Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR–Cas9-mediated IFITM-mutant cell lines, we demonstrate that human IFITM1, IFITM2 and IFITM3 act cooperatively and function in a dose-dependent fashion in interferon-stimulated cells. Through site-specific fluorophore tagging and live-cell imaging studies, we show that IFITM3 is on endocytic vesicles that fuse with incoming virus particles and enhances the trafficking of this pathogenic cargo to lysosomes. IFITM3 trafficking is specific to restricted viruses, requires S-palmitoylation and is abrogated with loss-of-function mutants. The site-specific protein labeling and live-cell imaging approaches described here should facilitate the functional analysis of host factors involved in pathogen restriction as well as their mechanisms of regulation.
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Bailey, C. C., Zhong, G., Huang, I. C. & Farzan, M. IFITM-family proteins: the cell’s first line of antiviral defense. Annu. Rev. Virol. 1, 261–283 (2014).
Brass, A. L. et al. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139, 1243–1254 (2009).
Perreira, J. M., Chin, C. R., Feeley, E. M. & Brass, A. L. IFITMs restrict the replication of multiple pathogenic viruses. J. Mol. Biol. 425, 4937–4955 (2013).
Savidis, G. et al. The IFITMs inhibit Zika virus replication. Cell Rep. 15, 2323–2330 (2016).
Monel, B. et al. Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells. EMBO J. 36, 1653–1668 (2017).
Warren, C. J. et al. The antiviral restriction factors IFITM1, 2 and 3 do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus. PLoS One 9, e96579 (2014).
Zhao, X. et al. Interferon induction of IFITM proteins promotes infection by human coronavirus OC43. Proc. Natl. Acad. Sci. USA 111, 6756–6761 (2014).
Zhao, X. et al. Identification of residues controlling restriction versus enhancing activities of IFITM proteins on the entry of human coronaviruses. J. Virol. 92, e01535-17 (2018).
Ranjbar, S., Haridas, V., Jasenosky, L. D., Falvo, J. V. & Goldfeld, A. E. A role for IFITM proteins in restriction of Mycobacterium tuberculosis infection. Cell Rep. 13, 874–883 (2015).
Wakim, L. M., Gupta, N., Mintern, J. D. & Villadangos, J. A. Enhanced survival of lung tissue-resident memory CD8+ T cells during infection with influenza virus due to selective expression of IFITM3. Nat. Immunol. 14, 238–245 (2013).
Bailey, C. C., Huang, I. C., Kam, C. & Farzan, M. Ifitm3 limits the severity of acute influenza in mice. PLoS Pathog. 8, e1002909 (2012).
Everitt, A. R. et al. IFITM3 restricts the morbidity and mortality associated with influenza. Nature 484, 519–523 (2012).
Wu, X. et al. Intrinsic immunity shapes viral resistance of stem cells. Cell 172, 423–438.e425 (2018).
Yount, J. S., Karssemeijer, R. A. & Hang, H. C. S-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (IFITM3)-mediated resistance to influenza virus. J. Biol. Chem. 287, 19631–19641 (2012).
Bailey, C. C., Kondur, H. R., Huang, I. C. & Farzan, M. Interferon-induced transmembrane protein 3 is a type II transmembrane protein. J. Biol. Chem. 288, 32184–32193 (2013).
Weston, S. et al. A membrane topology model for human interferon inducible transmembrane protein 1. PLoS One 9, e104341 (2014).
Ling, S. et al. Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3. Sci. Rep. 6, 24029 (2016).
Chesarino, N. M. et al. IFITM3 requires an amphipathic helix for antiviral activity. EMBO Rep. 18, 1740–1751 (2017).
Chesarino, N. M., McMichael, T. M. & Yount, J. S. Regulation of the trafficking and antiviral activity of IFITM3 by post-translational modifications. Future Microbiol. 9, 1151–1163 (2014).
Peng, T. & Hang, H. C. Site-specific bioorthogonal labeling for fluorescence imaging of intracellular proteins in living cells. J. Am. Chem. Soc. 138, 14423–14433 (2016).
