FIG 2.

Y20 of IFITM3 plays a critical role in modulating the entry of 229E, MERS-CoV, SARS-CoV, and NL63 in both 293 and Huh7.5 cells. (A) FLIP-IN T Rex cells expressing CAT or the indicated wild-type or mutant IFITM proteins were cultured in the presence of 1 μg/ml of Tet for 24 h. Expression of FLAG-tagged IFITM mutants was detected by Western blotting assay using anti-FLAG monoclonal antibody. β-Actin served as a loading control. (B to E) The above-mentioned FLP-IN T Rex-derived cell lines were cultured in the presence or absence of Tet for 24 h and then infected with HCoV-OC43pp (B), SARSpp or NL63pp (C), 229Epp (D), or MERSpp and LASVpp (E). Luciferase activities were determined at 48 hpi. Relative infection efficiency is the ratio of the luciferase activity in cells cultured in the presence of Tet over that in cells cultured in the absence of Tet. The error bars indicate standard deviations (n = 6). (F) Huh7.5 cells were stably transduced with an empty retroviral vector (pQCXIP) or vectors expressing wild-type or mutant IFITM3 proteins. Expression of FLAG-tagged IFITM proteins was detected by Western blotting assay using an anti-FLAG monoclonal antibody. β-Actin served as a loading control. (G to K) The above-mentioned Huh7.5-derived cell lines were infected with OC43pp (G), SARSpp and NL63pp (H), 229Epp (I), MERSpp (J), or LASVpp (K). Luciferase activities were determined at 48 hpi. Relative infection is the ratio of the luciferase activity of cells expressing the indicated IFITM protein over that of cells transduced with the empty vector. The error bars indicate standard deviations (n = 6).