Thermostable hepatitis C virus JFH1-derived variant isolated by adaptation to Huh7.5.1 cells

Hepatitis C virus (HCV) infection and propagation in cultured cells have mainly been investigated using the infectious clinical clone JFH1. However, its infectivity is not high enough for infection to be detected easily. In this study, we attempted to isolate HCV-JFH1 variants adapted to human hepatoma Huh7.5.1 cells. By performing serial passages of the wild-type HCV-JFH1 in Huh7.5.1 cells, we obtained a variant that was capable of inducing severe cytopathic effects and showed approximately 700-fold higher infectivity than the wild-type HCV-JFH1. Further, when highly permissive Huh7.5.1-8 cells were infected with this variant, viral particles were produced at >10¹¹ copies ml^-1, making this variant one of the most efficient HCV production systems. Two adaptive mutations were noted in the variant genome: a1994c (K74T) in the core protein region and t3014c (I414T) in the E2 protein region. Both mutations contributed to enhanced infectivity and their combination showed synergistic effects in this regard. An examination of recombinant viruses carrying K74T, I414T and K74T/I414T mutations revealed that none of the mutations had an effect on the steps after viral entry (genome replication, particle assembly and egress), but led to the viral infection becoming less dependent on scavenger receptor class B type I, changes of the infectious particles to a broader and lower range of densities, and enhanced thermal stability of the infectious viruses. Thus, this Huh7.5.1-adapted HCV-JFH1 variant with higher and stable infectivity should be a valuable tool for studying the molecular mechanisms behind the life cycle of HCV and for antiviral screening.