated with a poor clinical and immunologic progression of the disease and it may facilitate the emergence of mutant strains in children treated with zidovudine.

Acute hepatitis e viral infection in pregnancy and maternal morbidity

International Nuclear Information System (INIS)

Khaskheli, M.N.; Baloch, S.

2015-01-01

To determine the maternal morbidity in pregnant women with acute hepatitis E viral infection. Study Design: Observational, cross-sectional study. Place and Duration of Study: Departments of Obstetrics and Gynaecology and Medicine, Liaquat University of Medical and Health Sciences, Jamshoro, Red Crescent General Hospital and Saint Elizabeth Hospital, Hyderabad, from January 2011 to December 2013. Methodology: The study population was pregnant women with acute hepatitis E infection confirmed by ELIZA technique. Pregnant women with other hepatic viral infections were excluded. All medical and obstetric conditions, and mortality were noted on the predesigned proforma. Results: Out of the total 45 admitted pregnant women with hepatitis E viral infection, 22 women (48.9%) had severe morbidity. The most common were hepatic coma in 8 (36.36%) cases and disseminated intravascular coagulation in 14 (63.63%) cases. Highest mortality rate was seen in women with hepatic coma (100%), while in those with disseminated intravascular coagulation, one out of the 14 cases (7.14%) died. Conclusion: The acute viral hepatitis E infection in pregnant women is associated with maternal morbidities and high mortality rate. (author)

Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

International Nuclear Information System (INIS)

Fradet-Turcotte, Amelie; Brault, Karine; Titolo, Steve; Howley, Peter M.; Archambault, Jacques

2009-01-01

The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.

Mutants of Escherichia coli K-12 with enhanced resistance to ionizing radiation. 4. Peculiarities of recombination in Gamsup(r) mutants

International Nuclear Information System (INIS)

Bresler, S.E.; Kalinin, V.L.; Laneeva, N.I.

1984-01-01

Radioresistant mutant Gam sup(r) 444 differs from a wild type and from Gam sup(r) 445 mutant in decreased frequency of long episome heritage ORF 1 (pur E + -tsx + -proC + -lac + ) and F 14 (ilv + -argE + ), containing hot points of RecRecF - depending recombination and in increased frequency of chromosome mobilization and integrative suppression of temperature sensitive dna A46 mutation by sexual factor F. In this respect Gam sup(r) 444 mutant resembles rec BC sbs B mutant with RecF - recombination type

Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

Science.gov (United States)

Karita, M; Etterbeek, M L; Forsyth, M H; Tummuru, M K; Blaser, M J

1997-10-01

Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced. This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins. In E. coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to L-diaminopimelic acid (L-DAP) and succinate. When wild-type H. pylori strains were transformed to select for dapE mutagenesis, mutants were present when plates were supplemented with DAP but not with lysine; orf2 mutants were selected without DAP supplementation. Consistent with the finding that GidA is essential in Escherichia coli, we were unable to obtain a gidA mutant in H. pylori despite evidence that insertional mutagenesis had occurred. The positions of gidA, dapE, and orf2 suggest that they form an operon, which was supported by slot blot RNA hybridization and reverse transcriptase PCR studies. The data imply that the H. pylori dapE mutant may be useful as a conditionally lethal vaccine.

Hearing loss associated with enlarged vestibular aqueduct and zero or one mutant allele of SLC26A4.

Science.gov (United States)

Rose, Jane; Muskett, Julie A; King, Kelly A; Zalewski, Christopher K; Chattaraj, Parna; Butman, John A; Kenna, Margaret A; Chien, Wade W; Brewer, Carmen C; Griffith, Andrew J

2017-07-01

To characterize the severity and natural history of hearing loss, and the prevalence of having a cochlear implant in a maturing cohort of individuals with enlarged vestibular aqueduct (EVA) and zero or one mutant allele of SLC26A4. Prospective cohort study of subjects ascertained between 1998 and 2015 at the National Institutes of Health Clinical Center. Study subjects were 127 individuals (median age, 8 years; range, 0-59 years) with EVA in at least one ear. Ears with EVA and zero or one mutant allele of SLC26A4 had mean 0.5/1/2/4-kHz pure-tone averages of 62.6 and 52.9 dB HL, respectively, in contrast to EVA ears with two mutant alleles of SLC26A4 (88.1 dB HL; P zero, one, and two mutant alleles, respectively (P = .00833). This association was not independent (P = .534) but reflected underlying correlations with age at time of first audiogram (P = .003) or severity of hearing loss (P = .000). Ears with EVA and zero or one mutant allele of SLC26A4 have less severe hearing loss, no difference in prevalence of fluctuation, and a lower prevalence of cochlear implantation in comparison to ears with two mutant alleles of SLC26A4. NA Laryngoscope, 127:E238-E243, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

Science.gov (United States)

Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

2004-05-01

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Mutational Analysis of the Hypervariable Region of Hepatitis E Virus Reveals Its Involvement in the Efficiency of Viral RNA Replication ▿

OpenAIRE

Pudupakam, R. S.; Kenney, Scott P.; Córdoba, Laura; Huang, Yao-Wei; Dryman, Barbara A.; LeRoith, Tanya; Pierson, F. William; Meng, Xiang-Jin

2011-01-01

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication...

Contribution of viral recombinants to the study of the immune response against the Epstein-Barr virus.

Science.gov (United States)

Delecluse, Henri-Jacques; Feederle, Regina; Behrends, Uta; Mautner, Josef

2008-12-01

Over the past two decades, Epstein-Barr virus (EBV) mutants have become valuable tools for the analysis of viral functions. Several experimental strategies are currently used to generate recombinant mutant genomes that carry alterations in one or several viral genes. The probably most versatile approach utilizes bacterial artificial chromosomes (BAC) carrying parts or the whole EBV genome, which permits extensive genetic manipulations in Escherichia coli cells. The 'mini-EBVs', for example, which contain roughly half of the wild type viral information, efficiently transform primary B cells and have been used as gene vectors for foreign antigens. After expression in lymphoblastoid cell lines (LCLs), these antigens are efficiently presented on MHC molecules and recognized by antigen-specific T cells. These vectors, however, cannot undergo lytic replication and require a helper cell line for efficient replication and DNA packaging. Further experimental systems include the complete viral genome cloned onto a BAC. These mutants can typically be complemented by expression plasmids, some of which are expressed on EBV-derived vectors and can be propagated without requirement of a helper cell line. Over the last years, these viral recombinants have been utilized increasingly to analyse different aspects of the immune response against EBV. Immunological applications are manifold and steadily growing and include crude screening of T cell clones for their specificity towards latent versus lytic antigens, or more detailed analyses in which the exact specificity of T cells is determined using EBV mutants that lack a single viral antigen. Other applications include detailed analysis of protein domains important for immune recognition, e.g. Gly-Ala repeats in the EBV nuclear antigen 1 (EBNA1) protein, expansion of T cell clones directed against virion structures using virus-like particles and phenotypic analysis of virus mutants defective in infection. Future developments might

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

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Velasquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

2016-07-11

mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

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Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

2017-06-13

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription

Fitness ranking of individual mutants drives patterns of epistatic interactions in HIV-1.

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Javier P Martínez

Full Text Available Fitness interactions between mutations, referred to as epistasis, can strongly impact evolution. For RNA viruses and retroviruses with their high mutation rates, epistasis may be particularly important to overcome fitness losses due to the accumulation of deleterious mutations and thus could influence the frequency of mutants in a viral population. As human immunodeficiency virus type 1 (HIV-1 resistance to azidothymidine (AZT requires selection of sequential mutations, it is a good system to study the impact of epistasis. Here we present a thorough analysis of a classical AZT-resistance pathway (the 41-215 cluster of HIV-1 variants by fitness measurements in single round infection assays covering physiological drug concentrations ex vivo. The sign and value of epistasis varied and did not predict the epistatic effect on the mutant frequency. This complex behavior is explained by the fitness ranking of the variants that strongly depends on environmental factors, i.e., the presence and absence of drugs and the host cells used. Although some interactions compensate fitness losses, the observed small effect on the relative mutant frequencies suggests that epistasis might be inefficient as a buffering mechanism for fitness losses in vivo. While the use of epistasis-based hypotheses to make general assumptions on the evolutionary dynamics of viral populations is appealing, our data caution their interpretation without further knowledge on the characteristics of the viral mutant spectrum under different environmental conditions.

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

OpenAIRE

Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

2015-01-01

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates f...

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

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Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription

The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment.

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Harris, Leanne; McFarlane-Majeed, Laura; Campos-León, Karen; Roberts, Sally; Parish, Joanna L

2017-01-01

In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2 Y131A ) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2 Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2 WT ), the chromatin-bound pool of E2 Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2 Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2 WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2 Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection

Ethanol production using engineered mutant E. coli

Science.gov (United States)

Ingram, Lonnie O.; Clark, David P.

1991-01-01

The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

Science.gov (United States)

Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

2006-06-01

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Genetic and biochemical studies of the lipid-containing bacteriophage PR4

International Nuclear Information System (INIS)

Vanden Boom, T.J.

1989-01-01

Bacteriophage PR4 is a lipid-containing bacterial virus able to infect Escherichia coli and Salmonella typhimurium. The icosahedral virion consists of an external protein capsid layer which surrounds a membrane vesicle enclosed ds DNA genome. The author has analyzed the time course of phage PR4 protein synthesis and have identified at least 34 proteins present in phage infected cells not detected in uninfected control cultures. In addition, he has isolated a more extensive set of conditional-lethal nonsense mutants of this virus. This collection of mutants permitted the identification of seven additional phage PR4 gene products, including the terminal genome protein and an accessory lytic factor. The present collection of phage PR4 mutants has been assigned to 19 distinct genetic groups on the basis of genetic complementation tests and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the proteins produced in mutant-infected UV-irradiated cells. A restriction endonuclease map of the phage PR4 genome was constructed which includes 59 sites for ten restriction endonucleases. In addition, he has constructed a collection of recombinant plasmids containing subgenomic DNA fragments of bacteriophage PR4. He has used this collection of plasmids to generate a physical-genetic map of the PR4 genome. The physical-genetic map localizes mutations in 13 phage PR4 genetic groups on the viral DNA molecule. To investigate the role of phosphatidylglycerol (PG) in phage assembly and infectivity, he propagated PR4 on an E. coli mutant defective in PG synthesis. The PG content of phage PR4 grown on the mutant host accounted for 0.4% of the total viral phospholipids, representing a 90-fold decrease in PG relative to the PG content of phage grown on a wild type host

Transient nephritis during resolution phase of acute virale hepatitis E

OpenAIRE

Arden, Amir David

2009-01-01

Hepatitis E Virus is a causative agent of hepatitis. Viral E hepatitis is responsible for various clinical manifestations. However, immune reactions due to hepatitis E virus are rarely encountered. A case of membranoproliferative glomerulonephritis associated with hepatitis E virus is reported her.

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

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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.

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

International Nuclear Information System (INIS)

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J.

2016-01-01

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.

Integration, viral charge and E2 mRNA levels in the progresion of intraepitelial cervical lesions

Directory of Open Access Journals (Sweden)

Esperanza Trujillo

2018-01-01

Full Text Available It is important to distinguish among squamous intraepithelial lesions (SIL those associated with increased risk cervical cancer. Our aim was to evaluate if the expression level of gen E2 in women with SIL and evidence of viral integration is associated to the grade of lesion. Cervical scrapes HPV16 positive from 19 women with normal histology, 45 women with low-grade SIL (LSIL and 45 women with high-grade SIL (HSIL were analyzed. Real-time PCR was used to quantify the mRNA of E2 and E2 and E6 genes to calculate viral load (E6 and the ratio E2/E6 to assess viral integration. Similar frequencies of E2 expression were found in LSIL and HSIL15/45 (33 %, the frequency in women without SIL was lower 3/19 (15.8 %, and all cases with E2 gene expression had mixed episomal and integrated viral DNA. The viral load increased significantly with the grade of SIL (p= 0.049, while E2/E6 ratio decreased (p=0.049. The ROC analysis showed low capacity of the three viral parameters analyzed to distinguish between low and high grade SIL. In conclusion although SIL with mixed and integrated viral DNA with E2 expression could be at lower risk of progression, and viral load and integration were associated with higher severity of the lesion, its clinical value as biomarkers of HSIL is limited.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

Science.gov (United States)

Yue, Chen-Li; Shi, Jie-Ran; Shi, Chang-Hong; Zhang, Hai; Zhao, Lei; Zhang, Ting-Fen; Zhao, Yong; Xi, Li

2008-10-01

To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta.

Science.gov (United States)

Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I; Mäkinen, Kristiina

2011-09-01

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.

Potyviral VPg Enhances Viral RNA Translation and Inhibits Reporter mRNA Translation In Planta▿

Science.gov (United States)

Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I.; Mäkinen, Kristiina

2011-01-01

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5′ untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation. PMID:21697470

Hepatitis A through E (Viral Hepatitis)

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... Treatment Eating, Diet, & Nutrition Clinical Trials Wilson Disease Hepatitis (Viral) View or Print All Sections What is Viral Hepatitis? Viral hepatitis is an infection that causes liver inflammation ...

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif.

Science.gov (United States)

Ala-Poikela, Marjo; Goytia, Elisa; Haikonen, Tuuli; Rajamäki, Minna-Liisa; Valkonen, Jari P T

2011-07-01

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.

Deletions of the hypervariable region (HVR) in open reading frame 1 of hepatitis E virus do not abolish virus infectivity: evidence for attenuation of HVR deletion mutants in vivo.

Science.gov (United States)

Pudupakam, R S; Huang, Y W; Opriessnig, T; Halbur, P G; Pierson, F W; Meng, X J

2009-01-01

Hepatitis E virus (HEV) is an important human pathogen, although little is known about its biology and replication. Comparative sequence analysis revealed a hypervariable region (HVR) with extensive sequence variations in open reading frame 1 of HEV. To elucidate the role of the HVR in HEV replication, we first constructed two HVR deletion mutants, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a genotype 1 human HEV replicon. Evidence of HEV replication was detected in Huh7 cells transfected with RNA transcripts from mutant hHVRd2, as evidenced by expression of enhanced green fluorescent protein. To confirm the in vitro results, we constructed three avian HEV mutants with various HVR deletions: mutants aHVRd1, with deletion of aa 557 to 585 (Delta557-585); aHVRd2 (Delta612-641); and aHVRd3 (Delta557-641). Chickens intrahepatically inoculated with capped RNA transcripts from mutants aHVRd1 and aHVRd2 developed active viral infection, as evidenced by seroconversion, viremia, and fecal virus shedding, although mutant aHVRd3, with complete HVR deletion, was apparently attenuated in chickens. To further verify the results, we constructed four additional HVR deletion mutants using the genotype 3 swine HEV as the backbone. Mutants sHVRd2 (Delta722-781), sHVRd3 (Delta735-765), and sHVRd4 (Delta712-765) were shown to tolerate deletions and were infectious in pigs intrahepatically inoculated with capped RNA transcripts from the mutants, whereas mutant sHVRd1 (Delta712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype in infected pigs. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

Science.gov (United States)

Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

2013-01-01

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Cytotoxic CD4 T Cells—Friend or Foe during Viral Infection?

Science.gov (United States)

Juno, Jennifer A.; van Bockel, David; Kent, Stephen J.; Kelleher, Anthony D.; Zaunders, John J.; Munier, C. Mee Ling

2017-01-01

CD4 T cells with cytotoxic function were once thought to be an artifact due to long-term in vitro cultures but have in more recent years become accepted and reported in the literature in response to a number of viral infections. In this review, we focus on cytotoxic CD4 T cells in the context of human viral infections and in some infections that affect mice and non-human primates. We examine the effector mechanisms used by cytotoxic CD4 cells, the phenotypes that describe this population, and the transcription factors and pathways that lead to their induction following infection. We further consider the cells that are the predominant targets of this effector subset and describe the viral infections in which CD4 cytotoxic T lymphocytes have been shown to play a protective or pathologic role. Cytotoxic CD4 T cells are detected in the circulation at much higher levels than previously realized and are now recognized to have an important role in the immune response to viral infections. PMID:28167943

Mutant E. coli strain with increased succinic acid production

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Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

2001-09-25

A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

Mutant E. coli strain with increased succinic acid production

Science.gov (United States)

Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

1998-01-01

A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

Metabolism goes viral.

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Miyake-Stoner, Shigeki J; O'Shea, Clodagh C

2014-04-01

Viral and cellular oncogenes converge in targeting critical protein interaction networks to reprogram the cellular DNA and protein replication machinery for pathological replication. In this issue, Thai et al. (2014) show that adenovirus E4ORF1 activates MYC glycolytic targets to induce a Warburg-like effect that converts glucose into nucleotides for viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

VIRAL HEPATITIS A TO E IN SOUTH MEDITERRANEAN COUNTRIES

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Sanaa M. Kamal

2010-02-01

Full Text Available Viral hepatitis represents an important health problem in the South Mediterranean countries, Egypt, Libya, Tunisia, Algeria and Morocco.  Emerging natural history and epidemiological information reveal differences in the overall epidemiology, risk factors and modes of transmission of viral hepatitis A, B, C, D, E infections in the South Mediterranean region. The differences in the in incidence and prevalence of viral hepatitis across North African countries is attributed to variations in health care  and sanitation standards, risk factors and immunization strategies. The active continuous population movement through travel, tourism and migration from and to the South Mediterranean countries contribute to the spread of infections due to hepatitis viruses across borders leading to outbreaks and emergence of new patterns of infection or introduction of uncommon genotypes in other countries, particularly in Europe.

Survival and mutant production induced by mutagenic agents in Metarhizium anisopliae Sobrevivência e obtenção de mutantes induzidos por agentes mutagênicos em Metarhizium anisopliae

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V. Kava - Cordeiro

1995-12-01

Full Text Available A wild strain of Metarhizium anisopliae, an entomopathogenic fungus, was submitted to three mutagenic agents: gamma radiation, ultraviolet light and nitrous acid. Survival curves were obtained and mutants were selected using different mutagenic doses which gave 1 to 5% survival. Morphological and auxotrophic mutants were isolated. Morphological mutants were grouped in a class with yellow conidia and other with pale vinaceous conidia as opposed to the green wild type conidia. Auxotrophic mutants had requirements for vitamin and aminoacid biosynthesis. More than 58% of the total auxotrophk mutants required proline/aipnine. Gamma radiation showed to be the most efficient mutagenic agent giving 0.2% of auxotrophk mutants followed by ultraviolet light (0.12% and nitrous acid (0.06%.The conidial colour and auxotrophk mutants isolated until now from M. anisopliae were reviewed.Uma linhagem selvagem do fungo entomopatogênico Metarhizium anisopliae foi submetida à ação de três agentes mutagênicos: radiação gama, luz ultravioleta e ácido nitroso. Curvas de sobrevivência foram obtidas para cada mutagênicos utilizado e mutantes foram selecionados a partir de doses dos mutagênicos que proporcionassem de 1 a 5% de sobrevivência. Mutantes morfológicos para a coloração de conídios e mutantes auxotróficos foram isolados. Mutantes para coloração de conidios foram agrupados em duas classes, uma com conídios amarelos e outra com conídios vinho pálido. Os mutantes auxotróficos obtidos foram deficientes para aminoácidos e vitaminas e mais de 58% deles eram auxotróficos para prolina/argmina. Radiação gama foi o mutagênico mais eficiente com uma porcentagem de obtenção de mulantes auxotróficos de aproximadamente 0,2%, seguido pela luz ultravioleta (0.12% e pelo ácido nitroso (0.06%.Os mulantes morfológicos e auxotróficos obtidos até o momento em Metarhizium anisopliae foram revistos.

Herpes simplex virus mutant generation and dual-detection methods for gaining insight into latent/lytic cycles in vivo.

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Sawtell, Nancy M; Thompson, Richard L

2014-01-01

Two important components to a useful strategy to examine viral gene regulation in vivo are (1) a highly efficient protocol to generate viral mutants that limits undesired mutation and retains full replication competency in vivo and (2) an efficient system to detect and quantify viral promoter activity in rare cells in vivo. Our strategy and protocols for generating, characterizing, and employing HSV viral promoter/reporter mutants in vivo are provided in this two-part chapter.

The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

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Blaise Ndjamen

2014-03-01

Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

E1B-55K mediated regulation of RNF4 STUbL promotes HAdV gene expression.

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Müncheberg, Sarah; Hay, Ron T; Ip, Wing H; Meyer, Tina; Weiß, Christina; Brenke, Jara; Masser, Sawinee; Hadian, Kamyar; Dobner, Thomas; Schreiner, Sabrina

2018-04-25

HAdV E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in non-permissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 Ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established.RNF4, a cellular SUMO-targeted Ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM, and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNAi resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies. IMPORTANCE Daxx is a PML-NB-associated transcription factor, which was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 Ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain productive viral life

ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response.

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Malakhova, Oxana A; Zhang, Dong-Er

2008-04-04

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in anti-viral responses. The interferon-inducible ubiquitin-like protein ISG15 is expressed in cells in response to a variety of stress conditions like viral or bacterial infection and is present in its free form or is conjugated to cellular proteins. In addition, protein ubiquitination plays a regulatory role in the immune system. Many viruses modulate the ubiquitin (Ub) pathway to alter cellular signaling and the antiviral response. Ubiquitination of retroviral group-specific antigen precursors and matrix proteins of the Ebola, vesicular stomatitis, and rabies viruses by Nedd4 family HECT domain E3 ligases is an important step in facilitating viral release. We found that Nedd4 is negatively regulated by ISG15. Free ISG15 specifically bound to Nedd4 and blocked its interaction with Ub-E2 molecules, thus preventing further Ub transfer from E2 to E3. Furthermore, overexpression of ISG15 diminished the ability of Nedd4 to ubiquitinate viral matrix proteins and led to a decrease in the release of Ebola VP40 virus-like particles from the cells. These results point to a mechanistically novel function of ISG15 in the enhancement of the innate anti-viral response through specific inhibition of Nedd4 Ub-E3 activity. To our knowledge, this is the first example of a Ub-like protein with the ability to interfere with Ub-E2 and E3 interaction to inhibit protein ubiquitination.

Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

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Mostafa, Heba H; Thompson, Thornton W; Konen, Adam J; Haenchen, Steve D; Hilliard, Joshua G; Macdonald, Stuart J; Morrison, Lynda A; Davido, David J

2018-04-01

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39 , which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo , we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39 mut ), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection. IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in

Viral and cellular requirements for the budding of Feline Endogenous Retrovirus RD-114

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Fukuma Aiko

2011-12-01

Full Text Available Abstract Background RD-114 virus is a feline endogenous retrovirus and produced as infectious viruses in some feline cell lines. Recently, we reported the contamination of an infectious RD-114 virus in a proportion of live attenuated vaccines for dogs and cats. It is very difficult to completely knock out the RD-114 proviruses from cells, as endogenous retroviruses are usually integrated multiply into the host genome. However, it may be possible to reduce the risk of contamination of RD-114 virus by regulating the viral release from cells. Results In this study, to understand the molecular mechanism of RD-114 virus budding, we attempted to identify the viral and cellular requirements for RD-114 virus budding. Analyses of RD-114 L-domain mutants showed that the PPPY sequence in the pp15 region of Gag plays a critical role in RD-114 virus release as viral L-domain. Furthermore, we investigated the cellular factors required for RD-114 virus budding. We demonstrated that RD-114 virus release was inhibited by overexpression of dominant negative mutants of Vps4A, Vps4B, and WWP2. Conclusions These results strongly suggest that RD-114 budding utilizes the cellular multivesicular body sorting pathway similar to many other retroviruses.

Mutants of alfalfa mosaic virus

International Nuclear Information System (INIS)

Roosien, J.

1983-01-01

In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

Identification of an ovine atadenovirus gene whose product activates the viral E2 promoter: possible involvement of E2F-1

International Nuclear Information System (INIS)

Kuemin, Daniel; Hofmann, Christian; Uckert, Wolfgang; Both, Gerald W.; Loeser, Peter

2004-01-01

Activation of the adenoviral E2 promoter is an early step in adenovirus gene expression. For members of the mast- and aviadenoviruses, this requires induction of the cellular transcription factor E2F by virally encoded gene products such as E1A, E4orf6/7 and orf22/GAM-1. The newly recognized genus atadenovirus, of which the ovine isolate OAdV is the prototype, lacks any sequence homology to those genes. To find a possible link between E2 promoter activation and OAdV gene expression, we utilized a screening method to search for genes within the OAdV genome that were capable of stimulating the viral E2 promoter. One such gene, E43, was identified within the proposed E4 region toward the right-hand end of the OAdV genome. The E43 gene product was also found to be capable of stimulating E2F-1-dependent gene expression. A closer inspection of the E2 promoter revealed the presence of a non-palindromic E2F binding site within the OAdV E2 promoter. Mutation of this site markedly reduced both E2F-1- and E43-dependent promoter activation. Moreover, a direct protein-protein interaction of the E43 gene product with E2F, but not with the retinoblastoma protein pRb, suggested a possible cooperation between these two proteins in activating the E2 promoter. The importance of the E43 gene product for virus replication is also underlined by the finding that an OAdV recombinant with a functionally inactivated E43 gene showed severely inhibited virus growth

Developmental delay in a Streptomyces venezuelae glgE null mutant is associated with the accumulation of α-maltose 1-phosphate.

Science.gov (United States)

Miah, Farzana; Bibb, Maureen J; Barclay, J Elaine; Findlay, Kim C; Bornemann, Stephen

2016-07-01

The GlgE pathway is thought to be responsible for the conversion of trehalose into a glycogen-like α-glucan polymer in bacteria. Trehalose is first converted to maltose, which is phosphorylated by maltose kinase Pep2 to give α-maltose 1-phosphate. This is the donor substrate of the maltosyl transferase GlgE that is known to extend α-1,4-linked maltooligosaccharides, which are thought to be branched with α-1,6 linkages. The genome of Streptomyces venezuelae contains all the genes coding for the GlgE pathway enzymes but none of those of related pathways, including glgC and glgA of the glycogen pathway. This provides an opportunity to study the GlgE pathway in isolation. The genes of the GlgE pathway were upregulated at the onset of sporulation, consistent with the known timing of α-glucan deposition. A constructed ΔglgE null mutant strain was viable but showed a delayed developmental phenotype when grown on maltose, giving less cell mass and delayed sporulation. Pre-spore cells and spores of the mutant were frequently double the length of those of the wild-type, implying impaired cross-wall formation, and spores showed reduced tolerance to stress. The mutant accumulated α-maltose 1-phosphate and maltose but no α-glucan. Therefore, the GlgE pathway is necessary and sufficient for polymer biosynthesis. Growth of the ΔglgE mutant on galactose and that of a Δpep2 mutant on maltose were analysed. In both cases, neither accumulation of α-maltose 1-phosphate/α-glucan nor a developmental delay was observed. Thus, high levels of α-maltose 1-phosphate are responsible for the developmental phenotype of the ΔglgE mutant, rather than the lack of α-glucan.

Evaluation of some mutant lines of rice induced by gamma radiation treatment 1. mean performance of rice mutants in M4 generation

International Nuclear Information System (INIS)

El-Banna, M.N.; El-Wakil, H.M.F.; Ebaid, R.A.; Sallam, R.A.

2009-01-01

Grains of eight rice mutants; SC 1, SC 6, RTY 1, RTY 3, HY 14, HYI 17, EH 4 and HYPI 22 were secured from Botany Department Faculty of Agriculture Cairo university. The procedures and the methodology for induction these mutants as well as the original mean performance of such mutants are presented else where; Sabbour, (1989) and Sabbour etal. (2002). Grains were sown (M4 generation) at the experimental farm in Itai EI-Baroud Agricultural Research Station Behaira Governorate Agricultural Research Center (ARC) in the summer season (2007). The mean performance of such mutants was studied during M4 generation. The most exciting results were as follows: the selected line SC 1 showed in M4 generation superior agronomic and yield traits. Sc 1 mutant line is not bred truly and it need more generations to reach stability. SC 6 in M4 generation showed considerable number of individuals scored low mean values toward the negative direction and lowering the overall trait mean performance. The rice lines RTY 1 and RTY 3 proved that, the average number of fertile tillers per plant of the selected lines maintained previously recorded mean values of M3 generation in M4. The traits showed significant differences among their progeny that recorded high CV% values as compared with those showed no significant differences. The rice lines HY 14 and HYI 17 showed a true breeding signs and no more breeding generations are required. Rice lines EH 4, showed a considerable reduction in number of days elapsed from date of cultivation till harvest. As, this mutant maintained 86.58 days till heading. Rice mutant line HYPI 22 did not bred truly for the original selected traits (high yield and high protein content) and it still need more generations of selection to reach considerable stability

ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

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Pellet, J; Tafforeau, L; Lucas-Hourani, M; Navratil, V; Meyniel, L; Achaz, G; Guironnet-Paquet, A; Aublin-Gex, A; Caignard, G; Cassonnet, P; Chaboud, A; Chantier, T; Deloire, A; Demeret, C; Le Breton, M; Neveu, G; Jacotot, L; Vaglio, P; Delmotte, S; Gautier, C; Combet, C; Deleage, G; Favre, M; Tangy, F; Jacob, Y; Andre, P; Lotteau, V; Rabourdin-Combe, C; Vidalain, P O

2010-01-01

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

Using viral e-mails to distribute tobacco control advertisements: an experimental investigation.

