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Open AccessArticle

Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates

1
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary
2
Doctoral School of Molecular Cell and Immune Biology, University of Debrecen, 4032 Debrecen, Hungary
3
MTA-DE Laboratory of Protein Dynamics, Department of Biochemistry and Molecular Biology, University of Debrecen, 4032 Debrecen, Hungary
4
Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(20), 7686; https://doi.org/10.3390/ijms21207686
Received: 5 August 2020 / Revised: 13 October 2020 / Accepted: 14 October 2020 / Published: 16 October 2020
(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes)
The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1′, and P2′ variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1′ amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses. View Full-Text
Keywords: VEEV; Venezuelan equine encephalitis virus; non-structural protein; nsP2; protease; alphavirus; alphaviral protease; Group IV virus; specificity VEEV; Venezuelan equine encephalitis virus; non-structural protein; nsP2; protease; alphavirus; alphaviral protease; Group IV virus; specificity
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Bozóki, B.; Mótyán, J.A.; Hoffka, G.; Waugh, D.S.; Tőzsér, J. Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates. Int. J. Mol. Sci. 2020, 21, 7686.

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