IFITM1 and IFITM3 cooperate to restrict virus entry in endolysosomes

Interferon-induced transmembrane (IFITM) proteins are potent innate immune factors that restrict an array of viruses at the entry stage of infection. We previously characterized a GxxxG motif in the CD225 domain of human IFITM3 that mediates its multimerization, which is essential for the reduction of membrane fluidity by IFITM3 and for its antiviral activity against Influenza A virus. Here, using an unbiased approach coupling immunoprecipitation with mass spectrometry, we show that the GxxxG motif is also important for the interaction of IFITM3 with other proteins, including IFITM1. While IFITM1 is primarily regarded as a cell surface protein that restricts the entry of viruses fusing at the plasma membrane, this model is based mostly on overexpression studies and is at odds with some studies showing that it can restrict endocytic viruses. Here, we show that endogenous IFITM1 and IFITM3 co-reside in membranes of acidic late endosomes and lysosomes (endolysosomes) and form a protein-protein complex as determined by co-immunoprecipitation and proximity ligation assay. Knockdown of endogenous IFITM3 resulted in enhanced localization of IFITM1 at the plasma membrane, indicating that IFITM3 promotes IFITM1 localization to endolysosomes. To assess the antiviral protection conferred by endogenous IFITM1 and IFITM3 against viruses fusing at endolysosomal membranes, we measured cell entry mediated by the Influenza A fusogen hemagglutinin (HA). While knockdown of IFITM3 significantly boosted HA-mediated entry, combined knockdown of both IFITM3 and IFITM1 boosted entry even further. These results suggest that endogenous IFITM1 restricts Influenza A virus entry in a manner that is non-redundant with IFITM3, and that IFITM1 and IFITM3 inhibit virus entry in a cooperative manner.