Analysis of hepatitis C virus Core/NS5A co-localization using novel cell culture systems expressing Core-NS2 and NS5A of genotype 1-7

Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. Apparently the HCV Core and NS5A proteins co-localize on cytoplasmic lipid droplets and are involved in particle production. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among HCV genotypes. We previously developed recombinant viruses with genotype-specific Core-NS2 or NS5A. These represented important tools for analyzing the effects of antibodies and antivirals on diverse HCV genotypes. However, they did not permit genotype-specific analysis of interactions between NS5A and structural proteins. Here we developed novel JFH1-based recombinants expressing Core-NS2 and NS5A from genotypes 1-7, and analyzed Core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of Core-NS2/NS5A recombinants, and putative adaptive mutations were analyzed by reverse genetics. Adapted recombinants produced infectivity titers of 10(2.5)-10(4.5) FFU mL(-1). Co-localization analysis demonstrated that Core and NS5A from all genotypes co-localized extensively, with Pearson's coefficients of 0.73-0.82. One-way ANOVA statistical analysis indicated no significant difference in co-localization among different genotypes (p=0.66). In summary, we developed a panel of HCV genotype 1-7 Core-NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting Core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of Core/NS5A interactions during assembly and release of HCV particles of all major genotypes.