In Vivo Determination of Substrate Specificity of Hepatitis C Virus NS3 Protease: Genetic Assay for Site-Specific Proteolysis

149

189

Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.The hepatitis C virus (HCV) is an enveloped virus of the genus Hepacivirus within the family Flaviviridae (52). HCV isolates can be classified into six major genotypes that differ in their nucleotide sequence by 30 to 35%, and within these genotypes, several subtypes can be defined (47). Viral infection most often becomes persistent and causes acute and chronic liver disease (22,46). At present, more than 170 million people suffer from chronic hepatitis C (44). Current treatment consists of a combination of polyethylene glycol-conjugated alpha interferon and ribavirin (44), but success rates are limited and the outcome of therapy is very dependent on the genotype of the infecting virus (13).The genome of HCV is a linear, single-stranded RNA molecule of positive polarity with a size of ϳ9.6 kb, and it is flanked at the 5Ј and 3Ј ends by noncoding regions (NCRs). Both NCRs play an important role in the initiation of RNA synthesis by the viral RNA-dependent RNA polymerase (RdRp) NS5B (16,18,27). The RNA genome carries a long open reading frame of about 3,000 amino acids (aa) that is coand posttranslationally cleaved by cellular and viral proteases into the structural proteins core, E1, and E2, followed by p7 and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (3). Expression of the polyprotein is initiated at an internal ribosome entry site, comprising most of the 5Ј NCR and the 5Ј-proximal core coding region (41). The core protein forms the viral nucleocapsid, which is surrounded by the lipid envel...

Section: Discussionsupporting

confidence: 93%

Cell Culture Adaptation of Hepatitis C Virus and In Vivo Viability of an Adapted Variant

Kaul

Woerz

Meuleman

et al. 2007

149

189

“…This alteration in the nucleotide sequence substitutes alanine for serine at the P1Ј position in the NS4B-NS5A cleavage site. However, alanine is present at the junction between NS4A and NS4B and, along with serine, is highly favored at the P1Ј position (33). Mutations in NS4B were generated using the QuikChange mutagenesis kit (Stratagene) and introduced into plasmid pGEM-NS4B CT .…”

Section: Methodsmentioning

confidence: 99%

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

Jones¹,

Patel²,

Targett-Adams³

et al. 2009

125

145

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.Recent estimates predict that the prevalence of hepatitis C virus (HCV) infection is approximately 2.2% worldwide, equivalent to about 130 million persons (22). The virus typically establishes a chronic infection that frequently leads to serious liver disease (1), and current models indicate that both morbidity and mortality as a consequence of HCV infection will continue to rise for about the next 20 years (10,11,29).HCV is the only assigned species of the Hepacivirus genus within the family Flaviviridae. The virus can be classified into six genetic groups or clades (numbered 1 to 6) and then further separated into subtypes (e.g., 1a, 1b, 2a, 2b, etc.) (53, 55). HCV has a single-stranded, positive-sense RNA genome that is approximately 9.6 kb in length (reviewed in reference 46). Genomic RNA carries a single open reading frame flanked by 5Ј and 3Ј nontranslated regions, which are important for both replication and translation (19,20,34,47,56). Viral RNA is translated by the host ribosomal machinery, and the resultant polyprotein is co-and posttranslationally cleaved to generate the mature viral proteins. The structural proteins (core, E1, and E2) and a small hydrophobic polypeptide called p7 are produced by the cellular proteases signal peptidase and signal peptide peptidase (28,45,54). Two virus-encoded proteases, the NS2-3 autoprotease and th...

“…NS3-4Ap specificity has been defined by identification (17,44) and mutagenesis (5,23,49,53) of the natural cleavage sites and selection of optimized cleavage sites using peptide libraries (21,41). The NS3/NS4A junction is cleaved in cis and tolerates substitutions at all positions except P1, where a threonine residue is found in all isolates.…”

mentioning

confidence: 99%

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Trozzi

Bartholomew

Ceccacci

et al. 2003

115

136

The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.Despite the introduction of blood-screening tests ϳ10 years ago, hepatitis C virus (HCV) is still the major cause of bloodborne chronic hepatitis, with nearly 200 million infected people worldwide. HCV infection often evolves into a chronic disease, which can lead to liver dysfunction and hepatocellular carcinoma. Current therapeutic regimens based on alpha interferon (IFN-␣) and the nucleoside analog ribavirin are only partially effective and are limited by the adverse effects of both agents (50). Given the high prevalence of this disease, developing new treatments is a major public health objective. Similarly to human immunodeficiency virus (HIV) research, most efforts to develop antiviral agents for HCV have focused on the inhibition of key viral enzymes, serine protease, helicase, and polymerase (2).The most extensively studied HCV target has been the NS3-4A serine protease, a heterodimeric enzyme comprising the N-terminal domain of the NS3 protein (amino acids 1 to 180) and the small hydrophobic NS4A protein (3). This protease cleaves the viral polyprotein at four junctions (NS3/ NS4A, NS5A/NS5B, NS4A/NS4B, and NS4B/NS5A), and its activity is necessary for viral replication (24). Although the NS3 protease domain possesses enzymatic activity, the 54-amino-acid NS4A protein is required for cleavage at the NS3/ NS4A and NS4B/NS5A sites and increases cleavage efficiency at the NS4A/NS4B and NS5A/NS5B junctions (4, 14, 28, 47). X-ray crystallography (20, 35, 51) and nuclear magnetic resonance (NMR) spectroscopy (1, 36) have shown that the NS3-4A structure is similar to that of other chymotrypsin-like serine proteases, with two domains, both composed of a ␤-barrel and two short ␣-helices. The catalytic triad comprises histidine 57, aspartate 81, and serine 139 and is located between the two domains. The central region of NS4A is an integral part of t...