skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

Abstract

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

Authors:
; ; ;
Publication Date:
OSTI Identifier:
22581703
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 496; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CLEAVAGE; ENCEPHALITIS; FORMATES; LUCIFERASE; PEPTIDES; SCREENING; SUBSTRATES; VIRUSES

Citation Formats

Campos-Gomez, Javier, Ahmad, Fahim, Rodriguez, Efrain, and Saeed, Mohammad F., E-mail: saeed@southernresearch.org. A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease. United States: N. p., 2016. Web. doi:10.1016/J.VIROL.2016.05.012.
Campos-Gomez, Javier, Ahmad, Fahim, Rodriguez, Efrain, & Saeed, Mohammad F., E-mail: saeed@southernresearch.org. A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease. United States. doi:10.1016/J.VIROL.2016.05.012.
Campos-Gomez, Javier, Ahmad, Fahim, Rodriguez, Efrain, and Saeed, Mohammad F., E-mail: saeed@southernresearch.org. Thu . "A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease". United States. doi:10.1016/J.VIROL.2016.05.012.
@article{osti_22581703,
title = {A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease},
author = {Campos-Gomez, Javier and Ahmad, Fahim and Rodriguez, Efrain and Saeed, Mohammad F., E-mail: saeed@southernresearch.org},
abstractNote = {The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.},
doi = {10.1016/J.VIROL.2016.05.012},
journal = {Virology},
number = ,
volume = 496,
place = {United States},
year = {Thu Sep 15 00:00:00 EDT 2016},
month = {Thu Sep 15 00:00:00 EDT 2016}
}