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Antiviral Res. 2014 Mar;103:17-24. doi: 10.1016/j.antiviral.2013.12.012. Epub 2014 Jan 4.

Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases.

Author information

1
Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences & Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
2
Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences & Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
3
Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
4
Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, and German Centre for Infection Research (DZIF), University of Lübeck, Lübeck, Germany.
5
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Graduate School of the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
6
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
7
Okapi Sciences NV, Heverlee, Belgium.
8
Veterinary and Agrochemical Research Centre, Brussels, Belgium.
9
Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences & Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
10
Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, and German Centre for Infection Research (DZIF), University of Lübeck, Lübeck, Germany; State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Graduate School of the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; Laboratory for Structural Biology of Infection and Inflammation, c/o DESY, Hamburg, Germany.
11
Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences & Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Electronic address: F.J.M.vanKuppeveld@uu.nl.

Abstract

Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases and peptide substrates. To deal with the disadvantages associated with in vitro assays, we modified a cell-based protease assay for picornavirus proteases. The assay is based on the induction of expression of a firefly luciferase reporter by a chimeric transcription factor in which the viral protease and cleavage sites are inserted between the GAL4 binding domain and the VP16 activation domain. Firefly luciferase expression is dependent on cleavage of the transcription factor by the viral protease. This biosafe assay enables testing the effect of compounds on protease activity in cells while circumventing the need for infection. We designed the assay for 3C proteases (3C(pro)) of various enteroviruses as well as of viruses of several other picornavirus genera, and show that the assay is amenable for use in a high-throughput setting. Furthermore, we show that the spectrum of activity of 3C(pro) inhibitor AG7088 (rupintrivir) not only encompasses enterovirus 3C(pro) but also 3C(pro) of foot-and-mouth disease virus (FMDV), an aphthovirus. In contrary, AG7404 (compound 1), an analogue of AG7088, had no effect on FMDV 3C(pro) activity, for which we provide a structural explanation.

KEYWORDS:

3C(pro); Cell-based assay; Inhibitor; Picornavirus; Protease

PMID:
24393668
DOI:
10.1016/j.antiviral.2013.12.012
[Indexed for MEDLINE]

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