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Carter, Owen B J; Donovan, Robert; Jalleh, Geoffrey

2011-08-01

The authors' objective was to conduct a trial of viral e-mail marketing as a distribution method for tobacco control advertisements. University students (n = 200) in the state of Western Australia were randomly allocated to receive 1 of 2 e-mails with hyperlinks to tobacco control advertisements ("Toilet" and "Rubbish") emphasizing the disgusting nature of smoking. Recipients followed a hyperlink to a Web page playing Toilet or Rubbish on endless loop. Viewers were encouraged to forward the e-mail to their friends and invited to complete an online survey about the advertisement. Unique downloads for each advertisement were identified by internet provider (IP) location and tallied by date and geographical location to assess subsequent dissemination beyond the initial 200 students. There were 826 unique viewings of the advertisements averaging 26.9 viewings per day for the first fortnight, followed by a lower average of 4.1 hits per day for the next 3.5 months. IP addresses identified hits from 3 other Australian states and 7 other countries. Online surveys were completed by 103 respondents (12.5% of total hits) but included few smokers (n = 9). Significantly more respondents rated Toilet as "funny" compared with Rubbish (40% vs. 11%; p e-mail sent but daily hits rapidly deteriorated over time. Entertainment appears to facilitate viral e-mails being forwarded onwards but only exceptionally compelling tobacco control materials are ever likely to become self-perpetuating.

CD4 T Cell Responses in Latent and Chronic Viral Infections

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Walton, Senta; Mandaric, Sanja; Oxenius, Annette

2013-01-01

The spectrum of tasks which is fulfilled by CD4 T cells in the setting of viral infections is large, ranging from support of CD8 T cells and humoral immunity to exertion of direct antiviral effector functions. While our knowledge about the differentiation pathways, plasticity, and memory of CD4 T cell responses upon acute infections or immunizations has significantly increased during the past years, much less is still known about CD4 T cell differentiation and their beneficial or pathological functions during persistent viral infections. In this review we summarize current knowledge about the differentiation, direct or indirect antiviral effector functions, and the regulation of virus-specific CD4 T cells in the setting of persistent latent or active chronic viral infections with a particular emphasis on herpes virus infections for the former and chronic lymphocytic choriomeningitis virus infection for the latter. PMID:23717308

Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex

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Jihui Lin

2017-08-01

Full Text Available Classical swine fever virus (CSFV is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation.

An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

International Nuclear Information System (INIS)

Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

2008-01-01

It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells

[Other viral food poisoning (hepatitis A and E)].

Science.gov (United States)

Yano, Kunio

2012-08-01

Hepatitis A and E viruses are spread via the fecal-oral route. In the endemic area, restaurant and school outbreaks due to contaminated water or food have been reported. The clinical signs and symptoms in patients with typical hepatitis A and E are similar to those seen with other forms of acute viral hepatitis. Hepatitis A tends to be more severe when acquired at older ages. Hepatitis E appears to be relatively severe compared with hepatitis A. Although both hepatitis are self-limited illness, severe hepatits are rarely observed. Hepatitis A and E can be prevented by improved sanitary conditions, handwashing, heating foods appropriately. Avoidance of water and foods from endemic areas is also effective.

Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

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Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

1990-12-01

Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

Inhibition of bovine viral diarrhea virus RNA synthesis by thiosemicarbazone derived from 5,6-dimethoxy-1-indanone.

Science.gov (United States)

Castro, Eliana F; Fabian, Lucas E; Caputto, María E; Gagey, Dolores; Finkielsztein, Liliana M; Moltrasio, Graciela Y; Moglioni, Albertina G; Campos, Rodolfo H; Cavallaro, Lucía V

2011-06-01

In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV).

Viral CTL escape mutants are generated in lymph nodes and subsequently become fixed in plasma and rectal mucosa during acute SIV infection of macaques.

Directory of Open Access Journals (Sweden)

Thomas H Vanderford

2011-05-01

Full Text Available SIV(mac239 infection of rhesus macaques (RMs results in AIDS despite the generation of a strong antiviral cytotoxic T lymphocyte (CTL response, possibly due to the emergence of viral escape mutants that prevent recognition of infected cells by CTLs. To determine the anatomic origin of these SIV mutants, we longitudinally assessed the presence of CTL escape variants in two MamuA*01-restricted immunodominant epitopes (Tat-SL8 and Gag-CM9 in the plasma, PBMCs, lymph nodes (LN, and rectal biopsies (RB of fifteen SIV(mac239-infected RMs. As expected, Gag-CM9 did not exhibit signs of escape before day 84 post infection. In contrast, Tat-SL8 escape mutants were apparent in all tissues by day 14 post infection. Interestingly LNs and plasma exhibited the highest level of escape at day 14 and day 28 post infection, respectively, with the rate of escape in the RB remaining lower throughout the acute infection. The possibility that CTL escape occurs in LNs before RBs is confirmed by the observation that the specific mutants found at high frequency in LNs at day 14 post infection became dominant at day 28 post infection in plasma, PBMC, and RB. Finally, the frequency of escape mutants in plasma at day 28 post infection correlated strongly with the level Tat-SL8-specific CD8 T cells in the LN and PBMC at day 14 post infection. These results indicate that LNs represent the primary source of CTL escape mutants during the acute phase of SIV(mac239 infection, suggesting that LNs are the main anatomic sites of virus replication and/or the tissues in which CTL pressure is most effective in selecting SIV escape variants.

The Effect of Vitamin E on the Survival Rate of unc-13 Caenorhabditis elegans mutants under Oxidative Stress

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Jessica Porcelan

2012-01-01

Full Text Available Caenorhabditis elegans unc-13 mutants express decreased neuronal activity and thus are a good model strain for examining defective nervous systems. These unc-13 mutants as well as wild type N2 strains, show rapid mortality when under oxidative stress. However, the antioxidant vitamin E may prolong survival in unc-13 mutant and N2 strains under oxidative stress. The addition of vitamin E to organisms under oxidative stress has a protective effect in both N2 and unc-13 C. elegans strains. Interestingly, vitamin E resulted in a greater increase in survival rate in N2 worms than with unc-13 mutant worms. While both strains displayed lower mortality rates with the addition of vitamin E, this finding suggests that vitamin E more efficiently increases survival rates of C. elegans with typical nervous system function. The efficacy of vitamin E implies that use of antioxidants may lessen the damage caused by oxidative stress in both N2 and mutant worms.

Diversity in viral anti-PKR mechanisms: a remarkable case of evolutionary convergence.

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Elena Domingo-Gil

Full Text Available Most viruses express during infection products that prevent or neutralize the effect of the host dsRNA activated protein kinase (PKR. Translation of Sindbis virus (SINV mRNA escapes to PKR activation and eIF2 phosphorylation in infected cells by a mechanism that requires a stem loop structure in viral 26S mRNA termed DLP to initiate translation in the absence of functional eIF2. Unlike the rest of viruses tested, we found that Alphavirus infection allowed a strong PKR activation and eIF2α phosphorylation in vitro and in infected animals so that the presence of DLP structure in mRNA was critical for translation and replication of SINV. Interestingly, infection of MEFs with some viruses that express PKR inhibitors prevented eIF2α phosphorylation after superinfection with SINV, suggesting that viral anti-PKR mechanisms could be exchangeable. Thus, translation of SINV mutant lacking the DLP structure (ΔDLP in 26S mRNA was partially rescued in cells expressing vaccinia virus (VV E3 protein, a known inhibitor of PKR. This case of heterotypic complementation among evolutionary distant viruses confirmed experimentally a remarkable case of convergent evolution in viral anti-PKR mechanisms. Our data reinforce the critical role of PKR in regulating virus-host interaction and reveal the versatility of viruses to find different solutions to solve the same conflict.

Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication

International Nuclear Information System (INIS)

Sriram, Bandi; Thakral, Deepshi; Panda, Subrat Kumar

2003-01-01

The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by ∼37 and ∼60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

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Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

2017-01-01

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

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Peng Li

2017-09-01

Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

Soybean promising mutant lines super early maturity Q-298 and 4-Psj

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Arwin; Mulyana, H.I.; Tarmizi; Masrizal; Faozi, K.; Adie, M.

2012-01-01

One of the efforts to increase the national soybean (Glycine max L. Merr.) production is by growing super early maturity with high yielding varieties, so that the planting time can be shortened to fill out the cropping pattern of ''rice-rice-soybean''. Such varieties can be developed through mutation breeding method using γ ray irradiation. In this research the seeds of Tidar variety were irradiated by 200 Gy γ ray from 60 Co. Irradiated seeds were planted in the field and selections with emphasis on early maturing character were conducted in M 2 generation. Selected plants were purified to M 7 generation and selected pure mutant lines were subjected to preliminary and advanced yield trials. Based on these results 5 promising mutant lines were selected to continue in multi location yield trials. A set of lines for multi location yield trials consist of 14 lines included 5 mutant lines from this experiment, 5 lines from UNSUD, 3 national leading varieties, Argomulyo, Gorobogan, Burangrang, as national control varieties and Tidar as an original of mutant lines. Based on the result of multi location yield trials, 2 mutant lines, Q-298 dan 4-Psj, have significant high productivities compared to productivities of other lines and varieties. The growth duration of these lines were only 66 days and 68 days, respectively with average productivities were 2.41 tons / ha and 2.42 tons / ha, respectively. Index stability of Q-298 and 4-Psj mutant lines were 0.84 and 0.79, respectively, it means that the productivities of these two lines were stable in all tested locations. Based on the results, the Q-298 and 4-Psj mutant lines were proposed to be released as new varieties with the names of Gamasugen 1 and Gamasugen 2, respectively. (author)

A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy.

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Swoboda, Ines; Bugajska-Schretter, Agnes; Linhart, Birgit; Verdino, Petra; Keller, Walter; Schulmeister, Ulrike; Sperr, Wolfgang R; Valent, Peter; Peltre, Gabriel; Quirce, Santiago; Douladiris, Nikolaos; Papadopoulos, Nikolaos G; Valenta, Rudolf; Spitzauer, Susanne

2007-05-15

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients' IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.

Analysis of fast neutron-generated mutants at the Arabidopsis thaliana HY4 locus

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Bruggemann, E.; Handwerger, K.; Essex, C.; Storz, G.

1996-01-01

Ionizing radiation is expected to produce mutants with deletions or other chromosomal rearrangements. These mutants are useful for a variety of purposes, such as creating null alleles and cloning genes whose existence is known only from their mutant phenotype; however, only a few mutations generated by ionizing radiation have been characterized at the molecular level in Arabidopsis thaliana. Twenty fast neutron-generated alleles of the Arabidopsis HY4 locus, which encodes a blue light receptor, CRY1, were isolated and characterized. Nine of the mutant alleles displayed normal genetic behavior. The other 11 mutant alleles were poorly transmitted through the male gametophyte and were lethal in homozygous plants. Southern blot analysis demonstrated that alleles of the first group generally contain small or moderate-sized deletions at HY4, while alleles of the second group contain large deletions at this locus. These results demonstrate that fast neutrons can produce a range of deletions at a single locus in Arabidopsis. Many of these deletions would be suitable for cloning by genomic subtraction or representational difference analysis. The results also suggest the presence of an essential locus adjacent to HY4. (author)

Barley yellow mosaic virus VPg is the determinant protein for breaking eIF4E-mediated recessive resistance in barley plants

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Huangai Li

2016-09-01

Full Text Available In this study, we investigated the barley yellow mosaic virus (BaYMV, genus Bymovirus factor(s responsible for breaking eIF4E-mediated recessive resistance genes (rym4/5/6 in barley. Genome mapping analysis using chimeric infectious cDNA clones between rym5-breaking (JT10 and rym5-non-breaking (JK05 isolates indicated that genome-linked viral protein (VPg is the determinant protein for breaking the rym5 resistance. Likewise, VPg is also responsible for overcoming the resistances of rym4 and rym6 alleles. Mutational analysis identified that amino acids Ser-118, Thr-120 and His-142 in JT10 VPg are the most critical residues for overcoming rym5 resistance in protoplasts. Moreover, the rym5-non-breaking JK05 could accumulate in the rym5 protoplasts when eIF4E derived from a susceptible barley cultivar was expressed from the viral genome. Thus, the compatibility between VPg and host eIF4E determines the ability of BaYMV to infect barley plants.

Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

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Kaushik, Nidhi; Subramani, Chandru; Anang, Saumya; Muthumohan, Rajagopalan; Shalimar; Nayak, Baibaswata; Ranjith-Kumar, C T; Surjit, Milan

2017-11-01

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used

UV irradiation analysis of complementation between, and replication of, RNA-negative temperature-sensitivie mutants of Newcastle disease virus

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Peeples, M.E.; Bratt, M.A.

1982-01-01

Random uv irradiation-induced lesions destroy the infectivity of Newcastle disease virus (NDV) by blocking downstream transcription from the single viral promoter. The nucleocapsid-associated polypeptides most likely to be involved in RNA synthesis are located at the extreme ends of the genome: NP and P are promoter proximal genes, and L is the most distal gene. We attempted to order the two temperature-sensitive (ts) RNA-negative (RNA-) mutant groups of NDV by determining the uv target sizes for the complementing abilities of mutants A1 and E1. After uv irradiation, E1 was unable to complement A1, a result compatible with the A mutation lying in the L gene. In contrast, after uv irradiation A1 was able to complement E1 for both virus production and viral protein synthesis, with a target size most consistent with the E mutation lying in the P gene. UV-irradiated virus was unable to replicate as indicated by its absence in the yields of multiply infected cells, either as infectious virus or as particles with complementing activity. After irradiation, ts mutant B1ΔP, with a non-ts mutation affecting the electrophoretic mobility of the P protein, complemented E1 in a manner similar to A1, but it did not amplify the expression of ΔP in infected cells. This too is consistent with irradiated virus being unable to replicate despite the presence of the components needed for replication of E1. At high uv doses, A1 was able to complement E1 in a different, uv-resistant manner, probably by direct donation of input polypeptides. Multiplicity reactivation has previously been observed at high-multiplicity infection by uv-irradiated paramyxoviruses. In this case, virions which are noninfectious because they lack a protein component may be activated by a protein from irradiated virions

Inhibition of Bovine Viral Diarrhea Virus RNA Synthesis by Thiosemicarbazone Derived from 5,6-Dimethoxy-1-Indanone▿

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Castro, Eliana F.; Fabian, Lucas E.; Caputto, María E.; Gagey, Dolores; Finkielsztein, Liliana M.; Moltrasio, Graciela Y.; Moglioni, Albertina G.; Campos, Rodolfo H.; Cavallaro, Lucía V.

2011-01-01

In the present work, we described the activity of the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone (TSC), which we previously characterized as a new compound that inhibits bovine viral diarrhea virus (BVDV) infection. We showed that TSC acts at a point of time that coincides with the onset of viral RNA synthesis and that it inhibits the activity of BVDV replication complexes (RCs). Moreover, we have selected five BVDV mutants that turned out to be highly resistant to TSC but still susceptible to ribavirin (RBV). Four of these resistant mutants carried an N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). The remaining mutant showed an A392E mutation within the same protein. Some of these mutants replicated slower than the wild-type (wt) virus in the absence of TSC, whereas others showed a partial reversion to the wt phenotype over several passages in the absence of the compound. The docking of TSC in the crystal structure of the BVDV RdRp revealed a close contact between the indane ring of the compound and several residues within the fingers domain of the enzyme, some hydrophobic contacts, and hydrogen bonds with the thiosemicarbazone group. Finally, in the mutated RdRp from resistant BVDV, these interactions with TSC could not be achieved. Interestingly, TSC inhibited BVDV replication in cell culture synergistically with RBV. In conclusion, TSC emerges as a new nonnucleoside inhibitor of BVDV RdRp that is synergistic with RBV, a feature that turns it into a potential compound to be evaluated against hepatitis C virus (HCV). PMID:21430053

Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament.

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Mikitas, Olga V; Ivin, Yuri Y; Golyshev, Sergey A; Povarova, Natalia V; Galkina, Svetlana I; Pletjushkina, Olga Y; Nadezhdina, Elena S; Gmyl, Anatoly P; Agol, Vadim I

2012-05-01

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

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Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

2015-01-01

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment

Science.gov (United States)

2012-01-01

Background Hepatitis B virus (HBV), because of its error-prone viral polymerase, has a high mutation rate leading to widespread substitutions, deletions, and insertions in the HBV genome. Deletions may significantly change viral biological features complicating the progression of liver diseases. However, the clinical conditions correlating to the accumulation of deleted mutants remain unclear. In this study, we explored HBV deletion patterns and their association with disease status and antiviral treatment by performing whole genome sequencing on samples from 51 hepatitis B patients and by monitoring changes in deletion variants during treatment. Clone sequencing was used to analyze preS regions in another cohort of 52 patients. Results Among the core, preS, and basic core promoter (BCP) deletion hotspots, we identified preS to have the highest frequency and the most complex deletion pattern using whole genome sequencing. Further clone sequencing analysis on preS identified 70 deletions which were classified into 4 types, the most common being preS2. Also, in contrast to the core and BCP regions, most preS deletions were in-frame. Most deletions interrupted viral surface epitopes, and are possibly involved in evading immuno-surveillance. Among various clinical factors examined, logistic regression showed that antiviral medication affected the accumulation of deletion mutants (OR = 6.81, 95% CI = 1.296 ~ 35.817, P = 0.023). In chronic carriers of the virus, and individuals with chronic hepatitis, the deletion rate was significantly higher in the antiviral treatment group (Fisher exact test, P = 0.007). Particularly, preS2 deletions were associated with the usage of nucleos(t)ide analog therapy (Fisher exact test, P = 0.023). Dynamic increases in preS1 or preS2 deletions were also observed in quasispecies from samples taken from patients before and after three months of ADV therapy. In vitro experiments demonstrated that preS2 deletions alone

Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

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Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

2005-01-28

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

Translation initiation factor AteIF(iso4E is involved in selective mRNA translation in Arabidopsis thaliana seedlings.

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Ana Valeria Martínez-Silva

Full Text Available One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5'end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso4E knockout mutant [(iso4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1, Sucrose transporter 3 (SUC3, ABC transporter-like with ATPase activity (MRP11 and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso4E is relevant for Arabidopsis root development under normal growth conditions.

Loss of Catalytic Activity in the E134D, H67A, and H349A Mutants of DapE: Mechanistic Analysis with QM/MM Investigation.

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Dutta, Debodyuti; Mishra, Sabyashachi

2016-11-17

In the fight against bacterial infections and antibiotic resistance, the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a potentially safe target enzyme. The role of the Glu134, His67, and His349 residues in the binding and hydrolysis of N-succinyl-l,l-diaminopimelic acid (SDAP) is investigated by employing molecular dynamics simulation and hybrid quantum mechanical-molecular mechanical (MM) calculations of the E134D, H67A, and H349A mutants of DapE. The free energy of substrate binding obtained from the MM-Poisson-Boltzmann surface area approach correctly reproduced the experimentally observed ordering of substrate affinity, that is, E134D > wt > H67A > H349A. The mechanism of catalytic action by the E134D mutant is elucidated by structurally and energetically characterizing the intermediates and the transition states along the reaction pathway. The rate-determining step in the general acid-base hydrolysis reaction by the E134D mutant is found to be the nucleophilic attack step, which involves an activation energy barrier 10 kcal/mol greater than that in the wild-type (wt)-DapE. This explains the 3 orders of magnitude decrease in the experimentally determined k cat value for the E134D mutant compared to that of wt-DapE. In the H67A and H349A mutants, the Glu134 residue undergoes a conformational change and exhibits a strong coordination with the metal centers. This not only results in a weaker substrate binding in the two histidine mutants but also hinders the activation of the catalytic water molecule, which constitutes the first step of the substrate hydrolysis by DapE. This leads to an effective quenching of the catalytic activity in the H67A and H349A mutants.

The eEF1A proteins: at the crossroads of oncogenesis, apoptosis and viral infections.

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Georges eHerbein

2015-04-01

Full Text Available Eukaryotic translation elongation factors 1 alpha, eEF1A1 and eEF1A2, are not only translation factors, but also pleiotropic proteins that are highly expressed in human tumors, including breast cancer, ovarian cancer and lung cancer. eEF1A1 modulates cytoskeleton, exhibits chaperone-like activity and also controls cell proliferation and cell death. By contrast eEF1A2 protein favors oncogenesis as shown by the fact that overexpression of eEF1A2 leads to cellular transformation and gives rise to tumors in nude mice. The eEF1A2 protein stimulates the phospholipid signaling and activates the Akt-dependent cell migration and actin remodeling that ultimately favors tumorigenesis. By contrast, inactivation of eEF1A proteins leads to immunodeficiency, neural and muscular defects, and favors apoptosis. Finally, eEF1A proteins interact with several viral proteins resulting in enhanced viral replication, decreased apoptosis and increased cellular transformation. This review summarizes the recent findings on eEF1A proteins indicating that eEF1A proteins play a critical role in numerous human diseases through enhancement of oncogenesis, blockade of apoptosis and increased viral pathogenesis.

Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P2.

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Kakisaka, Michinori; Yamada, Kazunori; Yamaji-Hasegawa, Akiko; Kobayashi, Toshihide; Aida, Yoko

2016-09-01

To be incorporated into progeny virions, the viral genome must be transported to the inner leaflet of the plasma membrane (PM) and accumulate there. Some viruses utilize lipid components to assemble at the PM. For example, simian virus 40 (SV40) targets the ganglioside GM1 and human immunodeficiency virus type 1 (HIV-1) utilizes phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]. Recent studies clearly indicate that Rab11-mediated recycling endosomes are required for influenza A virus (IAV) trafficking of vRNPs to the PM but it remains unclear how IAV vRNP localized or accumulate underneath the PM for viral genome incorporation into progeny virions. In this study, we found that the second intrinsically disordered region (IDR2) of NP regulates two binding steps involved in viral genome packaging. First, IDR2 facilitates NP oligomer binding to viral RNA to form vRNP. Secondly, vRNP assemble by interacting with PI(4,5)P2 at the PM via IDR2. These findings suggest that PI(4,5)P2 functions as the determinant of vRNP accumulation at the PM. Copyright © 2016 Elsevier Inc. All rights reserved.

Calcein represses human papillomavirus 16 E1-E2 mediated DNA replication via blocking their binding to the viral origin of replication.

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Das, Dipon; Smith, Nathan W; Wang, Xu; Richardson, Stacie L; Hartman, Matthew C T; Morgan, Iain M

2017-08-01

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolation and partial characterization of a mutant of Bacillus thuringiensis producing melanin Isolamento e caracterização parcial de um mutante de Bacillus thuringiensis produtor de melanina

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Gislayne T. Vilas-Bôas

2005-09-01

Full Text Available A mutant (407-P of Bacillus thuringiensis subsp. thuringiensis strain 407 producing a melanin was obtained after treatment with the mutagenic agent ethyl-methane-sulfonate. Several microbiological and biochemical properties of the two strains were analyzed and the results were similar. The mutant 407-P was also incorporated into non-sterilized soil samples, recovered, easily identified, and quantified, what enables its use in ecology of B. thuringiensis.Um mutante (407-P da linhagem Bacillus thuringiensis subsp. thuringiensis 407 produtor de melanina foi obtido após tratamento com o agente mutagênico etil-metano-sulfonato. Diversas propriedades microbiológicas e bioquímicas das duas linhagens foram analisadas e os resultados foram similares. O mutante 407-P foi incorporado em amostras de solo não esterilizado, recuperado, facilmente identificado e quantificado, possibilitando seu uso em estudos de ecologia de B. thuringiensis.

pH-dependent absorption spectra of rhodopsin mutant E113Q: On the role of counterions and protein

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Xie, Peng; Zhou, Panwang; Alsaedi, Ahmed; Zhang, Yan

2017-03-01

The absorption spectra of bovine rhodopsin mutant E113Q in solutions were investigated at the molecular level by using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. The calculations suggest the mechanism of the absorption variations of E113Q at different pH values. The results indicate that the polarizations of the counterions in the vicinity of Schiff base under protonation and unprotonation states of the mutant E113Q would be a crucial factor to change the energy gap of the retinal to tune the absorption spectra. Glu-181 residue, which is close to the chromophore, cannot serve as the counterion of the protonated Schiff base of E113Q in dark state. Moreover, the results of the absorption maximum in mutant E113Q with the various anions (Cl-, Br-, I- and NO3-) manifested that the mutant E113Q could have the potential for use as a template of anion biosensors at visible wavelength.

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

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Antony A, Charles; Alone, Pankaj V

2017-05-13

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5 G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui - ) phenotype due to its hyper GTPase activity. The eIF5 G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn - phenotype) in the eIF5 G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF. Copyright © 2017 Elsevier Inc. All rights reserved.

Anatomia foliar de microtomateiros fitocromo-mutantes e ultra-estrutura de cloroplastos Leaf anatomy of micro-tomato phytochrome-mutants and chloroplast ultra-structure

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Hyrandir Cabral de Melo

2011-02-01

Full Text Available Plantas fitocromo-mutantes têm sido utilizadas com o intuito de caracterizar isoladamente, dentre os demais fotorreceptores, a ação dos fitocromos sobre eventos ligados à fotomorfogênese. Raros são os estudos que relatam a ação dos fitocromos sobre aspectos estruturais, embora sejam fundamentais à compreensão do desenvolvimento das plantas. Neste trabalho, objetivou-se analisar características ultraestruturais de cloroplastos e aspectos anatômicos foliares dos microtomateiros (Solanum lycopersicum L. cv. Micro-Tom fitocromo-mutantes aurea (subexpressa fitocromos, hp1 e atroviolacea (ambos supra-responsivos a eventos mediados por fitocromo em plantas em estágio de floração. Observou-se que os fitocromos são responsáveis pela expressão de muitas características anatômicas da epiderme foliar, assim como do mesofilo e da ultraestrutura dos cloroplastos.Phytochrome-mutant plants have been used for phytochrome action characterization among all photoreceptors, in events of photomorphogenesis. Studies relating the phytochrome action on structural aspects, which are fundamental to the comprehension of plant development, are rare. The objective of this work was to analyze chloroplast ultra structure and leaf anatomical characteristics of micro-tomatos (Solanum lycopersicum L. cv. Micro-Tom phytochrome-mutants aurea (sub express phytochrome, hp1 and atroviolacea (both super express phytochrome events-mediated in plants in the flowering stage. The results show that phytochromes are responsible for the expression of many characteristics of leaf epidermis, mesophyll and chloroplast ultra-structure.

Manifestações estomatológicas, contagem de linfócitos T-CD4+ e carga viral de crianças brasileiras e norte-americanas infectadas pelo HIV

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GRANDO Liliane Janete

2002-01-01

Full Text Available A prevalência de manifestações estomatológicas associadas à infecção pelo HIV foi avaliada em 184 crianças de ambos os sexos, da faixa etária de zero a 13 anos de idade, atendidas nos Ambulatórios de AIDS Pediátrica do Hospital São Lucas (HSL da PUCRS, e do Hospital de Clínicas de Porto Alegre (HCPA da UFRGS, ambos em Porto Alegre, RS, Brasil e no Centro Pediátrico para Doenças Infecciosas do Hospital Universitário e Departamento de Odontopediatria da Faculdade de Odontologia da Universidade Estadual de Nova Iorque (SUNY em Stony Brook, Nova Iorque, EUA, no período de janeiro de 1999 a maio de 2000. A freqüência de crianças brasileiras (72,73% e norte-americanas (53,66% com manifestações estomatológicas associadas à infecção pelo HIV foi estatisticamente significativa, com destaque para linfadenopatia cérvico-facial, aumento de volume das parótidas, candidíase eritematosa, petéquias, gengivite, xerostomia e queilite angular. As crianças com manifestações estomatológicas apresentaram contagens médias de linfócitos T-CD4+ próximas à normalidade e carga viral alta.

Phenotypic characterization of adenovirus type 12 temperature-sensitive mutants in productive infection and transformation.

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Hama, S; Kimura, G

1980-01-01

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxylamine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for "initiation" of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutation in H12ts505 is required to maintain at least some aspects of the transformed state.

Synthesis, antiviral evaluation and molecular docking studies of N4-aryl substituted/unsubstituted thiosemicarbazones derived from 1-indanones as potent anti-bovine viral diarrhea virus agents.

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Soraires Santacruz, María C; Fabiani, Matías; Castro, Eliana F; Cavallaro, Lucía V; Finkielsztein, Liliana M

2017-08-01

A series of N 4 -arylsubstituted thiosemicarbazones derived from 1-indanones and a set of compounds lacking such substitution in the N 4 position of the thiosemicarbazone moiety were synthesized and evaluated for their anti-bovine viral diarrhea virus (BVDV) activity. Among these, derivatives 2 and 15 displayed high activity (EC 50 =2.7±0.4 and 0.7±0.1µM, respectively) as inhibitors of BVDV replication. Novel key structural features related to the anti-BVDV activity were identified by structure-activity relationship (SAR) analysis. In a previous study, the thiosemicarbazone of 5,6-dimethoxy-1-indanone (5,6-TSC) was characterized as a non-nucleoside inhibitor (NNI) of the BVDV RNA-dependent RNA polymerase. In the present work, cross-resistance assays were performed with the most active compounds. Such studies were carried out on 5,6-TSC resistant BVDV (BVDV-TSC r T1) carrying mutations in the viral polymerase. This BVDV mutant was also resistant to compound 15. Molecular docking studies and MM/PBSA calculations were performed to assess the most active derivatives at the 5,6-TSC viral polymerase binding site. The differences in the interaction pattern and the binding affinity of derivative 15 either to the wild type or BVDV-TSC r T1 polymerase were key factors to define the mode of action of this compound. Copyright © 2017 Elsevier Ltd. All rights reserved.

IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination.

