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Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease.
Antiviral Res. 2020 02; 174:104661.AR

Abstract

Coronavirus papain-like proteases (PLPs or PLpro), such as the one encoded in the genome of the infectious Middle East Respiratory Syndrome (MERS) virus, have multiple enzymatic activities that promote viral infection. PLpro acts as a protease and processes the large coronavirus polyprotein for virus replication. PLpro also functions as both a deubiquitinating (DUB) and deISGylating (deISG) enzyme and removes ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) from cellular proteins. Both DUB and deISG activities are implicated in suppressing innate immune responses; however, the precise role of each activity in this process is still unclear due in part to the difficulties in separating each activity. In this study, we determine the first structure of MERS PLpro in complex with the full-length human ISG15 to a resolution of 2.3 Å. This structure and available structures of MERS PLpro-Ub complexes were used as molecular guides to design PLpro mutants that lack either or both DUB/deISG activities. We tested 13 different PLpro mutants for protease, DUB, and deISG activitites using fluorescence-based assays. Results show that we can selectively modulate DUB activity at amino acid positions 1649 and 1653 while mutation of Val1691 or His1652 of PLpro to a positive charged residue completely impairs both DUB/deISG activities. These mutant enzymes will provide new functional tools for delineating the importance of DUB versus deISG activity in virus-infected cells and may serve as potential candidates for attenuating the MERS virus in vivo for modified vaccine design efforts.

Authors+Show Affiliations

Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.Department of Biological Sciences, Purdue University, West Lafayette, IN, USA; Department of Biochemistry, Purdue University, West Lafayette, IN, USA; Center for Cancer Research, Purdue University, West Lafayette, IN, USA. Electronic address: amesecar@purdue.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

31765674

Citation

Clasman, Jozlyn R., et al. "Decoupling deISGylating and Deubiquitinating Activities of the MERS Virus Papain-like Protease." Antiviral Research, vol. 174, 2020, p. 104661.
Clasman JR, Everett RK, Srinivasan K, et al. Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease. Antiviral Res. 2020;174:104661.
Clasman, J. R., Everett, R. K., Srinivasan, K., & Mesecar, A. D. (2020). Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease. Antiviral Research, 174, 104661. https://doi.org/10.1016/j.antiviral.2019.104661
Clasman JR, et al. Decoupling deISGylating and Deubiquitinating Activities of the MERS Virus Papain-like Protease. Antiviral Res. 2020;174:104661. PubMed PMID: 31765674.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease. AU - Clasman,Jozlyn R, AU - Everett,Renata K, AU - Srinivasan,Karthik, AU - Mesecar,Andrew D, Y1 - 2019/11/23/ PY - 2019/08/02/received PY - 2019/11/12/revised PY - 2019/11/18/accepted PY - 2019/11/26/pubmed PY - 2019/11/26/medline PY - 2019/11/26/entrez KW - Coronavirus KW - Crystal structure KW - ISG15 KW - PLpro KW - Papain-like protease KW - deISGylase SP - 104661 EP - 104661 JF - Antiviral research JO - Antiviral Res. VL - 174 N2 - Coronavirus papain-like proteases (PLPs or PLpro), such as the one encoded in the genome of the infectious Middle East Respiratory Syndrome (MERS) virus, have multiple enzymatic activities that promote viral infection. PLpro acts as a protease and processes the large coronavirus polyprotein for virus replication. PLpro also functions as both a deubiquitinating (DUB) and deISGylating (deISG) enzyme and removes ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) from cellular proteins. Both DUB and deISG activities are implicated in suppressing innate immune responses; however, the precise role of each activity in this process is still unclear due in part to the difficulties in separating each activity. In this study, we determine the first structure of MERS PLpro in complex with the full-length human ISG15 to a resolution of 2.3 Å. This structure and available structures of MERS PLpro-Ub complexes were used as molecular guides to design PLpro mutants that lack either or both DUB/deISG activities. We tested 13 different PLpro mutants for protease, DUB, and deISG activitites using fluorescence-based assays. Results show that we can selectively modulate DUB activity at amino acid positions 1649 and 1653 while mutation of Val1691 or His1652 of PLpro to a positive charged residue completely impairs both DUB/deISG activities. These mutant enzymes will provide new functional tools for delineating the importance of DUB versus deISG activity in virus-infected cells and may serve as potential candidates for attenuating the MERS virus in vivo for modified vaccine design efforts. SN - 1872-9096 UR - https://www.unboundmedicine.com/medline/citation/31765674/Decoupling_deISGylating_and_deubiquitinating_activities_of_the_MERS_virus_papain_like_protease_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0166-3542(19)30436-X DB - PRIME DP - Unbound Medicine ER -