Zhang, Y. H. et al. Interferon-induced transmembrane protein-3 genetic variant rs12252-C is associated with severe influenza in Chinese individuals. Nat. Commun. 4, 1418 (2013).
Wang, Z. et al. Early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal H7N9infection. Proc. Natl. Acad. Sci. USA 111, 769–774 (2014).
Yang, X. et al. Interferon-inducible transmembrane protein 3 genetic variant rs12252 and influenza susceptibility and severity: a meta-analysis. PLoS One 10, e0124985 (2015).
Zhang, Y. et al. Interferon-induced transmembrane protein-3 rs12252-C is associated with rapid progression of acute HIV-1 infection in Chinese MSM cohort. AIDS 29, 889–894 (2015).
López-Rodríguez, M. et al. IFITM3 and severe influenza virus infection. No evidence of genetic association. Eur. J. Clin. Microbiol. Infect. Dis. 35, 1811–1817 (2016).
Naderi, M. et al. Evaluation of interferon-induced transmembrane protein-3 (IFITM3) rs7478728 and rs3888188 polymorphisms and the risk of pulmonary tuberculosis. Biomed. Rep. 5, 634–638 (2016).
Randolph, A. G. et al. Evaluation of IFITM3 rs12252 association with severe pediatric influenza infection. J. Infect. Dis. 216, 14–21 (2017).
Williams, D. E. et al. IFITM3 polymorphism rs12252-C restricts influenza A viruses. PLoS One 9, e110096 (2014).
Makvandi-Nejad, S. et al. Lack of truncated IFITM3 transcripts in cells homozygous for the rs12252-C variant that is associated with severe influenza infection. J. Infect. Dis. 217, 257–262 (2018).
Allen, E. K. et al. SNP-mediated disruption of CTCF binding at the IFITM3 promoter is associated with risk of severe influenza in humans. Nat. Med. 23, 975–983 (2017).
Feeley, E. M. et al. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry. PLoS Pathog. 7, e1002337 (2011).
Wee, Y. S., Roundy, K. M., Weis, J. J. & Weis, J. H. Interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-ATPase localization and function. Innate Immun. 18, 834–845 (2012).
Huang, I. C. et al. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus. PLoS Pathog. 7, e1001258 (2011).
Li, K. et al. IFITM proteins restrict viral membrane hemifusion. PLoS Pathog. 9, e1003124 (2013).
Amini-Bavil-Olyaee, S. et al. The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry. Cell Host Microbe 13, 452–464 (2013).
Desai, T. M. et al. IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion. PLoS Pathog. 10, e1004048 (2014).
Desai, T. M., Marin, M., Mason, C. & Melikyan, G. B. pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion. J. Biol. Chem. 292, 7817–7827 (2017).
Shi, G., Schwartz, O. & Compton, A. A. More than meets the I: the diverse antiviral and cellular functions of interferon-induced transmembrane proteins. Retrovirology 14, 53 (2017).
Erazo-Oliveras, A. et al. Protein delivery into live cells by incubation with an endosomolytic agent. Nat. Methods 11, 861–867 (2014).
Eierhoff, T., Hrincius, E. R., Rescher, U., Ludwig, S. & Ehrhardt, C. The epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) into host cells. PLoS Pathog. 6, e1001099 (2010).
Spence, J. S., Krause, T. B., Mittler, E., Jangra, R. K. & Chandran, K. Direct visualization of ebola virus fusion triggering in the endocytic pathway. mBio 7, e01857–e15 (2016).
Wrensch, F. et al. Interferon-induced transmembrane protein-mediated inhibition of host cell entry of ebolaviruses. J. Infect. Dis. 212, S210–S218 (2015).
Jia, R. et al. The N-terminal region of IFITM3 modulates its antiviral activity by regulating IFITM3 cellular localization. J. Virol. 86, 13697–13707 (2012).