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Wijesundara, Danushka K; Jackson, Ronald J; Tscharke, David C; Ranasinghe, Charani

2013-09-23

We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of the double mutant e//e w//w and the culture medium on the productivity of Drosophila melanogaster

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Francisco Mora

2000-01-01

Full Text Available We investigated the effect of two culture media on the productivity of the double mutant ebony-white (e//e w//w of Drosophila melanogaster, aimed at improving the conditions for maintenance of Drosophila’s collection, Departamento de Biología, Universidad Nacional de Colombia. The results indicate that the productivity is affected by the culture medium, being the maize culture medium more productive than the wheat one; it was also shown that the productivity depends both, on the crosses type that is realize and on the mutant. The “+//+ +//+ x e//e w/ cross is more productive than its reciprocal cross, where the position of the ebony allele is the most important factor. With respect to the white allele, when carried by males it does not have effect on the productivity. In addition, we detected a negative effect of wheat culture medium on females +//e +//w.

Compostos fenólicos e capacidade antioxidante em frutos de tomateiros mutantes fotomorfogenéticos

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Tiago Dias

2015-05-01

Full Text Available Compostos fenólicos e capacidade antioxidante são mecanismos de defesa das plantas aos danos do estresse oxidativo. Os compostos fenólicos são sintetizados pela via dos fenilpropanoides, cuja enzima chave, fenilalanina amônia liase, é influenciada pela luz e ação de fotorreceptores, como o fitocromo. O objetivo do presente trabalho é avaliar a concentração de compostos fenólicos e a capacidade antioxidante de frutos de microtomateiro selvagem, cultivar "Micro-Tom" (MT, e seus mutantes fotomorfogenéticos high pigment 1 (hp1, super-responsivo a eventos mediados por luz e aurea (au, deficiente quantitativo em fitocromos. Vinte frutos maduros de cada genótipo (MT, hp1 e au foram utilizados para as análises, realizadas em triplicata. Para quantificação dos compostos fenólicos totais, foi utilizado o método de Folin-Ciocalteu e a capacidade antioxidante foi realizada pelos métodos Ferric Reducing Antioxidant Power (FRAP e 2,2-diphenyl-1-picrylhydrazyl (DPPH. Os frutos do mutante hp1 apresentaram maiores conteúdos de compostos fenólicos totais e também maior capacidade antioxidante em relação à cultivar selvagem ("MT" e ao mutante au, o qual não diferiu significativamente da cultivar "MT".

Unfinished stories on viral quasispecies and Darwinian views of evolution.

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Más, Antonio; López-Galíndez, Cecilio; Cacho, Isabel; Gómez, Jordi; Martínez, Miguel Angel

2010-04-09

Experimental evidence that RNA virus populations consist of distributions of mutant genomes, termed quasispecies, was first published 31 years ago. This work provided the earliest experimental support for a theory to explain a system that replicated with limited fidelity and to understand the self-organization and adaptability of early life forms on Earth. High mutation rates and quasispecies dynamics of RNA viruses are intimately related to both viral disease and antiviral treatment strategies. Moreover, the quasispecies concept is being applied to other biological systems such as cancer research in which cellular mutant spectra can be also detected. This review addresses some of the unanswered questions regarding viral and theoretical quasispecies concepts as well as more practical aspects concerning resistance to antiviral treatments and pathogenesis. (c) 2010 Elsevier Ltd. All rights reserved.

Four-tiered π interaction at the dimeric interface of HIV-1 integrase critical for DNA integration and viral infectivity

International Nuclear Information System (INIS)

Al-Mawsawi, Laith Q.; Hombrouck, Anneleen; Dayam, Raveendra; Debyser, Zeger; Neamati, Nouri

2008-01-01

HIV-1 integrase (IN) is an essential enzyme for viral infection. Here, we report an extensive π electron orbital interaction between four amino acids, W132, M178, F181 and F185, located at the dimeric interface of IN that is critical for the strand transfer activity alone. Catalysis of nine different mutant IN proteins at these positions were evaluated. Whereas the 3'-processing activity is predominantly strong, the strand transfer activity of each enzyme was completely dependent on an intact π electron orbital interaction at the dimeric interface. Four representative IN mutants were constructed in the context of the infectious NL4.3 HIV-1 viral clone. Whereas viruses with an intact π electron orbital interaction at the IN dimeric interface replicated comparable to wild type, viruses containing an abolished π interaction were non-infectious. Q-PCR analysis of viral DNA forms during viral replication revealed pleiotropic effects of most mutations. We hypothesize that the π interaction is a critical contact point for the assembly of functional IN multimeric complexes, and that IN multimerization is required for a functional pre-integration complex. The rational design of small molecule inhibitors targeting the disruption of this π-π interaction should lead to powerful anti-retroviral drugs

The Triticum Mosaic Virus 5' Leader Binds to Both eIF4G and eIFiso4G for Translation.

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Robyn Roberts

Full Text Available We recently identified a remarkably strong (739 nt-long IRES-like element in the 5' untranslated region (UTR of Triticum mosaic virus (TriMV, Potyviridae. Here, we define the components of the cap-binding translation initiation complex that are required for TriMV translation. Using bio-layer interferometry and affinity capture of the native translation apparatus, we reveal that the viral translation element has a ten-fold greater affinity for the large subunit eIF4G/eIFiso4G than to the cap binding protein eIF4E/eIFiso4E. This data supports a translation mechanism that is largely dependent on eIF4G and its isoform. The binding of both scaffold isoforms requires an eight base-pair-long hairpin structure located 270 nucleotides upstream of the translation initiation site, which we have previously shown to be crucial for IRES activity. Despite a weak binding affinity to the mRNA, eIFiso4G alone or in combination with eIFiso4E supports TriMV translation in a cap-binding factor-depleted wheat germ extract. Notably, TriMV 5' UTR-mediated translation is dependent upon eIF4A helicase activity, as the addition of the eIF4A inhibitor hippuristanol inhibits 5' UTR-mediated translation. This inhibition is reversible with the addition of recombinant wheat eIF4A. These results and previous observations demonstrate a key role of eIF4G and eIF4A in this unique mechanism of cap-independent-translation. This work provides new insights into the lesser studied translation mechanisms of plant virus-mediated internal translation initiation.

Hepatitis C Virus: Viral Quasispecies and Genotypes.

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Tsukiyama-Kohara, Kyoko; Kohara, Michinori

2017-12-22

Hepatitis C virus (HCV) mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs), which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1

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Meijia Yang

2017-09-01

Full Text Available BRAF-V600E (1799T > A is one of the most frequently reported driver mutations in multiple types of cancers, and patients with such mutations could benefit from selectively inactivating the mutant allele. Near this mutation site, there are two TTTN and one NGG protospacer-adjacent motifs (PAMs for Cpf1 and Cas9 CRISPR nucleases, respectively. The 1799T > A substitution also leads to the occurrence of a novel NGNG PAM for the EQR variant of Cas9. We examined the editing efficacy and selectivity of Cpf1, Cas9, and EQR variant to this mutation site. Only Cpf1 demonstrated robust activity to induce specific disruption of only mutant BRAF, not wild-type sequence. Cas9 recognized and cut both normal and mutant alleles, and no obvious gene editing events were observed using EQR variant. Our results support the potential applicability of Cpf1 in precision medicine through highly specific inactivation of many other gain-of-function mutations. Keywords: Cpf1, targeted therapy, BRAF V600E

E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis.

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Park, Kyeong-Su; Kim, Ju Hee; Shin, Hee Won; Chung, Kyung-Sook; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

2015-10-26

Missense mutation of VHL gene is frequently detected in type 2 VHL diseases and linked to a wide range of pVHL functions and stability. Certain mutant pVHLs retain ability to regulate HIFs but lose their function by instability. In this case, regulating of degradation of mutant pVHLs, can be postulated as therapeutic method. The stability and cellular function of missense mutant pVHLs were determine in HEK293T transient expressing cell and 786-O stable cell line. Ubiquitination assay of mutant VHL proteins was performed in vitro system. Anticancer effect of adenovirus mediated shUCP expressing was evaluated using ex vivo mouse xenograft assay. Three VHL missense mutants (V155A, L158Q, and Q164R) are directly ubiquitinated by E2-EPF UCP (UCP) in vitro. Mutant pVHLs are more unstable than wild type in cell. Missense mutant pVHLs interact with UCP directly in both in vitro and cellular systems. Lacking all of lysine residues of pVHL result in resistance to ubiquitination thereby increase its stability. Missense mutant pVHLs maintained the function of E3 ligase to ubiquitinate HIF-1α in vitro. In cells expressing mutant pVHLs, Glut-1 and VEGF were relatively upregulated compared to their levels in cells expressing wild-type. Depletion of UCP restored missense mutant pVHLs levels and inhibited cell growth. Adenovirus-mediated shUCP RNA delivery inhibited tumor growth in ex vivo mouse xenograft model. These data suggest that targeting of UCP can be one of therapeutic method in type 2 VHL disease caused by unstable but functional missense mutant pVHL.

Search for C4 developmental mutants in Panicum maximum Jacq

International Nuclear Information System (INIS)

Fladung, M.

2001-01-01

Full text: Mutant plants are useful tools for studying developmental processes in defined genetic backgrounds by comparing them with their respective wild type forms. In this sense, developmental mutants or mutations involved in the establishment of certain leaf or flower specific traits are of special interest. In particular, the evolution of C 4 photosynthesis from C 3 precursors was accompanied by severe developmental changes in leaf morphology and anatomy. Our search of such mutants was followed by the idea to approach the evolution of the C 4 syndrome from a mutagenic point of view. Variants affecting normal development of the C 4 leaf anatomy may, in fact, represent possible regressive steps in C4 photosynthesis. Seeds of the C4 grass Panicum maximum Jacq. were mutagenized using ethylmethanesulfonate (EMS) and putative variants were isolated in the M2 generation by visual inspection. Main selection characteristics were whole plant, leaf morphology and pigmentation, and growth characteristics. The choice of a polyploid species for mutagenesis experiments was based on the need of detecting rare mutants, which are possibly lethal when using a diploid plant species. These variants could be of regulatory nature, affecting both morphology and physiology of C 4 photosynthesis early in leaf development. In total, nearly 100 variants were isolated and grown to maturity. Main isolated variants, which conforms to the prediction mentioned above, were as following: large interveinal space-1 and -3 (lis1, lis3), abnormal bundle sheath (abs), midribless (mbl) and variegated leaf -1 (var1). The variant lis1 was a short plant with leaves smaller than the wild type, and had a leaf lamina with a crinkly surface. Photosynthetically, lis1 indicates a clear regression from the C 4 to the C 3 photosynthesis type, which was correlated in the leaf lamina with an increase in the distance between small veins. The variant lis3 was not similar phenotypically to lis1, but it also had very

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

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Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

2009-03-09

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate

The development of the conditionally replication-competent adenovirus: replacement of E4 orf1-4 region by exogenous gene.

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Nam, Jae-Kook; Lee, Mi-Hyang; Seo, Hae-Hyun; Kim, Seok-Ki; Lee, Kang-Huyn; Kim, In-Hoo; Lee, Sang-Jin

2010-05-01

Tumor or tissue specific replicative adenovirus armed with a therapeutic gene has shown a promising anti-cancer therapeutic modality. However, because the genomic packaging capacity is constrained, only a few places inside it are available for transgene insertion. In the present study, we introduce a novel strategy utilizing the early E4 region for the insertion of therapeutic gene(s). We constructed the conditionally replication-competent adenovirus (CRAd), Ad5E4(mRFP) by: (i) replacing the E4/E1a promoter by the prostate-specific enhancer element; (ii) inserting mRFP inside the E4orf1-4 deletion region; and (iii) sub-cloning enhanced green fluorescent protein controlled by cytomegalovirus promoter in the left end of the viral genome. Subsequently, we evaluated its replication abilities and killing activities in vitro, as well as its in vivo anti-tumor efficacy in CWR22rv xenografts. When infected with Ad5E4(mRFP), the number and intensity of the mRFP gene products increased in a prostate cancer cell-specific manner as designed, suggesting that the mRFP gene and E4orfs other than E4orf1-4 were well synthesized from one transcript via alternative splicing as the recombinant adenovirus replicated. As expected from the confirmed virus replication capability, Ad5E4(mRFP) induced cell lysis as potent as the wild-type adenovirus and effectively suppressed tumor growth when tested in the CWR22rv xenografts in nude mice. Furthermore, Ad5E4(endo/angio) harboring an endostatin-angiostatin gene in E4orf1-4 was able to enhance CRAd by replacing mRFP with a therapeutic gene. The approach employed in the present study for the insertion of a therapeutic transgene in CRAd should facilitate the construction of CRAd containing multiple therapeutic genes in the viral genome that may have the potential to serve as highly potent cancer therapeutic reagents. Copyright (c) 2010 John Wiley & Sons, Ltd.

Replication-dependent 65R→K reversion in human immunodeficiency virus type 1 reverse transcriptase double mutant K65R + L74V

International Nuclear Information System (INIS)

Sharma, Prem L.; Nurpeisov, Viktoria; Lee, Kimberly; Skaggs, Sara; Di San Filippo, Christina Amat; Schinazi, Raymond F.

2004-01-01

Understanding of the mechanisms of interaction among nucleoside reverse transcriptase inhibitor (NRTI)-selected mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) coding sequence is essential for the design of newer drugs and for enhancing our vision of the structure function relationship among amino acids of the polymerase domain of HIV-1. Although several nucleoside reverse transcriptase inhibitors select RT mutations K65R and L74V, the combination of 65R + 74V is rare in clinics. A novel NRTI (-)-β-D-dioxolane-guanosine (DXG) is known to select in vitro either the 65R or 74V mutant virus (Antimicrob. Agents Chemother. 44 (2000) 1783). These mutations were not selected together during repeated passaging of the HIV-1 in the presence of this drug. To analyze the impact of these RT mutations on viral replication, a double mutant containing K65R + L74V was created by site-directed mutagenesis in a pNL4-3 background. Replication kinetic assays revealed that the mutant K65R + L74V is unstable, and 65R→K reversion occurs during replication of virus in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication kinetic assays in MT-2 cells demonstrated that double mutant 65R + 74V is highly attenuated for replication and the initiation of reversion is related to the increase in RT activity. Additionally, the suppression of viral replication in the presence of DXG or under suboptimal human recombinant interleukin-2 leads to minimal or no 65R→K reversion. These observations provide evidence that 65R→K reversion in the double mutant 65R + 74V is dependent on a specific rate of viral replication in a pNL4-3 background. A similar phenomenon may occur in vivo, which may have implications for treatment management strategies

Functional analysis of the cloverleaf-like structure in the 3' untranslated region of bamboo mosaic potexvirus RNA revealed dual roles in viral RNA replication and long distance movement

International Nuclear Information System (INIS)

Chen, I-H.; Meng Hsiao; Hsu, Y.-H.; Tsai, C.-H.

2003-01-01

The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop D, which in turn is followed by a pseudoknot E and a poly(A) tail. The coat protein accumulation level of the mutant, BaMV40A/ΔABC, lacking ABC domain was just 15% that of wild-type when inoculated into protoplasts of Nicotiana benthamiana. This suggested that ABC domain might play an important role in BaMV RNA replication. To define the precise role of each of the three stem-loops of ABC domain in RNA replication, three mutants BaMV40B and C each lacking stem-loop A, B, and C, respectively, were created. Our results showed that accumulation of viral products of mutants BaMV40B and C were not as efficient as wild-type. On the contrary, level of accumulation of viral products of BaMVA was similar to that of wild-type in protoplasts and inoculated leaves. Interestingly, the accumulation of viral products was not as efficient as that of wild-type in systemic leaves, implying that stem-loop A is dispensable for replication, but signifies a role in systemic accumulation. Using UV cross-linking and competition experiments, it was demonstrated that the E. coli expressed helicase domain of BaMV ORF1 can preferentially interact with the ABC domain

The double-stranded RNA-activated protein kinase mediates viral-induced encephalitis

International Nuclear Information System (INIS)

Scheuner, Donalyn; Gromeier, Matthias; Davies, Monique V.; Dorner, Andrew J.; Song Benbo; Patel, Rupali V.; Wimmer, Eckard J.; McLendon, Roger E.; Kaufman, Randal J.

2003-01-01

The double-stranded (ds) RNA-activated protein kinase (PKR) plays an important role in control of viral infections and cell growth. We have studied the role of PKR in viral infection in mice that are defective in the PKR signaling pathway. Transgenic mice were derived that constitutively express a trans-dominant-negative kinase-defective mutant PKR under control of the β-actin promoter. The trans-dominant-negative PKR mutant expressing transgenic mice do not have a detectable phenotype, similar to observations with PKR knock-out mice. The requirement for PKR in viral pathogenesis was studied by intracerebral infection of mice with a mouse-adapted poliovirus. Histopathological analysis revealed diffuse encephalomyelitis with severe inflammatory lesions throughout the central nervous system (CNS) in infected wild-type mice. In contrast, histopathological evaluation of virus-injected trans-dominant-negative PKR transgenic mice as well as PKR knock-out mice yielded no signs of tissue damage associated with inflammatory host responses. However, the virus did replicate in both models of PKR-deficient mice at a level equal to that observed in wild-type infected mice. Although the results indicate a clear difference in susceptibility to poliovirus-induced encephalitis, this difference manifests clinically as a slight delay in fatal neuropathy in trans-dominant-negative PKR transgenic and PKR knock-out animals. Our observations support the finding that viral-induced PKR activation may play a significant role in pathogenesis by mediating the host response to viral CNS infection. They support PKR to be an effective target to control tissue damage due to deleterious host responses to viral infection

Adenovirus structural protein IIIa is involved in the serotype specificity of viral DNA packaging.

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Ma, Hsin-Chieh; Hearing, Patrick

2011-08-01

The packaging of the adenovirus (Ad) genome into a capsid displays serotype specificity. This specificity has been attributed to viral packaging proteins, the IVa2 protein and the L1-52/55K protein. We previously found that the Ad17 L1-52/55K protein was not able to complement the growth of an Ad5 L1-52/55K mutant virus, whereas two other Ad17 packaging proteins, IVa2 and L4-22K, could complement the growth of Ad5 viruses with mutations in the respective genes. In this report, we investigated why the Ad17 L1-52/55K protein was not able to complement the Ad5 L1-52/55K mutant virus. We demonstrate that the Ad17 L1-52/55K protein binds to the Ad5 IVa2 protein in vitro and the Ad5 packaging domain in vivo, activities previously associated with packaging function. The Ad17 L1-52/55K protein also associates with empty Ad5 capsids. Interestingly, we find that the Ad17 L1-52/55K protein is able to complement the growth of an Ad5 L1-52/55K mutant virus in conjunction with the Ad17 structural protein IIIa. The same result was found with the L1-52/55K and IIIa proteins of several other Ad serotypes, including Ad3 and Ad4. The Ad17 IIIa protein associates with empty Ad5 capsids. Consistent with the complementation results, we find that the IIIa protein interacts with the L1-52/55K protein in vitro and associates with the viral packaging domain in vivo. These results underscore the complex nature of virus assembly and genome encapsidation and provide a new model for how the viral genome may tether to the empty capsid during the encapsidation process.

Involvement of C4 protein of beet severe curly top virus (family Geminiviridae in virus movement.

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Kunling Teng

Full Text Available BACKGROUND: Beet severe curly top virus (BSCTV is a leafhopper transmitted geminivirus with a monopartite genome. C4 proteins encoded by geminivirus play an important role in virus/plant interaction. METHODS AND FINDINGS: To understand the function of C4 encoded by BSCTV, two BSCTV mutants were constructed by introducing termination codons in ORF C4 without affecting the amino acids encoded by overlapping ORF Rep. BSCTV mutants containing disrupted ORF C4 retained the ability to replicate in Arabidopsis protoplasts and in the agro-inoculated leaf discs of N. benthamiana, suggesting C4 is not required for virus DNA replication. However, both mutants did not accumulate viral DNA in newly emerged leaves of inoculated N. benthamiana and Arabidopsis, and the inoculated plants were asymptomatic. We also showed that C4 expression in plant could help C4 deficient BSCTV mutants to move systemically. C4 was localized in the cytosol and the nucleus in both Arabidopsis protoplasts and N. benthamiana leaves and the protein appeared to bind viral DNA and ds/ssDNA nonspecifically, displaying novel DNA binding properties. CONCLUSIONS: Our results suggest that C4 protein in BSCTV is involved in symptom production and may facilitate virus movement instead of virus replication.

Hepatitis C Virus: Viral Quasispecies and Genotypes

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Kyoko Tsukiyama-Kohara

2017-12-01

Full Text Available Hepatitis C virus (HCV mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs, which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

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Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A

2016-10-01

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluating Andrographolide as a Potent Inhibitor of NS3-4A Protease and Its Drug-Resistant Mutants Using In Silico Approaches

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Vivek Chandramohan

2015-01-01

Full Text Available Current combination therapy of PEG-INF and ribavirin against the Hepatitis C Virus (HCV genotype-1 infections is ineffective in maintaining sustained viral response in 50% of the infection cases. New compounds in the form of protease inhibitors can complement the combination therapy. Asunaprevir is new to the drug regiment as the NS3-4A protease inhibitor, but it is susceptible to two mutations, namely, R155K and D168A in the protein. Thus, in our study, we sought to evaluate Andrographolide, a labdane-diterpenoid from the Andrographis paniculata plant as an effective compound for inhibiting the NS3-4A protease as well as its concomitant drug-resistant mutants by using molecular docking and dynamic simulations. Our study shows that Andrographolide has best docking scores of −15.0862, −15.2322, and −13.9072 compared to those of Asunaprevir −3.7159, −2.6431, and −5.4149 with wild-type R155K and D168A mutants, respectively. Also, as shown in the MD simulations, the compound was good in binding the target proteins and maintains strong bonds causing very less to negligible perturbation in the protein backbone structures. Our results validate the susceptibility of Asunaprevir to protein variants as seen from our docking studies and trajectory period analysis. Therefore, from our study, we hope to add one more option in the drug regiment to tackle drug resistance in HCV infections.

Tomato golden mosaic virus open reading frame AL4 is genetically distinct from its C4 analogue in monopartite geminiviruses.

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Pooma, W; Petty, I T

1996-08-01

Tomato golden mosaic virus (TGMV) is a bipartite geminivirus with six well-characterized genes. An additional open reading frame (ORF), AL4, lies within the essential AL1 gene. Recent studies of monopartite, dicot-infecting geminiviruses have revealed that mutations in their analogous C4 ORFs have host-specific effects on infectivity, symptomatology, virus movement and/or viral DNA accumulation. We have investigated whether TGMV has a similar host-specific requirement for AL4. The phenotypes of three TGMV al4 mutants were determined in a range of hosts, which included species that revealed c4 mutant phenotypes for monopartite geminiviruses. Each TGMV al4 mutant was indistinguishable from wild-type TGMV in all hosts tested. Additional analyses of double mutants revealed no evidence for functional redundancy between AL4 and the AL3, or AR1 genes. In contrast to the putative C4 proteins of monpartite geminiviruses, TGMV AL4, if it is expressed, is either non-functional, or functionally redundant with an essential TGMV gene product.

Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G.

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Chung, Liliane; Bailey, Dalan; Leen, Eoin N; Emmott, Edward P; Chaudhry, Yasmin; Roberts, Lisa O; Curry, Stephen; Locker, Nicolas; Goodfellow, Ian G

2014-08-01

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

International Nuclear Information System (INIS)

Groppo, Rachel; Brown, Bradley A.; Palmenberg, Ann C.

2011-01-01

Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3C pro . Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

Science.gov (United States)

Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P ras4B cell growth (P ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

Science.gov (United States)

Shcherbakov, Victor P; Kudryashova, Elena

2014-09-01

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i×j) and three-factor (i k×j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1×i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1-i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1×i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1×i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII(+) recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss

Experimental and computational studies on the DNA translocation mechanism of the T4 viral packaging motor

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Migliori, Amy; Arya, Gaurav; Smith, Douglas E.

2012-10-01

Bacteriophage T4 is a double stranded DNA virus that infects E.coli by injecting the viral genome through the cellular wall of a host cell. The T4 genome must be ejected from the viral capsid with sufficient force to ensure infection. To generate high ejection forces, the genome is packaged to high density within the viral capsid. A DNA translocation motor, in which the protein gp17 hydrolyzes ATP and binds to the DNA, is responsible for translocating the genome into the capsid during viral maturation of T4. This motor generates forces in excess of 60 pN and packages DNA at rates exceeding 2000 base pairs/second (bp/s)1. Understanding these small yet powerful motors is important, as they have many potential applications. Though much is known about the activity of these motors from bulk and single molecule biophysical techniques, little is known about their detailed molecular mechanism. Recently, two structures of gp17 have been obtained: a high-resolution X-ray crystallographic structure showing a monomeric compacted form of the enzyme, and a cryo-electron microscopic structure of the extended form of gp17 in complex with actively packaging prohead complexes. Comparison of these two structures indicates several key differences, and a model has been proposed to explain the translocation action of the motor2. Key to this model are a set of residues forming ion pairs across two domains of the gp17 molecule that are proposed to be involved in force generation by causing the collapse of the extended form of gp17. Using a dual optical trap to measure the rates of DNA packaging and the generated forces, we present preliminary mutational data showing that these several of these ion pairs are important to motor function. We have also performed preliminary free energy calculations on the extended and collapsed state of gp17, to confirm that these interdomain ion pairs have large contributions to the change in free energy that occurs upon the collapse of gp17 during the

The E4 protein; structure, function and patterns of expression

Energy Technology Data Exchange (ETDEWEB)

Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

2013-10-15

The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup ∧}E

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

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Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L.

2015-01-01

Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition. PMID:26317997

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway.

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Pierre-Emmanuel Joubert

2015-08-01

Full Text Available Chikungunya virus (CHIKV, the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K and MAP kinase-activated protein kinase (MnKs activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

Oxidant resistance in a yeast mutant deficient in the Sit4 phosphatase

DEFF Research Database (Denmark)

López-Mirabal, H Reynaldo; Winther, Jakob R; Kielland-Brandt, Morten C

2008-01-01

Resistance to thiol oxidation can arise from mutations altering redox homeostasis. A Saccharomyces cerevisiae sit4-110 mutant is here described, which was isolated as resistant to the thiol-specific oxidant dipyridyl disulfide (DPS) and which contains a single-residue substitution in the SIT4 gene...

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation

Science.gov (United States)

Yu, Alec; Zhu, Wandi; Silva, Jonathan R.; Ruben, Peter C.

2017-01-01

E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation. PMID:28898267

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation.

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Colin H Peters

Full Text Available E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation.

Comparative analysis of seven viral nuclear export signals (NESs reveals the crucial role of nuclear export mediated by the third NES consensus sequence of nucleoprotein (NP in influenza A virus replication.

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Nopporn Chutiwitoonchai

Full Text Available The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4 was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function

Arabidopsis C3HC4-RING finger E3 ubiquitin ligase AtAIRP4 positively regulates stress-responsive abscisic acid signaling.

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Yang, Liang; Liu, Qiaohong; Liu, Zhibin; Yang, Hao; Wang, Jianmei; Li, Xufeng; Yang, Yi

2016-01-01

Degradation of proteins via the ubiquitin system is an important step in many stress signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA)-insensitive RING protein 4 (AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4-RING finger domain in its C-terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T-DNA insertion mutant atairp4 effectively recovered the ABA-associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild-type and atairp4 mutant plants. In addition, the expression levels of ABA- and drought-induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild-type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA-mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis. © 2015 The Authors. Journal of Integrative Plant Biology published by Wiley Publishing Asia Pty Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.

Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

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Belhumeur Pierre

2008-11-01

Full Text Available Abstract Background HIV-1 integrase (IN is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s and/or motif(s required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype. Results Our study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant. Conclusion Overall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of

Analysis of a conserved RGE/RGD motif in HCV E2 in mediating entry

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Rong Lijun

2009-01-01

Full Text Available Abstract Background Hepatitis C virus (HCV encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors. It is clear that HCV uses a multi-receptor complex to gain entry into susceptible cells, however key elements of this complex remain elusive. In this study, the role of a highly conserved RGE/RGD motif of HCV E2 glycoprotein in viral entry was examined. The effect of each substitution mutation in this motif was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp. In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E2 proteins, CD81 binding profiles, and conformation of mutants were examined. Results Based on these characteristics, mutants either displayed wt characteristics (high infectivity [≥ 90% of wt HCVpp], CD81 binding, E1E2 expression, and incorporation into viral particles and proper conformation or very low infectivity (≤ 20% of wt HCVpp. Only amino acid substitutions of the 3rd position (D or E resulted in wt characteristics as long as the negative charge was maintained or a neutral alanine was introduced. A change in charge to a positive lysine, disrupted HCVpp infectivity at this position. Conclusion Although most amino acid substitutions within this conserved motif displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, and incorporation into HCVpp. Our results suggest that although RGE/D is a well-defined integrin binding motif, in this case the role of these three hyperconserved amino acids does not appear to be integrin binding. As the extent of conservation of this region extends well beyond these three amino acids, we speculate that this region may play an important role in the structure of HCV E2 or in mediating the interaction with other factor(s during

[Viral hepatitis in travellers].

