Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations

Studies of the Hepatitis C virus (HCV) replication cycle have been made pos sible with the development of subgenomic selectable RNAs that replicate aut onomously in cultured cells. In these replicons the region encoding the HCV structural proteins nas replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replica tion of these RNAs. Subsequent analyses revealed that, within selected cell s, HCV RNAs had acquired adaptive mutations that increased the efficiency o f colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we sho w that adaptive mutations enhance RNA replication. Transient-transfection a ssays that did not require selection of transfected cells demonstrated a cl ear correlation between the level of adaptation and RNA replication. The hi ghest replication level was found with an adapted replicon carrying two ami no acid substitutions located in NS3 and one in NS5A that acted synergistic ally. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and R NA replication was corroborated with replicons in which the selectable mark er gene was replaced by the gene encoding firefly luciferase. Upon transfec tion of naive Huh-7 cells, the levels of luciferase activity directly refle cted the replication efficiencies of the various replicon RNAs. These resul ts show that cell culture-adaptive mutations enhance HCV RNA replication.