Specificity and mechanism analysis of hepatitis C virus RNA-dependent RNA polymerase.
The RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene has been expressed as a nonfusion protein in bacterial cells and purified to homogeneity using sequential chromatographic columns. The purified NS5B protein exhibited RNA-dependent RNA polymerase activity using poly(A) template and the K(m) and V(max) were determined as 8.4 microM and 1976 pmol/mg-min, respectively. This full-length NS5B protein exhibited much stronger binding affinity toward the 30-mer poly(G) than other homopolymeric RNAs of the same size. For the first time, we demonstrate that the HCV NS5B was able to bind various ribonucleotides. Using a panel of oligonucleotides varying in length, we studied the NS5B catalytic efficiency and proposed the size of the NS5B active site to be 8-10 nucleotides. The multifunctional nature of NS5B protein is also discussed and compared with other viral RNA polymerases.
Johnson
R B
RB
Infectious Diseases Research, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Sun
X L
XL
Hockman
M A
MA
Villarreal
E C
EC
Wakulchik
M
M
Wang
Q M
QM
D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D011068:Poly G / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D013816:Thermodynamics
D002413:Cations, Divalent; C034477:RNA primers; D012367:RNA, Viral; D011994:Recombinant Proteins; D012265:Ribonucleotides; D011061:Poly A; D011068:Poly G; D012313:RNA; D012324:RNA-Dependent RNA Polymerase