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Abreu, Cândida

2007-01-01

Considering the geographical asymmetric distribution of viral hepatitis A, B and E, having a much higher prevalence in the less developed world, travellers from developed countries are exposed to a considerable and often underestimated risk of hepatitis infection. In fact a significant percentage of viral hepatitis occurring in developed countries is travel related. This results from globalization and increased mobility from tourism, international work, humanitarian and religious missions or other travel related activities. Several studies published in Europe and North America shown that more than 50% of reported cases of hepatitis A are travel related. On the other hand frequent outbreaks of hepatitis A and E in specific geographic areas raise the risk of infection in these restricted zones and that should be clearly identified. Selected aspects related with the distribution of hepatitis A, B and E are reviewed, particularly the situation in Portugal according to the published studies, as well as relevant clinical manifestations and differential diagnosis of viral hepatitis. Basic prevention rules considering enteric transmitted hepatitis (hepatitis A and hepatitis E) and parenteral transmitted (hepatitis B) are reviewed as well as hepatitis A and B immunoprophylaxis. Common clinical situations and daily practice "pre travel" advice issues are discussed according to WHO/CDC recommendations and the Portuguese National Vaccination Program. Implications from near future availability of a hepatitis E vaccine, a currently in phase 2 trial, are highlighted. Potential indications for travellers to endemic countries like India, Nepal and some regions of China, where up to 30% of sporadic cases of acute viral hepatitis are caused by hepatitis E virus, are considered. Continued epidemiological surveillance for viral hepatitis is essential to recognize and control possible outbreaks, but also to identify new viral hepatitis agents that may emerge as important global health

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

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Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-02-01

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Description of some characteristics of flowers and seeds of Arabidopsis thaliana - ecotype landsberg erecta and mutant NW4

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Leszek Trząski

2014-01-01

Full Text Available Flowers and seeds of Landsberg erecta (Ler ecotype and NW4 mutant were studied by light microscopy and scanning electron microscopy to reveal characteristic features of their structure. The NW4 mutant flowers differ from Ler mainly in presence of two bract-like sepals with complicated vasculature and a variable number of secondary flowers. In the two outer whorls of NW4 flower, variable number of transformed stamen-, petal-, sepal- and style-like elements also occur. The NW4 mutant seeds are characterized by the absence of mucilage around the surface and a deviating seed coat morphology.

Alterações dermatológicas em crianças com Aids e sua relação com categorias clínico-imunológicas e carga viral

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Carvalho Vânia Oliveira de

2003-01-01

Full Text Available FUNDAMENTOS: Desde o início da epidemia da Aids as dermatoses têm sido freqüentemente descritas em pacientes com essa doença, com relatos de casos atípicos e estudos de séries de pacientes adultos; no entanto, há poucas publicações sobre alterações cutâneas em pacientes pediátricos com Aids. OBJETIVOS: Estudo prospectivo para avaliar a presença de dermatoses em 40 pacientes pediátricos com Aids. MÉTODOS: Quarenta pacientes, com idade inferior a 13 anos e portadores de Aids, foram estudados por um período de seis meses para avaliação de: número de alterações dermatológicas; suas características clínicas; distribuição conforme as categorias clínico-imunológicas e o valor da carga viral. RESULTADOS: A prevalência de dermatoses foi de 82,4%, na primeira consulta, e, no acompanhamento longitudinal, 92,5% dos pacientes tiveram alterações dermatológicas, com proporção de cinco diagnósticos por doente. As crianças com classificação clínico-imunológica grave e carga viral acima de 100.000 cópias/ml apresentaram maior número de alterações dermatológicas quando comparadas àquelas das categorias clínico-imunológicas leves. A proporção de diagnósticos por paciente na categoria clínica C foi de 6,8 e na A de 3,6; na categoria imunológica grave, de sete, e na leve de 3,7; e na carga viral > 100.000 de 7,3, e na < 100.000 de 4,2 (todos com significância estatística. CONCLUSÃO: As dermatoses foram freqüentes nas crianças com Aids e ocorreram em maior número nos pacientes pertencentes às categorias graves. A elevada freqüência de alterações da pele nos pacientes pediátricos com a doença indica ser imprescindível sua avaliação dermatológica minuciosa e freqüente.

Hepatitis E Virus Mutations: Functional and Clinical Relevance

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Hoang van Tong

2016-09-01

Full Text Available Hepatitis E virus (HEV infection is a major cause of acute hepatitis and affects more than 20 million individuals, with three million symptomatic cases and 56,000 recognized HEV-related deaths worldwide. HEV is endemic in developing countries and is gaining importance in developed countries, due to increased number of autochthone cases. Although HEV replication is controlled by the host immune system, viral factors (especially specific viral genotypes and mutants can modulate HEV replication, infection and pathogenesis. Limited knowledge exists on the contribution of HEV genome variants towards pathogenesis, susceptibility and to therapeutic response. Nonsynonymous substitutions can modulate viral proteins structurally and thus dysregulate virus-host interactions. This review aims to compile knowledge and discuss recent advances on the casual role of HEV heterogeneity and its variants on viral morphogenesis, pathogenesis, clinical outcome and antiviral resistance.

Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg).

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Golsaz-Shirazi, Forough; Amiri, Mohammad Mehdi; Farid, Samira; Bahadori, Motahareh; Bohne, Felix; Altstetter, Sebastian; Wolff, Lisa; Kazemi, Tohid; Khoshnoodi, Jalal; Hojjat-Farsangi, Mohammad; Chudy, Michael; Jeddi-Tehrani, Mahmood; Protzer, Ulrike; Shokri, Fazel

2017-08-01

Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The "a" determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression.

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Shives, Katherine D; Massey, Aaron R; May, Nicholas A; Morrison, Thomas E; Beckham, J David

2016-10-18

West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7 GpppN m 5' cap with 2'- O -methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

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Katherine D. Shives

2016-10-01

Full Text Available West Nile virus (WNV is a (+ sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1 for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K and eukaryotic translation initiation factor 4E-binding protein (4EBP pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E interaction and eukaryotic initiation factor 4F (eIF4F complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6 and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4+ T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host.

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Jodi L McGill

Full Text Available Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4+ T cell proliferation and IFNγ production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs.

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Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D; Pelletier, Jerry; Ferraiuolo, Maria A; Sonenberg, Nahum

2008-07-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

The deoxyhypusine synthase mutant dys1-1 reveals the association of eIF5A and Asc1 with cell wall integrity.

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Fabio Carrilho Galvão

Full Text Available The putative eukaryotic translation initiation factor 5A (eIF5A is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1 and deoxyhypusine hydroxylase (Lia1 catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1 and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of m

Suppressor of cytokine signaling 4 (SOCS4 protects against severe cytokine storm and enhances viral clearance during influenza infection.

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Lukasz Kedzierski

2014-05-01

Full Text Available Suppressor of cytokine signaling (SOCS proteins are key regulators of innate and adaptive immunity. There is no described biological role for SOCS4, despite broad expression in the hematopoietic system. We demonstrate that mice lacking functional SOCS4 protein rapidly succumb to infection with a pathogenic H1N1 influenza virus (PR8 and are hypersusceptible to infection with the less virulent H3N2 (X31 strain. In SOCS4-deficient animals, this led to substantially greater weight loss, dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This was associated with impaired trafficking of influenza-specific CD8 T cells to the site of infection and linked to defects in T cell receptor activation. These results demonstrate that SOCS4 is a critical regulator of anti-viral immunity.

Increased sensitivity to salt stress in tocopherol-deficient Arabidopsis mutants growing in a hydroponic system

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Ellouzi, Hasna; Hamed, Karim Ben; Cela, Jana; Müller, Maren; Abdelly, Chedly; Munné-Bosch, Sergi

2013-01-01

Recent studies suggest that tocopherols could play physiological roles in salt tolerance but the mechanisms are still unknown. In this study, we analyzed changes in growth, mineral and oxidative status in vte1 and vte4 Arabidopsis thaliana mutants exposed to salt stress. vte1 and vte4 mutants lack α-tocopherol, but only the vte1 mutant is additionally deficient in γ-tocopherol. Results showed that a deficiency in vitamin E leads to reduced growth and increased oxidative stress in hydroponically-grown plants. This effect was observed at early stages, not only in rosettes but also in roots. The vte1 mutant was more sensitive to salt-induced oxidative stress than the wild type and the vte4 mutant. Salt sensitivity was associated with (i) high contents of Na+, (ii) reduced efficiency of PSII photochemistry (Fv/Fm ratio) and (iii) more pronounced oxidative stress as indicated by increased hydrogen peroxide and malondialdeyde levels. The vte 4 mutant, which accumulates γ- instead of α-tocopherol showed an intermediate sensitivity to salt stress between the wild type and the vte1 mutant. Contents of abscisic acid, jasmonic acid and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid were higher in the vte1 mutant than the vte4 mutant and wild type. It is concluded that vitamin E-deficient plants show an increased sensitivity to salt stress both in rosettes and roots, therefore indicating the positive role of tocopherols in stress tolerance, not only by minimizing oxidative stress, but also controlling Na+/K+ homeostasis and hormonal balance. PMID:23299430

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib.

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Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

2018-02-01

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.

Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells

International Nuclear Information System (INIS)

Labadie, Karine; Pelletier, Isabelle; Saulnier, Aure; Martin, Javier; Colbere-Garapin, Florence

2004-01-01

Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal Caco-2 cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost 2 years [J. Virol. 74 (2000) 3001], were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days postvaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 (S3) strain. In contrast, three VDPVs excreted 136, 442, and 637 days postvaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cell surface

Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

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Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

2015-09-08

It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

Effects of the deletion of early region 4 (E4 open reading frame 1 (orf1, orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

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Michael A Thomas

Full Text Available The global health burden engendered by human immunodeficiency virus (HIV-induced acquired immunodeficiency syndrome (AIDS is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1 through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and

Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

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Thomas, Michael A; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A; Venzon, David; Robert-Guroff, Marjorie

2013-01-01

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a

Asociación de LT-CD4 y carga viral con candidiasis bucal en pacientes VIH/SIDA en Talca, Chile Association between LT-CD4 and the viral load with oral candidiasis in HIV/AIDS patients in Talca, Chile

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Pedro Brevis Azocar

2009-12-01

Full Text Available La candidiasis bucal aparece con frecuencia en las personas infectadas por el virus de la inmunodeficiencia humana (VIH y puede causar diversas manifestaciones clínicas y complicaciones. En los pacientes VIH la carga viral es considerada uno de los principales predictores en la progresión de la enfermedad. Se realizó un estudio en 29 pacientes adultos VIH-positivos para evaluar los niveles de linfocitos T-CD4 y carga viral; se estableció una relación con las manifestaciones de la candidiasis bucal. El análisis estadístico mostró que hubo relación entre la manifestación de la candidiasis bucal y la carga viral, pues en pacientes con cargas virales superiores a 10 000 copias/mL, las lesiones fueron más frecuentes.Oral candidiasis is frequently appearing in persons infected by human immunodeficiency virus (HIV and may to cause many clinical and complications manifestations. In HIV patients the viral load is considered one of the main predictors in disease progression. A study was conducted in 29 HIV-positive adult patients to assess the T-CD4 lymphocyte levels and the viral load establishing a relationship with oral candidiasis manifestations. The statistical analysis showed that there was a relationship between the oral candidiasis manifestation and the viral load since in patients with viral load higher than 10 000 copies/mL, lesions were more frequent.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

OpenAIRE

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D.; Pelletier, Jerry; Ferraiuolo, Maria A.; Sonenberg, Nahum

2008-01-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5′-cap-binding protein, mediates the association of eIF4F with the mRNA 5′-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported...

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

OpenAIRE

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler Jr, Richard E.; Lalani, Alshad S.; Dent, Paul

2017-01-01

ABSTRACT The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFR...

The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

Science.gov (United States)

Tunnicliffe, Richard B.; Lockhart-Cairns, Michael P.; Levy, Colin; Mould, A. Paul; Jowitt, Thomas A.; Sito, Hilary; Baldock, Clair; Sandri-Goldin, Rozanne M.

2017-01-01

Abstract The transcription factor ICP4 from herpes simplex virus has a central role in regulating the gene expression cascade which controls viral infection. Here we present the crystal structure of the functionally essential ICP4 DNA binding domain in complex with a segment from its own promoter, revealing a novel homo-dimeric fold. We also studied the complex in solution by small angle X-Ray scattering, nuclear magnetic resonance and surface-plasmon resonance which indicated that, in addition to the globular domain, a flanking intrinsically disordered region also recognizes DNA. Together the data provides a rationale for the bi-partite nature of the ICP4 DNA recognition consensus sequence as the globular and disordered regions bind synergistically to adjacent DNA motifs. Therefore in common with its eukaryotic host, the viral transcription factor ICP4 utilizes disordered regions to enhance the affinity and tune the specificity of DNA interactions in tandem with a globular domain. PMID:28505309

Toll-like receptor 4 mutant and null mice retain morphine-induced tolerance, hyperalgesia, and physical dependence.

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Theresa Alexandra Mattioli

Full Text Available The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4. Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (- naloxone, an opioid receptor antagonist, and (+ naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence.

Molecular Genetic Identification Of Some Flax Mutants

International Nuclear Information System (INIS)

AMER, I.M.; MOUSTAFA, H.A.M.

2009-01-01

Five flax genotypes (Linum usitatissimum L.) i.e., commercial cultivar Sakha 2, the mother variety Giza 4 and three mutant types induced by gamma rays, were screened for their salinity tolerance in field experiments (salinity concentration was 8600 and 8300 ppm for soil and irrigation water, respectively). Mutation 6 was the most salt tolerant as compared to the other four genotypes.RAPD technique was used to detect some molecular markers associated with salt tolerance in flax (Mut 6), RAPD-PCR results using 12 random primers exhibited 149 amplified fragments; 91.9% of them were polymorphic and twelve molecular markers (8.1%) for salt tolerant (mutant 6) were identified with molecular size ranged from 191 to 4159 bp and only eight primers successes to amplify these specific markers. Concerning the other mutants, Mut 15 and Mut 25 exhibited 4.3% and 16.2% specific markers, respectively. The induced mutants exhibited genetic similarity to the parent variety were about 51%, 58.3% and 61.1% for Mut 25, Mut 6 and Mut 15, respectively. These specific markers (SM) are used for identification of the induced mutations and it is important for new variety registration.

Temporal and spatial alterations in mutant swarm size of St. Louis encephalitis virus in mosquito hosts.

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Ciota, Alexander T; Koch, Evan M; Willsey, Graham G; Davis, Lauren J; Jerzak, Greta V S; Ehrbar, Dylan J; Wilke, Claus O; Kramer, Laura D

2011-03-01

St. Louis encephalitis virus (SLEV; Flaviviridae; Flavivirus) is a member of the Japanese encephalitis serocomplex and a close relative of West Nile virus (WNV). Although SLEV remains endemic to the US, both levels of activity and geographical dispersal are relatively constrained when compared to the widespread distribution of WNV. In recent years, WNV appears to have displaced SLEV in California, yet both viruses currently coexist in Texas and several other states. It has become clear that viral swarm characterization is required if we are to fully evaluate the relationship between viral genomes, viral evolution, and epidemiology. Mutant swarm size and composition may be particularly important for arboviruses, which require replication not only in diverse tissues but also divergent hosts. In order to evaluate temporal, spatial, and host-specific patterns in the SLEV mutant swarm, we determined the size, composition, and phylogeny of the intrahost swarm within primary mosquito isolates from both Texas and California. Results indicate a general trend of decreasing intrahost diversity over time in both locations, with recent isolates being highly genetically homogeneous. Additionally, phylogenic analyses provide detailed information on the relatedness of minority variants both within and among strains and demonstrate how both geographic isolation and seasonal maintenance have shaped the viral swarm. Overall, these data generally provide insight into how time, space, and unique transmission cycles influence the SLEV mutant swarm and how understanding these processes can ultimately lead to a better understanding of arbovirus evolution and epidemiology. Copyright © 2010 Elsevier B.V. All rights reserved.

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products.

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Rattay, Stephanie; Trilling, Mirko; Megger, Dominik A; Sitek, Barbara; Meyer, Helmut E; Hengel, Hartmut; Le-Trilling, Vu Thuy Khanh

2015-08-01

Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted

Efficacy of hepatitis B vaccine against antiviral drug-resistant hepatitis B virus mutants in the chimpanzee model.

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Kamili, Saleem; Sozzi, Vitini; Thompson, Geoff; Campbell, Katie; Walker, Christopher M; Locarnini, Stephen; Krawczynski, Krzysztof

2009-05-01

Hepatitis B virus (HBV) mutants resistant to treatment with nucleoside or nucleotide analogs and those with the ability to escape from HBV-neutralizing antibody have the potential to infect HBV-vaccinated individuals. To address this potential serious public health challenge, we tested the efficacy of immunity induced by a commercial hepatitis B vaccine against a tissue culture-derived, clonal HBV polymerase mutant in HBV seronegative chimpanzees. The polymerase gene mutant contained a combination of three mutations (rtV173L, rtL180M, rtM204V), two of which resulted in changes to the overlapping viral envelope of the hepatitis B surface antigen (sE164D, sI195M). Prior to the HBV mutant challenge of vaccinated chimpanzees, we established virologic, serologic, and pathologic characteristics of infections resulting from intravenous inoculation of the HBV polymerase gene mutant and the sG145R vaccine-escape surface gene mutant. Cloning and sequencing experiments determined that the three mutations in the polymerase gene mutant remained stable and that the single mutation in the surface gene mutant reverted to the wild-type sequence. Immunological evidence of HBV replication was observed in the vaccinated chimpanzees after challenge with the polymerase gene mutant as well as after rechallenge with serum-derived wild-type HBV (5,000 chimpanzee infectious doses administered intravenously), despite robust humoral and cellular anti-HBV immune responses after hepatitis B vaccination. Our data showing successful experimental infection by HBV mutants despite the presence of high anti-HBs levels considered protective in the vaccinated host are consistent with clinical reports on breakthrough infection in anti-HBs-positive patients infected with HBV mutants. In the absence of a protective humoral immunity, adaptive cellular immune responses elicited by infection may limit HBV replication and persistence.

[Comparison of clinical and laboratory characteristics of viral hepatitis A and E in Montenegro].

Science.gov (United States)

Terzić, Dragica; Mijović, Gordana; Dupanović, Brankica; Drasković, Nenad; Svirtlih, Neda

2010-01-01

Hepatitis E has many similarities in with hepatitis A concerning clinical picture, route of transmission and nonexistence of chronicity. Comparison of clinical and laboratory parameters of patients with hepatitis A and E to estimate characteristics of these diseases. Total of 54 patients divided into two groups was investigated: 27 had hepatitis A, others had hepatitis E. Detailed history past, clinical examination, liver function tests and ultrasonography of the upper abdomen, were done in all patients. Aetiology of viral hepatitis was investigated serologically by enzyme immunoassay (ELISA) using commercial kits for following viruses: Hepatitis A-E viruses, cytomegalovirus, and Epstein-Barr virus. Asymptomatic infections (29.6%) and clinical forms without jaundice (59.3%) were more frequent in patients with hepatitis E. Splenomegaly was found more frequent in patients with hepatitis A than in hepatitis E (66.7% vs. 33.3%). Patients with hepatitis E had significantly lower activity of aminotransferases than patients with hepatitis A. A significant increase of gamma-glutamyltranspeptidase was found in patients with hepatitis E (mean value: 120 IU/L). Our results are in concordance with other reports that hepatitis E virus infection is more common asymptomatic disease than hepatitis A. In addition, hepatocyte necrosis in hepatitis E is less extensive than in hepatitis A measured by the activity of aminotransferases. Contrary to that the value of gamma-glutamyltranspeptidase is more increased in hepatitis E than in hepatitis A without exact explanation so far: Viral hepatitis E and A have differences in some clinical features and laboratory parameters although both diseases principally have resolved without consequences after 6-8 weeks.

Effects of vitamin D in the prevention of acute viral bronchiolitis: systematic review = Efeitos da vitamina D na prevenção da bronquiolite viral aguda: revisão sistemática

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Rueda, Angélica Maria Barba

2015-01-01

Full Text Available Objetivos: Evidências publicadas sugerem que a suplementação da vitamina D pode ter efeito protetor nas infecções do trato respiratório inferior. O objetivo desta revisão foi avaliar os efeitos da ingestão de vitamina D na prevenção da bronquiolite viral aguda em crianças. Métodos: Foram feitas buscas nas bases de dados Medline, EMBASE, Web of Science, LILACS e Cochrane Central Register of Controlled Trials, até dezembro de 2014, usando os descritores "Vitamin D" ou cholecalciferol ou ergocalciferol e "bronchiolitis, viral" ou "viral bronchiolitis" ou "bronchiolitides, viral" ou "viral bronchiolitides". Foram incluídos estudos que avaliaram o efeito da ingesta da vitamina D na prevenção da bronquiolite viral aguda em crianças. Estudos com intervenção menor que duas semanas e artigos de revisão foram excluídos. Resultados: A busca identificou 241 artigos, entre os quais 20 artigos foram selecionados para leitura na íntegra e dois artigos foram incluídos na revisão sistemática, incluindo 296 crianças. Nenhum estudo mediu os níveis séricos de vitamina D. Um dos estudos incluídos foi um ensaio clinico, no qual o número de episódios de bronquiolite foi significativamente menor nas crianças suplementadas com vitamina D (Grupo I: média 0, 6±0, 7 Grupo II: média 1, 4 ±0, 9; P =0, 001. No outro, um estudo de casos e controles, não se encontrou relação significativa entre casos de bronquiolite viral aguda e ingesta de vitamina D (odds ratio 1, 7 – intervalo de confiança 95% 0, 7 - 4, 0. Conclusões: As evidências científicas atuais são insuficientes para comprovar os benefícios clínicos da vitamina D na prevenção da bronquiolite viral aguda

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation

Science.gov (United States)

Schepetilnikov, Mikhail; Kobayashi, Kappei; Geldreich, Angèle; Caranta, Carole; Robaglia, Christophe; Keller, Mario; Ryabova, Lyubov A

2011-01-01

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator–viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV–TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP—a novel and specific target of TOR/S6K1—in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes. PMID:21343906

Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

International Nuclear Information System (INIS)

Kokusho, Ryuhei; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu

2016-01-01

Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

Energy Technology Data Exchange (ETDEWEB)

Kokusho, Ryuhei, E-mail: kokusho@ss.ab.a.u-tokyo.ac.jp; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu, E-mail: katsuma@ss.ab.a.u-tokyo.ac.jp

2016-11-15

Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

Generation and characterization of koi herpesvirus recombinants lacking viral enzymes of nucleotide metabolism.

Science.gov (United States)

Fuchs, Walter; Fichtner, Dieter; Bergmann, Sven M; Mettenleiter, Thomas C

2011-06-01

Koi herpesvirus (KHV) causes a fatal disease in koi and common carp, but no reliable and genetically characterized vaccines are available up to now. Therefore, we generated KHV recombinants possessing deletions within the viral ribonucleotide reductase (RNR), thymidine kinase (TK), dUTPase, or TK and dUTPase genes, and their corresponding rescuants. All KHV mutants were replication competent in cultured cells. Whereas plaque sizes and titers of RNR-negative KHV were reduced, replication of the other mutants was not affected. Experimental infection of carp indicated attenuation of TK- or dUTPase-deleted KHV, and PCR analysis of tissue samples permitted differentiation of mutant from wild-type virus.

Restriction of phage T4 internal protein I mutants by a strain of Escherichia coli

International Nuclear Information System (INIS)

Black, L.W.; Abremski, K.

1974-01-01

Phage T4 internal protein I(IPI), a small (ca, 10,000 MW), basic protein injected into the host with the phage DNA, is not required for infection of most hosts, but mutants defective in IPI are restricted by at least one naturally occurring strain of Escherichia coli, CT 596 (CT). Phages lacking IPI (IPI - ) appear to inject their DNA and bind it to the membrane of CT cells as well as wild-type phage T4 does, but shutoff of host protein synthesis, initiation of T4 protein synthesis, and cell killing are abnormal in the IPI - mutant infected CT host. The injection of IPI appears to be important in allowing T4 DNA to carry out early steps involved in takeover of this host. Restriction of IPI - phage growth by CT cells appears to be due, at least in part, to a defective prophage it harbors which renders the host resistant to successful infection by phage T4 which lack IPI or rII functions. Bacteria cured of this prophage can be infected by mutants defective in these functions. The resistance of CT cells to other coliphages, and the question of T-even phage internal protein diversity are discussed. (U.S.)

Waiting times for the appearance of cytotoxic T-lymphocyte escape mutants in chronic HIV-1 infection

International Nuclear Information System (INIS)

Liu Yi; Mullins, James I.; Mittler, John E.

2006-01-01

The failure of HIV-1 to escape at some cytotoxic T-lymphocyte (CTL) epitopes has generally been explained in terms of viral fitness costs or ineffective or attenuated CTL responses. Relatively little attention has been paid to the evolutionary time required for escape mutants to be detected. This time is significantly affected by selection, mutation rates, the presence of other advantageous mutations, and the effective population size of HIV-1 in vivo (typically estimated to be ∼10 3 in chronically infected patients, though one study has estimated it to be ∼10 5 ). Here, we use a forward simulator with experimentally estimated HIV-1 parameters to show that these delays can be substantial. For an effective population size of 10 3 , even highly advantageous mutants (s = 0.5) may not be detected for a couple of years in chronically infected patients, while moderately advantageous escape mutants (s = 0.1) may not be detected for up to 10 years. Even with an effective population size of 10 5 , a moderately advantageous escape mutant (s = 0.1) may not be detected in the population within 2 years if it has to compete with other selectively advantageous mutants. Stochastic evolutionary forces, therefore, in addition to viral fitness costs and ineffective or attenuated CTL responses, must be taken into account when assessing the selection of CTL escape mutations

Mutant prevention concentrations of four carbapenems against gram-negative rods.

Science.gov (United States)

Credito, Kim; Kosowska-Shick, Klaudia; Appelbaum, Peter C

2010-06-01

We tested the propensities of four carbapenems to select for resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii mutants by determining the mutant prevention concentrations (MPCs) for 100 clinical strains with various ss-lactam phenotypes. Among the members of the Enterobacteriaceae family and A. baumannii strains, the MPC/MIC ratios were mostly 2 to 4. In contrast, for P. aeruginosa the MPC/MIC ratios were 4 to > or =16. The MPC/MIC ratios for beta-lactamase-positive K. pneumoniae and E. coli isolates were much higher (range, 4 to >16 microg/ml) than those for ss-lactamase-negative strains.

Cooperative heteroassembly of the adenoviral L4-22K and IVa2 proteins onto the viral packaging sequence DNA.

Science.gov (United States)

Yang, Teng-Chieh; Maluf, Nasib Karl

2012-02-21

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

Disseminated HIV-Associated Kaposi’s Sarcoma With High CD4 Cell Count And Low Viral Load

Directory of Open Access Journals (Sweden)

Diana Pereira Anjos

2017-12-01

Full Text Available Kaposi’s sarcoma is considered an acquired immunodeficiency syndrome-defining illness and is caused by human herpesvirus 8. It has been associated with patients infected with human immunodeficiency virus (HIV who have CD4 T lymphocytes <200 cells/uL and high viral loads. We report a case of a 23-year old woman infected with HIV-1 and receiving antiretroviral treatment since diagnosis, with high CD4 cell count and low viral load that presented with disseminated Kaposi’s sarcoma. Clinicians should be aware of the occurrence of Kaposi’s sarcoma despite robust CD4 cell counts.

Food insecurity, CD4 counts, and incomplete viral suppression among HIV+ patients from Texas Children's Hospital: A pilot study

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Our goal was to determine the relationship between food insecurity and CD4 counts and viral suppression among pediatric HIV-positive patients. Food insecurity was assessed by validated survey. CD4 counts and viral load were abstracted from patients’ charts. We used linear regression for the dependen...

HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

Science.gov (United States)

Redmond, Catherine J.; Dooley, Katharine E.; Fu, Haiqing; Gillison, Maura L.; Akagi, Keiko; Symer, David E.; Aladjem, Mirit I.

2018-01-01

Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression. PMID:29364907

Persistence of Coxsackievirus B4 in pancreatic ductal-like cells results in cellular and viral changes.

Science.gov (United States)

Alidjinou, E K; Engelmann, I; Bossu, J; Villenet, C; Figeac, M; Romond, M-B; Sané, F; Hober, D

2017-10-03

Although known as cytolytic viruses, group B coxackieviruses (CVB) are able to establish a persistent infection in vitro and in vivo. Viral persistence has been reported as a key mechanism in the pathogenesis of CVB-associated chronic diseases such as type 1 diabetes (T1D). The impact of CVB4 persistence on human pancreas ductal-like cells was investigated. A persistent CVB4 infection was established in ductal-like cells. PDX-1 expression, resistance to CVB4-induced lysis and CAR expression were evaluated. The profile of cellular microRNAs (miRNAs) was investigated through miRNA-sequencing. Viral phenotypic changes were examined, and genomic modifications were assessed by sequencing of the viral genome. The CVB4 persistence in ductal-like cells was productive, with continuous release of infectious particles. Persistently infected cells displayed a resistance to CVB4-induced lysis upon superinfection and expression of PDX-1 and CAR was decreased. These changes were maintained even after virus clearance. The patterns of cellular miRNA expression in mock-infected and in CVB4-persistently infected ductal-like cells were clearly different. The persistent infection-derived virus (PIDV) was still able to induce cytopathic effect but its plaques were smaller than the parental virus. Several mutations appeared in various PIDV genome regions, but amino acid substitutions did not affect the predicted site of interaction with CAR. Cellular and viral changes occur during persistent infection of human pancreas ductal-like cells with CVB4. The persistence of cellular changes even after virus clearance supports the hypothesis of a long-lasting impact of persistent CVB infection on the cells.

Dissecting HIV Virulence: Heritability of Setpoint Viral Load, CD4+ T-Cell Decline, and Per-Parasite Pathogenicity.

