Analysis of mutant NS5B proteins encoded by isolates from chimpanzees chronically infected following clonal HCV RNA inoculation.

We hypothesized that mutations in the HCV NS5B polymerase, which occur during infection, may affect RNA-dependent RNA polymerase (RdRp) activity. NS5B proteins corresponding to a genotype 1a infectious clone and mutants identified in chimpanzees following inoculation with the clone were expressed and purified and their in vitro RdRp activity was compared to a NS5B genotype 1b control. A Gln-65-to-His mutation increased RdRp activity by 1.8-fold as compared to the infectious clone. Moreover, this NS5B1a protein had RdRp activity similar to the NS5B1b control. Three NS5B proteins representing mutations found in another animal had no in vitro RdRp activity. All mutations were maintained in the majority circulating virus for at least 216 weeks. The results demonstrate that some in vivo mutations of NS5B directly enhance in vitro RdRp activity. In addition, they suggest that the in vitro RdRp activity of NS5B may not always reflect in vivo activity within replication complexes.