Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

The non-structural 5A (NS5A) protein of hepatitis C virus (HCV) has been the subject of intensive research over the last decade. It is generally accepted that NS5A is a pleiotropic protein with key roles in both viral RNA replication and modulation of the physiology of the host cell. Our understanding of the role of NS5A in the virus life cycle has been hampered by the lack of a robust in vitro system for the study of HCV replication, although the recent development of the subgenomic replicon has at least allowed us to begin to dissect the involvement of NS5A in the process of viral RNA replication. Early studies into the effects of NS5A on cell physiology relied on expression of NS5A either alone or in the context of other non-structural proteins; the advent of the replicon system has allowed the extrapolation of these studies to a more physiologically relevant cellular context. Despite recent progress, this field is controversial, and there is much work to be accomplished before we fully understand the many functions of this protein. In this article, the current state of our knowledge of NS5A, discussing in detail its direct involvement in virus replication, together with its role in modulating the cellular environment to favour virus replication and persistence, are reviewed. The effects of NS5A on interferon signalling, and the regulation of cell growth and apoptosis are highlighted, demonstrating that this protein is indeed of critical importance for HCV and is worthy of further investigation.

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Combination of a Hepatitis C Virus NS3-NS4A Protease Inhibitor and Alpha Interferon Synergistically Inhibits Viral RNA Replication and Facilitates Viral RNA Clearance in Replicon Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4784-4792.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4784-4792 ◽

Cited By ~ 65

Author(s):

Kai Lin ◽

Ann D. Kwong ◽

Chao Lin

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Adverse Effects ◽

Protease Inhibitor ◽

Standard Of Care ◽

Rna Replication ◽

Viral Rna ◽

Alpha Interferon ◽

Hcv Rna

ABSTRACT The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-α) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-α synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.

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MATHEMATICAL MODEL FOR SUPPRESSION OF SUBGENOMIC HEPATITIS C VIRUS RNA REPLICATION IN CELL CULTURE

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720007002849 ◽

2007 ◽

Vol 05 (02b) ◽

pp. 593-609 ◽

Cited By ~ 8

Author(s):

ELENA L. MISHCHENKO ◽

KIRILL D. BEZMATERNYKH ◽

VITALY A. LIKHOSHVAI ◽

ALEXANDER V. RATUSHNY ◽

TAMARA M. KHLEBODAROVA ◽

...

Keyword(s):

Mathematical Model ◽

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Suppressive Effect ◽

Viral Rna ◽

Hepatitis C Virus Rna ◽

Virus Rna ◽

Ns3 Protease

A mathematical model for suppression of the hepatitis C virus RNA replicon replication in Huh-7 cell culture in the presence of potential drugs was built. There was a good agreement between the experimental and theoretical kinetic data for the decrease in the level of viral RNA in the cell in the presence of the competitive HCV NS3 protease inhibitor. Using the model, we verified the estimates for the efficiency of the effect of potential drugs on replication of viral RNA and viral protein processing. It was demonstrated that the tested drugs are most efficient at the replication step of viral RNA. The efficiency of the combined action of real and putative inhibitors target on the host and viral proteins was also studied. It was found that the action of the inhibitor at low concentrations on the host factors considerably enhances the suppressive effect on viral RNA replication in the presence of even the low affine NS3 protease inhibitors. The developed mathematical model may serve as a tool for the evaluation of the efficiency of potential drugs on the HCV genome.

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Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03534-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5386-5394 ◽

Cited By ~ 27

Author(s):

Constance N. Wose Kinge ◽

Christine Espiritu ◽

Nishi Prabdial-Sing ◽

Nomathamsaqa Patricia Sithebe ◽

Mohsan Saeed ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Antiviral Agents ◽

Rna Replication ◽

Firefly Luciferase ◽

Hcv Rna ◽

Virus Genotype ◽

Subgenomic Replicon

ABSTRACTHepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy.In vitroreplication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established anin vitroreplication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds.

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In Vitro Resistance Study of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00834-07 ◽

2007 ◽

Vol 52 (2) ◽

pp. 675-683 ◽

Cited By ~ 34

Author(s):

Stephanie T. Shi ◽

Koleen J. Herlihy ◽

Joanne P. Graham ◽

Shella A. Fuhrman ◽

Chau Doan ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

High Throughput Screening ◽

Subgenomic Replicon ◽

Replicon Cell ◽

Resistance Selection ◽

Polymerase Inhibitors ◽

Virus Rna ◽

Allosteric Sites

ABSTRACT A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 Å away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 μM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

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The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus

Journal of Virology ◽

10.1128/jvi.01885-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2163-2177 ◽

Cited By ~ 107

Author(s):

Daniel M. Jones ◽

Arvind H. Patel ◽

Paul Targett-Adams ◽

John McLauchlan

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Infectious Virus ◽

Virus Assembly ◽

Rna Replication ◽

Viral Rna ◽

Focus Formation ◽

Er Membrane

ABSTRACT Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.

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Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture

Journal of Biological Chemistry ◽

10.1074/jbc.m401067200 ◽

2004 ◽

Vol 279 (24) ◽

pp. 25474-25482 ◽

Cited By ~ 5

Author(s):

Yuanyuan Ma ◽

Tetsuro Shimakami ◽

Hong Luo ◽

Naoyuki Hayashi ◽

Seishi Murakami

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Mutational Analysis ◽

Subgenomic Replicon ◽

Replicon Cell

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Faculty Opinions recommendation of Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1023038.265674 ◽

2004 ◽

Author(s):

Robert Lanford

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Selective Inhibition ◽

Viral Rna

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Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013455 ◽

2020 ◽

Vol 295 (35) ◽

pp. 12426-12436 ◽

Cited By ~ 1

Author(s):

Lorillee Tallorin ◽

Valerie A. Villareal ◽

Chih-Yun Hsia ◽

Mary A. Rodgers ◽

Dominique J. Burri ◽

...

Keyword(s):

Hepatitis C Virus ◽

Lipid Metabolism ◽

Hepatitis C ◽

Molecular Mechanisms ◽

Rna Replication ◽

Hcv Rna ◽

Genome Replication ◽

Subgenomic Replicon ◽

Lipid Environment

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

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