Trans-complementation of HCV replication by non-structural protein 5A

Although establishment of the subgenomic replicon system has considerably facilitated genetic analysis of HCV replication, many details remain largely unknown. To initially test whether HCV replication could be affected in trans, complementation studies were conducted in which defective replicon RNAs carrying a luciferase reporter were introduced into stable cells bearing functional replicons. The NS3 protease and the NS5B viral polymerase genes on the transfected replicons were rendered null by active site mutations and shown not to be complemented in trans by functional proteins expressed from the endogenous replicons. A new strategy was also developed to examine whether adapted copies of NS4B and NS5A could enhance the replication of transfected replicons carrying non-adapted genes. The replication efficiency of a replicon carrying two adaptive mutations in NS3 (E1202G and T1280I) had previously been shown to be greatly enhanced by the presence of a third mutation in either NS4B (K1846T) or NS5A (S2197P). A partially adapted luciferase replicon carrying only the two NS3 mutations was used to transfect cells containing replicons bearing the adapted NS4B or NS5A. Using this approach, NS5A, but not NS4B, was found to trans-complement. In a final confirmatory study, ectopically expressed NS5A also complemented the HCV replicon genome bearing the non-adapted NS5A. These studies strongly suggest that HCV non-structural proteins, with the exception of NS5A, can only act in cis on the RNA from which they were translated.