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NS5B
About: NS5B is a research topic. Over the lifetime, 1314 publications have been published within this topic receiving 59534 citations.
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TL;DR: Three different genomic regions of four HCV strains isolated from Peruvian patients were sequenced in order to investigate the congruence of HCV genotyping for these three genomic regions, revealing that a crossover event had taken place in the NS5B protein.
Abstract: Hepatitis C virus (HCV) has high genomic variability and, since its discovery, at least six different types and an increasing number of subtypes have been reported. Genotype 1 is the most prevalent genotype found in South America. In the present study, three different genomic regions (5′UTR, core and NS5B) of four HCV strains isolated from Peruvian patients were sequenced in order to investigate the congruence of HCV genotyping for these three genomic regions. Phylogenetic analysis using 5′UTR–core sequences found strain PE22 to be related to subtype 1b. However, the same analysis using the NS5B region found it to be related to subtype 1a. To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossover event had taken place in the NS5B protein. We discuss the consequences of this observation on HCV genotype classification, laboratory diagnosis and treatment of HCV infection.
139 citations
01 Jan 2006
TL;DR: The HCV Genome and Life Cycle, Recombinant Vesicular Stomatitis Virus (VSV) and Other Strategies in HCV Vaccine Designs and Immunotherapy, and Animal Models for HCV Study are described.
Abstract: Introduction: Hepatitis C 1. HCV Genome and Life Cycle 2. HCV 5' and 3'UTR: When Translation Meets Replication 3. Assemble and Interact: Pleiotropic Functions of the HCV Core Protein 4. HCV Glycoproteins: Assembly of a Functional E1-E2 Heterodimer 5. HCV NS2/3 Protease 6. HCV NS3-4A Serine Protease 7. HCV Helicase: Structure, Function, and Inhibition 8. HCV NS4B: from Obscurity to Central Stage 9. HCV NS5A: A Multifunctional Regulator of Cellular Pathways and Virus Replication 10. Biochemical Activities of the HCV NS5B RNA-Dependent RNA Polymerase 11. HCV Replicon Systems 12. Animal Models for HCV Study 13. HCV Regulation of Host Defense 14. Regulation of Adaptive Immunity by HCV 15. Recombinant Vesicular Stomatitis Virus (VSV) and Other Strategies in HCV Vaccine Designs and Immunotherapy 16. Development of an Infectious HCV Cell Culture System
139 citations
TL;DR: The higher frequency of known NNI resistance mutations or polymorphisms known to affect their antiviral potency when compared with the lack of detection of resistance mutations to the nucleoside analogues suggests a potential for primary reduced responsiveness as well as faster development of clinically significant resistance.
Abstract: Methods: The sensitivity of genotype (GT) 1 HCV NS5B clinical isolates from treatment-naive patients to nucleoside and non-nucleoside polymerase inhibitors was assessed. The genetic diversity at the population level, as well as that of their quasispecies, was correlated with the observed reduced sensitivity to inhibitors. Results: R1479 and NM107 (nucleoside analogues that have entered Phase 2 clinical trials as prodrugs R1626 and NM283, respectively) were similarly active across the tested clinical isolates. Resistance mutations to nucleoside analogues were not observed in any of the isolates. However, the activity of the non-nucleoside thumb II inhibitor NNI-1, palm I inhibitors NNI-2 and NNI-3, and palm II inhibitor HCV-796 was reduced across different isolates. This reduction in inhibitory activity for non-nucleoside inhibitors (NNIs) was, in most cases, correlated with the existence of known NNI resistance mutations in the NS5B polymerase population of the clinical isolates, as detected by population sequencing. Resistance mutations to NNIs were also observed at a low frequency within the clinical isolates’ viral quasispecies that allowed for their rapid selection upon drug selective pressure. Conclusions: The higher frequency of known NNI resistance mutations or polymorphisms known to affect their antiviral potency when compared with the lack of detection of resistance mutations to the nucleoside analogues suggests a potential for primary reduced responsiveness as well as faster development of clinically significant resistance.
138 citations
TL;DR: These replicons were modified to express a chimeric fusion protein of firefly luciferase and neomycin phosphotransferase to yield stable replicon-expressing cells and became useful for high-throughput screening and evaluation of antiviral compounds.
Abstract: Despite recent advances in the treatment of hepatitis C, the quest for pan-genotype, effective, and well-tolerated inhibitors continues. To facilitate these efforts, it is desirable to have in vitro replication systems for all major HCV genotypes. However, cell culture replication systems exist for only genotypes 1a, 1b, and 2a. In this study, we generated G418-selectable subgenomic replicons for prototype strains of genotypes 3a (S52) and 4a (ED43). Production of G418-resistant colonies by S52 and ED43 in Huh-7.5 cells required the amino acid substitutions S2210I and R2882G, respectively, cell culture adaptive mutations originally reported for genotype 1b replicons. RNA replication was confirmed by quantitative reverse transcription-PCR and detection of viral protein. Sequencing of multiple independent replicon clones revealed the presence of additional nonsynonymous mutations. Interestingly, all potentially adaptive mutations mapped to the NS3 protein. These mutations, when introduced back into original constructs, substantially increased colony formation efficiency. To make these replicons useful for high-throughput screening and evaluation of antiviral compounds, they were modified to express a chimeric fusion protein of firefly luciferase and neomycin phosphotransferase to yield stable replicon-expressing cells. Using these constructs, the inhibitory effects of beta interferon (IFN-β), an NS3 protease inhibitor, and an NS5B nucleoside polymerase inhibitor were readily detected by monitoring luciferase activity. In conclusion, we have established functional replicons for HCV genotypes 3a and 4a, important new additions to the armamentarium required to develop inhibitors with a pan-genotype activity.
137 citations
TL;DR: The fidelity of the HCV RNA-dependent RNA polymerase NS5B is studied in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo, and the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.
Abstract: The development of resistance to direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) can compromise therapy. However, mechanisms that determine prevalence and frequency of resistance-conferring mutations remain elusive. Here, we studied the fidelity of the HCV RNA-dependent RNA polymerase NS5B in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo. Enzyme kinetic measurements revealed unexpectedly high error rates (approximately 10-3 per site) for G∶U/U∶G mismatches. The strong preference for G∶U/U∶G mismatches over all other mistakes correlates with a mutational bias in favor of transitions over transversions. Deep sequencing of HCV RNA samples isolated from 20 treatment-naive patients revealed an approximately 75-fold difference in frequencies of the two classes of mutations. A stochastic model based on these results suggests that the bias toward transitions can also affect the selection of resistance-conferring mutations. Collectively, the data provide strong evidence to suggest that the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.
137 citations