FIG 8.

Inhibition of CCoV-HuPn-2018 entry by IFITMs and LY6E. (A) The expression of LY6E and FLAG-tagged IFITMs in Huh7-derived stable cell lines was detected by a Western blot assay using a rabbit polyclonal antibody against LY6E and a monoclonal antibody against the FLAG tag. β-Actin served as a loading control. (B) Inhibition of the entry of CCoV-HuPn-2018pp by IFITMs and LY6E. Huh7 cells stably expressing N-terminally FLAG-tagged human IFITM proteins, LY6E protein, or transduced with an empty vector (pQCXIP) were infected with CCoV-HuPn-2018pp and other indicated pseudoviral particles. Luciferase activities were determined 24 h post-infection. Relative infection is the ratio of luciferase activity of cells expressing the indicated IFITM protein or LY6E protein over that of cells transduced with an empty vector. (C) The expression of a control protein CAT, LY6E protein, or FLAG-tagged IFITM proteins in Flp-In T-Rex 293 cells cultured in the absence or presence of 2 µg/mL of tet for 24 h was detected by a Western blot assay using a rabbit polyclonal antibody against LY6E and a monoclonal antibody against the FLAG tag. β-Actin served as a loading control. (D) Inhibition of APN-dependent entry of CCoV-HuPn-2018pp by IFITMs and LY6E. Flp-In T-Rex 293 cells expressing CAT, the indicated IFITM protein, or the LY6E protein were transfected with feline APN, cultured in the presence or absence of tet for 24 h, and then infected with CCoV-HuPn-2018pp and other indicated pseudoviral particles. Luciferase activities were determined 24 h post-infection. Relative infection efficiency is the ratio of luciferase activity in cells cultured in the presence of tet over that in cells cultured in the absence of tet. Error bars reveal the standard deviation of the means from three biological repeats. *, P < 0.05; **, P < 0.001 compared to the level of empty vector-transduced cells.