Mutations in hepatitis C virus RNAs conferring cell culture adaptation

As an initial approach to studying the molecular replication mechanisms of hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, we have recently developed selectable self-replicating RNAs. Thes e replicons lacked the region encoding the structural proteins and instead carried the gene encoding the neomycin phosphotransferase. Although the rep lication levels of these RNAs within selected cells were high, the number o f G418-resistant colonies was reproducibly low. In a search for the reason, we performed a detailed analysis of replicating HCV RNAs and identified se veral adaptive mutations enhancing the efficiency of colony formation by se veral orders of magnitude. Adaptive mutations were found in nearly every no nstructural protein but not in the 5' or 3' nontranslated regions. The most drastic effect was found with a single-amino-acid substitution in NS5B, in creasing the number of colonies similar to 500-fold. This mutation was cons erved with RNAs isolated from one cell line, in contrast to other amino aci d substitutions enhancing the efficiency of colony formation to a much less er extent. Interestingly, some combinations of these nonconserved mutations with the highly adaptive one reduced the efficiency of colony formation dr astically, suggesting that some adaptive mutations are not compatible.