Construction Of HIFITM3 Recombinant Adenovirus And Its Antiviral Activity Against Influenza Virus

Interferon-inducible transmembrane proteins (IFITMs), a class of host restriction factors expressed by various cells have been found to play roles in restricting replication of both enveloped viruses and non-enveloped viruses by blocking their entry. Among the IFITMs, IFITM3 can inhibit the replication of multiple pathogenic viruses including influenza virus, West Nile virus and dengue virus (DENV). IFITM3 can exert antiviral activity through multiple avenues. It is reported that IFITM3 can inhibit entry of pseudo type virus whose outer membranes were H1N1、H3N2、H7N9 or H5N1.To lucubrate the antivirus activity, we constructed shuttle plasmid harboring human IFITM3 by utilizing pacAd and then, co-transfected the shuttle plasmid and skeleton plasmid pAd5 into HEK-293 packaging recombinant adeno virus containing IFITM3. We analyzed the expression of IFITM3 in A549 and MDCK in protein level by western blot to select the appropriate cell model. The influence of recombinant adenovirus and influenza virus on the activity of A549 and MDCK were detected by MTS method respectively. The protein and RNA of A549 and MDCK inoculated with recombinant adenovirus were harvested at 12h、24h、36h、48h after infection of influenza virus, and then, the expression of influenza virus protein HA and gene M2 were detected by western blot and RT-qPCR respectively.The results of PCR and Western blot showed that the expression of IFITM3 of the new constructed recombinant adenovirus was significantly higher than that of A549 and MDCK, which demonstrated that the recombinant adenovirus containing IFITM3 was successfully constructed. MTS revealed that both racAd-IFITM3 and wtAd5 have a time and dose dependent inhibitory on the activity of the cells detected, however, no significant difference of the inhibitory effect between the racAd-IFITM3 group and wtAd5 group was found. Furthermore,we also ound that the influenza virus can significantly inhibit cell activity 24h post infection, and the inhibitory effect is also time and dose dependent.qPCR showed that the A549 and MDCK overexpressing IFITM3 has significant inhibitory effect on influenza virus in transcriptional level at different time post infection.In conclusion, we successfully constructed rAd-IFITM3 containing IFITM3. Furthermore, A549 and MDCK inoculated with rAd-IFITM3 can significantly inhibit the replication of influenza virus in transcriptional and translational levels, which laid foundation for the further study of the antiviral mechanism of IFITM3.