Comparison of HCV NS3 protease and NS5B polymerase inhibitor activity in 1a, 1b and 2a replicons and 2a infectious virus

Paulson, Matthew; Yang, Huiling; Shih, I-hung; Feng, Joy Y.; Mabery, Eric; Robinson, Margaret; Zhong, Weidong; Delaney, William E.

doi:10.1016/j.antiviral.2009.04.003

Cited by 20 publications

(15 citation statements)

References 38 publications

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“…Restriction enzymes were purchased from New England BioLabs (Ipswich, MA). A chimeric HCV genome consisting of the J6 sequence (genotype 2a) from core through NS2 (genotype 2a) followed by the JFH-1 sequence (genotype 2a) of the 5Ј untranscribed region (UTR) and then from NS3 through the 3Ј UTR with a FLAG tag inserted in the NS5A coding sequence was called J6/JFH-1 Rluc and was previously described (30). To create a nonreporter version of this genome, the Rluc gene was removed by using the flanking MluI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

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Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

Pokrovskii

Bush

Beran

et al. 2011

J Virol

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Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 ؋ 10 8 50% tissue culture infective doses (TCID 50 )/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus that has only recently been adapted to tissue culture (22,41,46). The full-length genome of isolate JFH-1 was demonstrated to be competent for viral particle production in tissue culture (22,41,46) by using Huh-7-derived cell lines that are permissive to HCV infection and replication (2,20). Several of these HCV cell culture (HCVcc) systems have been described, the most robust of which are based on chimeric J6/JFH-1 viruses or tissue culture-adapted strains of JFH-1 (1,3,4,17,18,26,36,47). However, the quantity of infectious virions these systems can produce is limited, presumably due to currently unidentified constraints on infectious virus particle production in tissue culture (5,16,26,47). After passage of tissue culture-grown J6/JFH-1 virus in animals, the resultant viruses exhibited a higher specific infectivity (23). Similarly, passage of HCV in primary human hepatocytes yielded virus with higher specific infectivity (33). Although Huh-7 cells are currently the most efficient system for culturing of HCV, these data suggest that virus particle production is suboptimal in these cells compared to that in bona fide hepatocytes.Serial passage of HCV in cell culture has yielded virus isolates with increased viral titers (9,10,32,37,44). Interestingly, these ...

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Section: Methodsmentioning

confidence: 99%

“…In vitro transcripts were generated as previously described (30). Briefly, plasmids were linearized with XbaI and purified by using a MinElute column (Qiagen, Valencia, CA).…”

Section: Methodsmentioning

confidence: 99%

Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

Pokrovskii

Bush

Beran

et al. 2011

J Virol

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“…Replicon antiviral assays were performed as described previously (22). Briefly, replicon cells were seeded into 96-well plates at a density of 5 ϫ 10 3 cells per well in 100 l of complete DMEM, excluding G418.…”

Section: Methodsmentioning

confidence: 99%

Selection of Clinically Relevant Protease Inhibitor-Resistant Viruses Using the Genotype 2a Hepatitis C Virus Infection System

