Replication of Hepatitis C Virus Genotype 3a in Cultured Cells

Saeed, Mohsan; Gondeau, Claire; Hmwe, Su-Su; Yokokawa, Hiroshi; Date, Tomoko; Suzuki, Tetsuro; Kato, Takanobu; Maurel, Patrick; Wakita, Takaji

doi:10.1053/j.gastro.2012.09.017

Cited by 46 publications

(44 citation statements)

References 20 publications

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“…The development of genotype 1 to 6 replicons has permitted replication studies (40)(41)(42)(43)(44)(45)(46). However, NS3 is also involved in later steps of the viral life cycle (12).…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

Serre

Jensen

Ghanem

et al. 2016

Antimicrob Agents Chemother

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bHepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research. With more than 100 million chronic infections causing approximately 500,000 deaths annually, hepatitis C virus (HCV) is a major global health and economic burden (1, 2). The six epidemiologically important genotypes differ in ϳ30% of their sequence and in their sensitivity to antiviral regimens (3-6). In Europe, the Americas, Asia, and Australasia, genotypes 1, 2, and 3 are most prevalent. While genotypes 4, 5, and 6 are more restricted to specific geographical regions in Africa and Asia, they account for 20% of global HCV infections and have spread beyond these primary geographical locations (1, 2, 7).The development of directly acting antivirals (DAAs) has revolutionized HCV therapy. The main components of interferonfree regimens introduced in the clinic are inhibitors of the HCV nonstructural (NS) proteins NS3 protease (NS3P), NS5A,8,9). Even though DAA-based therapy regimens could cure most patients in clinical trials, failure rates of 5 to 10% are to be expected in real life, due primarily to the development of DAA resistance (4,8). Given the large ...

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“…The development of genotype 1 to 6 replicons has permitted replication studies (40)(41)(42)(43)(44)(45)(46). However, NS3 is also involved in later steps of the viral life cycle (12).…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

Serre

Jensen

Ghanem

et al. 2016

Antimicrob Agents Chemother

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“…Viable JFH1-based intergenotypic recombinants, containing genotype-specific structural proteins, p7 and the complete or partial NS2, were generated for various genotypes of HCV (56,59,60). Although robust propagation systems of full-length HCV infectious clones of the H77 strain (genotype 1a) (61), TN strain (1a) (62), JFH-2 strain (2a) (34), and S310 strain (3a) (63) were established, the construction of infectious clones of other genotypes has not succeeded yet.…”

Section: Discussionmentioning

confidence: 99%

Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus

Mai

Fukuhara

Ono

et al. 2014

J Virol

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Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. Although the HCV life cycle has been clarified by studying laboratory strains of HCV derived from the genotype 2a JFH-1 strain (cell culture-adapted HCV [HCVcc]), the mechanisms of particle formation have not been elucidated. Recently, we showed that exogenous expression of a liver-specific microRNA, miR-122, in nonhepatic cell lines facilitates efficient replication but not particle production of HCVcc, suggesting that liver-specific host factors are required for infectious particle formation. In this study, we screened human cancer cell lines for expression of the liver-specific ␣-fetoprotein by using a cDNA array database and identified liver-derived JHH-4 cells and stomach-derived FU97 cells, which express liver-specific host factors comparable to Huh7 cells. These cell lines permit not only replication of HCV RNA but also particle formation upon infection with HCVcc, suggesting that hepatic differentiation participates in the expression of liver-specific host factors required for HCV propagation. HCV inhibitors targeting host and viral factors exhibited different antiviral efficacies between Huh7 and FU97 cells. Furthermore, FU97 cells exhibited higher susceptibility for propagation of HCVcc derived from the JFH-2 strain than Huh7 cells. These results suggest that hepatic differentiation participates in the expression of liver-specific host factors required for complete propagation of HCV. IMPORTANCEPrevious studies have shown that liver-specific host factors are required for efficient replication of HCV RNA and formation of infectious particles. In this study, we screened human cancer cell lines for expression of the liver-specific ␣-fetoprotein by using a cDNA array database and identified novel permissive cell lines for complete propagation of HCVcc without any artificial manipulation. In particular, gastric cancer-derived FU97 cells exhibited a much higher susceptibility to HCVcc/JFH-2 infection than observed in Huh7 cells, suggesting that FU97 cells would be useful for further investigation of the HCV life cycle, as well as the development of therapeutic agents for chronic hepatitis C. More than 170 million individuals worldwide are infected with hepatitis C virus (HCV), and the cirrhosis and hepatocellular carcinoma induced by HCV infection are life-threatening diseases (1). Current standard therapy combining pegylated-interferon (peg-IFN) and ribavirin (RBV) has achieved a sustained virological response (SVR) in 50% of individuals infected with HCV genotype 1 (2). Recently, directly acting antiviral (DAA) agents have been applied in a clinical setting (3). An SVR rate of over 80% has been realized by combination therapy with peg-IFN, RBV, and NS3/4A inhibitors in genotype 1 patients (4, 5). In addition, several DAAs, including inhibitors for NS3/4A protease, NS5A, and NS5B polymerase, are currently in clinical trials. Several reports have shown that in vitro replication of HCV RNA is significantly inhibited by treat...

