Structural And Functional Analysis Of Papain-like Protease Of Middle East Respiratory Syndrome Coronavirus

BackgroundIn September2012, a new emerging Middle East respiratory syndrome coronavirus(MERS-CoV) was first detected in Jeddah and Saudi Arabia and quickly spread to someEuropean countries. The epidemic pattern and clinical symptoms caused by MERS-CoVare much similar to the severe acute respiratory syndrome coronavirus (SARS-CoV).MERS-CoV can cause severe acute respiratory syndrome and renal failure. FromSeptember2012to28Feb.2014, MERS-CoV has infected at least184people with afatality rate about45%globally. The Middle East respiratory syndrome coronavirus(MERS-CoV) genome is a positive-sense, single-stranded, just over30kb RNA. TheORF1a sequence of MERS-CoV encodes two protease domains that conserved in allother coronaviruses, a papain-like protease (PLpro) in nonstructural proteins3(nsp3)and a3C-like protease (3CLpro) in nsp5. Unlike other coronavirus which encodes twopapain-like proteases (PLP1and PLP2), MERS-CoV only encodes one papain-likeprotease named PLpro. We previously reported that SARS-CoV PLpro acts as bothdeubiquitinase (DUB) and interferon antagonist, however the function of theMERS-CoV PLpro was poorly understood. In the study, we will try to determine thestructure and functions of PLpro of the new emerging MERS-CoV.ObjectiveTaking the membrane anchored form of PLpro of MERS-CoV (PLpro-TM) as themodel, we aim here to explore the structure and function of MERS-CoV PLpro-TM,and further study the molecular mechanisms that PLpro-TM regulates the antiviralinnate immunity pathway which will provide the theoretical base for the research ofpathogenic mechanism and development of new antiviral drugs. MethodsFirstly, the plasmids MERS-CoV PLpro-TM and substrates genes nsp1-2, nsp2-3and nsp3-4were synthesized. Secondly, the plasmid of PLpro, the catalytic triadmutants of PLpro-TM and the mutants of substrat proteins were constructed byPCR-based site-directed mutagenesis. In order to detect the processing activity of PLpro,the construct of PLpro-TM or its catalytic triads was cotransfected into HEK293T cellswith plasmid DNAs of substrates of nsp1-2, nsp2-3and nsp3-4. The cells wereharvested and subjected to Western blotting using indicated antibodies to detect theprocessed products. Moreover, we cotransfected plasmid DNAs encoding PLpro-TMwith wild-type or the corresponding mutants of the substrates of nsp1-2, nsp2-3ornsp3-4into HEK293T cells to examine whether each substitution of PLpro processed itssubstrates. Meanwhile, to determine if MERS-CoV PLpro was an IFN antagonist whichmay contribute to inhibiting the expression of type I interferon, HEK293T cells weretransfected with the plasmids expressing wild-type PLpro-TM or its catalytic mutantsseparately, along with IFN-β promoter-driven luciferase reporters, and Flag-RIG-IN,N-terminal helicase domain of RIG-I as its constitutively active mutant that applied toactivate IFN-β promoter activity. Western blotting and immunofluorescence assay wereconducted to detect the phosphorylation status and subcellular localization of IRF3inPLpro-TM transfected cells. Finally, we detected the DUB activity of MERS-CoVPLpro through Western blotting.Results1. Structural ananlysis of MERS-CoV PLpro-TM. The3D structure ofMERS-CoV PLpro was determined based on bioinformatic analysis, it indicated that theMERS-CoV PLpro consists of four distinctive domains of Ubl domains in theN-terminal domain, a palm, a thumb, and a finger domain. They form an extendedright-hand architecture that closely resembles a similar structure in SARS-CoV PLpro. The conformation of the predicted catalytic sites (C1592-H1758-D1774) of MERS-CoVPLpro is consistent with the catalytic triads that found in SARS-CoV PLpro.2. MERS-CoV PLpro-TM recognizes and cleaves substract proteinsspecifically. Wild-type PLpro-TM completely cleaved the substrate nsp2-3to producethe cleavage products, while it partially processed the substrates, nsp1-2and nsp3-4,although the processed products were detectable. When the catalytic traids ofPLpro-TM or the LXGG consensus site of susbstract proteins were altered, thecleavaged products were reduction, indicating that MERS-CoV PLpro-TM possessesthe protease activity and cleavages the susbstract proteins specially.3. MERS-CoV PLpro-TM is a new interferon antagonist which encoded bycoronavirus. We found that wild-type PLpro-TM and all its catalytic mutants hadinhibition effects on RIG-IN activated IFN-β promoter-driven luciferase activity. Theseresults demonstrated that MERS-CoV PLpro-TM was an IFN antagonist whichindependent on its protease activity. MERS-CoV PLpro-TM significantly inhibited thephosphorylation of IRF3which was activated by RIG-IN and nuclear translocation ofIRF3. These data suggests that MERS-CoV PLpro-TM inhibits the cellular interferonproduction through disrupts the phophorylation and activation of IRF3.4. MERS-CoV PLpro-TM is a new coronaviral deubiquitinases. We found thata dramatic reduction of Ub-conjugated proteins was detected. Interestingly, PLpro-TMcatalytic mutants led to a reduction of Ub-conjugated proteins compared to the controlwhile the mutants of PLpro do not, indicating MERS-CoV PLpro-TM is a viraldeubiquitinase and the DUB activity is dependent on the TM domain but independenton the catalytic activity of PLpro. In addition, we found that wild-type PLpro-TM andthe catalytic mutants led to a significant reduction at the level of both K48-and K63-linked proteins, indicating that MERS-CoV PLpro-TM exhibited DUB activity onubiquitinated proteins without any specificity for either K48-or K63-linkage. ConclusionThe papain-like protease (PLpro-TM) encoded by MERS-CoV, which possessesthe protease activity, cleavages the substract proteins specially, and the processingactivity was dependent on the catalytic traids. Meanwhile, MERS-CoV PLpro-TM is anovel interferon antagonist which encoded by MERS coronavirus, PLpro-TM inhibitsthe expression of cellular interferon through disrupting the phosphorylation, activationof IRF3and nuclear translocation of IRF3. Moreover, MERS-CoV PLpro-TM is a newcoronavirus deubiquitinase and removes the ubiquitin conjugation from ubiquitinatedproteins in host cells.