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 Microbiol. 2019; 27 (31262533): 878-89110.1016/j.tim.2019.06.002Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The earliest step, receptor binding, has been visualized with nanometer resolution by electron tomography of plastic sectioned cells (4Sougrat R. Bartesaghi A. Lifson J.D. Bennett A.E. Bess J.W. Zabransky D.J. Subramaniam S. Electron tomography of the contact between T cells and SIV/HIV-1: implications for viral entry.PLoS Pathog. 2007; 3 (17480119): e6310.1371/journal.ppat.0030063Crossref PubMed Scopus (145) Google Scholar), but subsequent steps have not been directly observed. Fusion of influenza virus with its much simpler pH-sensitive fusion trigger has previously been studied by observing the merging of viral membrane envelopes with artificial liposomes using cryo-electron tomography (cryoET) (5Gui L. Ebner J.L. Mileant A. Williams J.A. Lee K.K. Visualization and sequencing of membrane remodeling leading to influenza virus fusion.J. Virol. 2016; 90 (27226364): 6948-696210.1128/JVI.00240-16Crossref PubMed Scopus (38) Google Scholar, 6Lee K.K. Architecture of a nascent viral fusion pore.EMBO J. 2010; 29 (20168302): 1299-131110.1038/emboj.2010.13Crossref PubMed Scopus (93) Google Scholar, 7Chlanda P. Mekhedov E. Waters H. Schwartz C.L. Fischer E.R. Ryham R.J. Cohen F.S. Blank P.S. Zimmerberg J. The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.Nat. Microbiol. 2016; 1 (27572837): 1605010.1038/nmicrobiol.2016.50Crossref PubMed Scopus (67) Google Scholar), but HIV's requirement for two cell surface membrane proteins to trigger fusion has made in vitro reconstitution difficult. To examine fusion of HIV at the plasma membrane with cryoET, we have adapted giant plasma membrane vesicles (GPMVs or blebs) as model target membranes. Blebs are detached plasma membrane–derived vesicles that are much smaller than a cell and can easily be frozen in thin, vitreous ice required for cryoET. Blebs produced from cells expressing CD4 and CCR5 incorporate these full-length proteins along with native lipid and E. P. membrane structure and behavior using giant plasma membrane 2012; PubMed Scopus Google Scholar, HIV plasma membrane for cell 3 PubMed Scopus Google Scholar). that the fusion of virus HIV and plasma membrane blebs can be visualized in by cryo-electron microscopy HIV plasma membrane for cell 3 PubMed Scopus Google Scholar), and that this is to snapshots of 3D thus multiple of the HIV fusion process by cryoET. In this we a combination of cryoET and fluorescence microscopy with membrane blebs from HIV target cells to various steps of fusion between two the HIV envelope and plasma membranes of of pseudovirus particles with cell membrane blebs a of HIV fusion. Serinc3 and Serinc5 are viral restriction and HIV-1 and are by PubMed Scopus Google Scholar, A. A. S. R. A. J. M. HIV-1 by from PubMed Scopus Google that are thought to at cell the mechanism the of a of plasma membrane proteins M. M. an into membrane lipid Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in the of the HIV protein, can incorporate into viral particles and the of viral particles J. J. S. of as the for HIV-1 Virol. PubMed Scopus Google Scholar). Whereas Serinc3 and Serinc5 can of subsequent other has that act at the earliest steps of membrane fusion by the or of M. J. E. G.B. fluorescence that human immunodeficiency virus fusion by envelope Scholar), as by binding of to on viral particles B. A. S. A. M. to membranes and HIV-1 entry with HIV-1 restriction by PubMed Scopus Google Scholar, S. S. A. of HIV-1 on Virol. PubMed Scopus Google Scholar, M. A. M. G.B. HIV-1 fusion pore by functional of envelope Biol. Chem. Full Text Full Text PDF PubMed Scopus (69) Google Scholar). are membrane proteins that have been to be in lipid M. M. an into membrane lipid Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the structure of Serinc5 a lipid-binding of its and is by and A. R. M. A. A. structural the of HIV-1 restriction Biol. 27 PubMed Scopus Google Scholar). these when into viruses, not to the of the viral membrane B. H. J. B. M. A. B. The restriction HIV-1 without the lipid and of viral Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have that Serinc the of in the viral membrane A. M. as a restriction for human immunodeficiency virus and Virol. PubMed Scopus Google Scholar). have of to be of an on fusion pore that prevent of the viral into the and HIV-1 and are by PubMed Scopus Google Scholar, A. A. S. R. A. J. M. HIV-1 by from PubMed Scopus Google Scholar). view was supported by the of fusion pore using cell of HIV pseudovirus cell entry M. A. M. G.B. HIV-1 fusion pore by functional of envelope Biol. Chem. Full Text Full Text PDF PubMed Scopus (69) Google Scholar). To at with membrane we a combination of TIRF microscopy and cryoET to the of fusion of viral particles with and without and Serinc in TIRF microscopy, we examine the fusion behavior of and HIV pseudovirus particles