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Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells

Division of Microbiology, Kobe University Graduate School of Medicine

¹ E-mail: hotta{at}med.kobe-u.ac.jp

Robust production of infectious hepatitis C virus (HCV) in cell culture was realized using the JFH1 strain and the homologous chimeric J6/JFH1 in Huh-7.5 cells, a highly HCV-permissive subclone of Huh-7 cells. In this study, we aimed to establish a more efficient HCV production system and to gain some insight into the adaptation mechanisms of efficient HCV production. By serially passaging the J6/JFH1-infected Huh-7.5 cells, we obtained culture-adapted J6/JFH1 variants, designated as P-27, P-38, and P-47. Sequence analyses revealed that the adaptive mutant viruses P-27, P-38, and P-47 possessed 8 mutations (four in E2, two in NS2, one in NS5A, and one in NS5B), 10 mutations (two additional mutations in 5'-UTR and Core), and 11 mutations (three additional mutations in 5'-UTR, Core, and NS5B), respectively. We introduced amino acid substitutions into the wild-type J6/JFH1 clone, generated the recombinant viruses with adaptive mutations, and analyzed the infectivity and the ability to produce infectious viruses. The viruses with the adaptive mutations exhibited higher expression of the HCV proteins than did the wild-type in Huh-7.5 cells. Moreover, we provided evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level. We thus demonstrated that an efficient HCV production system could be obtained by introducing adaptive mutations into the J6/JFH1 genome. The J6/JFH1-derived mutant viruses presented here would be a good tool for producing HCV particles with enhanced infectivity and studying the molecular mechanism of HCV entry.

Received 11 February 2009; accepted 5 March 2009.