No abstract

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3-dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin-mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion.

Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection.

The interferon-induced transmembrane proteins (IFITMs) restrict infection by numerous viruses, yet the importance and regulation of individual isoforms remains unclear. Here, we report that murine IFITM1 (mIFITM1) is palmitoylated on one nonconserved cysteine and three conserved cysteines that are required for anti-influenza A virus activity. Additionally, palmitoylation of mIFITM1 regulates protein stability by preventing proteasomal degradation, and modification of the nonconserved cysteine at the mIFITM1 C terminus supports an intramembrane topology with mechanistic implications.T he interferon-induced transmembrane proteins (IFITMs) have been described to inhibit infection by a wide range of viruses, including important human pathogens such as influenza virus, West Nile virus, severe acute respiratory syndrome (SARS) coronavirus, human immunodeficiency virus, and dengue virus (1-6). The viruses that are restricted share the common ability to enter cells through endocytosis, and the restriction is indeed dependent upon the glycoproteins used for cellular entry (1, 2). Inhibition by IFITM3 occurs prior to influenza virus fusion with the endosome (7), which is a primary site of IFITM3 localization (2,7,8). IFITM3 appears to promote acidification or merging of endosomes with lysosomes, thereby exposing viral particles to a degradative environment (7-9). Consistent with this model, IFITM3 has recently been shown to reduce membrane fluidity and alter the curvature of membranes in such a way that virus hemifusion from within the endosome is inhibited while the fusion of opposing membranes possessing IFITMs may be enhanced (10).The mechanism by which IFITMs change membrane curvature and the endolysosomal compartment is unknown. Addressing this question, we have recently proposed that the predicted dual transmembrane topology for IFITM3 is incorrect and that the active protein instead contains intramembrane domains that do not fully span the lipid bilayer ( Fig. 1A and B) (8). Partial insertion of intramembrane domains into the membrane bilayer is well understood to promote curvature by virtue of the bilayer couple effect (Fig. 1B) (11). Evidence for this topology emerges largely from analysis of posttranslational modifications of IFITM3. Mass spectrometry and mutational analysis identified ubiquitination of lysine 24 (K24) on the IFITM3 N terminus (8). If the predicted topology is adopted, this residue would reside in the endoplasmic reticulum (ER) or endosomal lumen. However, known ubiquitin ligases are cytoplasmic, suggesting that the IFITM3 N terminus is localized in the cytoplasm. This is also supported by the discovery that phosphorylation of IFITM3 on tyrosine 20 (Y20) by the cytoplasmic kinase Fyn is required for IFITM3 antiviral activity (12). We further tested this alternative topology hypothesis by demonstrating that neither the N nor the C terminus nor the central loop domain of IFITM3 is exposed to the ER lumen glycosylation machinery but an antivirally active IFITM3 mutant engineered to possess myristoy...