Hepatitis C Virus Therapies: Current Treatments, Targets and Future Perspectives

Walker, Michelle; Appleby, T.C.; Zhong, Weidong; Lau, Johnson Y. N.; Hong, Zhi

doi:10.1177/095632020301400101

Cited by 50 publications

(43 citation statements)

References 175 publications

(275 reference statements)

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“…NS5B has no homologue in the human cell and is a validated target for antiviral drug design (36,37). During RNA replication, NS5B performs highly specialized tasks such as de novo initiation of RNA synthesis and elongation of the nascent genomic or antigenomic RNA strand.…”

Section: Discussionmentioning

confidence: 99%

A Relaxed Discrimination of 2′-O-Methyl-GTP Relative to GTP between de Novo and Elongative RNA Synthesis by the Hepatitis C RNA-dependent RNA Polymerase NS5B

Dutartre

Boretto²,

Guillemot³

et al. 2005

Journal of Biological Chemistry

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Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2 -O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2 -Omethyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step (ϳ150-fold) compared with ϳ2-fold at the elongation step. Interestingly, discrimination of the 2 -Omethyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2 -modified nucleotide analogues, because it is more permissive to inhibition than initiation.The Flaviviridae is an important virus family comprising three genera, namely flavivirus, pestivirus, and hepacivirus. Both flavivirus and hepacivirus genera comprise major human pathogens, such as Dengue virus, West Nile virus, Yellow Fever virus, and the Hepatitis C virus (HCV 1 ), respectively. The discovery of HCV in the late 1980s and analysis of its prevalence in the general population has revealed that HCV is the most common etiological agent of chronic liver disease (1). More than 170 millions persons are infected by HCV in the world, making these individuals at risk of developing liver cirrhosis and hepatocellular carcinoma (2). Unlike other viral diseases for which antiviral therapies exist (e.g. human immunodeficiency virus or herpes infections), a persistent HCV infection can be controlled (3, 4). In some cases, HCV can be eradicated from an infected patient (5), and this represents the first example of a complete success of antiviral therapy ever.Current antiviral therapies rely on the association of interferon ␣ to the nucleoside analogue ribavirin. Ribavirin is a broad spectrum antiviral agent discovered about 30 years ago.Although not effective on all HCV isolates and associated with undesirable side effects (6), ribavirin exerts its antiviral activity by at least two mechanisms that are intimately linked. First, it is intracellularly converted to ribavirin 5Ј-monophosphate, which is a potent inhibitor of the cellular inosine 5Ј-monophosphate dehydrogenase (IMPDH) (7,8). Inhibition of IMPDH depresses the intra...

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Section: Discussionmentioning

confidence: 99%

A Relaxed Discrimination of 2′-O-Methyl-GTP Relative to GTP between de Novo and Elongative RNA Synthesis by the Hepatitis C RNA-dependent RNA Polymerase NS5B

Dutartre

Boretto²,

Guillemot³

et al. 2005

Journal of Biological Chemistry

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“…To avoid the late sequelae of chronic HCV infections, millions of infected individuals await the development of effective antivirals (29). Efforts to explore all the possible drug targets for HCV, including virion assembly, have been hampered by the lack of robust in vitro cell culture systems that yield infectious virions (60). Much of our current knowledge on HCV virion assembly is derived from the study of virus-like particles (VLPs).…”

