期刊
ANTIVIRAL RESEARCH
卷 103, 期 -, 页码 17-24出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.antiviral.2013.12.012
关键词
Cell-based assay; Protease; 3C(pro); Picornavirus; Inhibitor
资金
- Convenant KU Leuven-Radboud University Nijmegen framework (FJMvK, JN)
- European Union [260644]
- NWO-VICI Grant [91812628]
- Chinese Academy of Sciences for a Visiting Professorship for Senior International Scientists [2010T1S6]
- KU Leuven geconcerteerde onderzoeksactie (JN) [GOA/10/014]
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 picoma-virus 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. (C) 2013 Elsevier B.V. All rights reserved.
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