Determination of replication cycle of hepatitis C virus

Abstract

The hepatitis C virus (HCV), which has infected 3% of the world population, leads to chronic infection and ultimately to liver cirrhosis and hepatocellular carcinoma. No vaccine is available to date and there is no effective therapy for all genotype of HCV. HCV possesses a positive-sense single-stranded RNA with a nucleotide length of 9.6 kb, which encodes a single large precursor polyprotein that is flanked by nontranslated regions at its 5′ and 3′ ends. The viral polyprotein is processed by cellular and viral proteases into structural and nonstructural proteins. Recently, cell culture systems for in vitro infectious-virus production based on the full-length HCV genome of a genotype 2a were established. However, infectivity of this infectious HCV clone was too low for mass production of HCV virions. In order to obtain a highly efficient HCV cultivation system, I found two cell culture-adapted mutations, located in the E2 and p7 coding regions, which were responsible for the increased viral infectivity. The E2 mutation increased the viability of infectious virus in culture media at 37C, and the p7 mutation increased virus production by augmenting an early step of virion production. These cell culture-adapted infectious viruses will facilitate HCV-related research.HCV cell culture-based model allows the study of all stages of the HCV life cycle, including entry, replication, translation, assembly, and release. However, the whole life cycle of HCV has not been well characterized. To elucidate this, I investigated one-step of HCV growth curve with high titer of viruses. The observed profiles of HCV RNA replication indicated that the synthesis of negative-strand RNAs occurred at 6 hours (h) after infection, followed by the active synthesis of positive-strand RNAs. The measurements of infectious virus production showed that the latent period of HCV was about 12 h. The specific infectivity of HCV particles (focus-forming unit per viral RNA molecule) secreted to the extracellular milieu early in infection was about 30-fold higher than that secreted later during infection. The buoyant densities of the infectious virion particles differed with the duration of infection, indicating changes in the compositions of the virion particles.The standard HCV therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response (SVR) rate in those infected with genotype 1 virus. Two kinds of viral NS3/4A protease inhibitors, boceprevir and telaprevir, recently approved as antiviral agents. Even though these protease inhibitors increased the SVR rate in patients with genotype 1 infection, the emergence of drug-resistant mutations are frequently observed shortly after starting therapy. Therefore, more effective and safer antiviral agents for hepatitis C are clearly still needed. To identify antiviral agents, I performed high-throughput screenings using chemical compounds. I found a potent antiviral agent targeted to NS5A which has picomolar half-maximum effective concentrations (EC50).