The Mechanism Of Severe Acute Respiratory Syndrome Coronavirus 2 Plpro Inhibiting Innate Immunity

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a member of the genus Beta-coronaviridae of the family Coronaviridae.It is a single-stranded positive-stranded RNA virus with a genome size of 29.9 kb.It is the pathogen of Coronavirus disease 2019(COVID-19).The SARS-CoV-2 genome encodes 28 proteins,including 4 structural proteins(Spike,Envelope,Membrane,and Nucleocapsid),16 nonstructural proteins(NSP1-NSP16)and 8 accessory proteins(ORF3a,ORF3 b,ORF6,ORF7 a,ORF7b,ORF8,ORF9 b,ORF14).The case fatality rate of COVID-19 is only 2.1%,but it is more transmissible than the SARS epidemic in 2003.The threat of SARS-CoV-2 is imminent,and there are still many gaps in the research on its pathogenesis.Viruses have a series of strategies against the host’s innate immune response during evolution,using host proteins to process and package themselves to avoid being recognized and cleared by the host’s immune system.Papain-like protease is a cysteine protease encoded by SARS-CoV-2 NSP3.It is a necessary proteolytic enzyme in the process of virus replication and completes the release of NSP1,NSP2 and NSP3 for SARS-CoV-2 replication.Previous studies have shown that SARS-CoV-2 can inhibit innate immune responses through proteins such as NSP1,NSP6,NSP12,and NSP13,and PLpro in SARS-CoV can also exert the same inhibitory effect,but the mechanism of action of SARS-CoV-2 PLpro is not clear.In order to elucidate the role of the SARSCoV-2 PLpro in the host type I interferon response(including the production of type I interferon and the signal transduction of type I interferon),and to analyze the molecular mechanism of its effect,firstly,the PLpro encoded by SARS-CoV-2 was cloned into a eukaryotic expression vector pc DNA3.1,transfected into HEK-293 T cells,and its expression was verified by Western blotting.It was found that SARS-CoV-2 PLpro could inhibit Poly(I:C)and Se V-induced IFN-β promoter activity in a dose-dependent manner by dual-luciferase reporter gene assay.The q RT-PCR test found that SARSCoV-2 PLpro could inhibit the expression of antiviral factors such as IFN-β,and ELISA test results also showed that PLpro could inhibit Se V-induced IFN-β secretion in the supernatant of A549 cells,suggesting that SARS-CoV-2 PLpro has the effect of inhibiting the secretion of type I interferon.Secondly,in order to explore the mechanism of SARS-CoV-2 PLpro’s intervention in innate immunity,the target of PLpro’s inhibition of innate immune signals was determined by dual luciferase reporter gene and Western blot detection,and it was found that PLpro can promote endogenous and exogenous MDA5 degradation,while inhibiting the phosphorylation of IRF3.In order to explore the effect of PLpro expression on the degradation of MDA5 protein,after using autophagy inhibitors or ubiquitinated proteasome degradation inhibitors,it was found that PLpro had no significant effect on the degradation of MDA5 after inhibiting autophagy,while the expression of MDA5 recovered after using proteasome inhibitor.Furthermore,we examined the effect of PLpro on the K48 ubiquitination level of MDA5,and the results showed that PLpro promoted the degradation of K48 ubiquitination of MDA5.It is suggested that PLpro degrades MDA5 through the ubiquitinated proteasome pathway to reduce the recognition of viral RNA by MDA5 and achieve viral immune escape.Meanwhile,the use of the proteasome degradation inhibitor MG132 was able to improve the survival rate of cells infected with SARS-CoV-2.Finally,in exploring the effect of SARS-CoV-2 PLpro on the downstream signal transduction of type I interferon,we used IFN-α to activate the downstream pathway and found that SARS-CoV-2 PLpro could inhibit the phosphorylation of STAT1 and STAT2.In conclusion,this study demonstrated that SARS-CoV-2 PLpro could inhibit IFN-β production by inhibiting IRF3 phosphorylation and promoting MDA5 degradation;preventing type I interferon downstream signaling by inhibiting STAT1 and STAT2 phosphorylation.This findings further deepen the understanding of the immune escape mechanism of SARS-CoV-2,and provided a theoretical basis for understanding the pathogenic mechanism of SARS-CoV-2.