Science.gov (United States)

Bertels, Frederic; Marzel, Alex; Leventhal, Gabriel; Mitov, Venelin; Fellay, Jacques; Günthard, Huldrych F; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Battegay, Manuel; Rauch, Andri; Cavassini, Matthias; Calmy, Alexandra; Bernasconi, Enos; Schmid, Patrick; Scherrer, Alexandra U; Müller, Viktor; Bonhoeffer, Sebastian; Kouyos, Roger; Regoes, Roland R

2018-01-01

Pathogen strains may differ in virulence because they attain different loads in their hosts, or because they induce different disease-causing mechanisms independent of their load. In evolutionary ecology, the latter is referred to as "per-parasite pathogenicity". Using viral load and CD4+ T-cell measures from 2014 HIV-1 subtype B-infected individuals enrolled in the Swiss HIV Cohort Study, we investigated if virulence-measured as the rate of decline of CD4+ T cells-and per-parasite pathogenicity are heritable from donor to recipient. We estimated heritability by donor-recipient regressions applied to 196 previously identified transmission pairs, and by phylogenetic mixed models applied to a phylogenetic tree inferred from HIV pol sequences. Regressing the CD4+ T-cell declines and per-parasite pathogenicities of the transmission pairs did not yield heritability estimates significantly different from zero. With the phylogenetic mixed model, however, our best estimate for the heritability of the CD4+ T-cell decline is 17% (5-30%), and that of the per-parasite pathogenicity is 17% (4-29%). Further, we confirm that the set-point viral load is heritable, and estimate a heritability of 29% (12-46%). Interestingly, the pattern of evolution of all these traits differs significantly from neutrality, and is most consistent with stabilizing selection for the set-point viral load, and with directional selection for the CD4+ T-cell decline and the per-parasite pathogenicity. Our analysis shows that the viral genotype affects virulence mainly by modulating the per-parasite pathogenicity, while the indirect effect via the set-point viral load is minor. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

An extra early mutant of pigeonpea

International Nuclear Information System (INIS)

Ravikesavan, R.; Kalaimagal, T.; Rathnaswamy, R.

2001-01-01

The redgram (Cajanus cajan (L.) Huth) variety 'Prabhat DT' was gamma irradiated with 100, 200, 300 and 400 Gy doses. Several mutants have been identified viz., extra early mutants, monostem mutants, obcordifoliate mutants and bi-stigmatic mutants. The extra early mutant was obtained when treated with 100 Gy dose. The mutant was selfed and forwarded from M 2 to M 4 generation. In the M 4 generation the mutant line was raised along with the parental variety. Normal cultural practices were followed and the biometrical observations were recorded. It was observed that for the characters viz., total number of branches per plant, number of pods per plants, seeds per pod, 100 seed weight and seed yield per plant there was no difference between the mutant and parent variety. Whereas, regarding the days to flowering and maturity the mutants were earlier than the parents. The observation was recorded from two hundred plants each. The mutant gives the same yield in 90 days as that of the parent variety in 107 days, which make it an economic mutant

Multiple dendritic cell populations activate CD4+ T cells after viral stimulation.

Directory of Open Access Journals (Sweden)

Adele M Mount

2008-02-01

Full Text Available Dendritic cells (DC are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+ T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+ T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+ and CD8(+ T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+ and CD4(+ T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+ T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+ T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+ T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.

ENGENHARIA DE SEGURANÇA E MEDIDAS DE CONTROLE DE UM LABORATÓRIO DE VACINA VIRAL VETERINÁRIA - ESTUDO DE CASO / ENGINEERING AND SAFETY ACTIONS OF A VETERINARY VIRAL VACCINE LABORATORY - A CASE STUDY

OpenAIRE

Glaucia Aparecida PRATES; Maria Cristina Pacheco do NASCIMENTO; Danielle GOVEIA; Cristiane Inácio de CAMPOS; Maristela GAVA

2013-01-01

O presente trabalho tem como objetivos avaliar as medidas de controle em um laboratório de vacina viral veterinária de Curitiba através de análise documental. Avaliar o uso de equipamentos de proteção individual (EPIs) e (EPCs) através de análise do Programa de Prevenção de Riscos Ambientais - PPRA e identificar a conformidade dos riscos biológico, químico e físico de acordo com as Normas Regulamentadoras 6 (NR 6) e 9 (NR 9) . O objeto de estudo foi o laboratório de vacina viral de uma instit...

Host Innate Immunity against Hepatitis E Virus and Viral Evasion Mechanisms.

Science.gov (United States)

Kang, Sangmin; Myoung, Jinjong

2017-10-28

Hepatitis E virus (HEV) infections cause epidemic or sporadic acute hepatitis, which are mostly self-limiting. However, viral infection in immunocompromised patients and pregnant women may result in serious consequences, such as chronic hepatitis and liver damage, mortality of the latter of which reaches up to 20-30%. Type I interferon (IFN)-induced antiviral immunity is known to be the first-line defense against virus infection. Upon HEV infection in the cell, the virus genome is recognized by pathogen recognition receptors, leading to rapid activation of intracellular signaling cascades. Expression of type I IFN triggers induction of a barrage of IFN-stimulated genes, helping the cells cope with viral infection. Interestingly, some of the HEV-encoded genes seem to be involved in disrupting signaling cascades for antiviral immune responses, and thus crippling cytokine/chemokine production. Antagonistic mechanisms of type I IFN responses by HEV have only recently begun to emerge, and in this review, we summarize known HEV evasion strategies and compare them with those of other hepatitis viruses.

Role of the viral and cellular encoded thymidine kinase in the repair of UV-irradiated herpes simplex virus

International Nuclear Information System (INIS)

Rainbow, A.J.; McMaster Univ., Hamilton, ON

1989-01-01

A strain of herpes simplex type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk + ) gene and a thymidine kinase deficient (tk - ) mutant strain (HSC-1:PTK3B) were used as probes to examine the repair of UV-damaged viral DNA in one tk - (143) and two tk + (R970-5 and AC4) human cell lines. UV survival for each HSC-1 strain was similar for infection of both tk - and tk + cells suggesting that the repair of viral DNA was not dependent on the expression of a functional cellular tk gene. In contrast, UV survival of HSV-1:PTK3B was substantially reduced compared to HSV-1:KOS when infecting all 3 human cell lines, as well as Vero monkey kidney cells and LPM1A mouse cells. Tjese results suggest that the repair of UV-irradiated HSV-1 in lytically infected mammalian cells depends, in part at least, on the expression of the viral encoded tk. (author). 20 refs.; 1 fig

Viral load, CD4+ T-lymphocyte counts and antibody titres in HIV-1 ...

African Journals Online (AJOL)

Viral load, CD4+ T-lymphocyte counts and antibody titres in HIV-1 infected untreated children in Kenya; implication for immunodeficiency and AIDS progression. Washingtone Ochieng, Dorington Ogoyi, Francis J Mulaa, Simon Ogola, Rachel Musoke, Moses G Otsyula ...

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus for simultaneous biosynthesis of xylanase and laccase under solid-state fermentation.

Science.gov (United States)

Dwivedi, Pallavi; Vivekanand, V; Pareek, Nidhi; Sharma, Amit; Singh, Rajesh P

2011-10-01

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching. Copyright © 2011 Elsevier B.V. All rights reserved.

Defective processing of methylated single-stranded DNA by E. coli alkB mutants

Science.gov (United States)

Dinglay, Suneet; Trewick, Sarah C.; Lindahl, Tomas; Sedgwick, Barbara

2000-01-01

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells. PMID:10950872

Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis

International Nuclear Information System (INIS)

Peterson, K.R.; Ganesan, A.K.; Mount, D.W.; Stanford Univ., CA)

1986-01-01

The SOS response is displayed following treatments which damage DNA or inhibit DNA replication. Two associated activities include enhanced capacity for DNA repair resulting from derepression of the recA, uvrA, uvrB and uvrD genes and increased mutagenesis due to derepression of recA, umuC and umuD. These changes are the consequence of the derepression of at least seventeen unlinked operons negatively regulated by LexA repressor. Following treatments that induce the SOS response, a signal molecule interacts with RecA protein, converting it to an activated form. Activated RecA protein facilitates the proteolytic cleavage of LexA repressor, which results in derepression of the regulon. The cell then enters a new physiological state during which time DNA repair processes are augmented. The lexA41 mutant of E. coli is a uv-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. The resultant protein is unstable. Lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistent with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the uv-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA - mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis. 17 refs., 4 figs., 1 tab

Crystallization and preliminary crystallographic analysis of an Escherichia coli-selected mutant of the nuclease domain of the metallonuclease colicin E7

International Nuclear Information System (INIS)

Czene, Anikó; Tóth, Eszter; Gyurcsik, Béla; Otten, Harm; Poulsen, Jens-Christian N.; Lo Leggio, Leila; Larsen, Sine; Christensen, Hans E. M.; Nagata, Kyosuke

2013-01-01

An N-terminally truncated mutant of the colicin E7 nuclease domain was crystallized and diffraction data set was collected to 1.6 Å resolution. The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity is attributed to the nonspecific nuclease domain (NColE7), which possesses the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus of NColE7 (444–576) surprisingly showed no or significantly reduced endonuclease activity [Czene et al. (2013 ▶), J. Biol. Inorg. Chem.18, 309–321]. The necessity of the N-terminal amino acids for the function of the C-terminal catalytic centre poses the possibility of allosteric activation within the enzyme. Precise knowledge of the intramolecular interactions of these residues that affect the catalytic activity could turn NColE7 into a novel platform for artificial nuclease design. In this study, the N-terminal deletion mutant ΔN4-NColE7-C* of the nuclease domain of colicin E7 selected by E. coli was overexpressed and crystallized at room temperature by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.6 Å resolution and could be indexed and averaged in the trigonal space group P3 1 21 or P3 2 21, with unit-cell parameters a = b = 55.4, c = 73.1 Å. Structure determination by molecular replacement is in progress

A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

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Gao Chen

2012-02-01

Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

Hepatitis E virus is the leading cause of acute viral hepatitis in Lothian, Scotland

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I. Kokki

2016-03-01

Full Text Available Acute viral hepatitis affects all ages worldwide. Hepatitis E virus (HEV is increasingly recognized as a major cause of acute hepatitis in Europe. Because knowledge of its characteristics is limited, we conducted a retrospective study to outline demographic and clinical features of acute HEV in comparison to hepatitis A, B and C in Lothian over 28 months (January 2012 to April 2014. A total of 3204 blood samples from patients with suspected acute hepatitis were screened for hepatitis A, B and C virus; 913 of these samples were also screened for HEV. Demographic and clinical information on patients with positive samples was gathered from electronic patient records. Confirmed HEV samples were genotyped. Of 82 patients with confirmed viral hepatitis, 48 (59% had acute HEV. These patients were older than those infected by hepatitis A, B or C viruses, were more often male and typically presented with jaundice, nausea, vomiting and/or malaise. Most HEV cases (70% had eaten pork or game meat in the few months before infection, and 14 HEV patients (29% had a recent history of foreign travel. The majority of samples were HEV genotype 3 (27/30, 90%; three were genotype 1. Acute HEV infection is currently the predominant cause of acute viral hepatitis in Lothian and presents clinically in older men. Most of these infections are autochthonous, and further studies confirming the sources of infection (i.e. food or blood transfusion are required.

Fitness and virulence of a coxsackievirus mutant that can circumnavigate the need for phosphatidylinositol 4-kinase class III beta

NARCIS (Netherlands)

Thibaut, Hendrik Jan; van der Schaar, Hilde M; Lanke, Kjerstin H W; Verbeken, Erik; Andrews, Martin; Leyssen, Pieter; Neyts, Johan; van Kuppeveld, Frank J M

2014-01-01

Coxsackieviruses require phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) for replication but can bypass this need by an H57Y mutation in protein 3A (3A-H57Y). We show that mutant coxsackievirus is not outcompeted by wild-type virus during 10 passages in vitro. In mice, the mutant virus proved as

A mutant of a mutant of a mutant of a ...: Irradiation of progressive radiation-induced mutants in a mutation-breeding programme with Chrysanthenum morifolium RAM

International Nuclear Information System (INIS)

Broertjes, C.; Koene, P.; Veen, J.W.H. van.

1980-01-01

Radiation-induced sports in Chrysanthemum morifolium RAM. have been reported for several years. It has become an everyday practice to produce flower-colour mutants from outstanding cross-breeding products, even before they are distributed for the commercial production of cut flowers. One of the most successful and recent examples is that of cv. Horim, of which hundreds of mutants were produced by successive use of radiation-induced mutants in the mutation-breeding programme. Over about 4 years a variety of flower-colour mutants was obtained, not only largely including the outstanding characteristics of the original cultivar but sometimes even with an appreciable improvement in quality and yield. It is expected that the latter types, the Miros group, will soon completely supersede the spontaneous or raditation-induced Horim sports and mutants and take over the leading position of the Horim group in the production of all-year-round (AYR) cut-flowers. (orig.)

Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma

Science.gov (United States)

Stenson, Mary J.; Maurer, Matthew J.; Wellik, Linda E.; Link, Brian; Hege, Kristen; Dogan, Ahmet; Sotomayor, Eduardo; Witzig, Thomas; Gupta, Mamta

2015-01-01

Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4EWT) but not cap-mutant eIF4E (eIF4Ecap mutant) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients. PMID:25839159

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

Science.gov (United States)

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K; McCormick, Frank; Graeber, Thomas G; Christofk, Heather R

2014-04-01

Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. Copyright © 2014 Elsevier Inc. All rights reserved.

Good Friends, Bad News - Affect and Virality in Twitter

DEFF Research Database (Denmark)

Hansen, Lars Kai; Arvidsson, Adam; Nielsen, Finn Årup

2011-01-01

The link between affect, defined as the capacity for sentimental arousal on the part of a message, and virality, defined as the probability that it be sent along, is of significant theoretical and practical importance, e.g. for viral marketing. The basic measure of virality in Twitter is the prob......The link between affect, defined as the capacity for sentimental arousal on the part of a message, and virality, defined as the probability that it be sent along, is of significant theoretical and practical importance, e.g. for viral marketing. The basic measure of virality in Twitter...

Genetic analyses of nonfluorescent root mutants induced by mutagenesis in soybean

International Nuclear Information System (INIS)

Sawada, S.; Palmer, R.G.

1987-01-01

Nonfluorescent root mutants in soybean [Glycine max (L.) Merr.] are useful as markers in genetic studies and in tissue culture research. Our objective was to obtain mutagen-induced nonfluorescent root mutants and to conduct genetic studies with them. Thirteen nonfluorescent mutants were detected among 154016 seedlings derived from soybean lines treated with six mutagens. One of these mutants, derived from Williams treated with 20 kR gamma rays, did not correspond to any of the known (standard) nonfluorescent spontaneous mutants. This is the first mutagen-induced nonfluorescent root mutant in soybean. It was assigned Genetic Type Collection no. T285 and the gene symbol fr5 fr5. The fr5 allele was not located on trisomics A, B, or C and was not linked to five chlorophyll-deficient mutants (y9, y11, y12, y13, and y20-k2) or flower color mutant w1. The remaining nonfluorescent root mutants were at the same loci as known spontaneous mutants; i.e., four had the fr1 allele, five had the fr2 allele, and three had the fr4 allele

PE2 cleavage mutants of Sindbis virus : Correlation between viral infectivity and pH-dependent membrane fusion activation of the spike heterodimer

NARCIS (Netherlands)

Smit, JM; Klimstra, WB; Ryman, KD; Bittman, R; Johnston, RE; Wilschut, J

2001-01-01

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in

Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana.

Science.gov (United States)

Zirak, P; Penzkofer, A; Moldt, J; Pokorny, R; Batschauer, A; Essen, L-O

2009-11-09

The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD(ox)), semiquinone form (FADH(.)), and anionic hydroquinone form (FAD(red)H(-)). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD(ox) to the anionic semiquinone form (FAD()(-)) with a quantum efficiency of about 2% and a back recovery time of about 10s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD(ox) to FADH(.), FADH(.) to FAD(red)H(-), and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.

Evaluation of rice mutant lines for resistance to brown planthopper, nilaparvata lugens stall

International Nuclear Information System (INIS)

Mugiono

1985-01-01

The most important and common insect in rice cultivation in South East Asia is brown planthopper, nilaparvata lugens stall. Seven rice mutant lines produced by the National Atomic Energy Agency, Indonesia, were tested at IRRI, the Philippines for resistance to brown planthopper. Those mutant lines were Atomita 1, 627/10-3/PsJ, Atomita 2 and 627/4-E/PsJ originated from Pelita 1/1 which was irradiated with 0.2 kGy of gamma rays and A227/2/PsJ, A227/3/PsJ and A227/5/PsJ, originated from early maturing mutant A23/PsJ/72K from irradiated Pelita 1/1 which was irradiated with 0.1 kGy of gamma rays. Evaluation of resistance was carried out by seedling bulk screening, honeydew excretion, survival and population build up tests by using brown planthopper biotype 1, 2 and 3. Results of these tests showed that the seven tested mutant lines were resistant to biotype 1 but susceptible to biotype 2. Reaction to biotype 3 showed that six mutant lines tested were moderately resistant and only one mutant of 627/4-E/PsJ was susceptible. Reactions of the mutant lines to biotype 1, 2 and 3 were different from the resistant varieties, Mudgo or ASD-7. This indicated that mutant lines might have gene(s) for resistance which differed from those of resistant varieties. The results showed that resistance to brown planthopper is possible to be introduced in Indonesian rice varieties by means of mutations. (author)

Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

International Nuclear Information System (INIS)

Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina; Ramanan, Parameshwaran; Nix, Jay C.; Wang, Tianjiao; Prins, Kathleen C.; Otwinowski, Zbyszek; Honzatko, Richard B.; Helgeson, Luke A.; Basler, Christopher F.; Amarasinghe, Gaya K.

2010-01-01

Three mutant forms of Ebola VP35 interferon inhibitory domain were crystallized in three different space groups. VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6 1 22, P2 1 2 1 2 1 and P2 1 , respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source

Data supporting mitochondrial morphological changes by SPG13-associated HSPD1 mutants

Directory of Open Access Journals (Sweden)

Yuki Miyamoto

2016-03-01

Full Text Available The data is related to the research article entitled “Hypomyelinating leukodystrophy-associated missense mutation in HSPD1 blunts mitochondrial dynamics” [1]. In addition to hypomyelinating leukodystrophy (HLD 4 (OMIM no. 612233, it is known that spastic paraplegia (SPG 13 (OMIM no. 605280 is caused by HSPD1’s amino acid mutation. Two amino acid mutations Val-98-to-Ile (V98I and Gln-461-to-Glu (Q461E are associated with SPG13 [2]. In order to investigate the effects of HSPD1’s V98I or Q461E mutant on mitochondrial morphological changes, we transfected each of the respective mutant-encoding genes into Cos-7 cells. Either of V98I or Q461E mutant exhibited increased number of mitochondria and short length mitochondrial morphologies. Using MitoTracker dye-incorporating assay, decreased mitochondrial membrane potential was also observed in both cases. The data described here supports that SPG13-associated HSPD1 mutant participates in causing aberrant mitochondrial morphological changes with decreased activities. Keywords: SPG13, HSPD1, Mitochondrion, Morphological change

Insight into the molecular mechanism of yeast acetyl-coenzyme A carboxylase mutants F510I, N485G, I69E, E477R, and K73R resistant to soraphen A

Science.gov (United States)

Gao, Jian; Liang, Li; Chen, Qingqing; Zhang, Ling; Huang, Tonghui

2018-02-01

Acetyl-coenzyme A carboxylases (ACCs) is the first committed enzyme of fatty acid synthesis pathway. The inhibition of ACC is thought to be beneficial not only for diseases related to metabolism, such as type-2 diabetes, but also for infectious disease like bacterial infection disease. Soraphen A, a potent allosteric inhibitor of BC domain of yeast ACC, exhibit lower binding affinities to several yeast ACC mutants and the corresponding drug resistance mechanisms are still unknown. We report here a theoretical study of binding of soraphen A to wild type and yeast ACC mutants (including F510I, N485G, I69E, E477R, and K73R) via molecular dynamic simulation and molecular mechanics/generalized Born surface area free energy calculations methods. The calculated binding free energies of soraphen A to yeast ACC mutants are weaker than to wild type, which is highly consistent with the experimental results. The mutant F510I weakens the binding affinity of soraphen A to yeast ACC mainly by decreasing the van der Waals contributions, while the weaker binding affinities of Soraphen A to other yeast ACC mutants including N485G, I69E, E477R, and K73R are largely attributed to the decreased net electrostatic (ΔE ele + ΔG GB) interactions. Our simulation results could provide important insights for the development of more potent ACC inhibitors.

Construindo Marcas Mutantes

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Elizete De Azevedo Kreutz

2012-09-01

Full Text Available O presente artigo é o resultado de estudos realizados desde 2000 e busca instrumentalizar os proñssionals para a construção de Marcas Mutantes, que é   uma tendência contemporânea nas estratégias comunicacionais e de branding. Embora esta estratégia ainda não esteja consolidada, observamos que a mesma tem obtido um crescimento constante e tem sido adotadas pelas mais diferentes categorias de marcas e não apenas por aquelas direcionadas aos jovens, ao esporte, ao entretenimento, como era no principia. Com base na Hermenêutica de Profundidade de Thompson (1995, alicerçada nas pesquisas bibliográficas, de intemet, entrevistas e análise semiótica, desenhamos um método de construção de Marcas Mutantes dividido em sete fases. Como resultado, esperamos que este estudo possa auxiliar na compreensão dos processos envolvidos, ao mesmo tempo que provoque a discussão sobreo mesmo e, por consequência, o seu aprimoramento.

Acute Pancreatitis in acute viral hepatitis

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S K.C.

2011-03-01

Full Text Available Introduction: The association of acute viral hepatitis and acute pancreatitis is well described. This study was conducted to find out the frequency of pancreatic involvement in acute viral hepatitis in the Nepalese population. Methods: Consecutive patients of acute viral hepatitis presenting with severe abdominal pain between January 2005 and April 2010 were studied. Patients with history of significant alcohol consumption and gall stones were excluded. Acute viral hepatitis was diagnosed by clinical examination, liver function test, ultrasound examination and confirmed by viral serology. Pancreatitis was diagnosed by clinical presentation, biochemistry, ultrasound examination and CT scan. Results: Severe abdominal pain was present in 38 of 382 serologically-confirmed acute viral hepatitis patients. Twenty five patients were diagnosed to have acute pancreatitis. The pancreatitis was mild in 14 and severe in 11 patients. The etiology of pancreatitis was hepatitis E virus in 18 and hepatitis A virus in 7 patients. Two patients died of complications secondary to shock. The remaining patients recovered from both pancreatitis and hepatitis on conservative treatment. Conclusions: Acute pancreatitis occurred in 6.5 % of patients with acute viral hepatitis. Cholelithiasis and gastric ulcers are the other causes of severe abdominal pain. The majority of the patients recover with conservative management. Keywords: acute viral hepatitis, acute pancreatitis, pain abdomen, hepatitis E, hepatitis A, endemic zone

A Novel, Highly Selective Inhibitor of Pestivirus Replication That Targets the Viral RNA-Dependent RNA Polymerase

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Paeshuyse, Jan; Leyssen, Pieter; Mabery, Eric; Boddeker, Nina; Vrancken, Robert; Froeyen, Matheus; Ansari, Israrul H.; Dutartre, Hélène; Rozenski, Jef; Gil, Laura H. V. G.; Letellier, Carine; Lanford, Robert; Canard, Bruno; Koenen, Frank; Kerkhofs, Pierre; Donis, Ruben O.; Herdewijn, Piet; Watson, Julia; De Clercq, Erik; Puerstinger, Gerhard; Neyts, Johan

2006-01-01

We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 ± 0.01 μM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 ± 0.02 μM) and production of infectious virus (EC50 = 0.074 ± 0.003 μM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was ∼2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed. PMID:16352539

Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid.

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Charlotte Montespan

2017-02-01

Full Text Available Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.

Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations.

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Elizalde, María Mercedes; Sevic, Ina; González López Ledesma, María Mora; Campos, Rodolfo Héctor; Barbini, Luciana; Flichman, Diego Martin

2018-01-01

In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

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Nannenga Brent L

2011-12-01

Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

Mutant Prevention Concentrations of Four Carbapenems against Gram-Negative Rods▿ †

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Credito, Kim; Kosowska-Shick, Klaudia; Appelbaum, Peter C.

2010-01-01

We tested the propensities of four carbapenems to select for resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii mutants by determining the mutant prevention concentrations (MPCs) for 100 clinical strains with various ß-lactam phenotypes. Among the members of the Enterobacteriaceae family and A. baumannii strains, the MPC/MIC ratios were mostly 2 to 4. In contrast, for P. aeruginosa the MPC/MIC ratios were 4 to ≥16. The MPC/MIC ratios for β-lactamase-positive K. pneumoniae and E. coli isolates were much higher (range, 4 to >16 μg/ml) than those for ß-lactamase-negative strains. PMID:20308376

Dwarf Rice Mutant Derived from 0.2 kGy Gamma Rays Irradiated Seeds of Atomita 4 Variety

International Nuclear Information System (INIS)

Sobrizal; Sutisna Sanjaya; Carkum; Mohamad Ismachin

2004-01-01

Dwarf rice mutant was obtained when Atomita 4 seeds were irradiated by 0.2 kGy gamma rays. The results of segregation analyses in F2 populations and F3 lines derived from reciprocal crosses of mutant and Atomita 4 suggested that the dwarf was controlled by a single recessive gene. This gene was not located on rice cytoplasmic genome but on nuclear genome. The gene for dwarf obtained in this study tentatively could be assumed as a new finding until the allelic relationships with other dwarf genes are verified. (author)

Dual function of the nuclear export signal of the Borna disease virus nucleoprotein in nuclear export activity and binding to viral phosphoprotein.

Science.gov (United States)

Yanai, Mako; Sakai, Madoka; Makino, Akiko; Tomonaga, Keizo

2017-07-11

Borna disease virus (BoDV), which has a negative-sense, single-stranded RNA genome, causes persistent infection in the cell nucleus. The nuclear export signal (NES) of the viral nucleoprotein (N) consisting of leucine at positions 128 and 131 and isoleucine at positions 133 and 136 overlaps with one of two predicted binding sites for the viral phosphoprotein (P). A previous study demonstrated that higher expression of BoDV-P inhibits nuclear export of N; however, the function of N NES in the interaction with P remains unclear. We examined the subcellular localization, viral polymerase activity, and P-binding ability of BoDV-N NES mutants. We also characterized a recombinant BoDV (rBoDV) harboring an NES mutation of N. BoDV-N with four alanine-substitutions in the leucine and isoleucine residues of the NES impaired its cytoplasmic localization and abolished polymerase activity and P-binding ability. Although an alanine-substitution at position 131 markedly enhanced viral polymerase activity as determined by a minigenome assay, rBoDV harboring this mutation showed expression of viral RNAs and proteins relative to that of wild-type rBoDV. Our results demonstrate that BoDV-N NES has a dual function in BoDV replication, i.e., nuclear export of N and an interaction with P, affecting viral polymerase activity in the nucleus.

Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells

International Nuclear Information System (INIS)

Jones, N.J.; Cox, R.; Thacker, J.

1987-01-01

The V79-4 Chinese hamster line was mutagenized and surviving clones screened for X-ray sensitivity using a replica microwell technique. One slightly sensitive clone and 3 clearly sensitive clones were isolated from approximately 5000 screened, and designated irs 1 to irs 4. The 3 more sensitive clones showed different responses to the genotoxic agents mitomycin C (MMC), ethyl methanesulphonate (EMS) and ultraviolet light (UV). irs 1 showed considerable sensitivity to all the agents tested, in the order MMC >> EMS > UV. irs 2 and irs 3 had similar sensitivities to EMS and to UV (EMS > UV) but irs 3 was more sensitive than irs 2 to MMC. None of these mutants is identical in phenotype to previously published mutants. (Auth.)

Assessment of Lupin Induced Mutants for Quality Traits and Susceptibility to Callosbruchus chinensis and Heliothis armigera insects

International Nuclear Information System (INIS)

Ragab, A.I.; Boshra, S.A.; Mehany, A.L.; Darwish, A.A.; Kharrab, M.M.

2008-01-01

This study was conducted to assess 23 induced mutants and two parental varieties Giza1 and Giza2 in the three generations (M3, M4, and M5) for seed quality traits, and susceptibility to two insects i.e C. chinnensis and H.armigera. The obtained results exhibited highly significant decrease for alkaloid content of mutants 20 and 23 as compared with the two local varieties. Most of mutants and Giza 2 showed marked increase for protein content as compared with Giza1, however, the increase did not reach the level of significance for the most mutants as compared with Giza2 in the three generations. Except of M4 generation. marked resistance for infestations with C.chinensis and H.armigera was obtained for mutants 1, 5 and 11 in the three generations. However, for total infestation with the two insects, resistance was obtained in mutants 4 and 10. Except of mutant lines 1, 5 and 11, all mutants showed higher loss percentage than that of the local varieties

Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

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Hassan Abdel-Latif A. Mohamed

2006-06-01

Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6, and rad9 of Saccharomyces cerevisiae. [nicking

Energy Technology Data Exchange (ETDEWEB)

Prakash, L [Rochester Univ., N.Y. (USA). Dept. of Radiation Biology and Biophysics; Rochester Univ., N.Y. (USA). School of Medicine and Dentistry)

1977-10-01

The ability to remove ultraviolet-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 uv-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.

High protein mutants of winter fodder barley induced by radiation and chemical mutagens

Energy Technology Data Exchange (ETDEWEB)

Yankulov, M.; Genchev, K.; Nikolov, Kh.

