Establishment of a New Hepatitis C Virus-1 b Replicon
Backgrounds:Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh7 cells (Lohmann, Science 1999). Their replications can be enhanced by introduction of certain cell culture-adaptive mutations in the viral NS5A region (Blight, Science 2000, Krieger, J Virol 2001). To extend the previous results to other isolated HCV clones, we have constructed another HCV replicon from HC-J4, one of the chimpanzee-infectious clones, and demonstrated efficient intracellular replications in Huh7 cells.
Materials and methods: An HCV-replicon (R-J4) was constructed from a chimpanzee-infectious clone, HC-J4 (Yanagi, Virology 1999), which is consisted of HCV-5’-UTR, neomycin phosphotransferase (Neo) gene, the encephalomyocarditis virus IRES, HCV-non-structural region, NS3 to NS5B, and the 3’-UTR. The reported adaptive mutations were introduced in the wild type replicon (R-J4/S2197I, R-J4/S2201del, R-J4/S2204P). A replicon R-J4/ISDRmut was constructed in which 6 amino acids mutations were introduced into the interferon sensitivity determining region (ISDR) of NS5A gene. As a negative control, deletion was introduced in the NS5B, RNA-dependent RNA polymerase catalytic domain. Replicon RNA was transfected into Huh7 cells, and stable replicon-expressing cells were established by G418 selection.
Results:After transfection, the R-J4 and R-J4/ISDRmut did not produce G418-resistant colony. In contrast, cells were transduced efficiently by introduction of R-J4/S2197I, R-J4/S2201del, and R-J4/S2204P (>300 colonies), while no colony was found after transfection of replicons with deletion in NS5B catalytic domain. Replication of HCV-RNA was also confirmed by northern blot analysis. RJ4/S2201del with ISDR mutations replicated most efficiently, suggesting that synergistic effect of mutations in the serine cluster region and ISDR in NS5A on HCV replication.
KeywordsHuh7 Cell Adaptive Mutation Interferon Sensitivity Determine Region Subgenomic Replicon Serum Viral Load
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