{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T18:40:13Z","timestamp":1773859213829,"version":"3.50.1"},"reference-count":45,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T00:00:00Z","timestamp":1773792000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH\/NIAID","award":["R37 AI150453"],"award-info":[{"award-number":["R37 AI150453"]}]},{"name":"NIH\/NIAID","award":["R01 AI190198"],"award-info":[{"award-number":["R01 AI190198"]}]},{"name":"ANRS|MIE","award":["AO-2022-2"],"award-info":[{"award-number":["AO-2022-2"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral factors that restrict the entry of many enveloped viruses, including HIV-1, by modifying host membrane properties and trapping fusion at the hemifusion stage. Beyond blocking entry in target cells, IFITMs also reduce the infectivity of virions produced from IFITM-expressing cells, a phenomenon termed \u201cnegative imprinting\u201d. Conserved motifs, such as the amphipathic helix and oligomerization motifs, have been reported to be essential for IFITM-mediated protection of target cells from viral infection. Yet, the impact of IFITM incorporation on progeny virion infectivity remains poorly defined. Here, we show that IFITM3 mutants defective in target cell protection activity still markedly impair HIV-1 fusion\/infection upon incorporating into virions, without affecting viral maturation or Env incorporation. Immunofluorescence studies suggest mislocalization of the IFITM3 mutants as the reason for the lack of antiviral activity in target cells. Testing the antiviral activity of chimeras between antiviral and non-antiviral IFITM orthologs failed to clearly identify a domain responsible for reduction of HIV-1 infectivity, suggesting that multiple domains may be required for negative imprinting. Interestingly, co-incorporation of non-antiviral dog IFITM1 with human IFITM3 did not interfere with IFITM3\u2019s negative imprinting activity, despite forming mixed hetero-oligomers. This finding implies a dominant, oligomerization-independent antiviral phenotype of IFITM3 in virions. Our findings suggest that IFITMs may protect target cells and negatively imprint progeny virions through distinct mechanisms, underscoring the need to further characterize the molecular basis for the reduced fusion competence of IFITM-containing HIV-1 particles.<\/jats:p>","DOI":"10.3390\/biom16030459","type":"journal-article","created":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T11:14:14Z","timestamp":1773832454000},"page":"459","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Divergent HIV-1 Restriction Phenotypes of IFITMs Expressed in Target Cells and Incorporated into Virions"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5229-9986","authenticated-orcid":false,"given":"Smita","family":"Verma","sequence":"first","affiliation":[{"name":"Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Prikryl","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mariana","family":"Marin","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ruben M.","family":"Markosyan","sequence":"additional","affiliation":[{"name":"Department of Physiology and Biophysics, Rush University, Chicago, IL 60612, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrea","family":"Cimarelli","sequence":"additional","affiliation":[{"name":"Centre International de Recherche en Infectiologie (CIRI),  Universit\u00e9 de Lyon, Inserm, U1111, Universit\u00e9 Claude Bernard Lyon 1, CNRS, UMR5308, \u00c9cole Nationale Sup\u00e9rieure de Lyon, 69007 Lyon, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5385-3013","authenticated-orcid":false,"given":"Gregory B.","family":"Melikyan","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA"},{"name":"Children\u2019s Healthcare of Atlanta, Atlanta, GA 30322, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"eadz7083","DOI":"10.1126\/sciadv.adz7083","article-title":"Restriction of HIV-1 infectivity by interferon and IFITM3 is counteracted by Nef","volume":"11","author":"Agarwal","year":"2025","journal-title":"Sci. Adv."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1243","DOI":"10.1016\/j.cell.2009.12.017","article-title":"The IFITM Proteins Mediate Cellular Resistance to Influenza A H1N1 Virus, West Nile Virus, and Dengue Virus","volume":"139","author":"Brass","year":"2009","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4937","DOI":"10.1016\/j.jmb.2013.09.024","article-title":"IFITMs Restrict the Replication of Multiple Pathogenic Viruses","volume":"425","author":"Perreira","year":"2013","journal-title":"J. Mol. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Wang, J., Luo, Y., Katiyar, H., Liang, C., and Liu, Q. (2024). The Antiviral Activity of Interferon-Induced Transmembrane Proteins and Virus Evasion Strategies. Viruses, 16.","DOI":"10.3390\/v16050734"},{"key":"ref_5","first-page":"e03347\u201303324","article-title":"IFITM3 variants point to a critical role in emergent virus infections","volume":"16","year":"2025","journal-title":"mBio"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1740","DOI":"10.15252\/embr.201744100","article-title":"IFITM3 requires an amphipathic helix for antiviral activity","volume":"18","author":"Chesarino","year":"2017","journal-title":"EMBO Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"8155","DOI":"10.1021\/acsnano.0c10567","article-title":"Interferon-Induced Transmembrane Protein 3 Blocks Fusion of Diverse Enveloped Viruses by Altering Mechanical Properties of Cell Membranes","volume":"15","author":"Guo","year":"2021","journal-title":"ACS Nano"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"7837","DOI":"10.1128\/JVI.00481-13","article-title":"The CD225 Domain of IFITM3 Is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication","volume":"87","author":"Savidis","year":"2013","journal-title":"J. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e58537","DOI":"10.7554\/eLife.58537","article-title":"Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state","volume":"9","author":"Rahman","year":"2020","journal-title":"eLife"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Huang, I.C., Bailey, C.C., Weyer, J.L., Radoshitzky, S.R., Becker, M.M., Chiang, J.J., Brass, A.L., Ahmed, A.A., Chi, X., and Dong, L. (2011). Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus. PLoS Pathog., 7.","DOI":"10.1371\/journal.ppat.1001258"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2323","DOI":"10.1016\/j.celrep.2016.05.074","article-title":"The IFITMs Inhibit Zika Virus Replication","volume":"15","author":"Savidis","year":"2016","journal-title":"Cell Rep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1016\/j.chom.2016.08.006","article-title":"Resistance of Transmitted Founder HIV-1 to IFITM-Mediated Restriction","volume":"20","author":"Foster","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"14105","DOI":"10.1038\/s41598-018-32350-0","article-title":"IFITM1 expression is crucial to gammaherpesvirus infection, in vivo","volume":"8","author":"Hussein","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Everitt, A.R., Clare, S., McDonald, J.U., Kane, L., Harcourt, K., Ahras, M., Lall, A., Hale, C., Rodgers, A., and Young, D.B. (2013). Defining the Range of Pathogens Susceptible to Ifitm3 Restriction Using a Knockout Mouse Model. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0080723"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e02003-18","DOI":"10.1128\/JVI.02003-18","article-title":"Interferon-Induced Transmembrane Protein 1 Restricts Replication of Viruses That Enter Cells via the Plasma Membrane","volume":"93","author":"Smith","year":"2019","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1016\/j.ymthe.2020.01.027","article-title":"Type 1 Interferon Responses Underlie Tumor-Selective Replication of Oncolytic Measles Virus","volume":"28","author":"Aref","year":"2020","journal-title":"Mol. Ther."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1186\/s12977-014-0103-y","article-title":"IFITM proteins are incorporated onto HIV-1 virion particles and negatively imprint their infectivity","volume":"11","author":"Tartour","year":"2014","journal-title":"Retrovirology"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Li, K., Markosyan, R.M., Zheng, Y.-M., Golfetto, O., Bungart, B., Li, M., Ding, S., He, Y., Liang, C., and Lee, J.C. (2013). IFITM Proteins Restrict Viral Membrane Hemifusion. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003124"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Desai, T.M., Marin, M., Chin, C.R., Savidis, G., Brass, A.L., and Melikyan, G.B. (2014). IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion. PLoS Pathog., 10.","DOI":"10.1371\/journal.ppat.1004048"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1016\/j.chom.2014.11.001","article-title":"IFITM Proteins Incorporated into HIV-1 Virions Impair Viral Fusion and Spread","volume":"16","author":"Compton","year":"2014","journal-title":"Cell Host Microbe"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/j.celrep.2015.08.055","article-title":"IFITM Proteins Restrict HIV-1 Infection by Antagonizing the Envelope Glycoprotein","volume":"13","author":"Yu","year":"2015","journal-title":"Cell Rep."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Tartour, K., Nguyen, X.-N., Appourchaux, R., Assil, S., Barateau, V., Bloyet, L.-M., Burlaud Gaillard, J., Confort, M.-P., Escudero-Perez, B., and Gruffat, H. (2017). Interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of IFITMs. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006610"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e01994-20","DOI":"10.1128\/JVI.01994-20","article-title":"Escape of HIV-1 Envelope Glycoprotein from Restriction of Infection by IFITM3","volume":"95","author":"Drouin","year":"2021","journal-title":"J. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e02441-16","DOI":"10.1128\/JVI.02441-16","article-title":"The V3 Loop of HIV-1 Env Determines Viral Susceptibility to IFITM3 Impairment of Viral Infectivity","volume":"91","author":"Wang","year":"2017","journal-title":"J. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1657","DOI":"10.15252\/embr.201642771","article-title":"Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity","volume":"17","author":"Compton","year":"2016","journal-title":"EMBO Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"01716","DOI":"10.1128\/JVI.01716-18","article-title":"Functional Mapping of Regions Involved in the Negative Imprinting of Virion Particle Infectivity and in Target Cell Protection by Interferon-Induced Transmembrane Protein 3 against HIV-1","volume":"93","author":"Appourchaux","year":"2019","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e0043921","DOI":"10.1128\/JVI.00439-21","article-title":"Functional Heterogeneity of Mammalian IFITM Proteins against HIV-1","volume":"95","author":"Marziali","year":"2021","journal-title":"J. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1896","DOI":"10.1128\/AAC.46.6.1896-1905.2002","article-title":"Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy","volume":"46","author":"Wei","year":"2002","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"11016","DOI":"10.1128\/JVI.01242-09","article-title":"A Quantitative Affinity-Profiling System That Reveals Distinct CD4\/CCR5 Usage Patterns among Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus Strains","volume":"83","author":"Nguyen","year":"2009","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Verma, S., Chen, Y.-C., Marin, M., Gillespie, S.E., and Melikyan, G.B. (2023). IFITM1 and IFITM3 Proteins Inhibit the Infectivity of Progeny HIV-1 without Disrupting Envelope Glycoprotein Clusters. Viruses, 15.","DOI":"10.20944\/preprints202309.0844.v1"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"6014","DOI":"10.1074\/jbc.M117.777714","article-title":"SERINC5 protein inhibits HIV-1 fusion pore formation by promoting functional inactivation of envelope glycoproteins","volume":"292","author":"Sood","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5677","DOI":"10.1074\/jbc.RA118.004579","article-title":"Cell\u2013cell and virus\u2013cell fusion assay\u2013based analyses of alanine insertion mutants in the distal \u03b19 portion of the JRFL gp41 subunit from HIV-1","volume":"294","author":"Yamamoto","year":"2019","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Hammonds, J., Chen, X., Zhang, X., Lee, F., and Spearman Vaccine, P. Advances in methods for the production, purification, and characterization of HIV-1 Gag\u2013Env pseudovirion vaccines. Vaccine, 25, 8036\u20138048.","DOI":"10.1016\/j.vaccine.2007.09.016"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1848","DOI":"10.1021\/cb3002478","article-title":"Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate","volume":"7","author":"Hall","year":"2012","journal-title":"ACS Chem. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1021\/acschembio.5b00753","article-title":"NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells","volume":"11","author":"Dixon","year":"2016","journal-title":"ACS Chem. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1091\/mbc.e02-09-0573","article-title":"HIV-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation","volume":"14","author":"Markosyan","year":"2003","journal-title":"Mol. Biol. Cell"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1111\/j.1365-2818.2006.01706.x","article-title":"A guided tour into subcellular colocalization analysis in light microscopy","volume":"224","author":"Bolte","year":"2006","journal-title":"J. Microsc."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"167759","DOI":"10.1016\/j.jmb.2022.167759","article-title":"Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity","volume":"434","author":"Rahman","year":"2022","journal-title":"J. Mol. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e00514","DOI":"10.1128\/JVI.00514-20","article-title":"Differential Pressures of SERINC5 and IFITM3 on HIV-1 Envelope Glycoprotein over the Course of HIV-1 Infection","volume":"94","author":"Beitari","year":"2020","journal-title":"J. Virol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1016\/S0092-8674(00)80225-1","article-title":"The Vesicle Docking Protein p115 Binds GM130, a cis-Golgi Matrix Protein, in a Mitotically Regulated Manner","volume":"89","author":"Nakamura","year":"1997","journal-title":"Cell"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1038\/ncb1201-1101","article-title":"The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment","volume":"3","author":"Marra","year":"2001","journal-title":"Nat. Cell Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e202101174","DOI":"10.26508\/lsa.202101174","article-title":"A novel domain within the CIL regulates egress of IFITM3 from the Golgi and reveals a regulatory role of IFITM3 on the secretory pathway","volume":"5","author":"Zhong","year":"2022","journal-title":"Life Sci. Alliance"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Prikryl, D., Zhang, Y., Verma, S., and Melikyan, G.B. (2025). Attenuation of IFITM proteins\u2019 antiviral activity through sequestration into intraluminal vesicles of late endosomes. Front. Immunol., 16.","DOI":"10.3389\/fimmu.2025.1647166"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1186\/s12977-017-0377-y","article-title":"More than meets the I: The diverse antiviral and cellular functions of interferon-induced transmembrane proteins","volume":"14","author":"Shi","year":"2017","journal-title":"Retrovirology"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Wilkins, J., Zheng, Y.-M., Yu, J., Liang, C., and Liu, S.-L. (2016). Nonhuman Primate IFITM Proteins Are Potent Inhibitors of HIV and SIV. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0156739"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/3\/459\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T11:42:19Z","timestamp":1773834139000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/3\/459"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,18]]},"references-count":45,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["biom16030459"],"URL":"https:\/\/doi.org\/10.3390\/biom16030459","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,18]]}}}