RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3-end. We have previously shown that NS5B can utilize the 3-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single-stranded region closest to the 3-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells.
Hepatitis C virus (HCV)1 is the etiological agent of non-A, non-B hepatitis, often causing liver diseases including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1-4). HCV has a positive-sense, single-stranded RNA genome of approximately 9700 nucleotides (nt) in length, which is terminated with a stretch (98 nt) of highly conserved sequence, termed the X region (5-11). The X region folds into a stable secondary structure consisting of three stem-loop domains (12, 13). Upstream of the X region is a stretch of poly(U-U/C)-rich sequences of variable length and highly variable sequences of about 30 -40 nt (5-11). Infectivity assays showed that the X region and U-U/C-rich sequences are required for viral infectivity, but the variable sequences are not (14). As implicated by sequence conservation among all HCV genotypes, the structure and/or sequence of the X region of HCV is important for minusstrand RNA synthesis and translational regulation (15, 16). The replication of HCV RNA is mediated by NS5B, which is an RNA-dependent RNA polymerase (RdRp) (16 -20).The initial step of viral RNA replication is recognition of the 3Ј-end of RNA template by RdRp, which may occur directly or indirectly with the help of cellular proteins (21, 22). For example, Q bacteriophage replicase recognizes the replicable RNA templates with the help of cellular factors, including ribosomal protein S1 and translation elongation factor Tu (23-25), which are also important for template recognition on certain in vitro...