Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

Journal of Virology - Tập 87 Số 21 - Trang 11955-11962 - 2013
Andy Kilianski1, Anna M. Mielech1, Xufang Deng1, Susan C. Baker1
1Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois, USA

Tóm tắt

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.

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Tài liệu tham khảo

10.1056/NEJMoa1211721

10.1128/JVI.01244-13

10.1056/NEJMoa1303729

10.1016/S0140-6736(13)60982-4

10.1056/NEJMoa1306742

10.1007/978-1-59745-181-9_1

CormanVMMullerMACostabelUTimmJBingerTMeyerBKreherPLattweinEEschbach-BludauMNitscheABleickerTLandtOSchweigerBDrexlerJFOsterhausADHaagmansBLDittmerUBoninFWolffTDrostenC. 2012. Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections. Euro Surveill. 17(49):pii=20334. http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20334.

10.1128/mBio.00473-12

10.1073/pnas.0506735102

10.1126/science.1087139

World Health Organization (WHO). 21 July 2013. Global alert and response (GAR). Middle East respiratory syndrome coronavirus (MERS-CoV)—update. WHO, Geneva, Switzerland.

Centers for Disease Control and Prevention (CDC). 2013. Update: severe respiratory illness associated with Middle East respiratory syndrome coronavirus (MERS-CoV)—worldwide, 2012–2013. MMWR Morb. Mortal. Wkly. Rep. 62:480–483.

10.1136/bmj.f3114

10.1016/S0140-6736(03)13412-5

10.2217/fvl.11.33

10.1038/nrmicro2147

10.1021/jm1004489

JacobsJGrum-TokarsVZhouYTurlingtonMSaldanhaSAChasePEgglerADawsonESBaez-SantosYMTomarSMielechAMBakerSCLindsleyCWHodderPMesecarAStaufferSR. 2013. Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease. J. Med. Chem. 56:534–546.

10.1042/BST0391371

10.1073/pnas.0805240105

10.1126/science.1085658

10.1016/j.peptides.2005.09.006

10.1128/JVI.02114-07

10.1371/journal.pbio.0030324

10.1371/journal.pone.0066248

10.1128/JVI.02220-08

10.1128/JVI.77.13.7376-7382.2003

10.1073/pnas.0510851103

10.1128/JVI.79.24.15189-15198.2005

10.1016/j.copbio.2009.02.013

10.1021/cb200248h

10.1016/j.antiviral.2011.02.002

10.1371/journal.pone.0027228

10.1007/s13238-013-2841-3

10.1016/j.bmcl.2008.08.082

10.1016/j.chembiol.2008.04.011

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Journal of Virology

Tập 87 Số 21

11955-11962

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