Efficient Replication of Genotype 3a and 4a Hepatitis C Virus Replicons in Human Hepatoma Cells

Abstract

Despite recent advances in the treatment of hepatitis C, the quest for pan-genotype, effective, and well-tolerated inhibitors continues. To facilitate these efforts, it is desirable to have in vitro replication systems for all major HCV genotypes. However, cell culture replication systems exist for only genotypes 1a, 1b, and 2a. In this study, we generated G418-selectable subgenomic replicons for prototype strains of genotypes 3a (S52) and 4a (ED43). Production of G418-resistant colonies by S52 and ED43 in Huh-7.5 cells required the amino acid substitutions S2210I and R2882G, respectively, cell culture adaptive mutations originally reported for genotype 1b replicons. RNA replication was confirmed by quantitative reverse transcription-PCR and detection of viral protein. Sequencing of multiple independent replicon clones revealed the presence of additional nonsynonymous mutations. Interestingly, all potentially adaptive mutations mapped to the NS3 protein. These mutations, when introduced back into original constructs, substantially increased colony formation efficiency. To make these replicons useful for high-throughput screening and evaluation of antiviral compounds, they were modified to express a chimeric fusion protein of firefly luciferase and neomycin phosphotransferase to yield stable replicon-expressing cells. Using these constructs, the inhibitory effects of beta interferon (IFN-␤), an NS3 protease inhibitor, and an NS5B nucleoside polymerase inhibitor were readily detected by monitoring luciferase activity. In conclusion, we have established functional replicons for HCV genotypes 3a and 4a, important new additions to the armamentarium required to develop inhibitors with a pan-genotype activity.A ccording to estimates of the World Health Organization (WHO), hepatitis C virus (HCV) currently infects at least 130 million people worldwide, which is 2.2% of the global population (33). HCV infection becomes chronic in 60% to 80% of infected adults and can progress to hepatic fibrosis, liver cirrhosis, and hepatocellular carcinoma (HCC) (11). There is no vaccine against HCV infection, and the standard of care until last year, consisting of pegylated alpha interferon (IFN-␣) and ribavirin, resulted in a sustained virological response (SVR) in only half of patients (43). The recent addition of the HCV protease inhibitors telaprevir and boceprevir has increased SVR rates to 70 to 80% (26,48,53). However, the efficacy of these inhibitors is limited by the emergence of resistance, challenging side effect management, and limited HCV genotype coverage (19,37,38,50,57,58). Thus, there continues to be an urgent need to find better and more-broadly acting anti-HCV drugs. To promote this goal, it is important to establish in vitro replication systems for all HCV genotypes that can be used as preclinical tools for screening and optimization of new inhibitors.HCV strains from different parts of the world show significant genetic heterogeneity, and on the basis of phylogenetic analysis, HCV has been classified into seven ge...

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

“…The genotype 2a replicon, JFH1/SG-Feo, and the genotype 3a replicon, S52/SGFeo(AII), have been previously described (12). The genotype 4a replicon, ED43/SG-Feo(VYG), was made by introducing a newly identified mutation, leading to an M1205V substitution in the NS3 protein of ED43/SGFeo(Y), as described by Saeed et al (12).…”

“…The quality of in vitro-transcribed RNA was assessed by agarose gel electrophoresis. Huh-7.5 cells were electroporated with RNA transcripts using a BTX ElectroSquarePorator as described previously (12). To obtain cell colonies carrying autonomously replicating HCV genomes, electroporated cells were exposed to G418 (final concentration of 750 g/ml) at 48 h postelectroporation, and medium was replaced every third day with a fresh medium containing 750 g/ml G418.…”

“…To facilitate in vitro transcription, a T7 promoter was cloned upstream of the HCV IRES and an XbaI runoff site was introduced downstream of the 3= UTR. The NPTII gene of SA1/SG-neo was replaced with a chimeric gene encoding firefly luciferase protein fused in-frame with NPTII to synthesize SA1/SG-Feo, as described elsewhere (12,23).…”

“…Analysis of HCV RNA replication in replicon cells. The isolation of colonies, RNA quantification, and flow cytometry were performed exactly as described previously (12).…”

“…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.…”