Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive- and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5′ and 3′ ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5′ end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5′ end dramatically reduced the efficiency of cell colony formation. Likewise, the 3′-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3′ end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5′-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5′ end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5′-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3′-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3′ ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3′ end. We also determined the 5′-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5′ end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP.
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