Establishment Of Cell Line Induced IFITM3 Expression And Antiviral Function Against Influenza Virus

IFITMs were a transmembrane type II proteins encoded by the ISGs,and also membrane-associated cellular factors.IFITM1,IFITM2 and IFITM3 were widely expressed in various tissues and cells to participate in a variety of physiological activities in vivo.The antiviral properties of the IFITMs were firstly discovered in 1996.Recently,its antiviral effect and mechanism have become a research hotspot.Many studies have shown that IFITM can inhibit the replication of multiple pathogenic viruses,including influenza A virus,Human Immunodeficiency Virus,newly emerging pathogens Zika virus and so on.As a member of the family,IFITM3 played a crucial role in inhibiting influenza A virus infection.It has been reported that IFITM3 inhibits entry of influenza virus by interfering with the clathrin-mediated viral-host cell membrane fusion process,but the exact mechanism is unknown.In this study,A eukaryotic expression plasmid pLVX-TRE3G-IFITM3 containing IFITM3 gene was constructed based on Tet-On 3G system and then cotranfected with the regulator vector pLVX-Tet3 G into MDCK cells.After 48 h,the cells were subjected to selection with G418 and puromycin,followed by treatment with or without doxycycline(Dox)to identify IFITM3 expression,continuing to Dox sensitivity analysis,IFITM3+ cell percentage and location analysis.These results indicated that Inducible IFITM3-expressing MDCK cell lines were obtained and its expression was correlated with the dose and induction time of Dox.The concentration of Dox was determined to be 2.5 g/mL,and IFITM3+ cell percentage was up to 75% or more after 12 h.And IFITM3 was distributed in late endosomes/lysosomes.Secondly,we amplified HA5,HA7 and NA9 genes respectively,ligated to the eukaryotic expression vector pcDNA3.1 and transiently transfected into 293 cells to identify the expression of the target gene at the protein level.Next,we optimized the third generation lentivirus packaging plasmid system to constructe influenza pseudotyped virus integrated HA5 and HA7,and luciferase was as the reporter gene.It was indentified through infecting HEK293 cells and luciferase activity analysis.Based on influenza pseudotyped virus and H5N1 subtype of avian influenza virus,we confirmed IFITM3 inhibited influenza virus HA mediated viral entry,membrane fusion,viral replication and infection in two aspects,through siRNA silencing,luciferase activity,real-time quantitative PCR detection method established by our team,flow cytometry and fluorescent molecule labelling virus tracer technique.In conclusion,IFITM3 can inhibit influenza virus HA5 and HA7 mediated viral entry,which was helpful continued to explore the specific mechanism.