July 1: Novel hepatitis C virus infectious culture systems: implications for basic research and drug development
Speaker：Yiping Li
Host: Prof. Mingyao Liu
When: 2013-7-1 9:30
Where: Conference Room 534, School of Life Sciences
Abstract：Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases, but treatment options are limited and largely affected by HCV genotype. Basic HCV research required for vaccine and drug development has been hampered by the inability to culture patient isolates. To date, only the JFH1 clone (genotype 2a) replicates spontaneously in hepatoma cells and releases infectious virus. Using the unique replication capacity of JFH1, we developed efficient JFH1-based HCV recombinant viruses expressing the regions spanning the 5’ untranslated region (5’UTR) to nonstructural protein 2 (NS2) from genotypes 1-6, the Core-NS2 from genotypes 1-7 with divergent 5’UTRs, and the Core-NS2 from genotypes 1a and 2a with heterotypic chimeric 5’UTR domains. We demonstrated the non-genotype-specific role of the 5’UTR, the requirement of adenine at the 5’-terminus, and the functional importance of the 5’UTR stem-loop I (SLI) in virus production in vitro. Lack of the SLI could be compensated for by cellular and viral RNA insertions. MicroRNA miR-122 antagonism had potent antiviral effect against HCV genotypes 1-6; however, the efficacy could be reduced by host RNA insertion or mutations in the 5’UTR. Interferon-α or 5’UTR-targeting small interfering RNAs efficiently inhibited the infection of genotype recombinants. Finally, we succeeded in developing robust full-length HCV infectious culture systems independent of JFH1 for genotypes 1a, 2a, and 2b. Through a systematic approach, we initially identified three mutations, designated LSG, that permitted the propagation of full-length J6 (2a) and eventual adaptation for efficient growth. Importantly, the LSG mutations enabled the adaptation of the genetically divergent isolates J8 (2b) and TN (1a) to cell culture. Moreover, we demonstrated that the leading NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs inhibited the infection of full-length J6 and TN viruses in a dose-dependent manner. The availability of these novel infectious culture systems facilitates HCV basic research and the testing of antiviral drugs in a genotype-specific manner. The full-length infectious culture systems represent a major research advance, and the approach used might permit culture development of other clinical isolates, with implications for improved individualized treatments and disease prevention.