1982-01-01

Several induced mutants of winter fodder barley with higher rpotein content are described. These mutants were produced by treating seeds of cvs. Vogelsaenger Gold, Ager and 468 with gamma-rays, sodium azide and ethyl methanesulfonate (alone and in combinations) and with ethylene and formamide. The gamma-ray induced mutants of winter fodder barley have 1-4% higher protein content. The mutant line 109 has, besides high protein content (17,37%), 5.96 lysine per 100 g protein, but its endosperm is wrinkeled. Mutants produced by chemical mutagens have 6-7% higher protein content than the initial cultivars. All induced mutants have 85-95 cm high stems, i.e. they are by 10-20 cm shorter than the initial cultivars. Some of these mutants are now resistant to the diseases Helminthosporium gramineum and Ustilago nuda. The recommended mutants could be successfully used in breeding programs for producing of higher protein content and quality in winter fodder barley.

High protein mutants of winter fodder barley induced by radiation and chemical mutagens

International Nuclear Information System (INIS)

Yankulov, M.; Genchev, K.; Nikolov, Kh.

1982-01-01

Several induced mutants of winter fodder barley with higher rpotein content are described. These mutants were produced by treating seeds of cvs. Vogelsaenger Gold, Ager and 468 with gamma-rays, sodium azide and ethyl methanesulfonate (alone and in combinations) and with ethylene and formamide. The gamma-ray induced mutants of winter fodder barley have 1-4% higher protein content. The mutant line 109 has, besides high protein content (17,37%), 5.96 lysine per 100 g protein, but its endosperm is wrinkeled. Mutants produced by chemical mutagens have 6-7% higher protein content than the initial cultivars. All induced mutants have 85-95 cm high stems, i.e. they are by 10-20 cm shorter than the initial cultivars. Some of these mutants are now resistant to the diseases Helminthosporium gramineum and Ustilago nuda. The recommended mutants could be successfully used in breeding programs for producing of higher protein content and quality in winter fodder barley

Accumulation of infectious mutants in stocks during the propagation of fiber-modified recombinant adenoviruses

International Nuclear Information System (INIS)

Ugai, Hideyo; Inabe, Kumiko; Yamasaki, Takahito; Murata, Takehide; Obata, Yuichi; Hamada, Hirofumi; Yokoyama, Kazunari K.

2005-01-01

In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3' end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions

[Hepatitis E virus infection in patients with clinical diagnosis of viral hepatitis in Colombia].

Science.gov (United States)

Peláez, Dioselina; Hoyos, María Cristina; Rendón, Julio César; Mantilla, Carolina; Ospina, Martha Cecilia; Cortés-Mancera, Fabián; Pérez, Olga Lucía; Contreras, Lady; Estepa, Yaneth; Arbeláez, María Patricia; Navas, María Cristina

2014-01-01

Hepatitis E virus (HEV) is an emergent virus of global importance; it is the etiological agent of sporadic cases and outbreaks of hepatitis. The epidemiology of this infection in Colombia is unknown. To determine the seropositivity for hepatitis E virus in Colombia in cases with clinical diagnosis of viral hepatitis. Serum samples from patients that were sent to the Instituto Nacional de Salud during the period 2005-2010 (group 1) and samples sent to the Laboratorio Departamental de Salud Pública de Antioquia during the 2008-2009 period were included in this study (group 2). Serum samples were analyzed by immunoassay with commercial kits. From the 344 analyzed samples, 8.7% were positive for anti-HEV; the frequency of anti-HEV IgM was 1.74% (6/344) and the frequency of anti-HEV IgG was 7.5% (26/344). A difference in frequency of anti-HEV between group 1 (6.3%) and group 2 (1.3%) was observed. The cases were identified in nine departments of Colombia. This is the first study of hepatitis E virus infection in patients with diagnosis of hepatitis in Colombia. The frequency of anti-HEV described in this population of patients in Colombia is similar to that described in other Latin American countries like Brazil, Perú and Uruguay. Considering the results of this study, it could be necessary to include hepatitis E virus infection serological markers in the differential diagnosis of viral hepatitis in Colombia.

Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages.

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Matthew Dale Woolard

2013-02-01

Full Text Available Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS induces macrophages to synthesize prostaglandin E2 (PGE2. Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain Francisella tularensis subspecies novicida U112 (U112 two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI. Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used

Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

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Jonathan M.O. Rawson

2014-09-01

Full Text Available Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved.

Hepatitis B viral factors and treatment responses in chronic hepatitis B

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Chih-Lin Lin

2013-06-01

Full Text Available Baseline and on-treatment hepatitis B viral factors are reported to affect treatment responses. A lower baseline hepatitis B virus (HBV DNA level is a strong predictor of the response to antiviral therapy. HBV genotype A/B patients have better responses to interferon-based therapy than those with genotypes C/D. Regarding the association of HBV mutants with responses to antiviral therapy, current evidence is limited. On-treatment viral suppression is the most important predictor of response to nucleoside analogs. On-treatment hepatitis B surface antigen decline is significantly associated with response to pegylated interferon. In the future, individualized therapy should be based on treatment efficacy, adverse effects, baseline and on-treatment predictors of antiviral therapy.

BRAF-V600E mutant papillary craniopharyngioma dramatically responds to combination BRAF and MEK inhibitors.

Science.gov (United States)

Roque, Ashley; Odia, Yazmin

2017-04-01

We present a patient with BRAF-V600E mutant papillary craniopharyngioma successfully treated with combination BRAF (dabrafenib 150 mg twice daily) and MEK (trametinib 2 mg daily) inhibitors after her unresectable tumor proved refractory to radiation. Serial brain MRIs and PET revealed marked tumor reduction with gradual neurological improvement and permanent panhypopituitarism.

Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation

DEFF Research Database (Denmark)

Hammer, Karin; Jensen, Kaj Frank; Poulsen, Peter

1987-01-01

Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage lambda cII protein were isolated. The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of c......II or the activation by cII of the lambda promoters pE and pI. The sck-1, sck-2, and sck-3 mutations modify transcription termination. The growth of lambda, but not of the N-independent lambda variant, lambda nin-5, is hindered by these mutations, which act either alone or in concert with the bacterial nusA1 mutation....... In contrast to their effect on lambda growth, the three mutations reduce transcription termination in bacterial operons. The E. coli pyrE gene, which is normally regulated by attenuation, is expressed constitutively in the mutant strains. The sck mutations appear to prevent pyrE attenuation by slowing...

PedonnanceofE3rly MatUring MutantS Derived from ''SuPa'~ Rice ...

African Journals Online (AJOL)

Vienna, Austria in 1994. The dry seeds were in-adiated with gamma rays using three doses (170, 210. --iifid 24OC;Y).frOm C.obalt 60 (lCO) in order shorten the plant height and maturity period. From the resulting mutant. PoPulations ortgindtiriifroni modified single seed descent method, five Jery early maturing lines plus the ...

Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD.

Science.gov (United States)

Zhang, Le; Willemse, Joost; Hoskisson, Paul A; van Wezel, Gilles P

2018-05-09

Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

Construction of a catalytically inactive cholesterol oxidase mutant: investigation of the interplay between active site-residues glutamate 361 and histidine 447.

Science.gov (United States)

Yin, Ye; Liu, Pingsheng; Anderson, Richard G W; Sampson, Nicole S

2002-06-15

Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-5-en-3-one and its subsequent isomerization into cholest-4-en-3-one. Two active-site residues, His447 and Glu361, are important for catalyzing the oxidation and isomerization reactions, respectively. Double-mutants were constructed to test the interplay between these residues in catalysis. We observed that the k(cat) of oxidation for the H447Q/E361Q mutant was 3-fold less than that for H447Q and that the k(cat) of oxidation for the H447E/E361Q mutant was 10-fold slower than that for H447E. Because both doubles-mutants do not have a carboxylate at position 361, they do not catalyze isomerization of the reaction intermediate cholest-5-en-3-one to cholest-4-en-3-one. These results suggest that Glu361 can compensate for the loss of histidine at position 447 by acting as a general base catalyst for oxidation of cholesterol. Importantly, the construction of the double-mutant H447E/E361Q yields an enzyme that is 31,000-fold slower than wild type in k(cat) for oxidation. The H447E/E361Q mutant is folded like native enzyme and still associates with model membranes. Thus, this mutant may be used to study the effects of membrane binding in the absence of catalytic activity. It is demonstrated that in assays with caveolae membrane fractions, the wild-type enzyme uncouples platelet-derived growth factor receptor beta (PDGFRbeta) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant does not. Thus maintenance of membrane structure by cholesterol is important for PDGFRbeta-mediated signaling. The cholesterol oxidase mutant probe described will be generally useful for investigating the role of membrane structure in signal transduction pathways in addition to the PDGFRbeta-dependent pathway tested.

Comparisons of protein, lipid, phenolics, γ-oryzanol, vitamin E, and mineral contents in bran layer of sodium azide-induced red rice mutants.

Science.gov (United States)

Jeng, Toong Long; Ho, Pei Tzu; Shih, Yi Ju; Lai, Chia Chi; Wu, Min Tze; Sung, Jih Min

2011-06-01

The bran part of red rice grain is concentrated with many phytochemicals, including proanthocyanidins, oryzanol and vitamin E, that exert beneficial effects on human health, but it contains low levels of essential minerals such as Fe and Zn. In the present study, the protein, lipid, phytochemicals and mineral contents in bran samples were compared among red rice SA-586 and its NaN₃-induced mutants. The plant heights of NaN₃-induced mutants were decreased. The contents of protein, lipid, total phenolics, total flavonoids, total anthocyanins, total proanthocyanidins, total γ-oryzanol, total tocopherols and total tocotrienols also varied among the tested mutants. The brans of mutants M-18, M-56 and M-50 contained more proanthocyanidins, γ-oryzanol, vitamin E than that of SA-586, respectively. M-54 accumulated more Fe content (588.7 mg kg⁻¹ bran dry weight) than SA-586 (100.1 mg kg⁻¹ bran dry weight). The brans of M-18, M-50 and M-56 are good sources of proanthocyanidins, vitamin E and γ-oryzanol, respectively, while the bran of M-54 is rich in Fe. Thus these mutants could be used to produce high-value phytochemicals or Fe byproducts from bran during rice grain milling or as genetic resources for rice improvement programs. Copyright © 2011 Society of Chemical Industry.

Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage.

Science.gov (United States)

Guan, Su Hua; Belsham, Graham J

2017-04-01

Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

Post-Transcriptional Regulation of KLF4 by High-Risk Human Papillomaviruses Is Necessary for the Differentiation-Dependent Viral Life Cycle.

Directory of Open Access Journals (Sweden)

Vignesh Kumar Gunasekharan

2016-07-01

Full Text Available Human papillomaviruses (HPVs are epithelial tropic viruses that link their productive life cycles to the differentiation of infected host keratinocytes. A subset of the over 200 HPV types, referred to as high-risk, are the causative agents of most anogenital malignancies. HPVs infect cells in the basal layer, but restrict viral genome amplification, late gene expression, and capsid assembly to highly differentiated cells that are active in the cell cycle. In this study, we demonstrate that HPV proteins regulate the expression and activities of a critical cellular transcription factor, KLF4, through post-transcriptional and post-translational mechanisms. Our studies show that KLF4 regulates differentiation as well as cell cycle progression, and binds to sequences in the upstream regulatory region (URR to regulate viral transcription in cooperation with Blimp1. KLF4 levels are increased in HPV-positive cells through a post-transcriptional mechanism involving E7-mediated suppression of cellular miR-145, as well as at the post-translational level by E6-directed inhibition of its sumoylation and phosphorylation. The alterations in KLF4 levels and functions results in activation and suppression of a subset of KLF4 target genes, including TCHHL1, VIM, ACTN1, and POT1, that is distinct from that seen in normal keratinocytes. Knockdown of KLF4 with shRNAs in cells that maintain HPV episomes blocked genome amplification and abolished late gene expression upon differentiation. While KLF4 is indispensable for the proliferation and differentiation of normal keratinocytes, it is necessary only for differentiation-associated functions of HPV-positive keratinocytes. Increases in KLF4 levels alone do not appear to be sufficient to explain the effects on proliferation and differentiation of HPV-positive cells indicating that additional modifications are important. KLF4 has also been shown to be a critical regulator of lytic Epstein Barr virus (EBV replication

Gamma ray induced mutants in Coleus

Energy Technology Data Exchange (ETDEWEB)

Vasudevan, K; Jos, J S [Central Tuber Crops Research Institute, Trivandrum, Kerala (India)

1988-07-01

The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M{sub 1}V{sub 1} generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m{sup 2} area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing.

Gamma ray induced mutants in Coleus

International Nuclear Information System (INIS)

Vasudevan, K.; Jos, J.S.

1988-01-01

The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M 1 V 1 generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m 2 area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing

Association between depressive symptoms, CD4 count and HIV viral suppression among HIV-HCV co-infected people.

Science.gov (United States)

Aibibula, Wusiman; Cox, Joseph; Hamelin, Anne-Marie; Moodie, Erica E M; Anema, Aranka; Klein, Marina B; Brassard, Paul

2018-05-01

Depressive symptoms are associated with poor HIV viral control and immune recovery among people living with HIV. However, no prior studies assessed this association exclusively among people co-infected with HIV-hepatitis C virus (HCV). While people with HIV only and those with HIV-HCV co-infection share many characteristics, co-infected people may become more susceptible to the effects of depressive symptoms on health outcomes. We assessed this association exclusively among people co-infected with HIV-HCV in Canada using data from the Food Security & HIV-HCV Sub-Study (FS Sub-Study) of the Canadian Co-Infection Cohort (CCC). Stabilized inverse probability weighted marginal structural model was used to account for potential time-varying confounders. A total of 725 participants were enrolled between 2012 and 2015. At baseline, 52% of participants reported depressive symptoms, 75% had undetectable HIV viral load, and median CD4 count was 466 (IQR 300-665). People experiencing depressive symptoms had 1.32 times (95% CI: 1.07, 1.63) the risk of having detectable HIV viral load, but had comparable CD4 count to people who did not experience depressive symptoms (fold change of CD4 = 0.96, 95% CI: 0.91, 1.03). Presence of depressive symptoms is a risk factor for incomplete short-term HIV viral suppression among people co-infected with HIV-HCV. Therefore, depressive symptoms screening and related counseling may improve HIV related health outcomes and reduce HIV transmission.

The value of point-of-care CD4+ and laboratory viral load in tailoring antiretroviral therapy monitoring strategies to resource limitations.

Science.gov (United States)

Hyle, Emily P; Jani, Ilesh V; Rosettie, Katherine L; Wood, Robin; Osher, Benjamin; Resch, Stephen; Pei, Pamela P; Maggiore, Paolo; Freedberg, Kenneth A; Peter, Trevor; Parker, Robert A; Walensky, Rochelle P

2017-09-24

To examine the clinical and economic value of point-of-care CD4 (POC-CD4) or viral load monitoring compared with current practices in Mozambique, a country representative of the diverse resource limitations encountered by HIV treatment programs in sub-Saharan Africa. We use the Cost-Effectiveness of Preventing AIDS Complications-International model to examine the clinical impact, cost (2014 US$), and incremental cost-effectiveness ratio [$/year of life saved (YLS)] of ART monitoring strategies in Mozambique. We compare: monitoring for clinical disease progression [clinical ART monitoring strategy (CLIN)] vs. annual POC-CD4 in rural settings without laboratory services and biannual laboratory CD4 (LAB-CD4), biannual POC-CD4, and annual viral load in urban settings with laboratory services. We examine the impact of a range of values in sensitivity analyses, using Mozambique's 2014 per capita gross domestic product ($620) as a benchmark cost-effectiveness threshold. In rural settings, annual POC-CD4 compared to CLIN improves life expectancy by 2.8 years, reduces time on failed ART by 0.6 years, and yields an incremental cost-effectiveness ratio of $480/YLS. In urban settings, biannual POC-CD4 is more expensive and less effective than viral load. Compared to biannual LAB-CD4, viral load improves life expectancy by 0.6 years, reduces time on failed ART by 1.0 year, and is cost-effective ($440/YLS). In rural settings, annual POC-CD4 improves clinical outcomes and is cost-effective compared to CLIN. In urban settings, viral load has the greatest clinical benefit and is cost-effective compared to biannual POC-CD4 or LAB-CD4. Tailoring ART monitoring strategies to specific settings with different available resources can improve clinical outcomes while remaining economically efficient.

Induction of Mutants in Durum Wheat

International Nuclear Information System (INIS)

AL-Ubaidi, M.; Ibrahim, I.; AL-Hadithi, A.

2002-01-01

This investigation presents a breeding program for induction and development of a new genotype of durum wheat, resistant to lodging with high yield, by irradiation durum wheat hybrids (F2) with gamma rays 100 Gy, during 1990-1997 cultivation seasons. This program involves: induction of variability, selection evaluation of the mutants at three locations: Twaitha (Baghdad) Latifya ( Babylon) and Swari (Kutt). All mutants showed resistance to lodging and there was a significant reduction in plant height. Mutant SIXIZ-22 surpassed other mutants and its origin in lodging resistance and plant height (83.5,82.8 and 89.4 cm) in the three locations at generation M5 and M6, respectively. Also, there were significant differences between mutant and their origin in the number of spikes/M 2 and grain yild during the two successive generation. On the other hand, mutant IZxCO-105 surpassed other mutants in the number of spikes/M 2 (231.8,242.3 and 292) and grain yield (4336,3376 and 5232 kg/ha) in all testing location, respectively . (authors) 14 refs., 4 tabs

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

Energy Technology Data Exchange (ETDEWEB)

Cornelis, J.; Su, Z.Z.; Dinsart, C.; Rommelaere, J. (Universite libre de Bruxelles, Rhode St Genese (Belgium))

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

Science.gov (United States)

Davis, William G; Blackwell, Jerry L; Shi, Pei-Yong; Brinton, Margo A

2007-09-01

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

3,4-Methylenedioxymethamphetamine (MDMA) alters acute gammaherpesvirus burden and limits Interleukin 27 responses in a mouse model of viral infection

Science.gov (United States)

Nelson, Daniel A.; Singh, Sam J.; Young, Amy B.; Tolbert, Melanie D.; Bost, Kenneth L.

2011-01-01

Aims To test whether 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) abuse might increase the susceptibility, or alter the immune response, to murine gammaherpesvirus 68 (HV-68) and/or bacterial lipopolysaccharide. Methods Groups of experimental and control mice were subjected to three day binges of MDMA, and the effect of this drug abuse on acute and latent HV-68 viral burden were assessed. In vitro and in vivo studies were also performed to assess the MDMA effect on IL-27 expression in virally infected or LPS-exposed macrophages and dendritic cells, and latently infected animals, exposed to this drug of abuse. Results Acute viral burden was significantly increased in MDMA-treated mice when compared to controls. However the latent viral burden, and physiological and behavioral responses were not altered in infected mice despite repeated bingeing with MDMA. MDMA could limit the IL-27 response of HV-68 infected or LPS-exposed macrophages and dendritic cells in vitro and in vivo, demonstrating the ability of this drug to alter normal cytokine responses in the context of a viral infection and/or a TLR4 agonist. Conclusion MDMA bingeing could alter the host’s immune response resulting in greater acute viral replication and reductions in the production of the cytokine, IL-27 during immune responses. PMID:21269783

Evaluation of the G145R Mutant of the Hepatitis B Virus as a Minor Strain in Mother-to-Child Transmission.

Directory of Open Access Journals (Sweden)

Haruki Komatsu

Full Text Available The role of the hepatitis B virus (HBV mutant G145R, with a single change in amino acid 145 of the surface protein, as a minor population remains unknown in mother-to-child transmission. The minor strain as well as the major strain of the G145R mutant were evaluated in three cohorts using a locked nucleic acid probe-based real-time PCR. The breakthrough cohort consisted of children who were born to HBV carrier mothers and became HBV carriers despite immnoprophylaxis (n = 25. The control cohort consisted of HBV carriers who had no history of receiving the hepatitis B vaccine, hepatitis B immunoglobulin or antiviral treatment (n = 126. The pregnant cohort comprised pregnant women with chronic HBV infection (n = 31. In the breakthrough cohort, 6 showed positive PCR results (major, 2; minor, 4. In the control cohort, 13 showed positive PCR results (major, 0; minor, 13. HBeAg-positive patients were prone to have the G145R mutant as a minor population. Deep sequencing was performed in a total of 32 children (PCR positive, n = 13; negative, n = 19. In the breakthrough cohort, the frequency of the G145R mutant ranged from 0.54% to 6.58%. In the control cohort, the frequency of the G145R mutant ranged from 0.42% to 4.1%. Of the 31 pregnant women, 4 showed positive PCR results (major, n = 0; minor, n = 4. All of the pregnant women were positive for HBeAg and showed a high viral load. Three babies born to 3 pregnant women with the G145R mutant were evaluated. After the completion of immunoprophylaxis, 2 infants became negative for HBsAg. The remaining infant became negative for HBsAg after the first dose of HB vaccine. G145R was detected in one-fourth of the children with immunoprophylaxis failure. However, the pre-existence of the G145R mutant as a minor population in pregnant women does not always cause breakthrough infection in infants.

Long-term Mortality in HIV-Positive Individuals Virally Suppressed for >3 Years With Incomplete CD4 Recovery

DEFF Research Database (Denmark)

Engsig, Frederik N; Zangerle, Robert; Katsarou, Olga

2014-01-01

BACKGROUND: Some human immunodeficiency virus (HIV)-infected individuals initiating combination antiretroviral therapy (cART) with low CD4 counts achieve viral suppression but not CD4 cell recovery. We aimed to identify (1) risk factors for failure to achieve CD4 count >200 cells/µL after 3 years...... of the suppressed period. Logistic regression was used to identify risk factors for incomplete CD4 recovery (≤200 cells/µL) and Cox regression to identify associations with mortality. RESULTS: Of 5550 eligible individuals, 835 (15%) did not reach a CD4 count >200 cells/µL after 3 years of suppression. Increasing...... age, lower initial CD4 count, male heterosexual and injection drug use transmission, cART initiation after 1998, and longer time from initiation of cART to start of the virally suppressed period were risk factors for not achieving a CD4 count >200 cells/µL. Individuals with CD4 ≤200 cells/µL after 3...

Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

Science.gov (United States)

Varela, Mariana; Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong; Palmarini, Massimo

2016-06-01

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc

Functionality screen of streptavidin mutants by non-denaturing SDS-PAGE using biotin-4-fluorescein.

Science.gov (United States)

Humbert, Nicolas; Ward, Thomas R

2008-01-01

Site-directed mutagenesis or directed evolution of proteins often leads to the production of inactive mutants. For streptavidin and related proteins, mutations may lead to the loss of their biotin-binding properties. With high-throughput screening methodologies in mind, it is imperative to detect, prior to the high-density protein production, the bacteria that produce non-functional streptavidin isoforms. Based on the incorporation of biotin-4-fluorescein in streptavidin mutants present in Escherichia coli bacterial extracts, we detail a functional screen that allows the identification of biotin-binding streptavidin variants. Bacteria are cultivated in a small volume, followed by a rapid treatment of the cells; biotin-4-fluorescein is added to the bacterial extract and loaded on an Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) under non-denaturing conditions. Revealing is performed using a UV transilluminator. This screen is thus easy to implement, cheap and requires only readily available equipment.

Potent inhibition of HIV-1 replication by a Tat mutant.

Directory of Open Access Journals (Sweden)

Luke W Meredith

Full Text Available Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.

The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication.

Directory of Open Access Journals (Sweden)

Jie Zhang

Full Text Available Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1-471 of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

International Nuclear Information System (INIS)

Kim, Seong K.; Kim, Seongman; Dai Gan; Zhang Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

2011-01-01

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: → We examine the functional domains of IR2P that mediates negative regulation. → IR2P inhibits at the transcriptional level. → DNA-binding mutant or TFIIB-binding mutant fails to inhibit. → C-terminal aa 707 to 1116 are required for full inhibition. → Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.

Sodium 4-phenylbutyrate ameliorates the effects of cataract-causing mutant gammaD-crystallin in cultured cells.

Science.gov (United States)

Gong, Bo; Zhang, Li-Yun; Lam, Dennis Shun-Chiu; Pang, Chi-Pui; Yam, Gary Hin-Fai

2010-06-04

gammaD-Crystallin (CRYGD) is a major structural lens crystallin and its mutations result in congenital cataract formation. In this study, we attempted to correct the altered protein features of G165fsX8 CRYGD protein with small chemical molecules. Recombinant FLAG-tagged mutants (R15C, R15S, P24T, R61C, and G165fsX8) of CRYGD were expressed in COS-7 cells and treated with small chemical molecules with reported protein chaperoning properties (sodium 4-phenylbutyrate [4-PBA], trimethylamine N-oxide [TMAO], and glycerol and DMSO [DMSO]). Protein solubility in 0.5% Triton X-100 and subcellular distribution was examined by western blotting and immunofluorescence, respectively. Apoptosis was assayed as the percentage of fragmented nuclei in transfected cells. Expression of heat-shock proteins (Hsp70 and Hsp90) was examined by reverse transcription-polymerase chain reaction analysis. Unlike WT and most mutants (R15C, R15S, P24T, and R61C) of CRYGD, G165fsX8 CRYGD was significantly insoluble in 0.5% Triton X-100. This insolubility was alleviated by dose-dependent 4-PBA treatment. The treatment relieved the mislocalization of G165fsX8 CRYGD from the nuclear envelope. Also, 4-PBA treatment reduced cell apoptosis and caused an upregulation of Hsp70. 4-PBA treatment reduced the defective phenotype of mutant G165fsX8 CRYGD and rescued the affected cells from apoptosis. This could be a potential treatment for lens structural protein and prevent lens opacity in cataract formation.

Comparison of the health-related quality of life, CD4 count and viral ...

African Journals Online (AJOL)

This study compared the level of CD4 count, viral load and health-related quality of life (HRQOL) between treatment-naı¨ve AIDS patients and a cohort of people living with HIV who have been on treatment for 12 months. This study is based on a secondary data analysis of the records of 642 people with HIV consisting of ...

Mutant matrix metalloproteinase-9 reduces postoperative peritoneal adhesions in rats.

Science.gov (United States)

Atta, Hussein; El-Rehany, Mahmoud; Roeb, Elke; Abdel-Ghany, Hend; Ramzy, Maggie; Gaber, Shereen

2016-02-01

Postoperative peritoneal adhesions continue to be a major source of morbidity and occasional mortality. Studies have shown that matrix metalloproteinase-9 (MMP-9) levels are decreased postoperatively which may limits matrix degradation and participate in the development of peritoneal adhesions. In this proof-of-principle study, we evaluated the effect of gene therapy with catalytically inactive mutant MMP-9 on postoperative peritoneal adhesions in rats. Adenovirus encoding mutant MMP-9 (Ad-mMMP-9) or saline was instilled in the peritoneal cavity after cecal and parietal peritoneal injury in rats. Expression of mutant MMP-9 transcript was verified by sequencing. Adenovirus E4 gene expression, adhesion scores, MMP-9, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β1 (TGF-β1) expression were evaluated at sacrifice one week after treatment. Both mutant MMP-9 transcripts and adenovirus E4 gene were expressed in Ad-mMMP-9 treated adhesions. Adhesions severity decreased significantly (p = 0.036) in the Ad-mMMP-9-treated compared with saline-treated adhesions. Expression of MMP-9 mRNA and protein were elevated (p = 0.001 and p = 0.029, respectively) in the Ad-mMMP-9-treated adhesions compared with saline-treated adhesions. While tPA levels were increased (p = 0.02) in Ad-mMMP-9 treated adhesions compared with saline-treated adhesions, TGF-β1 and PAI-1 levels were decreased (p = 0.017 and p = 0.042, respectively). No difference in mortality were found between groups (p = 0.64). Mutant MMP-9 gene therapy effectively transduced peritoneal adhesions resulting in reduction of severity of primary peritoneal adhesions. Copyright © 2016 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

Characteristics of 36C103- influx into nitrate reductase deficient mutant E1 pisum sativum seedlings: evidence for restricted ''induction'' by nitrate compared with wild type

International Nuclear Information System (INIS)

Deane-Drummond, C.E.; Jacobsen, E.

1986-01-01

The characteristics of nitrate uptake into seedlings of Pisum sativum L. cv. Rondo mutant E 1 defective for nitrate reductase (NR) and of its parent variety Rondo have been investigated using 36 C10 3 - as an analogue for nitrate. The apparent Michaelis Menten constants (K m ) for 36 ClO 3 - influx measured over 10 min were similar for mutant E 1 and the wild type (Wt). There was a 28% increase in 36 C10 3 - into Wt seedlings following nitrate pretreatment but this was not found when mutant seedlings were used. N starvation increased 36 C10 3 - influx into both mutant and Wt seedlings, and the rate of cycling E/I was also enhanced to a similar extent. The results are discussed in terms of current ideas on the regulation of nitrate uptake and assimilation. (author)

Root hair mutants of barley

International Nuclear Information System (INIS)

Engvild, K.C.; Rasmussen, K.

2005-01-01

Barley mutants without root hairs or with short or reduced root hairs were isolated among M 2 seeds of 'Lux' barley (Hordeum vulgare L.) after acidified sodium azide mutagenesis. Root hair mutants are investigated intensively in Arabidopsis where about 40 genes are known. A few root hair mutants are known in maize, rice, barley and tomato. Many plants without root hairs grow quite well with good plant nutrition, and mutants have been used for investigations of uptake of strongly bound nutrients like phosphorus, iron, zinc and silicon. Seed of 'Lux' barley (Sejet Plant Breeding, Denmark) were soaked overnight, and then treated with 1.5-millimolarsodium azide in 0.1 molar sodium phosphate buffer, pH 3, for 2.5 hours according to the IAEA Manual on Mutation Breeding (2nd Ed.). After rinsing in tap water and air-drying, the M 2 seeds were sown in the field the same day. Spikes, 4-6 per M 1 plant, were harvested. The mutation frequency was similar to that obtained with other barley cultivars from which low-phytate mutants were isolated [5]. Seeds were germinated on black filter paper in tap water for 3 or 4 days before scoring for root hair mutants

SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release.