Cheng

Chan

Yang

et al. 2011

Antimicrob Agents Chemother

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Treatment of patients infected with hepatitis C virus (HCV) with direct acting antivirals can lead to the emergence of drug-resistant variants that may pose a long-term threat to viral eradication. HCV replicons have been used to select resistance mutations; however, genotype 2a JFH-1-based viruses provide the opportunity to perform resistance selection in a bona fide infection system. In this study, we used a tissue culture-adapted J6/JFH-1 virus to select resistance to the NS3 protease inhibitors BILN-2061 and VX-950. Lunet-CD81 cells were infected with J6/JFH-1 virus and maintained in the presence of inhibitors until high-titer viral supernatant was produced. Viral supernatants were passaged over naive cells at escalating drug concentrations, and the resulting viruses were then characterized. Three NS3 resistance mutations were identified in BILN-2061-resistant viruses: A156G, D168A, and D168V. Interestingly, D168A, D168V, and A156T/V, but not A156G, were selected in parallel using a genotype 2a replicon. For VX-950, the T54A and A156S NS3 resistance mutations were identified in the virus selections, whereas only A156T/V emerged in genotype 2a replicon selections. Of note, VX-950 resistance mutations selected using the 2a virus (T54A and A156S) were also observed during VX-950 clinical studies in genotype 2 patients. We also performed viral fitness evaluations and determined that the mutations selected in the viral system did not confer marked reductions in virus production kinetics or peak titers. Overall, the HCV infection system is an efficient tool for drug resistance selections and has advantages for the rapid identification and characterization of clinically relevant resistance mutations.Chronic hepatitis C virus (HCV) infection represents a significant and immediate worldwide health burden (2, 40). Accordingly, tremendous resources have been directed toward discovering and developing novel therapies to treat HCV infection. Currently, a number of direct-acting antiviral agents (DAAs) are under clinical development (7,35,37). Various DAAs have been designed to target distinct HCV viral proteins (3, 4, 6), including NS3 protease, NS5A, and the NS5B RNA-dependent RNA polymerase. During short-term monotherapy studies in HCV-infected patients, many of these DAAs have elicited pronounced antiviral effects (e.g., Ն3 log HCV viral load reductions in as few as 3 days of treatment). However, even in these short-term studies the selection of viral resistance was apparent and thus poses a significant challenge to the long-term efficacy of these novel agents (6,11,32,35).The HCV replicon has been a useful in vitro tool for identifying and characterizing resistance mutations for multiple classes of DAAs (11,32,35). For example, the NS3 mutations R155K, A156T/V, and D168A/V were selected in replicons using the first clinically active NS3 protease inhibitor, BILN-2061 (a prototype noncovalent NS3 inhibitor) (16,19). Unfortunately, the resistance profile of BILN-2061 in the clinic has not been reported, making it imposs...

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“…A library of 450,000 compounds was screened against both infectious HCV(GT2a) (J6/JFH1) (24,25) and the HCV(GT2a) replicon (26). In brief, Huh7-Lunet-CD81 cells (22) or HCV(GT2a) stable replicon cells were plated on 384-well plates using a MicroFlo dispenser (BioTek, Winooski, VT) and incubated overnight.…”

Section: Methodsmentioning

confidence: 99%

A Small-Molecule Inhibitor of Hepatitis C Virus Infectivity

Bush

Pokrovskii

Saito

et al. 2014

Antimicrob Agents Chemother

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One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.O ne of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates. The standard of care for HCV patients over the past decade has been to treat with pegylated interferon combined with ribavirin. Relatively recently, the HCV NS3-4A protease inhibitors teleprevir and boceprevir were added to this standard of care and have improved the sustained virologic response (1). However, poor tolerability to treatments containing pegylated interferon has motivated researchers to attempt to develop a variety of other interferon-free treatments (2). An interferon-free cure will likely require a combination of at least two antivirals with differing modes of action (3, 4). Combining multiple antivirals with different resistance profiles increases the number of resistance mutations required for viral breakthrough (5). Studies to determine optimal antiviral combinations which take into account the probability of emergence of specific resistance mutations as well as the subsequent viral fitness (3, 6, 7) are under way.We recently demonstrated that the in vitro combination of HCV entry inhibitors with HCV replication inhibitors could prolong the efficacy compared to a single treatment with either inhibitor class. Specifically, we studied the entry inhibitor anti-CD81 anti...

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Comparison of HCV NS3 protease and NS5B polymerase inhibitor activity in 1a, 1b and 2a replicons and 2a infectious virus

Cited by 20 publications

References 38 publications

Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

Selection of Clinically Relevant Protease Inhibitor-Resistant Viruses Using the Genotype 2a Hepatitis C Virus Infection System

A Small-Molecule Inhibitor of Hepatitis C Virus Infectivity

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