S-Palmitoylation and Ubiquitination Differentially Regulate Interferon-induced Transmembrane Protein 3 (IFITM3)-mediated Resistance to Influenza Virus

Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2012; 287(23):19631-41. DOI: 10.1074/jbc.M112.362095
Source: PubMed

ABSTRACT The interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cellular restriction factor that inhibits infection by influenza virus and many other pathogenic viruses. IFITM3 prevents endocytosed virus particles from accessing the host cytoplasm although little is known regarding its regulatory mechanisms. Here we demonstrate that IFITM3 localization to and antiviral remodeling of endolysosomes is differentially regulated by S-palmitoylation and lysine ubiquitination. Although S-palmitoylation enhances IFITM3 membrane affinity and antiviral activity, ubiquitination decreases localization with endolysosomes and decreases antiviral activity. Interestingly, autophagy reportedly induced by IFITM3 expression is also negatively regulated by ubiquitination. However, the canonical ATG5-dependent autophagy pathway is not required for IFITM3 activity, indicating that virus trafficking from endolysosomes to autophagosomes is not a prerequisite for influenza virus restriction. Our characterization of IFITM3 ubiquitination sites also challenges the dual-pass membrane topology predicted for this protein family. We thus evaluated topology by N-linked glycosylation site insertion and protein lipidation mapping in conjunction with cellular fractionation and fluorescence imaging. Based on these studies, we propose that IFITM3 is predominantly an intramembrane protein where both the N and C termini face the cytoplasm. In sum, by characterizing S-palmitoylation and ubiquitination of IFITM3, we have gained a better understanding of the trafficking, activity, and intramembrane topology of this important IFN-induced effector protein.

1 Follower
18 Reads
  • Source
    • "The IFITM proteins are members of the CD225 protein superfamily and contain two intramembrane domains (IM1 and IM2), which traverse through the cytosolic-facing leaflet of the lipid bilayer and are joined by a conserved intracellular loop (Yount et al., 2012). IFITM3 plays a critical role in vivo because Ifitm3 À/À mice succumb to a normally mild IAV infection (Bailey et al., 2012; Everitt et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The IFITMs inhibit influenza A virus (IAV) replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB), prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon's protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both a possible mechanism for IFITM-mediated restriction and its negation by AmphoB. Notably, we reveal that mice treated with AmBisome succumbed to a normally mild IAV infection, similar to animals deficient in Ifitm3. Therefore, patients receiving antifungal therapy with clinical preparations of AmphoB may be functionally immunocompromised and thus more vulnerable to influenza, as well as other IFITM3-restricted viral infections.
    Cell Reports 11/2013; 5(4). DOI:10.1016/j.celrep.2013.10.033 · 8.36 Impact Factor
  • Source
    • "These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3-5], cell-to-cell adhesion (IFITM1) [6-8], antiviral activity (IFITM1-3) [9-17], and bone formation (IFITM5) [18-22], although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9-14]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.
    PLoS ONE 09/2013; 8(9):e75831. DOI:10.1371/journal.pone.0075831 · 3.23 Impact Factor
  • Source
    • "The IFITM isoforms are relatively small (130 amino acids) and share a common topology of two conserved transmembrane domains, a short highly conserved cytoplasmic region, and luminal amino-and carboxyl termini (Siegrist et al., 2011; Wee et al., 2012). Recent studies showed that S-palmitoylation and ubiquitination of IFITM3 play crucial roles in its function (Yount et al., 2010, 2012) and that IFITM3 may influence v-ATPase complex activity and facilitates the subcellular localization of clathrin (Wee et al., 2012). Three additional studies showed that the N-terminal 21 amino acid residues of IFITM3 are required for its antiviral activity against VSV and IAV (Everitt et al., 2012; Jia et al., 2012; Weidner et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Vesicle-membrane-protein-associated protein A (VAPA) and oxysterol-binding protein (OSBP) regulate intracellular cholesterol homeostasis, which is required for many virus infections. During entry, viruses or virus-containing vesicles can fuse with endosomal membranes to mediate the cytosolic release of virions, and alterations in endosomal cholesterol can inhibit this invasion step. We show that the antiviral effector protein interferon-inducible transmembrane protein 3 (IFITM3) interacts with VAPA and prevents its association with OSBP, thereby disrupting intracellular cholesterol homeostasis and inhibiting viral entry. By altering VAPA-OSBP function, IFITM3 induces a marked accumulation of cholesterol in multivesicular bodies and late endosomes, which inhibits the fusion of intraluminal virion-containing vesicles with endosomal membranes and thereby blocks virus release into the cytosol. Consequently, ectopic expression or depletion of the VAPA gene profoundly affects IFITM3-mediated inhibition of viral entry. Thus, IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry, further underscoring the importance of cholesterol in virus infection.
    Cell host & microbe 04/2013; 13(4):452-64. DOI:10.1016/j.chom.2013.03.006 · 12.33 Impact Factor
Show more