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

Abstract

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 HCVpermissive subclone of Huh-7 cells. In this study, we aimed to establish a more efficient HCVproduction 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 59-untranslated region (59-UTR) and core] and 11 mutations (three additional mutations in 59-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. INTRODUCTIONHepatitis C virus (HCV) is the main cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (Choo et al., 1989;Kuo et al., 1989;Saito et al., 1990). As more than 170 million people worldwide are infected chronically with HCV (Poynard et al., 2003) and because the current antiviral therapy, interferon and ribavirin, produces sustained virus clearance in ,50 % of treated patients (Manns et al., 2007), HCV infection is clearly a problem of major proportions. HCV is a single-stranded, positive-sense RNA virus that is classified in the genus Hepacivirus in the family Flaviviridae. The approximately 9.6 kb HCV genome encodes one large open reading frame (ORF) that is flanked at the 59 and 39 ends by untranslated regions (UTRs) (Choo et al., 1991). The HCV polyprotein is processed into at least 10 proteins by viral proteases and cellular signalases (Grakoui et al., 1993;Hijikata et al., 1993a;McLauchlan et al., 2002). The structural proteins core, E1 and E2 are located in the N terminus of the polyprotein, followed by p7 and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B (Bartenschlager & Sparacio, 2007).Study of the HCV life cycle and virus-host interaction has been hampered severely by the lack of a robust in vitro cellculture system and small-animal models of HCV infection (Bartenschlager & Sparacio, 2007). The development of HCV replicon systems has made...

HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis

“…We prepared the virus stock used in this study as described previously (Deng et al, 2008). A cell culture-adapted P-47 strain (Bungyoku et al, 2009;Deng et al, 2008) was used throughout the experiments. Virus infection was performed at an m.o.i.…”

Cell Culture Systems for Hepatitis C Virus

“…However, genetic incompatibility between JFH1 and the alternative HCV gen ome segment fused to it often limits production of infectious virus This restriction can be overcome by serial passage of the chimeras in cell culture which over time results in the accumulation of adaptive changes compensating the genetic differences between the fused genomes and thus increases virus yields (Abe, Ikeda et al 2007;Gottwein, Scheel et al 2007;McMullan, Grakoui et al 2007;Yi, Ma et al 2007;Jensen, Gottwein et al 2008;Scheel, Gottwein et al 2008;Bungyoku, Shoji et al 2009;Gottwein, Scheel et al 2009;Gottwein, Jensen et al 2011;Koutsoudakis, Perez-del-Pulgar et al 2011;Chan, Cheng et al 2012). For instance, Yi and colleagues demonstrated that mutations in E1, p7, NS2 and NS3 contribute to the ability of a H77/JFH1 chimeric genome to assemble and release high amounts of virus particles .…”

Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

“…The full-length genome of isolate JFH-1 was demonstrated to be competent for viral particle production in tissue culture (22,41,46) by using Huh-7-derived cell lines that are permissive to HCV infection and replication (2,20). Several of these HCV cell culture (HCVcc) systems have been described, the most robust of which are based on chimeric J6/JFH-1 viruses or tissue culture-adapted strains of JFH-1 (1,3,4,17,18,26,36,47). However, the quantity of infectious virions these systems can produce is limited, presumably due to currently unidentified constraints on infectious virus particle production in tissue culture (5,16,26,47).…”