Template requirement and initiation site selection by hepatitis C virus polymerase on a minimal viral RNA template.
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.
Oh
J W
JW
Howard Hughes Medical Institute and Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, California 90033-1054, USA.
Sheu
G T
GT
Lai
M M
MM
D020413:3' Untranslated Regions / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D020413:3' Untranslated Regions; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase