Only a small fraction of purified hepatitis C RNA-dependent RNA polymerase is catalytically competent: implications for viral replication and in vitro assays.
The enzymatic activity of a C-terminally truncated form of the RNA-dependent RNA polymerase, termed NS5B(Delta21), of the hepatitis C virus (strain BK) has been investigated using both homopolymeric and heteropolymeric RNA templates. Incorporation of nucleotides into a heteropolymeric RNA template as catalyzed by NS5B(Delta21) is characterized by biphasic reaction time courses. At high concentrations of nucleoside triphosphate in reactions allowing a preincubation of NS5B(Delta21) and RNA template, an initial rapid phase of the reaction is followed by a slower linear phase. The amplitude of the first phase of the reaction varies directly with the concentration of the enzyme in the reaction. It is shown here that full-length copies of the template are produced during the first phase of the reaction. Our results reveal that NS5B(Delta21) is processive but only a small fraction, less than 1%, of the purified enzyme present participates productively in the reaction. Most importantly, the turnover number for the hepatitis C NS5B(Delta21) is comparable to those observed for other polymerases such as the HIV-1 reverse transcriptase. The combined results reconcile in part the apparent discrepancy of the low, observed specific activity of the purified enzyme and the rapid generation of HCV in vivo.
Carroll
S S
SS
Department of Antiviral Research, Merck Research Labs, West Point, Pennsylvania, 19486, USA.
Sardana
V
V
Yang
Z
Z
Jacobs
A R
AR
Mizenko
C
C
Hall
D
D
Hill
L
L
Zugay-Murphy
J
J
Kuo
L C
LC
D002384:Catalysis; D017353:Gene Deletion; D016174:Hepacivirus / Q000201:enzymology* / Q000502:physiology; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011930:Reaction Time; D013698:Templates, Genetic; D014779:Virus Replication
D012324:RNA-Dependent RNA Polymerase