Mechanism Of Deubiquitase USP29 Promoting SARS-CoV-2 Infection By Stabilizing ORF9b

COVID-19(Corona Virus Disease 2019)is caused by Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)infection,the main clinical symptom is pneumonia.It is a serious threat to human health,normal life and public health safety infectious disease.SARS-CoV-2 is composed of structural and non-structural proteins,and different proteins differ greatly in structure and function.Previous studies have shown that ORF9 b protein,as an interferon regulatory gene,can regulate the host interferon response and is crucial for immune escape.Therefore,Elucidation of the interaction between SARS-CoV-2 and host is of great significance for the prevention and treatment of COVID-19.Protein post-translational modification can regulate the activity of many important cellular factors,ubiquitin-proteasome system(UPS)become one of the important reasons of proteolytic in eukaryotic cells,but protein ubiquitination is dynamic and reversible,deubiquitination can reverse the biological role of ubiquitin modification and recycle ubiquitin,deubiquitinating enzymes(DUBs)have a key role in the process of deubiquitination.Previous studies have found that ubiquitin signaling is critical for the proliferation of suppressive pathogens.However,the role of the ubiquitin mechanism in the pathogenicity of SARS-CoV-2 has not been explored.To determine the role of the ubiquitin mechanism in the pathogenicity of SARSCoV-2,the following experiments were conducted: ORF9 b capable of regulating the ubiquitin-proteasome pathway;IP assay confirmed the relationship between ORF9 b degradation and ubiquitin-proteasome pathway.USPs-HA and ORF9b-HA were cotransfected into HEK293 T cells,and the effect of USPs on the expression of ORF9 b was screened.USP29 and ORF9 b interact by experiment of IP;USP29 and ORF9 b interacting regions by experiment of IP;the ORF9 b ubiquitination site by experiment of IP;the effect of USP29 on ORF9b-mediated type I interferon signaling by luciferase reporter gene and RT-q PCR experiments,including: IFN-β,NF-κB and downstream genes;The effect of USP29 on the infectivity of VSV-e GFP and SARSCoV-2 trVLPs was determined by flow cytometry;the relationship between USP29 and COVID-19 by RT-q PCR.The experimental results show that ORF9 b is degraded by the ubiquitinproteasome pathway and ORF9 b is ubiquitinated by the unknown E3 ubiquitin ligase.The ubiquitination site of ORF9 b is mainly occurring on lys4 and lys40.USP29 as a host restriction factor prevents the degradation of the ORF9 b.USP29 was found to interact with the carboxyl terminus of ORF9 b,and USP29 was able to remove the polyubiquitin chain of ORF9 b,thereby inhibiting the production of IFN-β and NF-κB.We also demonstrated that USP29 enhances the infectivity of VSV-e GFP and SARSCoV-2 trVLPs by stabilizing ORF9 b.Moreover,we found that USP29 m RNA were highly expressed in COVID-19 patients through the serum analysis of COVID-19 patients.In conclusion,SARS-CoV-2 ORF9 b is degraded through the ubiquitinproteasome pathway,while USP29 is able to deubiquitinate ORF9 b in an enzyme activity-dependent manner,which inhibiting IFN-β and NF-κB signaling activation,thus enhancing the infection of VSV-e GFP and SARS-CoV-2 trVLPs.