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(Δ21), 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 ca...

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Bibliographic Details
Published in:Biochemistry (Easton) 2000-07, Vol.39 (28), p.8243-8249
Main Authors: Carroll, Steven S, Sardana, Vinod, Yang, Zhucheng, Jacobs, Amanda R, Mizenko, Christine, Hall, Dawn, Hill, Lorraine, Zugay-Murphy, Joan, Kuo, Lawrence C
Format: Article
Language:English
Subjects:
AIDS/HIV
Catalysis
Gene Deletion
Hepacivirus - enzymology
Hepacivirus - physiology
Hepatitis C virus
Human immunodeficiency virus 1
Reaction Time
RNA Replicase - genetics
RNA Replicase - isolation & purification
RNA Replicase - metabolism
Templates, Genetic
Virus Replication
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Summary:The enzymatic activity of a C-terminally truncated form of the RNA-dependent RNA polymerase, termed NS5B(Δ21), 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(Δ21) is characterized by biphasic reaction time courses. At high concentrations of nucleoside triphosphate in reactions allowing a preincubation of NS5B(Δ21) 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(Δ21) 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(Δ21) 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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi991992s