SARS-CoV-2 Protein Interactome Analysis Using Proximity Labeling

Background: Coronavirus Disease 2019(COVID-19)caused by Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)is a severe acute respiratory infectious disease.The main symptoms of COVID-19 are fever,dry cough,dyspnea,chest pain,myalgia and fatigue.Some patients may develop hypoxemia,acute respiratory distress syndrome(ARDS),shock,arrhythmia,acute heart injury,acute kidney injury and other serious complications,and even death.SARS-CoV-2 is an envelope-positive single-stranded RNA virus that is susceptible to people of all ages and can spread from person to person through the respiratory tract and other routes,thereby causing outbreaks.Its high infectivity and pathogenicity have serious impact on human health and socioeconomic balance.There has been rapid progress in the study of the structure and mechanism of SARS-CoV-2.However,there is no clinically proven specific antiviral drug to treat SARS-CoV-2 infection.To further understand the process of SARS-CoV-2 infection in human cells,and to provide more mechanistic explanations and targets for the treatment of COVID-19,this study uses proximity labeling combined with mass spectrometry to screen the SARS-CoV-2 protein interaction map,then investigating the biology of SARS-CoV-2.Objective: To screen the interaction protein profiles of 26 proteins encoded by SARS-CoV-2 genome by using the proximity labeling combined with mass spectrometry analysis to investigate the process of SARS-CoV-2 infection in human cells.These 26 proteins consist of 16non-structural proteins(NSP1-NSP16),4 structural proteins(M、E 、N、S),and 6 accessary proteins(orf3a、orf6、orf7a、orf7b、orf8、orf10).Methods: Flag and HA-Turbo ID tag proteins were fused with SARS-CoV-2 proteins,then detecting their subcellular localization and expression levels.Compare the subcellular localization of SARS-CoV-2-proteins-3x Flag and SARS-CoV-2-proteins-3x HA-Turbo ID plasmids after expression.Pick up SARS-CoV-2-proteins-3x HA-Turbo ID plasmids which has the same location with SARS-CoV-2-proteins-3x Flag.And then transfected them into HEK293 cells,which continued to coincubated with biotin after transfection for a certain period of time.Turbo ID,as a biotin ligase,is capable of biotinylate of all proteins that in the range of 10 nm surround the expressed SARS-CoV-2 protein,Then the modified proteins are extracted with streptavidin and analyzed by mass spectrometry.Results: In this study,the recombinant plasmids of SARS-CoV-2protein components fused with Flag and HA-Turbo ID were constructed successively.The distribution of the two label plasmids corresponding to each protein was compared by immunofluorescence,and 16 proteins were selected for sample preparation and mass spectrometry analysis,and a total of 952 proteins with high confidence were screened.Bioinformatics analysis show that these human proteins involved in multiple metabolic pathways and a variety of biological processes,including ribosome,coronavirus disease-COVID-19,cytoplasmic translation and regulation,ferroptosis,proteasome-mediated ubiquitindependent protein catabolic process,regulation of DNA damage response,signal transduction by p53 class mediator etc.Conclusion: In this study,we screened several SARS-CoV-2 virus protein interaction proteins,and these proteins were involved in various metabolic pathways and biological processes.Although the specific mechanism of these proteins remains to be further verified and discussed,it provides a new research direction for us to study the action process of SARS-CoV-2 infection in human cell.