Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

Silberstein, Erica; Mihalik, Kathleen; Ulitzky, Laura; Plant, Ewan P.; Puig, Montserrat; Gagneten, Sara; Yu, Meng-Lin; Kaushik‐Basu, Neerja; Taylor, Derek J.

doi:10.1371/journal.ppat.1000910

Cited by 14 publications

(28 citation statements)

References 66 publications

(113 reference statements)

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“…This breakthrough was not accomplished until 2005. Other HCV culture systems have been reported (42)(43)(44); however, none was found to release infectious virus at a level high enough to study the HCV life cycle. The H77-S (1a) system with H77 replicon mutations was the only genotype 1a isolate reported to be able to, albeit inefficiently, infect Huh7.5 cells and release infectious virus particles (15).…”

Section: Discussionmentioning

confidence: 99%

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

Ramírez

Jensen

et al. 2012

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

106

153

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Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture patient isolates representing HCV genotypes 1-7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5′ untranslated region (5′UTR)-NS5A and JFH1 NS5B-3′UTR; recovered viruses acquired two adaptive mutations located in NS3 and NS4B. Introduction of these changes into a replication-deficient TN full-length genome, harboring LSG, permitted efficient HCV production. Additional identified NS4B and NS5B mutations fully adapted the TN full-length virus. Thus, a TN genome with 8 changes (designated TN cell-culture derived, TNcc) replicated efficiently and released infectious particles of ∼5 log 10 focus-forming units per mL; passaged TNcc did not require additional changes. IFN-α and directly acting antivirals targeting the HCV protease, NS5A, and NS5B, each inhibited full-length TN infection dose-dependently. Given the unique importance of genotype 1 for pathogenesis, this infectious 1a culture system represents an important advance in HCV research. The approach used and the mutations identified might permit culture development for other HCV isolates, thus facilitating vaccine development and personalized treatment. H epatitis C virus (HCV) chronically infects an estimated 130-170 million people worldwide. The infection increases the risk of developing liver cirrhosis and liver cancer and results in more than 350,000 deaths annually. No HCV vaccine is available. Current standard treatment is based on IFN-α/ribavirin, which, however, has low efficacy against the most prevalent HCV variants (1). Incorporation of directly acting antivirals (DAAs) in treatment regimens improves sustained viral response rate, but a favorable outcome is challenged by fast emergence of drug resistance and differential responses of the different HCV genotypes (2). Thus, HCV infection continues to be a huge health and economic burden to the world population, and improved in vitro experimental systems would be important to permit additional studies of new antivirals and associated resistance patterns.HCV is a small enveloped virus belonging to the genus Hepacivirus in the family Flaviviridae. The HCV genome is a positivesense single-strand RNA (∼9.6 kb), consisting of a single ORF flanked by 5′ and 3′ untranslated regions (UTRs). The ORF encodes virus structural proteins (core, E1, and E2), p7, and six nonstructural (NS...

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Section: Discussionmentioning

confidence: 99%

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

Ramírez

Jensen

et al. 2012

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

106

153

View full text Add to dashboard Cite

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“…In the case of the genotype 1a isolate H77, a highly adapted variant optimized for high replication gave rise to only low viral titers (53). A recently described genotype 1b-based cell culture model unfortunately lacks a cloned sequence (40). For Con1, which was the first viral isolate ever shown to replicate in cell culture (29), the most efficient combinations of replication-enhancing mutations were shown to entirely abolish infectivity in chimpanzees (14) by impairing the generation of infectious virions (37).…”

Section: Vol 85 2011mentioning

confidence: 99%

A Comprehensive Structure-Function Comparison of Hepatitis C Virus Strain JFH1 and J6 Polymerases Reveals a Key Residue Stimulating Replication in Cell Culture across Genotypes

