Recombinant viral RdRps can initiate RNA synthesis from circular templates

Use of linear and circular RNA templates by several recombinant RdRps. (A) RNA synthesis from +1C-C by purified recombinant RdRps from HCV, BVDV, and GBV. Each reaction was performed with different GTP concentrations to show that RNA synthesis required higher levels of GTP. Radiolabeled [α-³²P]CTP, ATP, and UTP were present at 250 nM, 100 μM, and 100 μM, respectively. (B) Circularization of an RNA (CU4) from bacteriophage [phis]6. CU4 is a 22-nt RNA derived from the 3′-terminal sequence of the [phis]6 RNA genome (sequence: 5′-GGCCCCUUCGGGGGCUCUCUCU-3′). After ligation with T4 RNA ligase, the linear and circularized RNAs were purified from a denaturing gel and then used for 3′-terminal extension reaction in the presence of [α-³²P]ATP with poly(A) polymerase. (C) RNA synthesis by the [phis]6 RdRp from the linear and circularized versions of CU4. The amount of input template in each reaction is denoted above the gel image.