Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase a nd therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, w e demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or C;Mg, Enhan cement of RNA synthesis was found with various heteropolymeric RNA template s, with poly(C)-oligo(G)(12) but not with poly(A)-oligo(U)(12). Several ami no acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthes is. Most of these mutations, in particular those affecting the GDD motif (m otif C) strongly reduced or completely abolished activation by GTP, suggest ing that the same NTP-binding site is used for stimulation and RNA synthesi s. Since GTP did not affect the overall RNA binding properties or the elong ation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis, Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pe stivirus classical swine fever virus, but not with the 3D polymerase of pol iovirus, Thus, stimulation of RdRp activity by GTP is evolutionarily conser ved between the closely related hepaciviruses and pestiviruses but not betw een these and the more distantly related picornaviruses.