Cooperation between the Hepatitis C Virus p7 and NS5B Proteins Enhances Virion Infectivity

Abstract

The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly. IMPORTANCEHepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection. Hepatitis C virus (HCV), a positive-sense single-stranded RNA virus and a member of the Flaviviridae family, infects nearly 170 million people worldwide (1, 2). HCV infection is the leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (3). While the previous interferon-based therapies for treatment of hepatitis C were effective in only 40% of those infected with genotype 1 virus, the most prevalent HCV genotype in the United States (4), newly developed therapies for hepatitis C now use interferon-free direct-acting antiviral (DAA) regimens, with over 90% success rates (5). However, antiviral resistance is still a problem, and a vaccine is not yet available for prevention of HCV transmission.The HCV RNA genome is translated into a single polyprotein from an internal ribosome entry site located in the 5= untranslated region of the genome. The resu...

A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I

“…Several growth-adaptive mutations have been identified previously in E2, and studying the properties of these mutants is an established method of linking structural features of E2 with functions in the virus life cycle (37)(38)(39)(40)(41). Past attempts to identify growth-adaptive mutations in the HCV genome have relied on serial passage of a clonal virus stock for weeks or months until adaptive mutations arise (25,(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50). To identify adaptive mutations more expediently, we created a library of infectious HCV with a fully mutagenized E2 ectodomain (Fig.…”

“…Because of lipoprotein incorporation, infectious HCV particles have a lower density and broader infectivity peak (3,4). Some E2 growth variants have altered buoyant density profiles relative to their parental strains (37,40), and such differences may reflect alterations in lipoprotein incorporation or 50 were determined by nonlinear regression analysis. The panels on the left are representative dose-response curves for parental virus and the mutants.…”

“…HEPES, pH 7.3 (10 mM), was added to the virus-containing supernatants for storage at 4°C prior to concentration with Amicon Ultra 100,000-kDa-molecular-size-cutoff centrifugal filters (EMD Millipore). Concentrated virus stocks were stored at Ϫ80°C, and titers were determined using the 50% tissue culture infectious dose (TCID 50 ) method (27).…”

“…Seventyfive TCID 50 with chilled media, and cellular RNA was harvested using a Qiagen RNeasy minikit. Viral genome copies were measured by qRT-PCR as described above.…”

“…Statistical analyses were performed using GraphPad Prism software. Differences in mean EC 50 , relative infection levels, titers, or RNA-to-TCID 50 ratios between the mutants and parent virus were analyzed by one-way analysis of variance (ANOVA) followed by a Dunnett's test.…”