Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression |
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| Authors: | Ben A Bailey-Elkin Robert C M Knaap Garrett G Johnson Tim J Dalebout Dennis K Ninaber Puck B van Kasteren Peter J Bredenbeek Eric J Snijder Marjolein Kikkert Brian L Mark |
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| Institution: | From the ‡Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada and ;the §Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands |
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| Abstract: | Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PLpro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PLpro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PLpro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PLpro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PLpro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PLpro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PLpro domain was found to suppress IFN-β promoter activation, PLpro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PLpro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PLpro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PLpro as a viral DUB during MERS-CoV infection. |
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| Keywords: | Cysteine Protease Deubiquitylation (Deubiquitination) Innate Immunity Structural Biology Viral Immunology X-ray Crystallography Middle East Respiratory Syndrome Coronavirus PLpro |
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