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Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors.
J Virol. 2013 Nov; 87(21):11955-62.JV

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.

Authors+Show Affiliations

Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

23986593

Citation

Kilianski, Andy, et al. "Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-like and 3C-like Proteases Using Luciferase-based Biosensors." Journal of Virology, vol. 87, no. 21, 2013, pp. 11955-62.
Kilianski A, Mielech AM, Deng X, et al. Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors. J Virol. 2013;87(21):11955-62.
Kilianski, A., Mielech, A. M., Deng, X., & Baker, S. C. (2013). Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors. Journal of Virology, 87(21), 11955-62. https://doi.org/10.1128/JVI.02105-13
Kilianski A, et al. Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-like and 3C-like Proteases Using Luciferase-based Biosensors. J Virol. 2013;87(21):11955-62. PubMed PMID: 23986593.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors. AU - Kilianski,Andy, AU - Mielech,Anna M, AU - Deng,Xufang, AU - Baker,Susan C, Y1 - 2013/08/28/ PY - 2013/8/30/entrez PY - 2013/8/30/pubmed PY - 2013/12/16/medline SP - 11955 EP - 62 JF - Journal of virology JO - J Virol VL - 87 IS - 21 N2 - 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. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/23986593/Assessing_activity_and_inhibition_of_Middle_East_respiratory_syndrome_coronavirus_papain_like_and_3C_like_proteases_using_luciferase_based_biosensors_ L2 - http://jvi.asm.org/cgi/pmidlookup?view=long&pmid=23986593 DB - PRIME DP - Unbound Medicine ER -