ROLE OF NONSTRUCTURAL PROTEINS IN PESTIVIRUS ASSEMBLY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) has been shown to produce moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been previously shown that intragenotypic recombinants that encode from core up to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized several intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of na¿ve Huh7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover NTRNS2 chimeric virus, which is equivalent to the previously reported FL-J6/JFH chimera, showed a 10- fold enhancement of virus titers compared to CNS2. Since there were no significant differences in terms of translation and replication between these two clones, it is possible that these three mutations may affect post-translational processes leading to an increase in virion production. Sequence comparisons between these two clones showed that NTRNS2 had three nucleotide differences that differed from CNS2. These mutations residing in the 5'NTR and core coding sequence had no effect on virion production. Importantly, we demonstrated that cells replicating and producing Cp7 virus showed decreased cell growth and increased apoptosis compared with JFH1, an effect that correlated with robust virion production. These studies begin to unravel the requirements for robust virus replication and the relationship between increased virion production and host cell viability.