Cell Culture-Adaptive Mutations Promote Viral Protein-Protein Interactions and Morphogenesis of Infectious Hepatitis C Virus

Recent genetic studies suggested that viral nonstructural (NS) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis C virus (HCV). Adaptive and compensatory mutations occurring in different NS proteins were demon-strated to promote HCV production in cell culture. However, the underlying molecular mechanism of NS proteins in HCVmor-phogenesis is poorly understood.We have isolated a cell culture-adapted HCV of genotype 2a (JFH1) which grew to an infectious titer 3 orders of magnitude higher than that of wild-type virus. Sequence analysis identified a total of 16 amino acid mutations in core (C), E1, NS2, NS3, NS5A, and NS5B, with the majority of mutations clustered in NS5A. Reverse genetic analysis of these mu-tations individually or in different combinations demonstrated that amino acid mutations in NS2 and NS5Amarkedly enhanced HCV production. Additionally, mutations in C, E1, NS3, and NS5B synergistically promoted HCV production in the background of NS2 and NS5Amutations. Adaptive mutations in NS5A domains I, II, and III independently enhanced HCV production, sug-gesting that all three domains of NS5A are important for HCVmorphogenesis. More importantly, adaptive mutations greatly enhanced physical interactions among HCV structural and NS proteins, as determined by studies with coimmunoprecipitation andmammalian two-hybrid assays. Collectively, these findings demonstrate that adaptive mutations can enhance specific pro-tein-protein interactions among viral structural and NS proteins and therefore promote the assembly of infectious HCV particles. Hepatitis C virus (HCV) is a major cause of chronic liver dis-eases, affecting approximately 170 million people worldwid