Cell Culture Adaptation of Hepatitis C Virus and In Vivo Viability of an Adapted Variant

Kaul, Artur; Woerz, Ilka; Meuleman, Philip; Leroux‐Roels, Geert; Bartenschlager, Ralf

doi:10.1128/jvi.01362-07

Cited by 147 publications

(201 citation statements)

References 60 publications

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“…Cell culture-derived adaptive mutations can greatly improve the in vitro replication capacity of the JFH1 strain of HCV (6,(9)(10)(11)(12)(13). However, the mechanism of these adaptive mutations and the steps at which they exert their effects are difficult to ascertain with highly permissive cell lines such as Huh-7.5 cells that support multiple rounds of the complete viral life cycle.…”

Section: Discussionmentioning

confidence: 99%

“…Because mutations in E2, p7, and NS2 often have been associated with adaptation in other studies (6,(9)(10)(11)(12), the four mutations in these genes were cloned into the wild-type JFH1 genome in the absence (JFH-AM1) or presence (JFH-AM2) of the NS5A mutation, and the replication capacity of these recombinant viruses was compared with that of wild type. After transfection, at day 5, both viruses had produced 100-to 1,000-fold more infectious virus than had wild-type (data not shown).…”

Section: Selection Of Mutations During Serialmentioning

confidence: 99%

“…A number of recent studies have identified adaptive or compensatory mutations that enhance infectious virus production from either wild-type JFH1 (6,9,10) or intergenotypic chimeras (11)(12)(13). To date, cell culture-selected mutations have been found in nearly all of the structural and nonstructural proteins, but the majority of these have mapped to the core-NS2 coding region, with a noticeable preference for the p7 and NS2 proteins (11,12).…”

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confidence: 99%

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Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

Russell

Meunier²,

Takikawa³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

158

208

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The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast, both the p7 and NS2 mutations independently increased the amount of virus released.single-cycle growth ͉ CD81-dependent

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Section: Discussionmentioning

confidence: 99%

Section: Selection Of Mutations During Serialmentioning

confidence: 99%

mentioning

confidence: 99%

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Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

Russell

Meunier²,

Takikawa³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

158

208

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“…Further examination will be required to clarify the effects of the F2281L mutation on the infectivity of the virus. Kaul et al (2007) reported the V2941M mutation in NS5B in the context of the JFH1 genome. Lohmann et al (2001) reported the R2884G mutation in the context of Con1-based replicon cells.…”

Section: Discussionmentioning

confidence: 99%

“…Upon transfection of Huh-7 cells with the in vitrotranscribed HCV JFH1 genome or the chimera FL-J6/JFH1, infectious HCV particles were secreted in an envelope glycoprotein-dependent manner (Lindenbach et al, 2005;Wakita et al, 2005;Zhong et al, 2005). Using HCVproduction systems, adaptive or compensatory mutations that promote the production of infectious virus from wildtype JFH1 (Delgrange et al, 2007;Kaul et al, 2007;Russell et al, 2008;Zhong et al, 2006) or chimeric viruses (Gottwein et al, 2007;Yi et al, 2006Yi et al, , 2007 have been identified. However, the molecular mechanisms of adaptive mutations are poorly understood.…”

Section: Introductionmentioning

confidence: 99%

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

Bungyoku

Shoji

Makine

et al. 2009

Journal of General Virology

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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...

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Hepatitis C virus JFH-1 strain infection in chimpanzees is associated with low pathogenicity and emergence of an adaptive mutation

et al. 2008

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The identification of the hepatitis C virus (HCV) strain JFH-1 enabled the successful development of infectious cell culture systems. Although this strain replicates efficiently and produces infectious virus in cell culture, the replication capacity and pathogenesis in vivo are still undefined. To assess the in vivo phenotype of the JFH-1 virus, cell culture-generated JFH-1 virus (JFH-1cc) and patient serum from which JFH-1 was isolated were inoculated into chimpanzees. Both animals became HCV RNA-positive 3 days after inoculation but showed low-level viremia and no evidence of hepatitis. HCV viremia persisted 8 and 34 weeks in JFH-1cc and patient serum-infected chimpanzees, respectively. Immunological analysis revealed that HCV-specific immune responses were similarly induced in both animals. Sequencing of HCV at various times of infection indicated more substitutions in the patient serum-inoculated chimpanzee, and the higher level of sequence variations seemed to be associated with a prolonged infection in this animal. A common mutation G838R in the NS2 region emerged early in both chimpanzees. This mutation enhances viral assembly, leading to an increase in viral production in transfected or infected cells. Conclusion: Our study shows that the HCV JFH-1 strain causes attenuated infection and low pathogenicity in chimpanzees and is capable of adapting in vivo with a unique mutation conferring an enhanced replicative phenotype. (HEPATOLOGY 2008;48:732-740.) H epatitis C virus (HCV) infects approximately 170 million people worldwide and is a major causative agent of chronic liver diseases including cirrhosis and hepatocellular carcinoma. 1,2 However, the underlying biological mechanisms of pathogenesis and persistence are still not well understood. No vaccine protecting against HCV infection is currently available. 3 Therapy for HCV-related chronic hepatitis remains problematic, with limited efficacy, high cost, and substantial adverse effects. 1,4,5 Understanding the biology of this virus and the development of new therapies has been hampered by a lack of appropriate model systems for replication and infection of this virus.

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Cell Culture Adaptation of Hepatitis C Virus and In Vivo Viability of an Adapted Variant

Cited by 147 publications

References 60 publications

Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

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

Hepatitis C virus JFH-1 strain infection in chimpanzees is associated with low pathogenicity and emergence of an adaptive mutation

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