Investigation of the role of nonstructural 4B protein in Hepatitis C Virus replication complex formation and infectious virus production

The Hepatitis C Virus (HCV) NS4B protein is one of the least understood viral proteins in terms of contributions to the virus lifecycle. NS4B has many functions related to formation and function of the viral genome replication complex (RC), most notably for its role in inducing a novel membrane structure termed the membranous web which houses the RC. The membranous web is largely believed to be derived from ER membranes, and recent data has also suggested membranes from the early endosome (EE) compartment are part of this membrane structure. This was due to the observation that Rab5, a regulator of EE membrane transport, was required for HCV RNA replication and that Rab5 co-localizes with NS4B foci in replicon cells. NS4B foci are considered to be an immunofluorescence marker equivalent to the membranous web. Recent data has also implicated a new role for the C-terminal domain (CTD) of NS4B in facilitating infectious virus production. Both point mutations and entire CTD sequence swaps between different genotype viruses have highlighted this new and exciting role for NS4B in virus production.;To test the hypothesis that cellular Rabs are involved in NS4B-mediated HCV RC formation, we employed two main methods. First, we modified an existing protocol for NS4B antibody directed immunoisolation of a subcellular fraction enriched in the HCV RC components, and used this fraction for immunoblot analysis. Secondly, we examined Rab co-localization with NS4B foci in replicon cells to compare to the profile in the isolated RC fraction, which was competent for HCV RNA synthesis. Rabs from every cellular compartment tested were present at different levels in the isolated RC fraction, but only the endocytic Rab5 and 7 were found to co-localize with NS4B foci. Dominant negative mutants of Rab5 and 7 disrupt NS4B foci, and silencing of Rab5 or 7 resulted in a significant decrease in HCV RNA replication. Taken together, our data implicate endocytic Rabs as being important for NS4B-mediated HCV RC formation.;To test the hypothesis that the NS4B CTD is involved in virus assembly, we created chimeric virus genomes where the NS4B CTD from JFH1 virus (genotype 2a, gt2a) was replaced with sequences from Con1 (gt1b) or H77 (gt1a) virus. These chimeric genomes had little impact on RNA replication, but showed a significant defect in virus production, likely at the level of virus assembly. Investigation into the causes of lower virus titers showed that the chimeric viruses have lower NS5A protein levels and a decrease in the NS5A p58/p56 ratio relative to JFH1. Cell culture adapted viruses were generated that had similar titer levels to that of JFH1, showed higher levels of NS5A protein, but interestingly did not change in their NS5A p58/p56 ratio relative to JFH1. Sequencing of the adapted viruses revealed four mutations, two in the chimeric NS4B CTD region, one in domain III of NS5A and one adjacent to the cytosolic loop of the p7 protein. Introduction of these mutations back into the parental chimeric viruses rescued virus production, highlighting a genetic interaction between NS4B, NS5A and p7 proteins. These studies illuminate a role for the NS4B CTD in virus production, possibly through interaction or modification of other HCV proteins.