Genotype-specific expression system of Hepatitis C Virus glycoproteins for efficient viral particle complementation

Development of the JFH1-based HCV infection system has permitted analysis of the complete replication cycle in tissue culture. Chimeric HCV genotype 2a (GT2a) genomes like Fl-J6/JFH1” or “Jc1” consisting of J6 and JFH1 segments produce much higher virus titers than parental JFH1 indicating that viral determinants in the core to NS2 proteins modulate efficiency of virus production. However, the role of the HCV glycoproteins E1 and E2 in this regard and for assembly and release in general is not well defined. Likewise, lack of tissue culture systems for primary, patient-derived isolates continues to limit systematic functional studies of viral quasiespecies. In this study, we developed a trans-complementation system to investigate the function of different E1 and E2 variants in HCV assembly and release and create an efficient system for the expression of E1/E2 on authentic HCV particles. To this end the glycoproteins from all seven genotypes and from patients infected with clinically representative genotypes were cloned. Subsequently, the ability of these glycoproteins to restore virus production of a glycoprotein-deficient JFH1 genome in Huh-7.5 packaging cell lines constitutively expressing Jc1 core, p7 and NS2 was assessed by trans-complementation. Among all E1/E2 genes tested, those from GT 2 and 7 restored virus production most efficiently. The replacement of JFH1 (GT2a) core to NS2 with GT 1a-derived H77 genes allowed the efficient production of HCV particles harbouring GT 1a glycoproteins. The created trans-complemented particles were further characterized regarding their density distributions as well as their neutralization capacity. These results highlight that HCV glycoproteins support virus production in a genotype-specific fashion likely due to specific interactions with additional viral factors in the course of virus production. Moreover, this system allows creation of HCV particles carrying glycoprotein genes from primary patient isolates of GT 1 and 2 for functional studies in cell culture, thus presenting an alternative to the pseudoparticle system. This study should increase our understanding of HCV morphogenesis and ultimately permit analysis of patient-derived HCV glycoprotein genes of all major genotypes in tissue culture.