Science.gov (United States)

Raghava, Smita; Giorda, Kristina M; Romano, Fabian B; Heuck, Alejandro P; Hebert, Daniel N

2013-06-04

Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results revealed that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of 1-5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.

Modelling Hepatitis B Virus Antiviral Therapy and Drug Resistant Mutant Strains

Science.gov (United States)

Bernal, Julie; Dix, Trevor; Allison, Lloyd; Bartholomeusz, Angeline; Yuen, Lilly

Despite the existence of vaccines, the Hepatitis B virus (HBV) is still a serious global health concern. HBV targets liver cells. It has an unusual replication process involving an RNA pre-genome that the reverse transcriptase domain of the viral polymerase protein translates into viral DNA. The reverse transcription process is error prone and together with the high replication rates of the virus, allows the virus to exist as a heterogeneous population of mutants, known as a quasispecies, that can adapt and become resistant to antiviral therapy. This study presents an individual-based model of HBV inside an artificial liver, and associated blood serum, undergoing antiviral therapy. This model aims to provide insights into the evolution of the HBV quasispecies and the individual contribution of HBV mutations in the outcome of therapy.

Dabrafenib plus trametinib in patients with previously untreated BRAFV600E-mutant metastatic non-small-cell lung cancer: an open-label, phase 2 trial.

Science.gov (United States)

Planchard, David; Smit, Egbert F; Groen, Harry J M; Mazieres, Julien; Besse, Benjamin; Helland, Åslaug; Giannone, Vanessa; D'Amelio, Anthony M; Zhang, Pingkuan; Mookerjee, Bijoyesh; Johnson, Bruce E

2017-10-01

BRAF V600E mutation occurs in 1-2% of lung adenocarcinomas and acts as an oncogenic driver. Dabrafenib, alone or combined with trametinib, has shown substantial antitumour activity in patients with previously treated BRAF V600E -mutant metastatic non-small-cell lung cancer (NSCLC). We aimed to assess the activity and safety of dabrafenib plus trametinib treatment in previously untreated patients with BRAF V600E -mutant metastatic NSCLC. In this phase 2, sequentially enrolled, multicohort, multicentre, non-randomised, open-label study, adults (≥18 years of age) with previously untreated metastatic BRAF V600E -mutant NSCLC were enrolled into cohort C from 19 centres in eight countries within North America, Europe, and Asia. Patients received oral dabrafenib 150 mg twice per day plus oral trametinib 2 mg once per day until disease progression, unacceptable adverse events, consent withdrawal, or death. The primary endpoint was investigator-assessed overall response, defined as the percentage of patients who achieved a confirmed complete response or partial response per Response Evaluation Criteria In Solid Tumors version 1.1. The primary and safety analyses were by intention to treat in the protocol-defined population (previously untreated patients). The study is ongoing, but no longer recruiting patients. This trial is registered with ClinicalTrials.gov, number NCT01336634. Between April 16, 2014, and Dec 28, 2015, 36 patients were enrolled and treated with first-line dabrafenib plus trametinib. Median follow-up was 15·9 months (IQR 7·8-22·0) at the data cutoff (April 28, 2017). The proportion of patients with investigator-assessed confirmed overall response was 23 (64%, 95% CI 46-79), with two (6%) patients achieving a complete response and 21 (58%) a partial response. All patients had one or more adverse event of any grade, and 25 (69%) had one or more grade 3 or 4 event. The most common (occurring in more than two patients) grade 3 or 4 adverse events were

[Outbreaks of viral hepatitis E in the Czech Republic?].

Science.gov (United States)

Trmal, Josef; Pavlík, Ivo; Vasícková, Petra; Matejícková, Ladislava; Simůnková, Lenka; Luks, Stanislav; Pazderková, Jana

2012-05-01

Until recently, viral hepatitis E (VHE) has typically been an imported infection, related to travel to developing countries. A number of travel-unrelated VHE cases currently diagnosed in the Czech Republic. Outcomes of the epidemiological investigations of two VHE outbreaks associated with the consumption of pork and pork products at pig-slaughtering feasts are presented. Thirteen cases have been reported in the first outbreak and eight cases in the second outbreak. The epidemiological investigations are described and the experience gained in analysing suspected biological specimens is presented. The source of infection has not been identified in the first outbreak while in the other one, a link between human cases and infection in farm pigs was revealed for the first time. Although the epidemiological investigation may not always lead to the detection of the VHE source, it must be conducted in any outbreak and can only be successful when done in cooperation of the public health authorities with the veterinary health agency.

Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue shifted intermediate

NARCIS (Netherlands)

Derix, N.M.; Wechselberger, R.W.|info:eu-repo/dai/nl/304829005; van der Horst, M.A.; Hellingwerf, K.J.; Boelens, R.|info:eu-repo/dai/nl/070151407; Kaptein, R.|info:eu-repo/dai/nl/074334603; van Nuland, N.A.J.

2003-01-01

The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB

Active Site Dynamics in Substrate Hydrolysis Catalyzed by DapE Enzyme and Its Mutants from Hybrid QM/MM-Molecular Dynamics Simulation.

Science.gov (United States)

Dutta, Debodyuti; Mishra, Sabyashachi

2017-07-27

The mechanism of the catalytic hydrolysis of N-succinyl diaminopimelic acid (SDAP) by the microbial enzyme DapE in its wild-type (wt) form as well as three of its mutants (E134D, H67A, and H349A) is investigated employing a hybrid quantum mechanics/molecular mechanics (QM/MM) method coupled with molecular dynamics (MD) simulations, wherein the time evolution of the atoms of the QM and MM regions are obtained from the forces acting on the individual atoms. The free-energy profiles along the reaction coordinates of this multistep hydrolysis reaction process are explored using a combination of equilibrium and nonequilibrium (umbrella sampling) QM/MM-MD simulation techniques. In the enzyme-substrate complexes of wt-DapE and the E134D mutant, nucleophilic attack is found to be the rate-determining step involving a barrier of 15.3 and 21.5 kcal/mol, respectively, which satisfactorily explains the free energy of activation obtained from kinetic experiments in wt-DapE-SDAP (15.2 kcal/mol) and the 3 orders of magnitude decrease in the catalytic activity due to E134D mutation. The catalysis is found to be quenched in the H67A and H349A mutants of DapE due to conformational rearrangement in the active site induced by the absence of the active site His residues that prohibits activation of the catalytic water molecule.

The Arabidopsis aba4-1 mutant reveals a specific function for neoxanthin in protection against photooxidative stress.

Science.gov (United States)

Dall'Osto, Luca; Cazzaniga, Stefano; North, Helen; Marion-Poll, Annie; Bassi, Roberto

2007-03-01

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions.

The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

International Nuclear Information System (INIS)

Mu, J.-J.; Tsay, Y.-G.; Juan, L.-J.; Fu, T.-F.; Huang, W.-H.; Chen, D.-S.; Chen, P.-J.

2004-01-01

Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (S-HDAg and L-HDAg). The S-HDAg is essential for viral RNA replication while the L-HDAg is required for viral assembly. In this study, we demonstrated that HDAg are acetylated proteins. Metabolic labeling with [ 3 H]acetate revealed that both forms of HDAg could be acetylated in vivo. The histone acetyltransferase (HAT) domain of cellular acetyltransferase p300 could acetylate the full-length and the N-terminal 88 amino acids of S-HDAg in vitro. By mass spectrometric analysis of the modified protein, Lys-72 of S-HDAg was identified as one of the acetylation sites. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. The mutant reduced viral RNA accumulation and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and mutation of HDAg at Lys-72 modulates HDAg subcellular localization and may participate in viral RNA nucleocytoplasmic shuttling and replication

Identification and molecular characterization of the second Chlamydomonas gun4 mutant, gun4-II [v2; ref status: indexed, http://f1000r.es/1id

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Phillip B Grovenstein

2013-07-01

Full Text Available The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study oxygenic photosynthesis. Chlorophyll (Chl and heme are major tetrapyrroles that play an essential role in photosynthesis and respiration. These tetrapyrroles are synthesized via a common branched pathway that involves mainly enzymes, encoded by nuclear genes. One of the enzymes in the pathway is Mg chelatase (MgChel. MgChel catalyzes insertion of Mg2+ into protoporphyrin IX (PPIX, proto to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto, the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4 protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

BRD4 is associated with raccoon polyomavirus genome and mediates viral gene transcription and maintenance of a stem cell state in neuroglial tumour cells.

Science.gov (United States)

Church, Molly E; Estrada, Marko; Leutenegger, Christian M; Dela Cruz, Florante N; Pesavento, Patricia A; Woolard, Kevin D

2016-11-01

Polyomavirus infection often results in persistence of the viral genome with little or no virion production. However, infection of certain cell types can result in high viral gene transcription and either cytolysis or neoplastic transformation. While infection by polyomavirus is common in humans and many animals, major questions regarding viral persistence of most polyomaviruses remain unanswered. Specifically, identification of target cells for viral infection and the mechanisms polyomaviruses employ to maintain viral genomes within cells are important not only in ascribing causality to polyomaviruses in disease, but in understanding specific mechanisms by which they cause disease. Here, we characterize the cell of origin in raccoon polyomavirus (RacPyV)-associated neuroglial brain tumours as a neural stem cell. Moreover, we identify an association between the viral genome and the host cell bromodomain protein, BRD4, which is involved in numerous cellular functions, including cell cycle progression, differentiation of stem cells, tethering of persistent DNA viruses, and regulation of viral and host-cell gene transcription. We demonstrate that inhibition of BRD4 by the small molecule inhibitors (+)-JQ1 and IBET-151 (GSK1210151A) results in reduced RacPyV genome within cells in vitro, as well as significant reduction of viral gene transcripts LT and VP1, highlighting its importance in both maintenance of the viral genome and in driving oncogenic transformation by RacPyV. This work implicates BRD4 as a central protein involved in RacPyV neuroglial tumour cell proliferation and in the maintenance of a stem cell state.

Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV in cattle

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Loy John Dustin

2013-01-01

Full Text Available Abstract Background Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen. Findings Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection. Conclusions Replicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

Enterocin A mutants identified by saturation mutagenesis enhance potency towards vancomycin-resistant Enterococci.

Science.gov (United States)

McClintock, Maria K; Kaznessis, Yiannis N; Hackel, Benjamin J

2016-02-01

Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13 ± 3- and 18 ± 4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. © 2015 Wiley Periodicals, Inc.

Absence of mutation at the 5'-upstream promoter region of the TPM4 gene from cardiac mutant axolotl (Ambystoma mexicanum).

Science.gov (United States)

Denz, Christopher R; Zhang, Chi; Jia, Pingping; Du, Jianfeng; Huang, Xupei; Dube, Syamalima; Thomas, Anish; Poiesz, Bernard J; Dube, Dipak K

2011-09-01

Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter-reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.

Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase.

Science.gov (United States)

Chen, Qi; Cheng, Xiaolin; Wei, Dongqing; Xu, Qin

2015-03-01

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we herein use the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

Synthesis of (E-2,4-Dinitro-N-((2E,4E-4-phenyl-5-(pyrrolidin-1-ylpenta-2,4-dienylideneaniline

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Mostafa Fesanghari

2009-07-01

Full Text Available (E-2,4-Dinitro-N-((2E,4E-4-phenyl-5-(pyrrolidin-1-ylpenta-2,4-dienylidene aniline dye was prepared in one pot by reaction of premade N-2,4-dinitrophenyl-3-phenylpyridinium chloride (DNPPC and pyrrolidine in absolute MeOH.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

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Anna Lundin

2014-05-01

Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

High yielding mutants of blackgram variety 'PH-25'

International Nuclear Information System (INIS)

Misra, R.C.; Mohapatra, B.D.; Panda, B.S.

2001-01-01

Seeds of blackgram (Vigna mungo L.) variety 'PH-5' were treated with chemical mutagens ethyl methanesulfonate (EMS), nitrosoguanidine (NG), maleic hydrazide (MH) and sodium azide (NaN 3 ), each at 3 different concentrations. Thirty six mutant lines developed from mutagenic treatments along with parent varieties were tested in M 4 generation. The mutants showed wide variation in most of the traits and multivariante D 2 analysis showed genetic divergence among themselves. Twenty of the thirty mutants showed genetic divergence from parent. Ten selected high yielding mutants were tested in M 5 . Yield and other productive traits of five high yielding mutants in M 4 and M 5 are presented

Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii

International Nuclear Information System (INIS)

Soriano, Erika V.; McCloskey, Diane E.; Kinsland, Cynthia; Pegg, Anthony E.; Ealick, Steven E.

2008-01-01

The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction

New insights from an old mutant: SPADIX4 governs fruiting body development but not hyphal fusion in Sordaria macrospora.

Science.gov (United States)

Teichert, Ines; Lutomski, Miriam; Märker, Ramona; Nowrousian, Minou; Kück, Ulrich

2017-02-01

During the sexual life cycle of filamentous fungi, multicellular fruiting bodies are generated for the dispersal of spores. The filamentous ascomycete Sordaria macrospora has a long history as a model system for studying fruiting body formation, and two collections of sterile mutants have been generated. However, for most of these mutants, the underlying genetic defect remains unknown. Here, we investigated the mutant spadix (spd) that was generated by X-ray mutagenesis in the 1950s and terminates sexual development after the formation of pre-fruiting bodies (protoperithecia). We sequenced the spd genome and found a 22 kb deletion affecting four genes, which we termed spd1-4. Generation of deletion strains revealed that only spd4 is required for fruiting body formation. Although sterility in S. macrospora is often coupled with a vegetative hyphal fusion defect, Δspd4 was still capable of fusion. This feature distinguishes SPD4 from many other regulators of sexual development. Remarkably, GFP-tagged SPD4 accumulated in the nuclei of vegetative hyphae and fruiting body initials, the ascogonial coils, but not in sterile tissue from the developing protoperithecium. Our results point to SPD4 as a specific determinant of fruiting body formation. Research on SPD4 will, therefore, contribute to understanding cellular reprogramming during initiation of sexual development in fungi.

BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes.

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Laura J Marinelli

Full Text Available Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED, in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags.

Combined BRAF and HSP90 inhibition in patients with unresectable BRAF V600E mutant melanoma.

Science.gov (United States)

Eroglu, Zeynep; Chen, Yian Ann; Gibney, Geoffrey T; Weber, Jeffrey S; Kudchadkar, Ragini R; Khushalani, Nikhil I; Markowitz, Joseph; Brohl, Andrew S; Tetteh, Leticia F; Ramadan, Howida; Arnone, Gina; Li, Jiannong; Zhao, Xiuhua; Sharma, Ritin; Darville, Lancia N F; Fang, Bin; Smalley, Inna; Messina, Jane L; Koomen, John M; Sondak, Vernon K; Smalley, Keiran S M

2018-04-19

BRAF inhibitors are clinically active in patients with advanced BRAF V600 -mutant melanoma, although acquired resistance remains common. Preclinical studies demonstrated that resistance could be overcome using concurrent treatment with the HSP90 inhibitor XL888. Vemurafenib (960 mg PO BID) combined with escalating doses of XL888 (30, 45, 90 or 135 mg PO twice weekly) was investigated in 21 patients with advanced BRAF V600 -mutant melanoma. Primary endpoints were safety and determination of a maximum tolerated dose. Correlative proteomic studies were performed to confirm HSP inhibitor activity. Objective responses were observed in 15/20 evaluable patients (75%; 95% CI: 51-91%), with 3 complete and 12 partial responses. Median progression-free and overall survival were 9.2 months (95% CI: 3.8-not reached) and 34.6 months (6.2-not reached), respectively. The most common grade 3/4 toxicities were skin toxicities such as rash (n=4, 19%) and cutaneous squamous cell carcinomas (n=3, 14%), along with diarrhea (n=3, 14%). Pharmacodynamic analysis of patients' PBMCs showed increased day 8 HSP70 expression compared to baseline in the three cohorts with XL888 doses ≥45 mg. Diverse effects of vemurafenib-XL888 upon intratumoral HSP-client protein expression were noted, with the expression of multiple proteins (including ERBB3 and BAD) modulated on therapy. XL888 in combination with vemurafenib has clinical activity in patients with advanced BRAF V600 -mutant melanoma, with a tolerable side-effect profile. HSP90 inhibitors warrant further evaluation in combination with current standard-of-care BRAF plus MEK inhibitors in BRAF V600 -mutant melanoma. Copyright ©2018, American Association for Cancer Research.

Dwarf mutant of Papaver somniferum with high morphine content

International Nuclear Information System (INIS)

Chauhan, S.P.; Patra, N.K.; Srivastava, H.K.

1987-01-01

Opium poppy, Papaver somniferum L. is an important medicinal plant known for its morphine, codeine, and thebaine alkaloids. This Institute had earlier released two latex opium yielding poppy varieties, Shyama and Shweta, which are now cultivated by the farmers under the supervision of the Narcotic Department of the Government of India. However, both these varieties became susceptible to downy mildew (Peronospora arborescens). Lodging due to heavy capsule weight is another problem affecting latex yield. With these problems in mind, we undertook mutation breeding on the above mentioned two varieties employing gamma rays (5 kR, 15 kR, 20 kR) and EMS (0.2%, 0.4%, 0.6%) and combined mutagens (5 kR + 0.2% EMS, 5 kR + 0.4% EMS and 5 kR + 0.6% EMS). M 1 from the treated seeds (405 plants) was raised in winter 1984-85. M 2 generation of 13,500 plants (i.e. 270 M 1 progenies x 50 plants) was raised in winter 1985/86. A dwarf mutant with high morphine content was identified in M 2 from the variety Shweta treated with 5 kR + 0.4% EMS. The mutant differs by its dwarf stature, compact leaf arrangements, multilocular capsules, increased capsule number, and small capsule size. The mutant is under testing for its superior morphine production. It may be used as dwarf gene source in hybridization for improving lodging resistance. This mutant is a novel type, which was not available in our germplasm collection

Detection of Genotype 4 Swine Hepatitis E Virus in Systemic Tissues in Cross-Species Infected Rabbits

OpenAIRE

Wu, Qiaoxing; An, Junqing; She, Ruiping; Shi, Ruihan; Hao, Wenzhuo; Soomro, MajidHussain; Yuan, Xuerui; Yang, Jinling; Wang, Jingyuan

2017-01-01

Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal...

Microbiological diagnostics of viral hepatitis

OpenAIRE

HASDEMİR, Ufuk

2016-01-01

Viral hepatitis is an infection that primarily affects the liverbut may also have systemic clinical manifestations. The vastmajority of viral hepatitis are caused by one of five hepatotropicviruses: hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis D (delta) virus (HDV), andhepatitis E virus (HEV) (Table I) [1]. HBV, HCV, and HDValso cause chronic hepatitis, whereas HAV does not. HEVcauses acute hepatitis in normal hosts but can cause protractedand chronic he...

Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice.

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Chun Fu

Full Text Available After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance protein. Fanconi anemia (FA proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2-3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line.

A highly intensified ART regimen induces long-term viral suppression and restriction of the viral reservoir in a simian AIDS model.

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Iart Luca Shytaj

Full Text Available Stably suppressed viremia during ART is essential for establishing reliable simian models for HIV/AIDS. We tested the efficacy of a multidrug ART (highly intensified ART in a wide range of viremic conditions (10³-10⁷ viral RNA copies/mL in SIVmac251-infected rhesus macaques, and its impact on the viral reservoir. Eleven macaques in the pre-AIDS stage of the disease were treated with a multidrug combination (highly intensified ART consisting of two nucleosidic/nucleotidic reverse transcriptase inhibitors (emtricitabine and tenofovir, an integrase inhibitor (raltegravir, a protease inhibitor (ritonavir-boosted darunavir and the CCR5 blocker maraviroc. All animals stably displayed viral loads below the limit of detection of the assay (i.e. <40 RNA copies/mL after starting highly intensified ART. By increasing the sensitivity of the assay to 3 RNA copies/mL, viral load was still below the limit of detection in all subjects tested. Importantly, viral DNA resulted below the assay detection limit (<2 copies of DNA/5*10⁵ cells in PBMCs and rectal biopsies of all animals at the end of the follow-up, and in lymph node biopsies from the majority of the study subjects. Moreover, highly intensified ART decreased central/transitional memory, effector memory and activated (HLA-DR⁺ effector memory CD4⁺ T-cells in vivo, in line with the role of these subsets as the main cell subpopulations harbouring the virus. Finally, treatment with highly intensified ART at viral load rebound following suspension of a previous anti-reservoir therapy eventually improved the spontaneous containment of viral load following suspension of the second therapeutic cycle, thus leading to a persistent suppression of viremia in the absence of ART. In conclusion, we show, for the first time, complete suppression of viral load by highly intensified ART and a likely associated restriction of the viral reservoir in the macaque AIDS model, making it a useful platform for testing

Radiation induced mutants in cassava (Manihot esculenta Crantz)

International Nuclear Information System (INIS)

Nayar, G.G.; Rajendran, P.G.

1987-01-01

Full text: Stem cuttings and true seeds of three promising cultivars of cassava were exposed respectively to 1 to 5 kR and 10 to 50 kR acute gamma rays from a 60 Co source. Treatments of stem cuttings beyond 5 kR and seeds beyond 50 kR were lethal. One mutant each in the cultivars M4, H-165 and H-2304 was obtained from the stem irradiated populations. Another mutant was found in the seed irradiated progeny of H-2304. The mutant of M4 is characterised by light green (chlorina) leaves. The mutant of H-165 shows significantly shorter petiole (22,5 against 35.2 cm) and narrow leaf lobes, while the H-2304 mutant shows speckled leaves, branching and early flowering. The mutant found in the seed irradiated progeny of H-2304 is having yellow tuber flesh indicating the presence of carotene. The mutants may be useful in studies related to basic information as well as in practical breeding. The chlorina mutant in M4 showed slow growth and high HCN content in leaves. Late branching may be a useful trait in the traditionally non-branching clones of cassava to maintain the desirable leaf area index during high leaf fall period. Early flowering could be useful in a recombinant breeding programme. The tuber yield of the short petiole mutant in H-165 increased by 20% - 25% through closer planting. The narrow leaf lobes of this mutant permit better light penetration to lower leaves. (author)

Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants.

Science.gov (United States)

Glerum, D M; Tzagoloff, A

1997-08-04

The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

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

Impact of Chloroquine on Viral Load in Breast Milk

Science.gov (United States)

Semrau, Katherine; Kuhn, Louise; Kasonde, Prisca; Sinkala, Moses; Kankasa, Chipepo; Shutes, Erin; Vwalika, Cheswa; Ghosh, Mrinal; Aldrovandi, Grace; Thea, Donald M.

2006-01-01

Summary The anti-malarial agent chloroquine has activity against HIV. We compared the effect of chloroquine (n = 18) to an anti-malarial agent without known anti-HIV-activity, sulfadoxine-pyrimethamine (n = 12), on breast milk HIV RNA levels among HIV-infected breastfeeding women in Zambia. After adjusting for CD4 count and plasma viral load, chloroquine was associated with a trend towards lower levels of HIV RNA in breast milk compared with sulfadoxine-pyrimethamine (P 0.05). Higher breastmilk viral load was also observed among women receiving presumptive treatment = for symptomatic malaria compared with asymptomatic controls and among controls reporting fever in the prior week. Further research is needed to determine the potential role of chloroquine in prevention of HIV transmission through breastfeeding. Impacte de la chloroquine sur la charge virale dans le lait maternelle La chloroquine, agent antimalarique, a une activité contre le VIH. Nous avons comparé l’effet de la chloroquine à celui d’un autre agent antimalarique, la sulfadoxine-pyrimethamine, dont l’activité sur le VIH n’est pas connue, en mesurant les taux d’ARN de VIH dans le lait maternel de femmes allaitantes infectées par le VIH en Zambie. Après ajustement pour les taux de CD4 et la charge virale dans le plasma, la chloroquine comparée à la sulfadoxine pyrimethamine était associée à une tendance vers des teneurs plus bas en ARN de VIH dans le lait maternel (P = 0,05). Des charges virales plus élevées dans le lait maternel étaient aussi observées chez des femmes recevant un traitement présomptif pour des symptômes de malaria par rapport aux contrôles asymptomatiques et par rapport à des contrôles rapportant de la fièvre durant la première semaine. Des études supplémentaires sont nécessaires pour déterminer le rôle potentiel de la chloroquine dans la prévention de la transmission du VIH par l’allaitement maternel. mots clésVIH, malaria, allaitement maternel

Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

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Yang Lin

2013-02-01

Full Text Available Abstract Adeno-associated virus (AAV is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolution of viral vectors. We further attempted to evolve the AAV using DNA shuffling and in vivo biopanning in a mouse model. An AAVM41 mutant was characterized, which was found to have improved transduction efficiency and specificity in myocardium, an attribute unknown for any natural AAV serotypes. This review focuses on the development of AAV vector for cardiac gene transfer, the history of directed evolution of viral vectors, and our creation of a cardiotropic AAV, which might have implications for the future design and application of viral vectors.

Experimentally-induced immune activation in natural hosts of SIV induces significant increases in viral replication and CD4+ T cell depletion

Energy Technology Data Exchange (ETDEWEB)

Ribeiro, Ruy M [Los Alamos National Laboratory

2008-01-01

Chronically SIVagm-infected African green monkeys (AGMs) have a remarkably stable non-pathogenic disease course, with levels of immune activation in chronic SIVagm infection similar to those observed in uninfected monkeys and stable viral loads (VLs) for long periods of time. In vivo administration of lipopolysaccharide (LPS) or an IL-2/diphtheria toxin fusion protein (Ontak) to chronically SIVagm-infected AGMs triggered increases in immune activation and subsequently of viral replication and depletion of intestinal CD4{sup +} T cells. Our study indicates that circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating that immune activation and T cell prolifeation are key factors in AIDS pathogenesis.

The Arabidopsis aba4-1 Mutant Reveals a Specific Function for Neoxanthin in Protection against Photooxidative Stress[W

Science.gov (United States)

Dall'Osto, Luca; Cazzaniga, Stefano; North, Helen; Marion-Poll, Annie; Bassi, Roberto

2007-01-01

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions. PMID:17351115

Rescuing mutant CFTR: a multi-task approach to a better outcome in treating cystic fibrosis.

Science.gov (United States)

Amaral, Margarida D; Farinha, Carlos M

2013-01-01

Correcting multiple defects of mutant CFTR with small molecule compounds has been the goal of an increasing number of recent Cystic Fibrosis (CF) drug discovery programmes. However, the mechanism of action (MoA) by which these molecules restore mutant CFTR is still poorly understood, in particular of CFTR correctors, i.e., compounds rescuing to the cells surface the most prevalent mutant in CF patients--F508del-CFTR. However, there is increasing evidence that to fully restore the multiple defects associated with F508del-CFTR, different small molecules with distinct corrective properties may be required. Towards this goal, a better insight into MoA of correctors is needed and several constraints should be addressed. The methodological approaches to achieve this include: 1) testing the combined effect of compounds with that of other (non-pharmacological) rescuing strategies (e.g., revertants or low temperature); 2) assessing effects in multiple cellular models (non-epithelial vs epithelial, non-human vs human, immortalized vs primary cultures, polarized vs non polarized, cells vs tissues); 3) assessing compound effects on isolated CFTR domains (e.g., compound binding by surface plasmon resonance, assessing effects on domain folding and aggregation); and finally 4) assessing compounds specificity in rescuing different CFTR mutants and other mutant proteins. These topics are reviewed and discussed here so as to provide a state-of-the art review on how to combine multiple ways of rescuing mutant CFTR to the ultimate benefit of CF patients.

Establishment of screening technique for mutant cell and analysis of base sequence in the mutation

International Nuclear Information System (INIS)

Sofuni, Toshio; Nomi, Takehiko; Yamada, Masami; Masumura, Kenichi

2000-01-01

This research project aimed to establish an easy and quick detection method for radiation-induced mutation using molecular-biological techniques and an effective analyzing method for the molecular changes in base sequence. In this year, Spi mutants derived from γ-radiation exposed mouse were analyzed by PCR method and DNA sequence method. Male transgenic mice were exposed to γ-ray at 5,10, 50 Gy and the transgene was taken out from the genome DNA from the spleen in vivo packaging method. Spi mutant plaques were obtained by infecting the recovered phage to E. coli. Sequence analysis for the mutants was made using ALFred DNA sequencer and SequiTherm TM Long-Red Cycle sequencing kit. Sequence analysis was carried out for 41 of 50 independent Spi mutants obtained. The deletions were classified into 4 groups; Group 1 included 15 mutants that were characterized with a large deletion (43 bp-10 kb) with a short homologous sequence. Group 2 included 11 mutants of a large deletion having no homologous sequence at the connecting region. Group 3 included 11 mutants having a short deletion of less than 20 bp, which occurred in the non-repetitive sequence of gam gene and possibly caused by oxidative breakage of DNA or recombination of DNA fragment produced by the breakage. Group 4 included 4 mutants having deletions as short as 20 bp or less in the repetitive sequence of gam gene, resulting in an alteration of the reading frame. Thus, the synthesis of Gam protein was terminated by the appearance of TGA between code 13 and 14 of redB gene, leading to inactivation of gam gene and redBA gene. These results indicated that most of Spi mutants had a deletion in red/gam region and the deletions in more than half mutants occurred in homologous sequences as short as 8 bp. (M.N.)

Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase

Energy Technology Data Exchange (ETDEWEB)

Chen, Qi [Shanghai Jiao Tong Univ., Shanghai (China). State Key Lab. of Microbial Metabolism and College of Life Science and Biotechnology; Cheng, Xiaolin [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Molecular Biophysics; Univ. of Tennessee, Knoxville, TN (United States). Dept. of Biochemistry and Cellular and Molecular Biology; Wei, Dongqing [Shanghai Jiao Tong Univ., Shanghai (China). State Key Lab. of Microbial Metabolism and College of Life Science and Biotechnology; Xu, Qin [Shanghai Jiao Tong Univ., Shanghai (China). State Key Lab. of Microbial Metabolism and College of Life Science and Biotechnology

2014-11-06

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we use in this paper the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

Deletion mutants of region E1 a of AD12 E1 plasmids: Effect on oncogenic transformation

NARCIS (Netherlands)

Bos, J.L.; Jochemsen, A.G.; Bernards, R.A.; Schrier, P.I.; Ormondt, H. van; Eb, A.J. van der

1983-01-01

Plasmids containing the El region of Ad12 DNA can transform certain rodent cells into oncogenic cells. To study the role of the Ela subregion in the process of oncogenic transformation, Ad12 region El mutants carrying deletions in the Ela region were constructed. Deletion mutants pR7 and pR8 affect

TAF-4 is required for the life extension of isp-1, clk-1 and tpk-1 Mit mutants.

Science.gov (United States)

Khan, Maruf H; Ligon, Melissa; Hussey, Lauren R; Hufnal, Bryce; Farber, Robert; Munkácsy, Erin; Rodriguez, Amanda; Dillow, Andy; Kahlig, Erynn; Rea, Shane L

2013-10-01

While numerous life-extending manipulations have been discovered in the nematode Caenorhabditis elegans, one that remains most enigmatic is disruption of oxidative phosphorylation. In order to unravel how such an ostensibly deleterious manipulation can extend lifespan, we sought to identify the ensemble of nuclear transcription factors that are activated in response to defective mitochondrial electron transport chain (ETC) function. Using a feeding RNAi approach, we targeted over 400 transcription factors and identified 15 that, when reduced in function, reproducibly and differentially altered the development, stress response, and/or fecundity of isp-1(qm150) Mit mutants relative to wild-type animals. Seven of these transcription factors--AHA-1, CEH-18, HIF-1, JUN-1, NHR-27, NHR-49 and the CREB homolog-1 (CRH-1)-interacting protein TAF-4--were also essential for isp-1 life extension. When we tested the involvement of these seven transcription factors in the life extension of two other Mit mutants, namely clk-1(qm30) and tpk-1(qm162), TAF-4 and HIF-1 were consistently required. Our findings suggest that the Mit phenotype is under the control of multiple transcriptional responses, and that TAF-4 and HIF-1 may be part of a general signaling axis that specifies Mit mutant life extension.

Counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment.

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Celia Perales

Full Text Available BACKGROUND: Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, non-mutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin. METHODOLOGY AND PRINCIPAL FINDINGS: Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis. CONCLUSIONS: (i When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a

CD4 decline is associated with increased risk of cardiovascular disease, cancer, and death in virally suppressed patients with HIV.

Science.gov (United States)

Helleberg, Marie; Kronborg, Gitte; Larsen, Carsten S; Pedersen, Gitte; Pedersen, Court; Obel, Niels; Gerstoft, Jan

2013-07-01

The clinical implications of a considerable CD4 decline despite antiretroviral treatment and viral suppression are unknown. We aimed to test the hypothesis that a major CD4 decline could be a marker of cardiovascular disease or undiagnosed cancer. Patients with human immunodeficiency virus (HIV) were followed in the Danish nationwide, population-based cohort study in the period 1995-2010 with quarterly CD4 measurements. Associations between a CD4 decline of ≥30% and cardiovascular disease, cancer, and death were analyzed using Poisson regression with date of CD4 decline as a time-updated variable. We followed 2584 virally suppressed HIV patients for 13 369 person-years (PY; median observation time, 4.7 years). Fifty-six patients developed CD4 decline (incidence rate, 4.2/1000 PY [95% confidence interval {CI}, 3.2-5.4]). CD4 counts dropped from a median of 492 cells/µL to 240 cells/µL. CD8, CD3, and total lymphocyte counts dropped concomitantly. No HIV-related factors, apart from treatment with didanosine, were associated with CD4 decline. The risk of cardiovascular disease, cancer, and death increased markedly ≤6 months after CD4 decline (incidence rate ratio, 11.7 [95% CI, 3.6-37.4] and 13.7 [95% CI, 4.3-43.6], respectively, and mortality rate ratio 4.3 [95% CI, 1.1-17.6]). A major decline in CD4 count is associated with a marked increased risk of cardiovascular disease, cancer, and death among virally suppressed HIV patients.

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2'-O-Methylation Mutant.

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Bianca Schmid

2015-12-01

Full Text Available Dengue virus (DENV is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

Spectrum of induced floral mutants in Petunia

International Nuclear Information System (INIS)

Padmaja, V.; Sudhakar, P.

1987-01-01

A total of six floral mutants of garden Petunia isolated from the populations raised from the seed treatment with γ-rays, 2, 4-D and sodium azide are described. Five of the mutants viz. stellata, Campyloflora, Rubriflora mixed, Grandiflora and Albiflora mixed originated as segregants in M 2 generation while the chimeral floral phenotype was expressed in M 1 generation itself. Breeding behaviour of these horticulturally interesting altered floral phenotypes were studied in subsequent generations and appropriate conclusions were drawn regarding mode of inheritance of the mutant traits. 15 refs., 4 figures, 1 table. (author)

DIARREA VIRAL BOVINA: PATOGÉNESIS E INMUNOPATOLOGÍA

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Iang Rondón

2006-05-01

Full Text Available La diarrea viral bovina (DVB representa un problema de ámbito mundial que causa considerables pérdidastanto en ganado de carne como lechero, afectándolo de diversas formas las cuales están supeditadas ala edad del animal, estado inmunológico y momento de la gestación en el que se produce la infección.La DVB es causada por un virus ARN, género Pestivirus, familia Flaviviridae, el cual ha sido clasificado en2 biotipos (citopático y no citopático según su comportamiento en células de cultivo y en 2 genotipos (Iy II basados en su secuencia genética. Dependiendo de las cepas infectantes se presenta un cuadroclínico particular variando en severidad desde una forma subclínica, pasando por la forma clínica eincluso produciendo la fatal enfermedad de las mucosas o causando efectos deletéreos sobre el feto. Apesar de que en nuestro medio ya existen estudios sobre esta entidad, la implementación de metodologíasdiagnósticas constituye una limitante para el manejo de la misma. La presente revisión se enfoca en lapatogenia e inmunopatología de la DVB.

Molecular basis for increased risk for late-onset Alzheimer disease due to the naturally occurring L28P mutation in apolipoprotein E4.

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Argyri, Letta; Dafnis, Ioannis; Theodossiou, Theodossis A; Gantz, Donald; Stratikos, Efstratios; Chroni, Angeliki

2014-05-02

The apolipoprotein (apo) E4 isoform has consistently emerged as a susceptibility factor for late-onset Alzheimer disease (AD), although the exact mechanism is not clear. A rare apoE4 mutant, apoE4[L28P] Pittsburgh, burdens carriers with an added risk for late-onset AD and may be a useful tool for gaining insights into the role of apoE4 in disease pathogenesis. Toward this end, we evaluated the effect of the L28P mutation on the structural and functional properties of apoE4. ApoE4[L28P] was found to have significantly perturbed thermodynamic properties, to have reduced helical content, and to expose a larger portion of the hydrophobic surface to the solvent. Furthermore, this mutant is thermodynamically destabilized and more prone to proteolysis. When interacting with lipids, apoE4[L28P] formed populations of lipoprotein particles with structural defects. The structural perturbations brought about by the mutation were accompanied by aberrant functions associated with the pathogenesis of AD. Specifically, apoE4[L28P] promoted the cellular uptake of extracellular amyloid β peptide 42 (Aβ42) by human neuroblastoma SK-N-SH cells as well as by primary mouse neuronal cells and led to increased formation of intracellular reactive oxygen species that persisted for at least 24 h. Furthermore, lipoprotein particles containing apoE4[L28P] induced intracellular reactive oxygen species formation and reduced SK-N-SH cell viability. Overall, our findings suggest that the L28P mutation leads to significant structural and conformational perturbations in apoE4 and can induce functional defects associated with neuronal Aβ42 accumulation and oxidative stress. We propose that these structural and functional changes underlie the observed added risk for AD development in carriers of apoE4[L28P].

Genetical, cytological and physiological studies on the induced mutants with special regard to effective methods for obtaining useful mutants in perennial woody plant

International Nuclear Information System (INIS)

Kukimura, H.; Ikeda, F.; Fujita, H.; Maeta, T.; Nakajima, K.; Katagiri, K.; Nakahira, K.; Somegou, M.

1976-01-01

The plants studied included apple trees, cryptomeria (japanese cedar) and mulberry. In apple, dwarf and compact types of mutants from cv. Fuji were found to be graft incompatible on Maruba-kaido(Malus prunifolia) rootstock. In Sunki mandarin(Citrus sunki), the number of nucellar embryo per seed was affected by gamma-irradiation, and morphological mutants from nucellar seedlings were obtained at high rate by irradiation at floral bud stage with 2kR exposure. In Cryptomeria, re-irradiated waxless mutants by gamma-rays showed very high rate of somatic mutation when compared to other morphological mutants. Pollen sterility and pollen shaped PMC were found in the most of gamma-induced-mutants. Mutants forming pollen shaped PMC had a genetical tendency of continuous male flower bud formation for a longer term. With mulberry, time of sprouting of induced mutants differed from the originals. Ability of root initiation of semi-softwood cuttings in morphological mutants were tested. Cytochimera induction were found at considerably high rate when actively growing diploid plants were irradiated by gamma-rays. Eight kinds of cytochimeras were induced. Frequency of 2-4-4 was extremely high(approx. 50%), then 4-2-2 and 2-4-2 chimeras followed. Seven kinds were induced by semi-acute irradiation(200R/h), while 4 kinds by acute irradiation(5kR/h). By breeding test it was cleared that the elongate and entire leaf was sexually transmissible, whereas the 'dwarf' was not obvious and the 'marginally curledleaf' was not transmissible. Pyronin-methylgreen staining method proved to be useful in some morphological mutants to distinguish the histo-genetical differences which exist in the shoot apex.

Fighting Viral Infections and Virus-Driven Tumors with Cytotoxic CD4+ T Cells

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Muraro, Elena; Merlo, Anna; Martorelli, Debora; Cangemi, Michela; Dalla Santa, Silvia; Dolcetti, Riccardo; Rosato, Antonio

2017-01-01

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors. PMID:28289418

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

Science.gov (United States)

Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

2018-06-01

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2

International Nuclear Information System (INIS)

Zerler, B.R.; Wallace, S.S.

1984-01-01

The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair. There was no detecttable pyrimidine dimer-DNA N-glycosylase activity, and pyrimidine dimer removal appeared to occur by a pathway analogous to the Escherichia coli Uvr pathway. The UV- and MMS-sensitive mutants of A. espejiana included three groups, each containing at least one mutation involved with excision, recombination, or inducible repair. One group that was UV sensitive but not sensitive to MMS or X rays showed a decreased ability to excise pyrimidine dimers. Mutants in this group were also sensitive to psoralen plus near-UV light and were phenotypically analogous to the E. coli uvr mutants. A second group was UV and MMS sensitive but not sensitive to X rays and appeared to contain mutations in a gene(s) involved in recombination repair. These recombination-deficient mutants differed from the E. coli rec mutants, which are MMS and X-ray sensitive. The third group of A. espejiana mutants was sensitive to UV, MMS, and X rays. These mutants were recombination deficient, lacked inducible repair, and were phenotypically similar to E. coli recA mutants

Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

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Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

2017-08-01

Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A.

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Cole, C N; Tornow, J; Clark, R; Tjian, R

1986-01-01

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional

A review of hepatitis viral infections in Pakistan

International Nuclear Information System (INIS)

Bosan, A.; Qureshi, H.; Bile, K.M.; Ahmad, I.; Hafiz, R.

2010-01-01

A review of published literature on viral hepatitis infections in Pakistan is presented. A total of 220 abstracts available in the Pakmedinet and Medline have been searched. All relevant articles were reviewed to determine the prevalence of hepatitis viral infections in Pakistan. Two hundred and three (203) relevant articles/abstracts including twenty nine supporting references are included in this review. Of the articles on prevalence of hepatitis infection, seven were related to Hepatitis A, fifteen to Hepatitis E while the remaining articles were on frequency of hepatitis B and C in different disease and healthy population groups. These included eight studies on healthy children, three on vertical transmission, nineteen on pregnant women, fifteen on healthy individuals, six on army recruits, thirty one on blood donors, thirteen on health care workers, five on unsafe injections, seventeen on high risk groups, five on patients with provisional diagnosis of hepatitis, thirty three on patients with chronic liver disease, four on genotypes of HBV and five on genotypes of HCV. This review highlights the lack of community-based epidemiological work as the number of subjects studied were predominantly patients, high risk groups and healthy blood donors. High level of Hepatitis A seroconversion was found in children and this viral infection accounts for almost 50%- 60% of all cases of acute viral hepatitis in children in Pakistan. Hepatitis E is endemic in the country affecting mostly the adult population and epidemic situations have been reported from many parts of the country. The mean results of HBsAg and Anti-HCV prevalence on the basis of data aggregated from several studies was calculated which shows 2.3% and 2.5% prevalence of HBsAg and Anti-HCV in children, 2.5% and 5.2% among pregnant women, 2.6% and 5.3% in general population, 3.5% and 3.1% in army recruits, 2.4% and 3.6% in blood donors, 6.0% and 5.4% in health care workers, 13.0% and 10.3% in high risk groups

Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Chen, Hang [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110122 (China); Hsiao, Yung-Chin [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Chiang, Sum-Fu [Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Wu, Chia-Chun; Lin, Yu-Tsun [Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Liu, Hsuan [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Zhao, Hong [Experimental Center of Functional Subjects, College of Basic Medicine, China Medical University, Shenyang, Liaoning 110122 (China); Chen, Jinn-Shiun [Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Chang, Yu-Sun [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Otolaryngology, Chang Gung Memorial Hospital, Linkou, Taiwan (China); and others

2016-08-24

The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides ({sup 592}IGDFGLATVK{sup 601} and {sup 592}IGDFGLATEK{sup 601}, respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuring WT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32–1.66 ng) and the V600E BRAF in two cases (0.1–0.13 ng; mutant-to-WT ratio, 0.16–0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies

Studies of viral DNA packaging motors with optical tweezers: a comparison of motor function in bacteriophages φ29, λ, and T4

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Smith, Douglas E.; Fuller, Derek N.; Raymer, Dorian M.; Rickgauer, Peter; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Catalano, Carlos E.; Kottadiel, Vishal; Rao, Venigalla B.

2007-09-01

A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.

Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication.

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Bryant, Kevin F; Yan, Zhipeng; Dreyfus, David H; Knipe, David M

2012-06-01

Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.

Studies on reduced height mutants in rice

International Nuclear Information System (INIS)

Narahari, P.; Bhagwat, S.G.

1984-01-01

Two cross-bred derivatives of the mutant TR5xTR17 and TR21 continued to show promise and were advanced to wider scale testing. TR5 was found to carry a semi-dwarfing gene different from that in IR8. New semi-dwarf mutants were screened from M 2 through M 4 from two separate radiation experiments. The gibberellin response of seedlings of mutant and tester strains was evaluated and crosses of tester stocks and mutant semi-dwarfs were made for genetic analyses. (author)

Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy

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Marcin P Komorowski

2016-01-01

Full Text Available Ovarian cancer remains the most lethal gynecologic malignancy owing to late detection, intrinsic and acquired chemoresistance, and remarkable heterogeneity. Here, we explored approaches to inhibit metastatic growth of murine and human ovarian tumor variants resistant to paclitaxel and carboplatin by oncolytic vaccinia virus expressing a CXCR4 antagonist to target the CXCL12 chemokine/CXCR4 receptor signaling axis alone or in combination with doxorubicin. The resistant variants exhibited augmented expression of the hyaluronan receptor CD44 and CXCR4 along with elevated Akt and ERK1/2 activation and displayed an increased susceptibility to viral infection compared with the parental counterparts. The infected cultures were more sensitive to doxorubicin-mediated killing both in vitro and in tumor-challenged mice. Mechanistically, the combination treatment increased apoptosis and phagocytosis of tumor material by dendritic cells associated with induction of antitumor immunity. Targeting syngeneic tumors with this regimen increased intratumoral infiltration of antitumor CD8+ T cells. This was further enhanced by reducing the immunosuppressive network by the virally-delivered CXCR4 antagonist, which augmented antitumor immune responses and led to tumor-free survival. Our results define novel strategies for treatment of drug-resistant ovarian cancer that increase immunogenic cell death and reverse the immunosuppressive tumor microenvironment, culminating in antitumor immune responses that control metastatic tumor growth.

Generation and characterization of pigment mutants of ...

African Journals Online (AJOL)

Compared to the wild CC-124, these mutants are characterized by a decrease in chlorophyll a & b content and an increase in carotenoids. The lowest decrease in chlorophyll a was 3 to 4 folds, while the highest increase in carotenoids was 2 to 4 folds. The result of bio-test, using the resulting pigment mutant of C. reinhardtii ...

Identification of Bombyx mori bidensovirus VD1-ORF4 reveals a novel protein associated with viral structural component.

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Li, Guohui; Hu, Zhaoyang; Guo, Xuli; Li, Guangtian; Tang, Qi; Wang, Peng; Chen, Keping; Yao, Qin

2013-06-01

Bombyx mori bidensovirus (BmBDV) VD1-ORF4 (open reading frame 4, ORF4) consists of 3,318 nucleotides, which codes for a predicted 1,105-amino acid protein containing a conserved DNA polymerase motif. However, its functions in viral propagation remain unknown. In the current study, the transcription of VD1-ORF4 was examined from 6 to 96 h postinfection (p.i.) by RT-PCR, 5'-RACE revealed the transcription initiation site of BmBDV ORF4 to be -16 nucleotides upstream from the start codon, and 3'-RACE revealed the transcription termination site of VD1-ORF4 to be +7 nucleotides downstream from termination codon. Three different proteins were examined in the extracts of BmBDV-infected silkworms midguts by Western blot using raised antibodies against VD1-ORF4 deduced amino acid, and a specific protein band about 53 kDa was further detected in purified virions using the same antibodies. Taken together, BmBDV VD1-ORF4 codes for three or more proteins during the viral life cycle, one of which is a 53 kDa protein and confirmed to be a component of BmBDV virion.

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

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Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

2017-04-06

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

Dissociation of eIF4E-binding protein 2 (4E-BP2 from eIF4E independent of Thr37/Thr46 phosphorylation in the ischemic stress response.

Directory of Open Access Journals (Sweden)

María I Ayuso

Full Text Available Eukaryotic initiation factor (eIF 4E-binding proteins (4E-BPs are translational repressors that bind specifically to eIF4E and are critical in the control of protein translation. 4E-BP2 is the predominant 4E-BP expressed in the brain, but their role is not well known. Here, we characterized four forms of 4E-BP2 detected by two-dimensional gel electrophoresis (2-DGE in brain. The form with highest electrophoretic mobility was the main form susceptible to phosphorylation at Thr37/Thr46 sites, phosphorylation that was detected in acidic spots. Cerebral ischemia and subsequent reperfusion induced dephosphorylation and phosphorylation of 4E-BP2 at Thr37/Thr46, respectively. The induced phosphorylation was in parallel with the release of 4E-BP2 from eIF4E, although two of the phosphorylated 4E-BP2 forms were bound to eIF4E. Upon long-term reperfusion, there was a decrease in the binding of 4E-BP2 to eIF4E in cerebral cortex, demonstrated by cap binding assays and 4E-BP2-immunoprecipitation experiments. The release of 4E-BP2 from eIF4E was without changes in 4E-BP2 phosphorylation or other post-translational modification recognized by 2-DGE. These findings demonstrated specific changes in 4E-BP2/eIF4E association dependent and independent of 4E-BP2 phosphorylation. The last result supports the notion that phosphorylation may not be the uniquely regulation for the binding of 4E-BP2 to eIF4E under ischemic stress.

Baicalin benefits the anti-HBV therapy via inhibiting HBV viral RNAs.

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Huang, Hai; Zhou, Wei; Zhu, Haiyan; Zhou, Pei; Shi, Xunlong

2017-05-15

Although current antiviral treatments (nucleoside analogs, NAs) for chronic hepatitis B virus (HBV) infection are effective in suppressing HBV-DNA replication, their clinical outcomes can be compromised by the increasing drug resistance and the inefficiency in promoting HBsAg/HBeAg seroconversion. In this study, we will explore possible effects and mechanism of a natural product baicalin (BA) with the anti-HBV efficacy of entecavir (ETV), a first-line anti-HBV drug, in HBV-DNA, HBsAg/HBeAg seroconversion and drug-resistance. The co-effects of BA and ETV were conducted in wild-type/NA-resistance mutant HBV cell lines and DHBV-infected duckling models. HBV-DNA/RNAs, HBsAg/HBeAg, host factors (hepatocyte nuclear factors) were explored for possible anti-HBV mechanism. BA could significantly enhance and reduced HBsAg and HBeAg in hepG2.2.15, a wild-type HBV cell line. Co-treatment of BA and ETV had a more dramatic effect in NA-resistant HBV rtM204V/rtLl80M transfected hepG2 cells. Our study further revealed that BA mainly inhibited the production of HBV RNAs (3.5, 2.4, 2.1kb), the templates for viral proteins and HBV-DNA synthesis. BA blocked HBV RNAs transcription possibly by down-regulating transcription and expression of HBV replication dependent hepatocyte nuclear factors (HNF1α and HNF4α). Thus, BA may benefit the anti-HBV therapy via inhibiting HBV viral RNAs. Copyright © 2017 Elsevier Inc. All rights reserved.

General epidemiological parameters of viral hepatitis A, B, C, and E in six regions of China: a cross-sectional study in 2007.

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Lu, Jian; Zhou, Yongdong; Lin, Xiaojing; Jiang, Yongzhen; Tian, Ruiguang; Zhang, Yonghui; Wu, Jia; Zhang, Fengwei; Zhang, Yong; Wang, Yue; Bi, Shengli

2009-12-24

Viral hepatitis is a serious health burden worldwide. To date, few reports have addressed the prevalence of hepatitis A, B, C, and E in China. Therefore, the general epidemiological parameters of viral hepatitis remain unknown. In this cross-sectional study, we performed a serological prevalence analysis of viral hepatitis A, B, C, and E in 8,762 randomly selected Chinese subjects, which represented six areas of China. The overall prevalence of anti-Hepatitis C virus antibody (anti-HCV) was 0.58%, which was much lower than was estimated by WHO. The prevalences of Hepatitis B virus surface antigen (HBsAg), anti-Hepatitis B virus surface protein antibody (HBsAb), and anti-Hepatitis B virus core protein antibody (HBcAb) were 5.84%, 41.31%, and 35.92%, respectively, whereas in the group of subjects less than 5 years old, these prevalences were 1.16%, 46.77%, and 8.69% respectively, which suggests that the Hepatitis B virus (HBV)-carrier population is decreasing, and the nationwide HBV vaccine program has contributed to the lowered HBV prevalence in the younger generation in China. Meanwhile, a large deficit remains in coverage provided by the national HBV immune program. In addition, our data suggested the possibility that HBsAb may not last long enough to protect people from HBV infection throughout life. The overall prevalence of anti-Hepatitis A virus antibody (anti-HAV) and anti-Hepatitis E virus antibody (anti-HEV) were as high as 72.87% and 17.66%, respectively. The indices increased with age, which suggests that a large proportion of Chinese adults are protected by latent infection. Furthermore, the pattern of HEV infection was significantly different among ethnic groups in China. Our study provided much important information concerning hepatitis A, B, C, and E prevalence in China and will contribute to worldwide oversight of viral hepatitis.

Phenotypic Analysis and Virulence of Candida albicans LIG4 Mutants

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Andaluz, Encarnación; Calderone, Richard; Reyes, Guadalupe; Larriba, Germán

2001-01-01

In previous studies, we reported the isolation and preliminary characterization of a DNA ligase-encoding gene of Candida albicans. This gene (LIG4) is the structural and functional homologue of both yeast and human ligase IV, which is involved in nonhomologous end joining (NHEJ) of DNA double-strand breaks. In the present study, we have shown that there are no other LIG4 homologues in C. albicans. In order to study the function of LIG4 in morphogenesis and virulence, we constructed gene deletions. LIG4 transcript levels were reduced in the heterozygote and were completely absent in null strains. Concomitantly, the heterozygote showed a pronounced defect in myceliation, which was slightly greater in the null strain. This was true with several solid and liquid media, such as Spider medium, medium 199, and 2% glucose–1% yeast extract–2% Bacto Peptone, at several pHs. Reintroduction of the wild-type allele into the null mutant partially restored the ability of cells to form hyphae. In agreement with the positive role of LIG4 in morphogenesis, we detected a significant rise in mRNA levels during the morphological transition. LIG4 is not essential for DNA replication or for the repair of DNA damage induced by ionizing radiation or UV light, indicating that these lesions are repaired primarily by homologous recombination. However, our data show that the NHEJ apparatus of C. albicans may control morphogenesis in this diploid organism. In addition, deletion of one or both copies of LIG4 resulted in attenuation of virulence in a murine model of candidiasis. PMID:11119499

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

Science.gov (United States)

Chambouvet, Aurélie; Milner, David S.; Attah, Victoria; Terrado, Ramón; Lovejoy, Connie; Moreau, Hervé; Derelle, Évelyne; Richards, Thomas A.

2017-01-01

Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer. PMID:28827361

X-ray-induced mutants resistant to 8-azaguanine

International Nuclear Information System (INIS)

Carver, J.H.; Dewey, W.C.; Hopwood, L.E.

1976-01-01

Asynchronous Chinese hamster ovary cells were irradiated and colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 μg/ml 8-Azaguanine (AG). Data indicated that a reproducible assay for the system was dependent upon controlling cell density at least two days prior to induction as well as throughout the expression period. Generally, spontaneous and radiation-induced mutant frequencies decreased when cell densities exceeded a critical density of 3-6 x 10 4 cells/cm 2 . Infrequently, the critical density was exceeded by a factor of two with no observed decrease, possibly correlated with a longer cell doubling time. Drug depletion artifacts can occur because of drug degradation, or because wild-type cells utilize the drug or produce conditions which reduce uptake of the drug. Thus, as the effective drug concentration is lowered, the observed mutant frequency increases because a spectrum of mutants resistant to only low concentrations can now survive. In fact, refeeding with AG at intervals during the incubation period lowered spontaneous and radiation-induced frequencies approx. 5-fold. Therefore, to standardize conditions, cells were trypsinized at the end of the expression time and replated at a constant cell number for mutant selection by AG. Over two generations of growth during the expression period were required for optimal manifestation of induced mutants, and when densities were kept below 4 x 10 4 cells/cm 2 at all times, observed mutant frequencies did not change significantly over a period between 80 and 140 h post-induction (over 4 generations for irradiated cells and over 6 generations for controls). Previous reports of observed mutant frequencies decreasing beyond three generations may be due to cell interaction prior to mutant selection

Prevalence of Oral Manifestations and Their Association with CD4/CD8 Ratio and HIV Viral Load in South India

Directory of Open Access Journals (Sweden)

Sharma Gaurav

2011-01-01

Full Text Available The objective of the present research was to determine the prevalence of oral manifestations in an HIV infected population from south India and evaluate their association with HIV viral load and CD4/CD8 ratio. Intraoral examination of 103 patients, whose CD4/CD8 ratio was available, were conducted. HIV viral loads were available for thirty patients only. The prevalence of oral manifestations was 80.6% (83/103. The most common oromucosal lesion was erythematous candidiasis (EC (38.8% followed by melanotic hyperpigmentation (35.9%. Patients having any oral manifestation had a mean CD4/CD8 ratio of 0.24. EC had positive predictive value of 85.0% for CD4/CD8 ratio 20,000 copies/mL (20,000 copies/mL.

Semi-dwarf mutants in triticale and wheat breeding

International Nuclear Information System (INIS)

Driscoll, C.J.

1984-01-01

The triticale lines Beagle and DR-IRA have been subjected to ionizing irradiation and chemical mutagenesis in order to produce semi-dwarf mutants. Beagle is 100 cm tall and DR-IRA 80 cm under average field conditions. A bulk then pedigree method is currently represented by 158 single plots of M 6 (or in some cases M 7 ) mutants that are from 5 to 35 cm shorter than the control variety. The shortest mutants are 65 cm in height. Forty of these mutants are also earlier flowering than the control varieties. Replicated yield testing will be conducted on confirmed mutants in 1983. Response to gibberellic acid of these mutants will also be determined. The Cornerstone male-sterility mutant (ms1c) on chromosome arm 4Aα has been combined with the GA-insensitive/reduced height gene Gai/Rht1 which is also on chromosome arm 4Aα. The ms1c mutant has also been combined with Gai/Rht2 on chromosome 4D and with both Gai/Rht1 and Gai/Rht2. The combination ms1c and Gai/Rht1 has been chosen as the basis of a composite cross. Thirteen varieties were tested with GA 3 and seven (Warigal, Aroona, Oxley, Banks, Avocet, Matipo and Toquifen) which contain Gai/Rht1 were crossed with ms1c Gai/Rht1 and entered into an interpollinating F 2 . The entire composite is homozygous for this semi-dwarf allele and selection will be practiced for increased height on a GA-insensitive background. (author)

Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal

OpenAIRE

Narayanan, Krishna; Chen, Chun-Jen; Maeda, Junko; Makino, Shinji

2003-01-01

For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into vi...