Only a small fraction of purified hepatitis C RNA-dependent RNA polymerase is catalytically competent: implications for viral replication and in vitro assays.

Abstract

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.

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@article{Carroll2000OnlyAS, title={Only a small fraction of purified hepatitis C RNA-dependent RNA polymerase is catalytically competent: implications for viral replication and in vitro assays.}, author={Steven S. Carroll and Vinod Sardana and Zheng-guan Yang and Aviva R. Jacobs and C Mizenko and Dawn L Hall and Lindsay Knauf Hill and Joan Zugay-Murphy and Laurence C Kuo}, journal={Biochemistry}, year={2000}, volume={39 28}, pages={8243-9} }