Avian Interferon-Inducible Transmembrane Protein Family Effectively Restricts Avian Tembusu Virus Infection

Chen, Shilong; Wang, Long; Chen, Jieying; Zhang, Lanlan; Wang, Song; Goraya, Mohsan U.; Chi, Xiaojuan; Na, Yang; Shao, Wenhan; Yang, Zhou; Zeng, Xiancheng; Chen, Shaoying; Chen, Ji-Long

Results presented above revealed that ATMUV infection effectively induced the expression of particular type I, type III IFNs, and IFITMs in vivo and in vitro. Furthermore, we tested that whether these IFITMs functioned in defense against the virus infection. For this, we generated stable DF-1 cell lines expressing specific shRNAs targeting IFITM1, IFITM2, IFITM3, or luciferase control, respectively. These cells were then infected with ATMUV and harvested at indicated times (24, 36, and 48 hpi) . Interference efficiency of the shRNAs was examined by qRT-PCR and viral load in cell culture supernatants was titrated via TCID 50 assay. Compared with the control shRNA targeting luciferase, the specific shRNAs caused decreased expression of IFITM1, IFITM2, or IFITM3 after ATMUV infection, respectively (Figures 5A-C) . Interestingly, silencing endogenous IFITM1 or IFITM3 resulted in significant increase in viral load, as evidenced by higher viral titers in culture supernatants from the IFITM1 or IFITM3 knockdown cells than those in supernatants of luciferase control cells ( Figure 5D) . However, knockdown of IFITM2 only slightly enhanced ATMUV replication ( Figure 5D ). To confirm these findings, DF-1 cell lines expressing specific shRNA targeting each of these IFITMs were infected with ATMUV and harvested at 36 hpi, followed by qRT-PCR assay to detect mRNA expression of viral genes. Consistent with the results from TCID 50 assay, data of qRT-PCR showed that mRNA expression of viral envelope and NS5 genes was clearly increased in IFITM1 or IFITM3 knockdown cells as compared to the control cells. However, no significant difference was observed in viral load between IFITM2 knockdown and luciferase control cells after infection with ATMUV ( Figure 5E ). This data suggests that disrupting the endogenous expression of chicken IFITM1 and IFITM3 but not IFITM2 can strongly enhance the ATMUV replication in DF-1 cells.