Fig. 4: Tyr 112 and Leu 104 on IFITM1 are responsible for the inhibition of EphA2-mediated EBV infection.
a, Three-dimensional structural model of the interactions between IFITM1 (green), EphA2 (yellow), gH (light blue) and gL (grey). A distance between atoms of the distinct protein backbones of less than 6.0 Å was considered to indicate interfacial amino acids, as indicated by the red arrows and as listed in the figure (IFITM1: Tyr 112, Leu 104; EphA2: Val 161, Asn 60, Met L59; gH/gL: Arg 130, Ala 32). b–e, Effect on the EBV infection efficiency when the predicted interfacial amino acids on IFITM1 (Tyr 112, Leu 104) were mutated. All assays were performed on cells containing the empty lentivirus or EphA2-overexpressed cells (NP69 and HEK293) with a single-site mutation (IFITM1m1 or IFITM1m2) or dual mutations (IFITM1m1+2) of IFITM1, and control cells (IFITM1w). EBV copy numbers were measured by TaqMan−qPCR when cells were incubated with cell-free EBV-GFP for 3 h (b, NP69; c, HEK293). d,e, Quantitative and qualitative analysis of EBV-GFP-positive cells after 72 h of EBV exposure in transformed HEK293 cells. d, Representative flow cytometric scatterplots and the corresponding quantitative analysis of EBV-GFP-positive cells. e, Representative images of cells infected with EBV-GFP. f, Co-IP assays showed that IFITM1 influences the binding of EphA2 to gH/gL or gB. A total of 3 combinations of plasmids were designed and 3 different plasmids were transfected into each group at a ratio of 1:1:1. Briefly, the plasmids were transfected into HEK293 cells for 8 h and the cells were collected at 48 h after transfection. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting analysis with anti-EphA2, Myc and IFITM1 antibodies. All results are expressed as mean ± s.e.m. of 3 biological replicates (n = 3, b–d). *P < 0.05, **P < 0.01, two-tailed t-test.
