The Role And Mechanism Of Host Factor IFITM3 In The Regulation Of Enveloped Virus Infection

Interferon-induced transmembrane protein 3（IFITM3）is a broad-spectrum antiviral protein encoded by Interferon-stimulated genes（ISGs）.Recent studies have found that IFITM3 not only prevents virus from infecting cells,but also can be coerced by some viruses（such as SARS-Co V-2）to improve the viruses’ability to infect cells.For its mechanism,it is generally believed that IFITM3 mainly functions by changing the physical properties of cell membrane.It was reported that IFITM3 could increase membrane lipid content in cells membrane（increasing rigidity and decreasing fluidity）,thereby increasing the difficulty of fusion of the viral envelope and the host cell membrane.And the amino acids in the intramembranous region of IFITM3 are amphipathic,thus they can change the stiffness and curvature of the cell membrane to inhibit the formation of the fusion micropores between the virus envelop and the host cell membrane.However,these viewpoints explain its mechanism from the perspective of the relationship between IFITM3and host cells,but they cannot fully explain why IFITM3 regulates and controls the functional differences on different viruses in the process of infecting host cells.Therefore,this study is based on the characteristics of the main viral membrane fusion protein in the process of envelope virus entering cells.The Nipah Virus（Ni V）,Influenza A virus（IAV）and Severe Fever with Thrombocytopenia Syndrome Virus（SFTSV）were selected as research objects to explore the role and mechanism of host factor IFITM3 in regulating the viral infection.The results are as follows:1.In order to facilitate the research on virus infection,a novel pseudotyped virus packaging system was established.Compared with the traditional pseudotyped virus packaging system,the expression level of the Luciferase reporter gene of this system increased by about 15 times.Then,based on this platform,the packaging systems of Ni V,H5N9,H7N9,SFTSV and VSV pseudoviruses were improved.Through cell infection test,it was confirmed that these pseudoviruses had the ability to infect target cells and expressed Luciferase reporter gene.In addition,an inducible IFITM3 overexpression cell line MDCK-Tet3G-IFITM3 was constructed by lentivirus infection.The cell line could express IFITM3 protein only when Dox was added.The best induction condition was Dox 5.0μg/ml and induction time was 24 h.At this time,the positive rate of IFITM3 overexpression cells was 90.7%.The establishment of the above two platforms has laid a foundation for the follow-up research.2.Based on the pseudovirus and MDCK-Tet3G-IFITM3 cell line packaged in the first part,the related studies of pseudovirus infection through MDCK-Tet3G-IFITM3 cell line were carried out.The results showed that when IFITM3 was overexpressed,the virus mediated by IAV HA protein and SFTSV M protein was significantly inhibited from entering host cells.While as for Ni Vpv,this fuction was on the contrary.When IFITM3 was overexpressed,the level of Ni Vpv mediated by Ni V G protein and F protein entering MDCK cells was relatively higher.3.Subcellular co-localization and immunoprecipitation assay were used to analyze whether there was a direct correlation between IFITM3 and virus fusion protein.The results showed that IFITM3 could have obvious subcellular co-localization and physical interaction with Ni V F protein,HA₂ subunit of IAV HA protein and GC subunit of SFTSV M protein.While IFITM3 has no related relationship with Ni V G protein,HA₁ subunit of IAV HA protein and Gn subunit of SFTSV M protein.4.In order to further explore the specific domain of the interaction between IFITM3and viral fusion protein,we truncated IFITM3,Ni V F protein,IAV HA protein and SFTSV M protein respectively,and analyzed them by subcellular co-localization and immunoprecipitation assay.The results showed that the intramembranous and transmembrane domains of IFITM3 interacted with the fusion peptide of Ni V F protein.And the transmembrane domain of IFITM3 interacted with the transmembrane domain of IAV HA₂ Subunit.The intramembranous region of IFITM3 interacted with SFTSV Gc subunit.And all of the three interactions had subcellular co-localization relationship.In conclusion,IFITM3 had different effects on virus entry mediated by different fusion proteins.This study found that IFITM3 can promote virus entry mediated by Ni V G/F,but inhibit virus entry mediated by SFTSV M and IAV HA proteins.The interaction study confirmed that IFITM3 interacted with the fusion subunits of three virus envelope proteins.However,the specific domains of the interaction between IFITM3 and the envelope proteins of the three viruses were not completely coincident,which might be one of the reasons for the different abilities or degrees of IFITM3 to regulate different viruses entering host cells.This study revealed a new mechanism of IFITM3 regulating virus infection,further improved the cognition of IFITM3’s function and mechanism in the body’s natural immune response,broadened the application scope of IFITM3 and provided a new target for finding and mining drugs for virus infection.