The hepatitis C virus NS5B RNA-dependent RNA polymerase activity and susceptibility to inhibitors is modulated by metal cations

The aim of this study was to understand the effect of metal cations on the hepatitis C virus (HCV) NS5B in vitro RNA-dependent RNA polymerase (RdRp) activity and its susceptibility to various inhibitors. A recombinant full-length HCV NS5B protein was expressed in insect cells and purified to homogen...

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Published inJournal of human virology Vol. 3; no. 6; p. 306
Main Authors Alaoui-Lsmaili, M H, Hamel, M, L'Heureux, L, Nicolas, O, Bilimoria, D, Labonté, P, Mounir, S, Rando, R F
Format Journal Article
LanguageEnglish
Published United States 01.11.2000
Subjects
Animals
Baculoviridae - genetics
Cations, Divalent - pharmacology
Cells, Cultured
Chlorides - pharmacology
Enzyme Inhibitors - pharmacology
Hepacivirus - enzymology
Hepacivirus - genetics
Hepacivirus - metabolism
Humans
Magnesium Chloride - pharmacology
Manganese Compounds - pharmacology
Recombinant Proteins - metabolism
RNA-Dependent RNA Polymerase - antagonists & inhibitors
RNA-Dependent RNA Polymerase - genetics
RNA-Dependent RNA Polymerase - metabolism
Spodoptera
Viral Nonstructural Proteins - genetics
Viral Nonstructural Proteins - metabolism
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Summary:The aim of this study was to understand the effect of metal cations on the hepatitis C virus (HCV) NS5B in vitro RNA-dependent RNA polymerase (RdRp) activity and its susceptibility to various inhibitors. A recombinant full-length HCV NS5B protein was expressed in insect cells and purified to homogeneity. RdRp activity was assessed using standard filtration or polyacrylamide gel-based assays. Efficient inhibition of the HCV NS5B RdRp activity by gliotoxin, as well as by various substrate analogs, occurs in the presence of Mn2+, but not of Mg2+. Assays performed in the presence of both cofactors suggest that, in vitro, the enzyme's affinity for Mn2+ is higher than that for Mg2+. In addition, the RdRp activity, displayed in the presence of heteropolymeric templates, is significantly increased when the metal cofactor consists of Mn2+. Finally, steady state kinetics showed that the velocity of the reaction, as well as the affinity of the enzyme for its substrate, could both be affected by the nature of the divalent metal cation used.
ISSN:1090-9508
1537-4491