{"doi":"10.1074/jbc.ra120.014466","title":"HIV-cell membrane fusion intermediates are restricted by Serincs as revealed by cryo-electron and TIRF microscopy","abstract":"To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein. To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein. To establish an infection, HIV must enter the cell by fusing its lipid membrane with a host cell membrane (1Melikyan G.B. Membrane fusion mediated by human immunodeficiency virus envelope glycoprotein.Curr. Top. Membr. 2011; 68 (21771496): 81-10610.1016/B978-0-12-385891-7.00004-0Crossref PubMed Scopus (46) Google Scholar). HIV membrane fusion is mediated by conformational rearrangements in the viral envelope protein, Env, that are induced by binding to receptor, CD4, and co-receptor, CCR5 or CXCR4 (2Blumenthal R. Durell S. Viard M. HIV entry and envelope glycoprotein-mediated fusion.J. Biol. Chem. 2012; 287 (23043104): 40841-4084910.1074/jbc.R112.406272Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The process of HIV membrane fusion is thought to proceed through defined intermediate steps: receptor binding, hemifusion, and fusion pore opening and widening, similar to other enveloped viruses, such as influenza (3Chen B. Molecular mechanism of HIV-1 entry.Trends ","journal":"Journal of Biological Chemistry","year":2020,"id":59846,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":65,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.954,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":315223,"name":"Volker Kiessling","orcid":"0000-0002-9388-5703","position":1,"is_corresponding":false},{"id":255584,"name":"Owen Pornillos","orcid":"0000-0001-9056-5002","position":2,"is_corresponding":false},{"id":315224,"name":"Judith M. 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