Host FAF2 Interacts With SARS-CoV-2 ORF6 And Inhibits Virus Replication

Since the outbreak in late 2019,the novel coronavirus,SARS-Co V-2,has caused more than 700 million infections and over six million deaths worldwide,posing a serious threat to human health and public safety.Therefore,a detailed understanding of the life cycle,immune evasion,and pathogenesis of SARS-Co V-2 is certainly needed.Viral infection relies heavily on a complex network of interactions to interact specifically with host proteins.However,some of the host proteins that interact with viral proteins are still unclear,which hinders the development of antiviral drugs and treatment of COVID-19.The aim of this project is to verify the interaction between the ORF6 protein of SARS-Co V-2 and candidate cell host proteins and to investigate the role of this interacting factor in SARS-Co V-2 infection.The host proteins co-localized with SARS-Co V-2 ORF6 were firstly screened by immunofluorescence technique,and then the interactions between the host proteins and ORF6 were further confirmed by pull-down assays.Subsequently,the interaction of representative host protein apoptosis-associated factor family member 2(FAF2)with ORF6 was investigated in detail,laying the foundation for further studies on the mechanism of the role of host factors in viral replication,which is of great value in the search for novel antiviral therapies targeting virus-host interactions.Here we firstly performed the conservation analysis of SARS-Co V-2 ORF6 in several SARS-Co V-2 variants,and found that ORF6 was relatively conserved in these variants,suggesting that ORF6 might play an important role in the life cycle of the virus.Then,the previously reported proteomic dataset of SARS-Co V-2 ORF6protein-protein interactors(PPI)was integrated with the data obtained from our laboratory for comparative analysis.Based on the results of affinity purification-mass spectrometry of SARS-Co V-2 ORF6 in our laboratory in previous,16 host factors were selected as potential interaction candidates,and the presence of co-localization of these proteins with the ORF6 was observed by immunofluorescence technique,and the presence of protein interactions was further confirmed by a pull-down assays.By using lipid droplet marker,ADRP protein and Lipi-Green probe,we further demonstrated the ORF6-FAF2 colocalization in the lipid droplet.Further,we constructed N-terminally and C-terminally deleted mutants of FAF2 for mapping analyses of its key ORF6-interacting domains.The results suggest that the UAS domain plays a major role in the process of FAF2 targeting of ORF6.Moreover,FAF2-specific si RNA was used to knock down FAF2 and explore the subsequent effects of FAF2 on the viral infection,and it was found that knocking down FAF2 protein promotes viral replication and proliferation,suggesting that FAF2 is directly or indirectly involved in the antiviral response.Since FAF2 protein was also reported to be a regulator in lipid droplet degradation,two drugs targeting the lipid metabolism,Ebelactone A(Eba)and Tetrahydrolipstatin(Orlislat),were selected to explore the role of lipid metablolism in SARS-Co V-2 infection.These preliminary results showed that Eba promoted viral replication at high concentrations,while Orlislat may have some antiviral effects.In summary,I confirmed that FAF2 is a host interactor of SARS-Co V-2 ORF6.FAF2 can target ORF6 in lipid droplets and participate directly or indirectly in the antiviral response.These findings provide new insights into the host proteins that interact with SARS-Co V-2 and may inform the development of antiviral interventions in the future.Further experiments and analysis are needed to clarify the mechanism of FAF2 action and the roles of the related lipid metabolism in SARS-Co V-2 infection.