The Effect Of SARS-CoV-2 ORF10 Protein Interaction With Cellular ALIX On Cell Apoptosi

Objectives:The widespread transmission of SARS-CoV-2 globally has caused immeasurable losses to humanity.Although various countries have invested a lot of resources in the prevention and control of the COVID-19 epidemic and have widely vaccinated against it,there is still a risk of large-scale spread of new mutant strains.Therefore,we still need long-term in-depth research on SARS-CoV-2.There is still much to be studied about the accessory proteins of SARS-CoV-2,especially ORF10,an accessory protein located at the end of the SARS-CoV-2 sequence that does not exist in SARS-CoV and MERS-CoV coronaviruses.There is a knowledge gap regarding the study of this protein and its homologs.The accessory proteins have received less attention compared to other viral proteins such as structural proteins,and there are relatively few related studies.However,accessory proteins often play an important role in the infection process and participate in various pathological processes.There are still many functions of accessory genes that are not yet clear.For this special accessory protein-ORF10,there are still many questions that need to be answered.The purpose of this study is to explore the expression of the ORF10 protein in cells,identify the cellular proteins that interact with ORF10,and finally verify the impact of the interaction between ORF10 and the target protein on cell apoptosis.Methods:Firstly,the ORF10 expression system was constructed in 293T cells,and the effect of expressed ORF10 protein on cells was observed.Secondly,Pull-down experiment was used to detect the proteins possibly related to ORF10,and possible functional proteins were selected through GO analysis for further study.Thirdly,Co-IP experiment and immunofluorescence experiment were used to verify the proteins that interacted with ORF10.Finally,the function and mechanism of ORF10 involved were verified based on the known functions of the target protein.Results:1.ORF10-FLAG-pcDNA3.1(+)plasmid and ORF10-Flag-pAcGFP1-C1 plasmid were constructed and successfully expressed in cells.A 3×ORF10-FLAGpcDNA3.1(+)plasmid with three ORF10s in tandem was constructed,and the 3×ORF10 protein was successfully expressed and detected by immunoblotting.2.Pull-down experiment was used to collect possible proteins that interact with ORF10,and 1171 differentially expressed proteins were identified as potential interacting proteins.After GO analysis and selection,ALIX and AVEN were selected for further analysis.3.Co-IP experiment successfully confirmed the interaction between ALIX and ORF10,but the interaction between AVEN and ORF10 was not detected.Immunofluorescence experiment found that ORF10 and ALIX were co-localized in cells.4.Cell apoptosis detection showed that ORF10 could promote cell apoptosis.The total apoptotic cell proportion was significantly increased compared to the control group,while co-overexpression of ALIX could reduce the promoting effect of ORF10 on cell apoptosis to a certain extent,leading to a decrease in the total apoptotic cell proportion.However,this inhibitory effect on cell apoptosis did not appear in the group with ALIX overexpression alone.Discussion:Our study initially showed that ORF10 could promote the process of cell apoptosis,and there is an interaction between ALIX and ORF10 in cells that can inhibit the promoting effect of ORF10 on cell apoptosis.The interaction between ORF10 and ALIX may be one of the mechanisms by which ORF10 promotes cell apoptosis.The ultimate result of viral infection is cell death,and different modes of cell death have different biological significance for viruses.Among them,apoptosis is a relatively mild cell death process,usually considered immunologically silent and does not cause a strong immune response.We speculate that the promoting effect of ORF10 on cell apoptosis may help the virus evade the host immune response,thereby facilitating virus replication in the host during the early stage of infection.