Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells.
Robust production of infectious hepatitis C virus (HCV) in cell culture was realized by using the JFH1 strain and the homologous chimeric J6/JFH1 strain 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 serial passaging of J6/JFH1-infected Huh-7.5 cells, we obtained culture-adapted J6/JFH1 variants, designated P-27, P-38 and P-47. Sequence analyses revealed that the adaptive mutant viruses P-27, P-38 and P-47 possessed eight mutations [four in E2, two in NS2, one in NS5A and one in NS5B), 10 mutations [two additional mutations in the 5'-untranslated region (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 recombinant viruses with adaptive mutations and analysed their infectivity and ability to produce infectious viruses. The viruses with the adaptive mutations exhibited higher expression of HCV proteins than did the wild type in Huh-7.5 cells. Moreover, we provide evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level. We thus demonstrate 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 for studying the molecular mechanism of HCV entry.
Bungyoku
Yasuaki
Y
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Shoji
Ikuo
I
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Makine
Tatsuhiko
T
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Adachi
Tetsuya
T
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Hayashida
Kazumi
K
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Nagano-Fujii
Motoko
M
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Ide
Yoshi-Hiro
YH
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Deng
Lin
L
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Hotta
Hak
H
Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
D020121:5' Untranslated Regions; D000220:Adaptation, Biological; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D016174:Hepacivirus / Q000235:genetics* / Q000254:growth & development*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D009154:Mutation; D020125:Mutation, Missense; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D012692:Serial Passage; D014759:Viral Envelope Proteins / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
D020121:5' Untranslated Regions; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C097010:NS2 protein, Hepatitis C virus; C091015:glycoprotein E2, Hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase