IFITM1 and IFITM3 cooperate to restrict virus entry in endolysosomes

Interferon-induced transmembrane (IFITM) proteins restrict virus infections at the stage of cellular entry. 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 the 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. IFITM1 is primarily regarded as a cell surface protein that restricts the entry of viruses fusing at the plasma membrane, but 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 and interact, as determined by co-immunoprecipitation and proximity ligation assay. Knockdown of endogenous IFITM3 resulted in enhanced localization of IFITM1 to 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 cellular infection by influenza A virus. While individual knockdown of IFITM1 or IFITM3 significantly increased infection, combined knockdown of both IFITM1 and IFITM3 boosted infection to a much greater extent. These results suggest that endogenous IFITM1 and IFITM3 restrict Influenza A virus entry in endolysosomes in a cooperative manner. IMPORTANCE: There exist multiple IFITM proteins encoded in the human genome, and the IFITM locus has expanded by gene duplication in multiple species for reasons that are poorly understood. Here, we show that two human IFITM proteins known for performing antiviral roles during virus infection, IFITM1 and IFITM3, interact with one another in endolysosomes. Using RNA interference, we found that knockdown of IFITM1 or IFITM3 resulted in elevated susceptibility to Influenza A virus infection. However, combined knockdown of both IFITM1 and IFITM3 led to even greater infection. These results demonstrate that both IFITM1 and IFITM3 act as barriers to Influenza A virus entry in endolysosomes. Furthermore, we found that IFITM3 is necessary for the trafficking of IFITM1 to endolysosomes. Overall, we reveal that IFITM proteins, which are related to one another through gene duplication, evolved to work together to restrict virus infection.