title: NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase.
abstract: Hepatitis C virus (HCV) RNA-dependent RNA polymerase replicates the viral genomic RNA and is a primary drug target for antiviral therapy. Previously, we described the purification of an active and stable polymerase-primer-template elongation complex. Here, we show that, unexpectedly, the polymerase elongation complex can use NTPs to excise the terminal nucleotide in nascent RNA. Mismatched ATP, UTP, or CTP could mediate excision of 3'-terminal CMP to generate the dinucleoside tetraphosphate products Ap(4)C, Up(4)C, and Cp(4)C, respectively. Pre-steady-state kinetic studies showed that the efficiency of NTP-mediated excision was highest with ATP. A chain-terminating inhibitor, 3'deoxy-CMP, could also be excised through this mechanism, suggesting important implications for nucleoside drug potency and resistance. The nucleotide excision reaction catalyzed by recombinant hepatitis C virus polymerase was 100-fold more efficient than the corresponding reaction observed with HIV reverse transcriptase.
authors: Virology Discovery, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA. zhinan.jin2@gmail.com
authors: Zhinan
authors: Z
authors: Jin
authors: Vincent
authors: V
authors: Leveque
authors: Han
authors: H
authors: Ma
authors: Kenneth A
authors: KA
authors: Johnson
authors: Klaus
authors: K
authors: Klumpp
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003568:Cytidine Monophosphate / Q000737:chemistry / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008957:Models, Genetic; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
chemical_list: D015226:Dinucleoside Phosphates; D009711:Nucleotides; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D054303:HIV Reverse Transcriptase; D003568:Cytidine Monophosphate; D014544:Uridine Triphosphate