Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate

• Published in: Biochemical journal Vol. 398; no. 3; pp. 557 - 567
• Main Authors: Bougie, Isabelle, Bisaillon, Martin
• Format: Journal Article
• Language: English
• Published: England Portland Press Ltd 15.09.2006
• Subjects: Acid Anhydride Hydrolases - antagonists & inhibitors; Acid Anhydride Hydrolases - metabolism; Chlorella virus; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Viral; Paramecium bursaria; Paramecium bursaria chlorella virus; Plant Viruses - enzymology; Protein Binding; RNA - metabolism; RNA, Viral - metabolism; Vanadates - pharmacology; Viral Proteins - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20060198

Paramecium bursaria chlorella virus, a large DNA virus that replicates in unicellular Chlorella-like algae, encodes an RNA triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5' end of the viral mRNAs. The Chlorella virus RNA triphosphatase is the smallest member of the metal-dependent RNA triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and CD studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent non-competitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both CD and guanidinium chloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture was occurring upon decavanadate binding. However, both fluorescence spectroscopy and CD experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA triphosphatases by decavanadate.