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

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

Virology ◽

10.1006/viro.2002.1461 ◽

2002 ◽

Vol 297 (2) ◽

pp. 298-306 ◽

Cited By ~ 35

Author(s):

I.Wayne Cheney ◽

Suhaila Naim ◽

Vicky C.H. Lai ◽

Shannon Dempsey ◽

Daniel Bellows ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Enzymatic Activity ◽

Rna Replication ◽

Viral Rna ◽

Subgenomic Replicon ◽

Replicon Cell ◽

Ns5b Polymerase

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Hepatitis C virus NS5A: tales of a promiscuous protein

Journal of General Virology ◽

10.1099/vir.0.80204-0 ◽

2004 ◽

Vol 85 (9) ◽

pp. 2485-2502 ◽

Cited By ~ 261

Author(s):

Andrew Macdonald ◽

Mark Harris

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Replication ◽

Rna Replication ◽

Viral Rna ◽

Cell Physiology ◽

Subgenomic Replicon ◽

Cellular Environment ◽

Cellular Context

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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Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle

Journal of Virology ◽

10.1128/jvi.03188-14 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5362-5370 ◽

Cited By ~ 26

Author(s):

David R. McGivern ◽

Takahiro Masaki ◽

William Lovell ◽

Chris Hamlett ◽

Susanne Saalau-Bethell ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Active Site ◽

Rna Synthesis ◽

Virus Assembly ◽

Rna Replication ◽

Helicase Domain ◽

Polyprotein Processing ◽

Virus Life Cycle

ABSTRACTHepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (<12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing.IMPORTANCEThe NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of protease activity can block RNA replication by preventing expression of mature replicase components. Like many viral proteins, NS3 is multifunctional, but how PIs affect stages of the HCV life cycle beyond polyprotein processing has not been well studied. Using cell-based assays, we show here that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.

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Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

Journal of Virology ◽

10.1128/jvi.00525-19 ◽

2019 ◽

Vol 93 (19) ◽

Cited By ~ 1

Author(s):

Philipp Schult ◽

Maren Nattermann ◽

Chris Lauber ◽

Stefan Seitz ◽

Volker Lohmann

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

Rna Viruses ◽

Rna Replication ◽

Rna Dependent Rna Polymerase ◽

Internal Initiation ◽

Positive Strand Rna

ABSTRACT Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5′ untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

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Hepatitis C Virus (HCV) NS5B Nonnucleoside Inhibitors Specifically Block Single-Stranded Viral RNA Synthesis Catalyzed by HCV Replication Complexes In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00498-06 ◽

2006 ◽

Vol 51 (1) ◽

pp. 338-342 ◽

Cited By ~ 3

Author(s):

Wengang Yang ◽

Yongnian Sun ◽

Avinash Phadke ◽

Milind Deshpande ◽

Mingjun Huang

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Viral Rna ◽

Double Stranded Rna ◽

Major Species ◽

Nonnucleoside Inhibitors ◽

Hcv Ns5b

ABSTRACT Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

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Quantitative Analysis of the Hepatitis C Virus Replication Complex

Journal of Virology ◽

10.1128/jvi.79.21.13594-13605.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13594-13605 ◽

Cited By ~ 211

Author(s):

Doris Quinkert ◽

Ralf Bartenschlager ◽

Volker Lohmann

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Nonstructural Protein ◽

Viral Proteins ◽

Viral Rna ◽

Nonstructural Proteins ◽

Positive Strand ◽

Negative Strand

ABSTRACT The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.

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A visualizable hepatitis A virus and hepatitis C virus coinfection model in vitro: coexistence of two hepatic viruses under limited competition in viral RNA synthesis

10.1101/709071 ◽

2019 ◽

Author(s):

Wang Jiang ◽

Pengjuan Ma ◽

Gang Long

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Synthesis ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Viral Rna ◽

Viral Interference ◽

Hcv Coinfection ◽

Virus Interactions

AbstractHepatitis A virus (HAV) and hepatitis C virus (HCV) coinfection in patients usually leads to HCV suppression or clearance. Whether this suppression/clearance is caused by direct virus-virus interaction or indirect interactions is still unknown. Here, we present a robust and visualizable HAV/HCV coinfection model for investigating their interactions in vitro. We find that HAV super infects HCV-persistently-infected Huh-7.5.1 cells without obvious virus-virus exclusion and vice versa, while there is a mild reciprocal viral interference in coinfection. Through single-cell scale confocal microscopy analysis and treating co-infected cells with rNTPs or antivirals e.g. Sofosbuvir, Simeprevir, and Ledipasvir, we find that HAV and HCV exploit cellular machinery in a compatible manner, but with reciprocal competition for rNTPs in their RNA synthesis. In conclusion, our findings reveal the absence of direct HAV-HCV interaction but the presence of indirect interaction, which may be due to limited competition in viral RNA synthesis. Therefore, we propose that suppression/clearance of HCV in HAV/HCV coinfected patients is probably due to indirect interactions e.g. viral competition or immunological interactions.Author summaryVirus-virus interactions could usually be categorized in three aspects: (1) direct interaction induced by interaction of viral gene or gene products; (2) indirect interaction mediated by immune response or (3) host environmental alteration. Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two important causatives of human hepatitis. In patients, their coinfection often induces HCV suppression/clearance, and sometimes may lead to fulminant hepatitis. In this study, we find that a limited reciprocal viral interference occurs in HAV/HCV coinfection in vitro, which results from a viral competition of rNTPs in viral RNA synthesis. Our study provides a new paradigm of studying virus-virus interactions in single-cell scale. To our knowledge, we identify the rNTPs competition in virus coinfection for the first time by experiment, though several mathematical modelings have predicted the resource competition in virus coinfection.

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Hepatitis C Virus RNA Replication Occurs on a Detergent-Resistant Membrane That Cofractionates with Caveolin-2

Journal of Virology ◽

10.1128/jvi.77.7.4160-4168.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4160-4168 ◽

Cited By ~ 203

Author(s):

Stephanie T. Shi ◽

Ki-Jeong Lee ◽

Hideki Aizaki ◽

Soon B. Hwang ◽

Michael M. C. Lai

Keyword(s):

Protein Synthesis ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Raft ◽

Rna Synthesis ◽

De Novo ◽

Rna Replication ◽

Viral Rna ◽

Hcv Rna ◽

Huh7 Cells

ABSTRACT The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed that the newly synthesized HCV RNA was localized to distinct speckle-like structures, which also contain all of the HCV nonstructural (NS) proteins. These speckles are distinct from lipid droplets and are separated from the endoplasmic reticulum (ER), where some HCV NS proteins also reside. Membrane flotation analysis demonstrated that almost all of the NS5A and part of the NS5B proteins and all of the viral RNA were present in membrane fractions which are resistant to treatment with 1% NP-40 at 4°C. They were cofractionated with caveolin-2, a lipid-raft-associated intracellular membrane protein, in the presence or absence of the detergent. In contrast, the ER-resident proteins were detergent soluble. These properties suggest that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes. The protein synthesis inhibitors cycloheximide and puromycin did not inhibit viral RNA synthesis, indicating that HCV RNA replication does not require continuous protein synthesis. We suggest that HCV RNA synthesis occurs on a lipid raft membrane structure.

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