Establishment and Functional Analysis of MDCK Cell Line Induced IFITM3 Expression Based on Tet-On 3G System

To explore the antiviral mechanisms of IFITM3, the eukaryotic expression plasmid pTRE3G-IFITM3 containing IFITM3 gene was constructed based on Tet-On 3G system and then cotranfected with the regulator vector pLVX-Tet3G into MDCK cells. After 48 h, the cells were subjected to select 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. And then, infection by lentiviruses pseudotyped with avian influence virus H5 or H7 hemagglutinin or VSV G proteins was performed to detect luciferase activities. The results indicated that inducible IFITM3-expressing MDCK cell lines were obtained and expression level of IFITM3 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 and efficiently suppressed the avian influence virus H5 or H7 hemagglutinin or VSV G-mediated viral entry, to lay a foundation for further research into the inhibition mechanism.