Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3

Ling, Shenglong; Zhang, Chengwei; Wang, Wei; Cai, Xiaoying; Yu, Lu; Wu, Fangming; Zhang, Longhua; Tian, Changlin

doi:10.1038/srep24029

Cited by 50 publications

(88 citation statements)

References 34 publications

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“…To confirm these observations, cells were stained either intact, or following permeabilization, and analyzed by flow cytometry to determine levels of each IFITM protein at the cell surface. Antibodies against the C‐terminal HA‐tag were used as we, and others, have shown the IFITM proteins to have a type‐II transmembrane topology with the C‐terminal domain facing the extracellular space . The mean fluorescence intensity (MFI) of labeled OS‐IFITM1‐HA, P1‐IFITM1‐HA and P2‐IFITM3‐Y20A‐HA was essentially equivalent between intact and permeabilized cells, suggesting the HA‐tag is accessible at the cell surface (Figure B).…”

Section: Resultsmentioning

confidence: 95%

Alphavirus Restriction by IFITM Proteins

Weston

Czieso

White

et al. 2016

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Interferon inducible transmembrane proteins (IFITMs) are broad‐spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo‐, flavi‐, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid‐induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH‐induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV.

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Section: Resultsmentioning

confidence: 95%

Alphavirus Restriction by IFITM Proteins

Weston

Czieso

White

et al. 2016

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“…IFITM3 comprises two S-fatty-acylation sites (C71 and C72) in proximity of a intramembrane domain, which were identified by our direct method (Figure 5B), and another site adjacent to a transmembrane domain (C105). 10,48 Our recent analysis of IFITM3 S-fatty-acylation by acyl-PEG exchange (APE) and alk-16 labeling revealed that C72A was the major site of S-fatty-acylation. 30 Since trypsin digest of IFITM3 results in large hydrophobic peptides (>29 amino acids), it appeared advantageous to digest IFITM3 with chymotrypsin, yielding 8–10 amino acid long peptides (SI Figure 18).…”

Section: Resultsmentioning

confidence: 99%

“…10,48 (B) MS/MS spectra of IFITM3 C71-ca, C72-ca was obtained following chymotrypsin digest. (C) MS/MS spectra of IFITM3 modified peptide showing S-palmitoylation on C72.…”

Section: Figurementioning

confidence: 99%

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites

et al. 2018

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S-fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins, and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

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“…IFITMs have no catalytic subunit but share similar domain architectures consisting of a short N-terminal domain, two antiparallel domains, a conserved intracellular loop, and a hydrophobic C-terminal domain (4,5). The topology of IFITM3 has been clarified by electron paramagnetic and nuclear magnetic resonance analyses; the N-terminal domain is located inside the cell, whereas the antiparallel domains reside as intramembrane ␣-helices, followed by the transmembrane C-terminal domain (6). Although IFITM1, -2, and -3 all have reported antiviral activity, IFITM3 exhibits the greatest protection against the broadest range of viruses, including influenza A virus (IAV), flaviviruses (dengue, West Nile [WNV], and Japanese encephalitis viruses), hepaciviruses (hepatitis C virus), filoviruses (Ebola and Marburg viruses), bunyaviruses (Rift Valley fever and La Crosse viruses), rhabdoviruses (vesicular stomatitis virus), coronaviruses (severe acute respiratory syndrome coronavirus [SARS-CoV]), paramyxoviruses (respiratory syncytial virus [RSV]), and reoviruses (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

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confidence: 99%

The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses

Poddar

Hyde

Gorman

et al. 2016

J Virol

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Host cells respond to viral infections by producing type I interferon (IFN), which induces the expression of hundreds of interferon-stimulated genes (ISGs). Although ISGs mediate a protective state against many pathogens, the antiviral functions of the majority of these genes have not been identified. IFITM3 is a small transmembrane ISG that restricts a broad range of viruses, including orthomyxoviruses, flaviviruses, filoviruses, and coronaviruses. Here, we show that alphavirus infection is increased in Ifitm3 ؊/؊ and Ifitm locus deletion (Ifitm-del) fibroblasts and, reciprocally, reduced in fibroblasts transcomplemented with Ifitm3. Mechanistic studies showed that Ifitm3 did not affect viral binding or entry but inhibited pH-dependent fusion. In a murine model of chikungunya virus arthritis, Ifitm3 ؊/؊ mice sustained greater joint swelling in the ipsilateral ankle at days 3 and 7 postinfection, and this correlated with higher levels of proinflammatory cytokines and viral burden. Flow cytometric analysis suggested that Ifitm3 ؊/؊ macrophages from the spleen were infected at greater levels than observed in wild-type (WT) mice, results that were supported by experiments with Ifitm3 ؊/؊ bone marrow-derived macrophages. Ifitm3 ؊/؊ mice also were more susceptible than WT mice to lethal alphavirus infection with Venezuelan equine encephalitis virus, and this was associated with greater viral burden in multiple organs. Collectively, our data define an antiviral role for Ifitm3 in restricting infection of multiple alphaviruses. IMPORTANCE The interferon-induced transmembrane protein 3 (IFITM3) inhibits infection of multiple families of viruses in cell culture.Compared to other viruses, much less is known about the antiviral effect of IFITM3 on alphaviruses. In this study, we characterized the antiviral activity of mouse Ifitm3 against arthritogenic and encephalitic alphaviruses using cells and animals with a targeted gene deletion of Ifitm3 as well as deficient cells transcomplemented with Ifitm3. Based on extensive virological analysis, we demonstrate greater levels of alphavirus infection and disease pathogenesis when Ifitm3 expression is absent. Our data establish an inhibitory role for Ifitm3 in controlling infection of alphaviruses. The type I interferon (IFN) response is a critical factor that orchestrates innate protection against viral pathogens. Upon detection of pathogen-associated molecular patterns (PAMPs), host cells produce type I IFN, which in turn induces expression of hundreds of IFN-stimulated genes (ISGs). ISGs can inhibit multiple steps of the viral life cycle (e.g., entry, protein translation, assembly, or egress) or modulate the immune response, such as by enhancing the recruitment of leukocytes or promoting B and T cell maturation (1).IFN-induced transmembrane (IFITM) proteins 1, 2, and 3 were among the first IFN-stimulated genes (ISGs) to be identified (2) and initially were studied for their roles in germ cell homing and maturation. IFITM proteins are approximately 130 amino acids in l...

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Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3

Cited by 50 publications

References 34 publications

Alphavirus Restriction by IFITM Proteins

Alphavirus Restriction by IFITM Proteins

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites

The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses

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