The hepatitis C virus (HCV) NS5B protein contains the RNA dependent RNA polymerase (RdRp) activity that catalyzes the synthesis of the viral genome with other host and viral factors. NS5A is an HCV-encoded protein previously shown to localize to the replisome and be necessary for viral replication. However, its role in replication has not been defined. Using an in vitro biochemical assay, we detected a stimulatory effect of NS5A on the NS5B replication reaction with minimal natural templates. NS5A stimulates replication by NS5B on two templates derived from the 3’ end of the RNA genome (4 fold ± 1.3 fold). A pre-incubation step with the two proteins prior to the replication reaction and substoichiometric levels of NS5A are required for detecting stimulation. With a template derived from the 3’end complementary to the RNA genome (the negative strand) no stimulation was observed. Furthermore, with a synthetic template that allows studying different phases of replication, NS5A stimulates NS5B during elongation. These findings suggest that NS5A stimulates NS5B during synthesis of the complementary (i.e., negative) strand of the RNA genome.
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