The hepatitis C virus core protein can modulate RNA-dependent RNA synthesis by the 2a polymerase

RNA replication enzymes are multi-subunit protein complexes whose activity can be modulated by other viral and cellular factors. For genotype 1b Hepatitis C virus (HCV), the RNA-dependent RNA polymerase (RdRp) subunit of the replicase, NS5B, has been reported to interact with the HCV Core protein to decrease RNA synthesis (Kang et al., 2009). Here we used a cell-based assay for RNA synthesis to examine the Core-NS5B interaction of genotype 2a HCV. Unlike the 1b NS5B, the activity of the 2a NS5B was stimulated by the Core protein. Using the bimolecular fluorescence complementation assay, the 2a Core co-localized with 2a NS5B when they were transiently expressed in cells. The two proteins can form a co-immunoprecipitable complex. Deletion analysis showed that the N-terminal 75 residues of 2a Core were required to contact 2a NS5B to modulate its activity. The C-terminal transmembrane helix of 2a NS5B also contributes to the interaction with the 2a Core. To determine the basis for the differential effects of the Core-RdRp interaction, we found that the 2a RdRp activity was enhanced by both the 1b Core and 2a Core. However, the 1b NS5B activity was slightly inhibited by either Core protein. The replication of the 2a JFH-1 replicon was increased by co-expressed 2a Core while the genotype 1b Con1 replicon was not significantly affected by the corresponding Core. Mutations in 2a NS5B that affected the closed RdRp structure were found to be less responsive to 2a Core. Finally, we determined that RNA synthesis by the RdRps from genotypes 2a, 3a and 4a HCV were increased by the Core proteins from HCV of genotypes 1-4. These results reveal another difference between RNA syntheses by the different genotype RdRps and add additional examples of a viral structural protein regulating viral RNA synthesis.