Mechanisms Of SARS-CoV-2 And MERS-CoV Multiple Accessory Proteins’ Degradation Via Ubiquitination

Background:The outbreak of novel coronavirus has drawn attention to coronaviruses and accelerated the exploration in the field of coronavirus infection and the corresponding host immune mechanisms.Among the seven coronaviruses known to infect humans,SARS-CoV,MERS-CoV,and SARS-CoV-2 can cause severe respiratory syndromes,which eventually leading to death in severely ill individuals.Among them,MERS-CoV has the highest lethality rate of 34.5%,while SARS-CoV-2has the largest number of confirmed cases and deaths and is causing a worldwide pandemic.The genome of coronaviruses can encode three classes of proteins,namely structural proteins,nonstructural proteins and accessory proteins.Structural proteins mainly constitute the viral capsid,are involved in the assembly of viral particles and mediate viral invasion into host cells;nonstructural proteins contain a variety of proteases that are involved in viral assembly and replication;and accessory proteins are more involved in the effects on host intrinsic immunity,apoptosis,autophagy and other pathways,which are mainly associated with viral pathogenicity.Compared to structural and nonstructural proteins,viral accessory proteins have been less studied.So the studies on accessory proteins can explore the pathogenicity mechanisms of coronaviruses more comprehensively.The most direct way for the host to attenuate the pathogenic function of viral proteins is to attenuate their expression or remove them.Ubiquitinproteasome system-mediated degradation of proteins is the most common mechanism for removing harmful or foreign proteins in eukaryotic cells.At present,the main research direction in coronavirus infection and pathogenesis is to explore how viruses induce deubiquitination or degradation of key host proteins,but the ubiquitination of viral proteins is still largely unexplored.Therefore,the studies on ubiquitination of coronavirus accessory proteins are promising and of great value.Objectives:First,we want to screen the accessory proteins of SARS-CoV-2 and MERS-CoV to identify the proteins that can undergo ubiquitination and degradation.Then we want to further identify the key E3 ligases of the host that regulate the ubiquitination of SARS-CoV-2 and MERS-CoV accessory proteins based on the phenomenon of proteasome-mediated degradation,and elucidate the specific mechanisms.Finally,we aim to validate the antiviral ability of these host proteins at the live virus level.This study provides new targets and strategies for the development of drugs against SARS-CoV-2 and MERS-CoV.Methods:Firstly,protein synthesis was inhibited under CHX treatment and the half-lives of SARS-CoV-2 and MERS-CoV accessory proteins were analyzed by western blot.Next,the functions of these viral proteins were determined by using luciferase reporter gene analysis,q PCR and Flow cytometry assay.The host interacting factors of viral proteins were identified by mass spectrometry and bioinformatics analysis was performed to initially predict the relevant properties of the viral proteins.In terms of mechanism validation,the key E3 ligases that might regulate the stability of these viral accessory proteins were then screened and identified using CO-Immunoprecipitation,in vivo and in vitro ubiquitination assays,protein half-life analysis,knockdown of the target protein by si RNA,constructing stable cell lines by lentivirus infection and point mutations.Among the related studies on MERS-CoV ORF8 b protein,two-dimensional gel electrophoresis and in vitro deamidation analysis were also applied to demonstrate the deamidation of ORF8 b protein in host cells.Finally,based on mechanism studies,whether these host E3 ligases and deamidases can act as antagonists of SARS-CoV-2 or MERS-CoV replication was verified at the live virus level.Results:1.For the first time,the SARS-CoV-2 ORF9b protein was found to be unstable in host cells with a short half-life and could form K48 typed polyubiquitinated chains,which in turn was recognized and degraded by the proteasome.Among the multiple lysine residues of ORF9 b,K67 was identified to be the key site for its ubiquitination.After screening,an E3 ligase,Cullin5,was found to induce ubiquitination and degradation of ORF9 b protein,while HSP90 could also bind to ORF9 b protein and enhanced its stability.Further studies revealed that TOM70 played a role as a substrate recognition receptor in the ubiquitinated degradation of ORF9 b protein.TOM70-HSP90-Cullin5 formed a complex that jointly regulated the stability of ORF9 b protein.The HSP90 inhibitors could target to enhance the degradation of ORF9 b protein and attenuated its ability to inhibit the production of antiviral cytokines,thus acting as an antagonist of SARS-CoV-2 replication.2.For the first time,the MERS-CoV ORF3 protein was found to induce apoptosis in host cells,which could also be recognized and degraded intracellularly by the ubiquitin-proteasome system.After screening,we identified that the host E3 ligase HUWE1 could bind to ORF3 protein and then specifically induced its ubiquitination and degradation,also identified the ubiquitination site K45 of ORF3 protein.HUWE1 could induce the degradation of ORF3 protein and thus diminish its ability to induce apoptosis.3.For the first time,the MERS-CoV ORF4b protein was found to be an unstable protein in host cells and could be degraded through the ubiquitin-proteasome system.ORF4 b protein could attenuate the activation of the innate immune pathway by inhibiting the phosphorylation of IRF3,and this pathological ability was dose-manner dependent.Further screening identified UBR5,a key E3 ligase that regulated the ubiquitination of ORF4 b protein,and identified K36 as the ubiquitination site of ORF4 b.UBR5 could locate to nucleus by its nuclear localization signals,and induced degradation of ORF4 b protein in both the cytoplasm and nucleus.Under MERS-CoV infection conditions,compared to the control group,the replication capacity of the virus was significantly enhanced when UBR5 was knocked down.4.For the first time,MERS-CoV ORF8b protein was found to undergo successive deamidation and ubiquitination in host cells.ORF8 b protein was firstly deamidated at the N105 site under the regulation of deamidase PFAS.The deamidated ORF8 b protein could then bind to the host E3 ligase TRIM21 and was induced by TRIM21 to undergo ubiquitination at the K60 site,which was then recognized and degraded by the proteasome.Both PFAS and TRIM21 synergistically mediated the degradation of ORF8 b protein by the ubiquitin-proteasome system,thereby attenuating the anti-immunity function of ORF8 b protein.Under MERSCoV infection conditions,knockdown of either PFAS or TRIM21 could enhance MERS-CoV replication.Conclusions:By analyzing the stability of SARS-CoV-2 and MERS-CoV accessory proteins,we found that SARS-CoV-2 ORF9 b protein,MERS-CoV ORF3,ORF4 b and ORF8 b proteins were unstable proteins and could be degraded by the ubiquitin-proteasome system.Moreover,we discovered the mechanisms related to the co-regulation of ORF9 b protein stability by TOM70-HSP90-Cullin5 complex,regulation of ORF3 protein stability by E3 ligase HUWE1,regulation of ORF4 b protein stability by E3 ligase UBR5,and co-regulation of ORF8 b protein stability by deamidase PFAS and E3 ligase TRIM21.These host proteins can be used as potential targets to develop new anti-SARS-CoV-2 and MERS-CoV therapeutic agents with great clinical prospect and social significance.