A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA

All of the previously reported recombinant RNA-dependent RNA polymerases (R dRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We ha ve now expressed a recombinant NS5B that is able to synthesize a full-lengt h HCV genome in a template-dependent and primer-independent manner. The kin etics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicati ng that it did not use the copy-back RNA as a primer. This RdRp was also ab le to accept heterologous viral RNA templates, including poly(A)- and non-p oly(A)-tailed RNA, in a primer-independent manner, but the products in thes e cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3'-end 98 nucleotides (nt) o f HCV RNA, which is conserved in all genotypes of HCV, as a template, a dis tinct RNA product was generated. Truncation of 21 nt from the 5' end or 45 nt from the 3' end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3'-end variable sequence region a nd the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3' end of minus-strand RNA of HCV genome also s erved as a template, and it required a minimum of 239 nt from the 3' end. T hese data defined the cis-acting sequences for HCV RNA synthesis at the 3' end of HCV RNA in both the plus and minus senses. This is the first recombi nant HCV RdRp capable of copying the full-length HCV RNA in the primer-inde pendent manner expected of the functional HCV RNA polymerase.