Development of robust genotype 1a hepatitis C replicons harboring adaptive mutations for facilitating the antiviral drug discovery and study of virus replication

• Published in: Journal of virological methods 2018-09, Vol.259, p.10-17
• Main Authors: Lin, Hui-Mei, Wu, Pei-Shan, Hu, Han-Shu, Chang, Wan-Chun, Wu, Ren-Huang, Tian, Jia-Ni, Chern, Jyh-Haur, Yueh, Andrew
• Format: Article
• Language: English
• Subjects: Adaptive mutation; Genotype 1a; HCV replicon; Replication capacity
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2018.05.010

•Novel adaptive mutations within NS3, NS4A, and NS4B were found in cells harboring stable replication HCV1a replicon.•One D1431Y adaptive mutation was identified in all stable HCV1a replicon cells.•Two novel HCV1a mutant replicons displayed robust replication either in transient and stable expression.•Robust HCV1a replicons exhibited no impairment in the susceptibility of replicon activity to known HCV inhibitors. The hepatitis C virus (HCV) subgenomic replicon is a valuable tool for studying virus replication and HCV drug development. Despite the fact that HCV genotype 1a (HCV1a) is the most prevalent genotype in the United States, few HCV1a reporter replicon constructs have been reported, and their replication capacities are not as efficient as those of HCV1b or 2a, especially in transient expression. In this study, we selected efficient HCV1a replicons and characterized the novel adaptive mutations derived from stable HCV1a (strain H77) replicon cells after G418 selection. These novel adaptive mutations were scored in NS3 (A1065V, C1073S, N1227D, D1431Y, and E1556G), NS4A (I1694T and E1709V), and NS4B (G1871C). The D1431Y mutation alone or combinations of other adaptive mutations introduced into the parental HCV1a replicon construct was observed to differentially enhance either transient or stable expression of replicon. In particular, two replicon mutants VDYG (A1065V, N1227D, D1431Y, and E1556G within NS3) and VDYGRG, VDYG with two additional adaptive mutations (NS4A-K1691R and NS4B-E1726G), displayed robust replication and exhibited no impairment in the susceptibility of replicon activity to various known HCV inhibitors.