mentioning

confidence: 99%

Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly

Ansari

Chen

Liang

et al. 2004

J Virol

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A novel mutant of bovine viral diarrhea virus (BVDV) was found with a virion assembly phenotype attributable to an insertion into the NS5B polymerase locus. This mutant, termed 5B-741, was engineered by reverse genetics to express NS5B with a C-terminal peptide tag of 22 amino acids. Electroporation of bovine cells with genomic RNA from this mutant showed levels RNA synthesis which were regarded as sufficient for infectivity, yet infectious virions were not produced. Pseudorevertants of mutant 5B-741 that released infectious virions and formed plaques revealed a single nucleotide change (T12369C). This change resulted in a leucine-toproline substitution within the NS5B tag (L726P). Genetic analysis revealed that indeed a single nucleotide change encoding proline at NS5B position 726 in the pseudorevertant polyprotein mediated recovery of virion assembly function without improving genomic RNA accumulation levels. A subgenomic BVDV reporter replicon (rNS3-5B) was used to analyze the consequences of alterations of the genomic region encoding the NS5B C terminus on replication and assembly. Interestingly, rNS3-5B-L726P (revertant) replicated with the same efficiency as the rNS3-5B-741 mutant but produced 10 times more virions in a trans-packaging assay. These results indicated that impairment of assembly function in 5B-741 was independent of RNA accumulation levels and agreed with the observations from the full-length mutant and revertant genomes. Finally, we recapitulated the packaging defect of 5B-741 with a vaccinia virus expression system to eliminate possible unwanted interactions between the helper virus and the packaged replicon. Taken together, these studies revealed an unexpected role of NS5B in infectious virion assembly.Virion assembly and release from the host cell constitute the final stages of the viral life cycle. These events are as important for viral pathogenesis in the host as the widely studied mechanisms of virus entry or genome replication. Enveloped virion assembly represents the convergence of viral proteins, nucleic acid, and host lipids to build an infectious particle. The process of virus budding, which characterizes enveloped viruses, is difficult to study because it involves a living cell. No cell-free virus budding systems have been developed to dissect this process.Hepatitis C virus (HCV) is an important human pathogen that causes persistent infections leading to chronic hepatitis, which can result in hepatocellular carcinoma (1, 2). To avoid the late sequelae of chronic HCV infections, millions of infected individuals await the development of effective antivirals (29). Efforts to explore all the possible drug targets for HCV, including virion assembly, have been hampered by the lack of robust in vitro cell culture systems that yield infectious virions (60). Much of our current knowledge on HCV virion assembly is derived from the study of virus-like particles (VLPs). Numerous reports support the concept that viral structural proteins carry enough structural information to direct the as...

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“…Within 10 to 20 years of infection, 20 to 30% of chronic carriers develop cirrhosis, making HCV infection one of the major reasons for liver transplantation. Current therapies are limited to interferon treatment, either alone or in combination with ribavirin (23,38), but patient response for certain genotypes is still unsatisfactory. This unmet medical need has created an urgent demand for the development of new drugs to treat chronic hepatitis C. However, the lack of an in vitro infection system has hampered HCV drug development.…”

mentioning

confidence: 99%

Establishment of a Subgenomic Replicon for Bovine Viral Diarrhea Virus in Huh-7 Cells and Modulation of Interferon-Regulated Factor 3-Mediated Antiviral Response

Horscroft

Bellows

Ansari

et al. 2005

J Virol

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We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N pro , and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2-␤-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N pro of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system. Hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) are both members of the Flaviviridae family and share many molecular and virological similarities (20, 27). They both have single-strand RNA genomes that replicate via negativestrand intermediates and produce a single polyprotein that is cleaved into individual proteins by a combination of host and viral proteases. Although HCV is a hepacivirus and BVDV is a pestivirus, there is a low degree of sequence homology between their respective nonstructural proteins and both RNAdependent RNA polymerases are structurally very similar (9).HCV is a major etiological agent for viral hepatitis. The World Health Organization estimates that 170 million people are chronically infected with HCV worldwide, and of those, 4 million are in the United States. Within 10 to 20 years of infection, 20 to 30% of chronic carriers develop cirrhosis, making HCV infection one of the major reasons for liver transplantation. Current therapies are limited to interferon treatment, either alone or in combination with ribavirin (23, 38), but patient response for certain genotypes is still unsatisfactory. This unmet medical need has created an urgent demand for the development of new dr...

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Hepatitis C Virus Therapies: Current Treatments, Targets and Future Perspectives

Cited by 50 publications

References 175 publications

A Relaxed Discrimination of 2′-O-Methyl-GTP Relative to GTP between de Novo and Elongative RNA Synthesis by the Hepatitis C RNA-dependent RNA Polymerase NS5B

A Relaxed Discrimination of 2′-O-Methyl-GTP Relative to GTP between de Novo and Elongative RNA Synthesis by the Hepatitis C RNA-dependent RNA Polymerase NS5B

Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly

Establishment of a Subgenomic Replicon for Bovine Viral Diarrhea Virus in Huh-7 Cells and Modulation of Interferon-Regulated Factor 3-Mediated Antiviral Response

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