Yount, J. S. et al. Palmitoylome profiling reveals S-palmitoylation-dependent antiviral activity of IFITM3. Nat. Chem. Biol. 6, 610–614 (2010).
Percher, A. et al. Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation. Proc. Natl. Acad. Sci. USA 113, 4302–4307 (2016).
Thinon, E., Fernandez, J. P., Molina, H. & Hang, H. C. Selective enrichment and direct analysis of protein S-palmitoylation sites. J. Proteome. Res. 17, 1907–1922 (2018).
John, S. P. et al. The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication. J. Virol. 87, 7837–7852 (2013).
McMichael, T. M. et al. The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity. J. Biol. Chem. 292, 21517–21526 (2017).
Narayana, S. K. et al. The Interferon-induced transmembrane proteins, IFITM1, IFITM2, and IFITM3 inhibit Hepatitis C virus entry. J. Biol. Chem. 290, 25946–25959 (2015).
Tsukamoto, T. et al. Role of S-palmitoylation on IFITM5 for the interaction with FKBP11 in osteoblast cells. PLoS One 8, e75831 (2013).
Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).
Jones, C. T. et al. Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system. Nat. Biotechnol. 28, 167–171 (2010).
McGee, C. E. et al. Infection, dissemination, and transmission of a West Nile virus green fluorescent protein infectious clone by Culex pipiens quinquefasciatus mosquitoes. Vector Borne Zoonotic Dis. 10, 267–274 (2010).
Schoggins, J. W. et al. Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro. Proc. Natl. Acad. Sci. USA 109, 14610–14615 (2012).
Petrakova, O. et al. Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells. J. Virol. 79, 7597–7608 (2005).
Brault, A. C. et al. Infection patterns of o’nyong nyong virus in the malaria-transmitting mosquito, Anopheles gambiae. Insect Mol. Biol. 13, 625–635 (2004).
Verdoes, M. et al. Improved quenched fluorescent probe for imaging of cysteine cathepsin activity. J. Am. Chem. Soc. 135, 14726–14730 (2013).
Charron, G. et al. Robust fluorescent detection of protein fatty-acylation with chemical reporters. J. Am. Chem. Soc. 131, 4967–4975 (2009).
E.T. acknowledges support a Marie Skłodowska-Curie postdoctoral fellowship. T.D. is supported by the Tri-Institutional Program in Chemical Biology at The Rockefeller University. H.-H.H. and C.M.R. acknowledge support from NIH R01AI091707. We thank W. Wei (Peking University) for sharing pCAS9 plasmid and gRNA vector (pGL3-U6). We thank Y.-C. Wang (The Rockefeller University) for the synthesis of dfTAT. We thank J. Yount and members of the Hang laboratory for helpful comments and discussion of the paper. T.P. acknowledges support from the National Natural Science Foundation of China (No. 21778010), the Shenzhen Science and Technology Innovation Committee (JCYJ20170412150832022), and Shenzhen Peacock Plan (KQTD2015032709315529). K.C. acknowledges grant support from NIH-NIAID R56AI088027. H.C.H. acknowledges grant support from NIH-NIGMS R01GM087544.
Supplementary Tables 1–2, Supplementary Figures 1–18
Example of DiD-IAV dequenching in a LAMP-GFP-expressing HeLa IFITM2/3-KO cell. The white arrow marks the particle of interest, and the red arrow indicates the onset of dequenching.
Example of DiD-IAV dequenching following colocalization with IFITM3-F8-BODIPY in a HeLa IFITM2/3-KO cell. The white arrow marks the particle of interest, and the red arrow indicates the onset of dequenching.
Example of DiD-IAV dequenching prior to colocalization with IFITM3-F8-BODIPY in a HeLa IFITM2/3-KO cell. The white arrow marks the particle of interest, and the red arrow indicates the onset of dequenching.
Example of LASV GPC-pseudotyped DiD-VSV dequenching following colocalization with IFITM3-F8-BODIPY in a HeLa IFITM2/3-KO cell. The white arrow marks the particle of interest, and the red arrow indicates the onset of dequenching.