The Mechanisms Of Artertivirus 3CL^proand Coronavirus 3CL^pro-mediated NEMO Cleavage

Both arterivirus and coronavirus belong to the same order Nidovirales.The members from these two families can encoded a 3C-like protease(3CL^pro),also known as main protease,which are responsible for cleaving the viral polyprotein precursors into multiple mature non-structural proteins,thus playing a key role in the viral replication process.Meanwhile,this protease can also target host proteins,especially the key molecules in IFN production and signal transduction,to regulate the immune response.Previous studies have shown that both Arteritivirus and Coronavirus encoded-3CL^pro can cleave NEMO(NF-?B essential modulator),a key kinase of type I IFN pathway,but different arterivirus or coronavirus-mediated NEMO cleavage exhibit distinct cleavage sites.To systematically understand the differences on the recognition and cleavage mechnism of different arteritivirus/coronavirus-mediated NEMO cleavage,this study focuses on the point of artertivirus and coronavirus 3CL^pro cleaving NEMO,choosing porcine reproductive and respiratory syndrome virus(PRRSV),equine arteritis virus(EAV),SARS coronavirus(SARS-CoV,SARS-CoV-2)and murine coronavirus(MHV),which pose a threat to animals or human health,as the research object,discussing the mechanism on the aboved viruses encoded-3CL^proregulate immune response.The detailed points are as follows:1.PRRSV nsp4 and EAV nsp4 inhibit IFN production by targeting NEMO at multiple sitesNF-?B essential regulatory protein(NEMO)is a key molecule in the interferon pathway,and a previous study has reported that PRRSV nsp4(encoded 3CL^pro)inhibits IFN production by cleaving E349 site of NEMO.However,in this study,we found that the cleavage fragment NEMO(1-349)still possessed the ability to induce IFN production,and PRRSV nsp4 could further inhibit NEMO(1-349)activated-IFN production,suggesting that the NEMO cleavage at E349 mediated by PRRSV nsp4 are not sufficient to disrupt NEMO-induced IFN-?production,and there may be another cleavage site.Western blot data further confirmed that both PRRSV nsp4 and EAV nsp4could cleavage multiple sites of NEMO,and both PRRSV and EAV infection reduced endogenous NEMO expression.By contructing a series of NEMO mutants,we demonstrated that both PRRSV nsp4 and EAV nsp4 cleaved NEMO at E166,E171 and E349.In addition,EAV nsp4 could target an additional site Q205 of NEMO.As expected,except for NEMO(1-349),other cleaved fragments lost the ability of inducing IFN production.The structural simulation analysis revealed that the S1 pocket of PRRSV nsp4 and EAV nsp4 was different,thus we speculated that the S1 pocket maybe responsible for the difference on substrate recognition.In conclusion,the present study confirmed that both PRRSV nsp4 and EAV nsp4 antagonize IFN-?production by cleaving NEMO at multiple sites.2.SARS-CoV-2 nsp5 and SARS-CoV nsp5 exhibits distinct cleavage efficient onNEMOPrevious studies found that alpha coronavirus PEDV nsp5 and delta coronavirus PDCoV nsp5 cleave NEMO at a single site Q231.However,whether?coronavirus also cleave NEMO remains unclear.In this study,we found that SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleaved NEMO at three sites(E152,Q205 and Q231).Notably,SARS-CoV-2 nsp5 exhibited a stronger ability to cleave NEMO than SARS-CoV nsp5.By sequence alignment and structural comparision,we found that there existed only one residue differs in the region near the S2 substrate recognition pocket of nsp5(amino acid position 44-50).This residue in SARS-CoV-2 nsp5 showed a serine(S),while an alanine(A)occupied in the corresponding position of SARS-CoV nsp5.The difference at this site may lead to the difference on NEMO cleavage efficiency between the two proteases.Further mutation experiments and enzyme activity experiment in vitro confirmed that the mutant S46A showed poorer cleavage ability compared with SARS-CoV-2 nsp5 WT,while a SARS-CoV nsp5 mutant A46S significantly enhanced the catalytic activity of the SARS-CoV nsp5 WT,suggesting that residue 46 played an important role in regulating the catalytic activity of proteases.Owning to stronger cleavage efficiency on NEMO,SARS-CoV-2 nsp5 is speculated to posseess a stronger ability on interferon antagonism than SARS-CoV nsp5.Dual luciferase assays confirmed this hypothesis,and found that residue 46 was responsible for these differences.In addition,further studies showed that different viral proteases from coronavirus exhibited distinct recognition sites on NEMO.Another two viral proteases,belonging to the lineage A of?-cornavirus,showed different substrate recognition on NEMO compared with SARS-CoV-2 nsp5.3.MHV nsp5 suppresses IFN-?production by cleaving NEMO and MDA5We further analyzed NEMO cleavage mediated by MHV nsp5(belonging to the lingeal A of?-cornavirus),which belongs to different branches but same genus with SARS-CoV-2 nsp5.Suprisingly,we found that mouse hepatitis virus(MHV)nsp5cleaved NEMO at five sites(Q145,E152,Q180,Q205,Q231)and its catalytic activity contributed to this cleavage,suggesting that the substrate pocket of MHV nsp5 was more tolerant and could recognize more different substrates for cleavage.Structural analysis also confirmed this hypothesis.As expected,MHV nsp5 significantly inhibited SEV-induced IFN production,and this inhibition was evaded upon expression of inactive mutant of MHV nsp5.Notably,in addition to cleaving NEMO,MHV nsp5 also target pattern recognition receptor MDA5,at Q818,to inhibited interferon production.The cleaved MDA5 fragments,MDA5(1-818)and MDA5(818-1025),both lose the ability to induce IFN-?production.These results suggest that MHV nsp5 cleaves NEMO in a more complex mode,and can also cleave other molecule in the IFN pathway,thus synergistically antagonizing IFN production.In conclusion,this study systematically elaborated the differences on NEMO cleavage of 3C-like proteases of arteritivirus(PRRSV,EAV)and coronavirus(SARS-CoV,SARS-CoV-2,MHV)and their relationship with innate immune pathways,revealed the molecular basis of different 3CL^pro recognizing host protein,providing new clues for elucidating viral evolution,pathogenesis and drug development.