Schmitt

Scrima

Radujkovic

et al. 2011

J Virol

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The hepatitis C virus (HCV) genotype 2a isolate JFH1 represents the only cloned HCV wild-type sequence capable of efficient replication in cell culture as well as in vivo. Previous reports have pointed to NS5B, the viral RNA-dependent RNA polymerase (RdRp), as a major determinant for efficient replication of this isolate. To understand the contribution of the JFH1 NS5B gene at the molecular level, we aimed at conferring JFH1 properties to NS5B from the closely related J6 isolate. We created intragenotypic chimeras in the NS5B regions of JFH1 and J6 and compared replication efficiency in cell culture and RdRp activity of the purified proteins in vitro, revealing more than three independent mechanisms conferring the role of JFH1 NS5B in efficient RNA replication. Most critical was residue I405 in the thumb domain of the polymerase, which strongly stimulated replication in cell culture by enhancing overall de novo RNA synthesis. A structural comparison of JFH1 and J6 at high resolution indicated a clear correlation of a closed-thumb conformation of the RdRp and the efficiency of the enzyme at de novo RNA synthesis, in accordance with the proposal that I405 enhances de novo initiation. In addition, we identified several residues enhancing replication independent of RdRp activity in vitro. The functional properties of JFH1 NS5B could be restored by a few single-nucleotide substitutions to the J6 isolate. Finally, we were able to enhance the replication efficiency of a genotype 1b isolate with the I405 mutation, indicating that this mechanism of action is conserved across genotypes.The hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae (47). The genome of HCV encompasses a single ϳ9,600-nucleotide (nt)-long RNA molecule carrying one large open reading frame (ORF), flanked by nontranslated regions (NTRs), that is translated primarily into one polyprotein. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different products (for a review, see reference 5). The nonstructural proteins NS3 to NS5B are necessary and sufficient for autonomous RNA replication. They form a membrane-associated replication complex, in which NS5B is the RNA-dependent RNA polymerase (RdRp), the key enzyme of viral RNA replication. Purified NS5B can initiate RNA synthesis in vitro by a primer-dependent mechanism or de novo (7,28,30,55). De novo initiation at the 3Ј end of the viral positiveand negative-strand RNA is likely to be the physiological mode of initiation of RNA synthesis in infected cells. The crystal structures of several viral RdRps that initiate RNA synthesis de novo have been reported, including that of HCV NS5B (3,13,25), the first such structure to be solved, and more recently those of other Flaviviridae polymerases (15, 31, 51). All of these enzymes are homologous and for all of them the "fingers" and "thumb" subdomains are connected (through the so-called "fingertips") and cluster around the central, catalytic "palm" s...

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“…Among other culture systems reported (41)(42)(43)(44)(45), only H77-S (genotype 1a) carrying mutations in NS3, NS4A, and NS5A, identified in the H77 replicon system, was found to enable studies of the HCV life cycle (44), and its infectivity was improved by an E2 mutation (46). Nevertheless, the extensive heterogeneity of HCV and genotype specificities in virus production (33,34) and in response to antivirals (7,(31)(32)(33) and neutralizing antibodies (23,24,27,34) argue for a critical need for developing full-length culture systems for other HCV isolates.…”

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confidence: 99%

Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains

Ramírez

Gottwein

et al. 2012

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

164

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Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases worldwide, but treatment options are limited. Basic HCV research required for vaccine and drug development has been hampered by inability to culture patient isolates, and to date only the JFH1 (genotype 2a) recombinant replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5′ end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six adaptive mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A-and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture adaptation of the genetically divergent isolate J8 (genotype 2b), which differed from the J6 nucleotide sequence by 24%. The most efficient recombinant, J8cc, had nine adaptive mutations and was genetically stable after viral passage. The availability of these robust JFH1-independent genotype 2a and 2b culture systems represents an important advance, and the approach used might permit culture development of other isolates, with implications for improved individualized treatments of HCV patients and for development of broadly efficient vaccines.epatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The outcome of infection is associated with genetic variability of HCV and host factors (1). No vaccine is available, and current IFNbased treatment is suboptimal, with many side effects, with low efficacy against the most prevalent HCV variants (2-4), and with differential influence from host factors (5). Directly acting antivirals (DAA) might improve treatment outcome but also have differential efficacy in treatment of patients with different HCV genotypes (6). The HCV positive sense single-strand RNA genome (∼9.6 kb) contains a single ORF flanked by 5′ and 3′ untranslated regions (UTRs). The ORF encodes virus structural proteins (Core, E1, and E2), p7, and six nonstructural (NS) proteins (7). HCV isolates are classified into seven major genotypes and numerous subtypes differing by 31-33% and 20-25%, respectively (8).The high heterogeneity of HCV and the lack of representative culture systems have hampered HCV vaccine development, preclinical drug testing, assessment of neutralizing antibodies, and basic HCV research. Although a number of HCV full-length genomes were shown to be infectious in chimpanzees (9-15), to date only the JFH1 strain (genotype 2a) could replicate auton...

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Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

Cited by 14 publications

References 66 publications

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

A Comprehensive Structure-Function Comparison of Hepatitis C Virus Strain JFH1 and J6 Polymerases Reveals a Key Residue Stimulating Replication in Cell Culture across Genotypes

Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains

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