Development of a robust luciferase reporter 1b/2a hepatitis C virus (HCV) for characterization of early stage HCV life cycle inhibitors.

The development of JFH1 based intergenotypic recombinants that exploit the unique replication characteristics of JFH1 has made it possible to study infectious hepatitis C virus (HCV) encoding the structural genes of additional HCV genotypes. To facilitate the study of 1b structural proteins, we aimed to develop a robust 1b/2a chimera encoding a humanized Renilla luciferase reporter gene (1b/2a hRluc). The unadapted genome replicated efficiently but produced very low titers of infectious virus. Adaptation by continuous passage over a novel Huh-7 Lunet clone improved viral titers approximately 100-fold but caused an unexpected decline in luciferase activity, limiting the utility of the reporter-containing virus. Genotypic analysis revealed 17 adenosine to guanosine (A to G) nucleotide mutations in the luciferase gene and two potential adaptive mutations. To overcome the problems of low viral titers and editing of the luciferase gene during viral adaptation, six adaptive mutations previously identified in a non-reporter 1b/2a HCV genome were introduced into the 1b/2a hRluc genome. This resulted in the immediate production of high-titer viral stocks (approximately 1000-fold greater than the parental virus) that could efficiently infect naïve cells and generate robust luciferase signals. The improved sensitivity of the luciferase reporter also facilitated time of addition studies validating the utility of this system for characterizing the early steps of HCV infection. Thus, the development of the 1b/2a hRluc reporter virus described here provides a versatile tool for discovery of inhibitors targeting the early steps of the viral life cycle and genotype 1b structural genes.