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“…2B). We have previously shown that some of the cell culture-adaptive changes, originally described for genotypes 1a and 1b replicons, can support replication of other genotypes (11,12). Based on this observation, we generated three mutants containing single-amino-acid substitutions: (i) SA1/SG-neo(i), containing a T1281I substitution in NS3 (corresponding to T1280I in genotype 1b strain Con1; GenBank accession no.…”

Section: Resultsmentioning

confidence: 99%

“…The same conditions were used for both rounds of PCR: initial denaturation at 98°C for 5 min, 35 cycles of PCR (98°C for 30 s, 55°C for 30 s, 72°C for 2 min), and a final elongation step at 72°C for 10 min. The 5= rapid amplification of cDNA ends (RACE) to amplify the terminal 5= end of the viral genome was performed exactly as described by Saeed et al (11). The terminal 3=-end sequence was determined by 3= RACE.…”

Section: Methodsmentioning

confidence: 99%

“…Of the six major HCV genotypes, replication systems for HCV genotypes 1 and 2 were developed over a decade ago (8)(9)(10) and provided a strong foundation for the development of currently approved antivirals. Recently, replication systems for genotypes 3, 4, and 6 have also been reported (11)(12)(13)(14). However, a similar system for genotype 5 is still lacking.…”

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confidence: 99%

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Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

Kinge

Espiritu

Prabdial-Sing³

et al. 2014

Antimicrob Agents Chemother

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cHepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pangenotype anti-HCV compounds. H epatitis C virus (HCV) currently infects approximately 185 million people worldwide, increasing their risk of developing liver cirrhosis and hepatocellular carcinoma (1). No vaccine is available against HCV infection, and the standard of care until 2011, consisting of PEGylated interferon (IFN) and ribavirin, cured only 50% of patients. However, the addition of direct-acting antiviral agents (DAAs) over the past few years has revolutionized HCV treatment, with cure rates now approaching 80 to 90% (2-5). The recently published results of phase 3 clinical trials report even better regimens with Ͼ95% cure rates (6, 7). These are exciting developments, yet the issues of drug resistance, side effects, and drug-drug interactions will remain a challenge. Therefore, the quest to find safe drugs with a pan-genotype activity and a high barrier to drug resistance will continue. To aid these efforts, it is important to have cell culture replication systems for all HCV genotypes. Of the six major HCV genotypes, replication systems for HCV genotypes 1 and 2 were developed over a decade ago (8-10) and provided a strong foundation for the development of currently approved antivirals. Recently, replication systems for genotypes 3, 4, and 6 have also been reported (11)(12)(13)(14). However, a similar system for genotype 5 is still lacking. This genotype is restricted mainly to South Afri...

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Replication of Hepatitis C Virus Genotype 3a in Cultured Cells

Cited by 46 publications

References 20 publications

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus

Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

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