Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (-) RNA synthesis have been identified in the 3' non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (-) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3'-end of the (-) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.