Establishment of 293 cell line inducibly expressing human IFITM3 and its inhibition to viral entry of H1N1 influenza virus

Abstract

Objective To establish FLP-IN T Rex 293 cell line inducibly expressing human interferon-inducible transmembrane protein 3 (IFITM3) and provide a cell model to explore the mechanism of IFITM3 inhibiting vial entry of influenza virus. Methods Full-length IFITM3 cDNA was cloned into pcDNA5/FRT/TO expression vector. Then 293 cell line inducibly expressing human IFITM3 was established by co-transfection of IFITM3/pcDNA5/FRT/TO expression vector and pOG44 plasmid and following antibiotic screening. The inducible expression of IFITM3 in 293 cells was tested by Western blot and immunofluorescence. The inhibitory effect of viral entry by IFITM3 was examined with H1N1 influenza virus pesudotyped virus (H1N1pp). Results The established FLP-IN T Rex 293 cell line of IFITM3 was able to express the target protein well, and the induced IFITM3 protein decreased the infection rate of H1N1 influenza pseudovirus by 97% (t = 38.08, P < 0.001), and the infection rate of vesicular stomatitis pseudovirus (VSVpp) decreased by 68% (t = 54.56, P < 0.001). Negative control group mice leukemia pseudovirus (MLVpp) infection rate (t = 1.282, P = 0.2208) and Lashapseudovirus (LASVpp) infection rate (t = 0.4814, P = 0.6377) had no significant influence. Conclusions FLP-IN T Rex 293 cell line inducibly expressing human IFITM3 is a pivotal restriction factor inhibiting H1N1 influenza virus entry. This IFITM3-inducible expression cell line can provide a useful platform to study IFITM3 antiviral mechanism. Key words: Interferon-induced transmembrane protein 3; H1N1 influenza virus; Pseudoviruses; Inducible expression; Restriction factor