Biological Function-study On 3C-like Protease Of Middle East Respiratory Syndrome Coronavirus

Background The Middle East Respiratory Syndrome coronavirus(MERS),an emerging coronavirus,was isolated by Zaki group from a 60-year-old male patient who was died of respiratory and renal failure in Saudi Arabia and diagnosed as MERS in Sep,2012.Since then 1952 people are infected,including 693 deaths with the mortality rate up to 36%,posing a serious threat to human life and health.The formation of the MERS replicase complex(RC)depends on Papain-like protease(PLpro)and 3C-like protease(3CLpro)and thus viral replication can be effectively blocked by suppressing the activation of these two kinds of proteases.Previous studies have indicated that PLpro can play an important role in the regulation of host antiviral innate immune response through deubiquitination.However,3CLpro has seldom been studied and its relationship with host antiviral innate immune response is not clear.Thus,the aim of the present study is to determine the functions of MERS-Co V 3CLpro and its relationships with the host innate immunity.Objective To reveal the mechnism by which MERS 3CLpro regulates IFN expression and determine whether 3CLpro induces autophagy.We aim to find the new functions of Co V 3CLpro,which will provide the theoretical basis for the study of pathogenic mechanism and the development of new antiviral drugs.Methods Firstly,the MERS-Co V/SARS-Co V/NL63-Co V 3CLpro were synthesized,and Cys/His on 3CLpro protease catalytically active site was mutated to Ala by PCR-based site-directed mutagenesis to obtain the 3CLpro catalytic mutants.Secondly,Western blotting,Luciferase reporter gene assay,ELISA and real-time PCR ware utilized to determine the influence of MERS-Co V 3CLpro and its mutants on the intracellular proteinic ubiquitination level.Moreover,we detected the DUB activity of MERS-Co V 3CLpro by Western blotting.Furthermore,we used immunoprecipitation and immunoblotting to determine the ubiquitination status of the key molecule of RIG-I signaling pathway.Finally,to gain insights into the relationship between coronavirus3 CLpro and cells autophagy,MERS-Co V 3CLpro and e GFP-LC3 B were co-transfected into HEK 293 T cells,and then the immunofluorescence was performed to monitor the punctas of e GFP-LC3 B,and the western blotting was used to detect the expression level of LC3-II.The transmission electron microscopy was further used to confirm whether3 CLpro induces autophagy.To further explore the mechanism,the western blotting assay was used to detect the expression of autophagic substrate p62,and the positioning of GFP-LC3 and the m RFP-LC3 autophagosome accumulation was visualized by confocal microscopy.Results 1.3CLpro acts as an IFN antagonist.(1)To determine whether IFN expression was negatively regulated by 3CLpro,the Luciferase reporter gene assay was performedand we found that the RIG-mediated and sendai virus stimuli induced intracellular activation of IFN-β can be inhibited by 3CLpro from MERS,SARS,and NL63,which was further evidenced by enzyme-linked immunosorbent assay(ELISA)and real-time PCR.The results showed that MERS 3CLpro antagonizes IPS-1/ERIS/TBK1/IKKε/IRF3/IRF7-mediated IFN-β transcription by interfering with the activation of RIG-I pathway.(2)We found that all catalytic mutants of 3CLpro were incapable of suppressing the RIG-IN-activated IFN-β promoter-driven luciferase activity.2.MERS 3CLpro has deubiquitinating activity.(1)HEK-293 T cells were transfected with plasmid DNA encoding either WT or catalytic mutants(H3288A and C3359A)of3CLpro along with pc DNA HA-Ub,K48-and K63-linked ubiquitin,we found MERS3 CLpro has potent DUB activity and can remove ubiquitin conjugates from many cellular substrates.Moreover,the DUB activity of 3CLpro is dependent on its catalytic activity.We further confirmed the DUB activity of 3CLpro with the in vitro DUB assay.(2)MERS-Co V 3CLpro had de ISGylating/de Sumoylating activity,which was dependent on intact catalytic residues H3288 A and C3359 A.3.The molecular mechanism of IFN inhibitation by MERS 3CLpro.We found that MERS 3CLpro acts as an IFN antagonist through blocking the ubiquitination of RIG-I,TBK1 or IRF3.Our results suggested that the DUB activity of MERS 3CLpro was pivotal for the inhibition of type I IFN induction,which may depend on 3CLpro catalytic sites of H3288 and C3359.4.3CLpro is a new type of autophagy induction protein encoded by coronavirus:(1)MERS 3CLpro can induce the punctas accumulation of e GFP-LC3 B and 3CLpro also induced up-regulation of LC3-II expression,indicating that 3CLpro induces the autophagy.The double-membrane structures of autophagosome were observed in the3CLpro-transfected HEK 293 T cells by transmission electron microscopy,which further confirmed that 3CLpro can induced the autophagy.(2)Our results demonstrated that MERS-Co V 3CLpro induced the incomplete autophagic process,in which 3CLpro activated autophagosome formation,but blocked its fusion with lysosomes.In addition,autophagy induced by 3CLpro was in a time-dependent mannerand independent of3 CLpro protease activity.Further study indicated that autophagy can be induced by3 CLpro from other two coronavirus,SARS and NL63 suggesting that induction of cell autophagy may be an attributeshared by various coronavirus.Conclusion In the present study,we revealed the new functions of 3CLpro in regulations of antiviral innate immune response and demonstrated that Co V 3CLpro is a kind of multifunctional protease.Our data confirmed that MERS 3CLpro acts as a new IFN antagonist and a DUB,which were both dependent on the catalytic sites of H3288 A and C3359 A.Meanwhile,3CLpro inhibits the activation of type I IFN signaling by cleaving ubiquitin chains from RIG-I,TBK1 and IRF3.Moreover,for the first time,we found that MERS coronavirus 3C-like protease can induce autophagy.These results may collectively contribute to reveal the mechanisms of coronavirus replication and regulation of antiviral innate immunity.