Kazi Rahman

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.

The CD225/Dispanin superfamily contains membrane proteins that regulate vesicular transport and membrane fusion events required for neurotransmission, glucose transport, and antiviral immunity. However, how the CD225 domain controls membrane trafficking has remained unknown. Here we show that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. Proline-rich transmembrane protein 2 (PRRT2) encodes a SNARE-like motif that enables interaction with neuronal SNARE proteins; mutations in this region disrupt SNARE binding and are linked to neurological disease. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), protects cells against influenza A virus infection. IFITM3 interacts with SNARE proteins that mediate late endosome-late endosome (homotypic) fusion and late endosome-lysosome (heterotypic) fusion. IFITM3 binds to syntaxin 7 (STX7) in cells and in vitro, and mutations that abrogate STX7 binding cause loss of antiviral activity against influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic fusion and accelerates the trafficking of endosomal cargo to lysosomes. Our results suggest that SNARE modulation plays a previously unrecognized role in the diverse functions performed by CD225 proteins.

The interferon-induced transmembrane ( IFITM ) gene family performs multiple functions in immunity, including inhibition of virus entry into cells. The IFITM repertoire varies widely between species and consists of protein-coding genes and pseudogenes.

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. Mutation of proline-rich transmembrane protein 2 (PRRT2), a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 (and to a lesser extent, glycine-91) disrupted IFITM3 oligomerization and reduced its antiviral activity against Influenza A virus. An oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent and amphipathic helix-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, Amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

The viral envelope glycoprotein, known as “Env” in Retroviridae , is found on the virion surface and facilitates virus entry into cells by mediating cell attachment and fusion. Env is a major structural component of retroviruses and is targeted by all arms of the immune response, including adaptive and innate immunity. Less is known about how cell-intrinsic immunity prevents retrovirus replication at the level of individual cells. Here, we show that cellular IFITM3 and IFITM2 inhibit the fusion potential of retroviral virions by inhibiting Env protein via a two-pronged mechanism. IFITM proteins inhibit Env abundance in cells and also impair its function when levels are low. The posttranslational block of retroviral Env function by IFITM proteins is likely to impede both exogenous and endogenous retrovirus replication. In support of a relevant role for IFITM3 in retrovirus control, the retroviral accessory protein glycoGag counteracts IFITM3 function to promote virus infectivity.