to bleb-derived and supported plasma membranes and that Serinc3 and but not to fusion at the hemifusion and at the pore Serinc3 and but not the progression of fusion and a of structures not in the of The combination of cryoET and TIRF microscopy on plasma membranes from cells to be a to 3D of membrane structures and HIV fusion and thus provide structural as as functional into HIV entry into model target membrane for HIV fusion receptor, CD4, and co-receptor, CCR5 or in a lipid of and to fluorescence and cryo-electron of CD4 and CCR5 into such a model membrane but be technically and lipid of the target membrane. of or blebs from cells is E. P. membrane structure and behavior using giant plasma membrane 2012; PubMed Scopus Google Scholar), and such vesicles can be from cells that CD4 and CCR5 HIV plasma membrane for cell 3 PubMed Scopus Google Scholar). Blebs have the of the native lipid of the plasma membrane and full-length proteins with appropriate have previously that blebs and are for fusion of virus particles with HIV in the of HIV plasma membrane for cell 3 PubMed Scopus Google Scholar). To the fusion of HIV viral particles with and without for structural by cryoET, we first to by TIRF blebs from cells expressing CD4 and CCR5 as blebs were from into an by first a lipid on a the by of the blebs on that HIV plasma membrane for cell 3 PubMed Scopus Google The was as a target membrane with to fusion TIRF microscopy fusion by HIV pseudovirus particles that HIV and incorporate a M. G.B. Visualization of from cell HIV-1 particles an 2016; PubMed Scopus Google Scholar). such particles were into an from we a of by the of in binding of the to the The and of the in are in The fluorescence of the was and to fluorescence to fusion and of into the between the supported membrane and the such were to the of by a to the and was to a model as in is into the and allowed to the of of to other previously of viral fusion of influenza virus membrane PubMed Scopus Google and fusion B. P. S. R. J.D. of of 3 PubMed Scopus Google to supported we such as in 1 to binding by membrane fusion and of viral the particles that to a fusion to and membranes than to membranes in the same this process is HIV has HIV particles to a T cell with multiple in between (4Sougrat R. Bartesaghi A. Lifson J.D. Bennett A.E. Bess J.W. Zabransky D.J. Subramaniam S. Electron tomography of the contact between T cells and SIV/HIV-1: implications for viral entry.PLoS Pathog. 2007; 3 (17480119): e6310.1371/journal.ppat.0030063Crossref PubMed Scopus (145) Google Scholar), but to has directly subsequent steps of HIV membrane fusion and cell entry by cryoET. To detailed snapshots of HIV pseudovirus particles in the act of membrane we as target membranes for HIV fusion by cryoET. To the of observing fusion intermediates between and blebs and HIV we fusion by to blebs at the to for and the for cryoET. fusion of multiple pseudovirus particles and blebs the structure to be than the is by the in The of particles as the of pseudovirus and was for a was by the of the to the of HIV the and of Env, and the that is required for fusion S. H. J. S. S. from the HIV fusion the HIV-1 entry by multiple in and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with its mechanism of only showed binding a and without that were for showed multiple structures that we as binding, hemifusion, and fusion that were for showed only binding binding at the later pseudovirus particles that are of fusion and thus to the when other have and are from The of of to for showed particles that we fusion is with of of of and showed viral and target membranes in but with a between the the of membranes. from the viral membrane surface with a of HIV from the membrane that the are such in the of of the two membranes. similar was in of virus and without and leading us to such of virus and as binding, an of HIV fusion. In frozen we was between the membrane but the and of the of the viral and membranes were and not and on the of these to in of influenza fusion with liposomes by cryoET P. Mekhedov E. Waters H. Schwartz C.L. Fischer E.R. Ryham R.J. Cohen F.S. Blank P.S. Zimmerberg J. The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.Nat. Microbiol. 2016; 1 (27572837): 1605010.1038/nmicrobiol.2016.50Crossref PubMed Scopus (67) Google Scholar), we these as a of a lipid of two with a of the lipid the between The of the smaller is of the in and extend from the membrane the smaller that be HIV were previously for influenza membrane fusion with liposomes and as an of fusion pore K.K. Architecture of a nascent viral fusion pore.EMBO J. 2010; 29 (20168302): 1299-131110.1038/emboj.2010.13Crossref PubMed Scopus (93) Google Scholar), leading us to these as an fusion a when the fusion pore is and the viral is were fusion to the plasma membrane of whole cells B. 3D intermediates of membrane fusion in virus 1 S. A. PubMed Scopus Google Scholar). In the at for we virus particles binding to blebs but we such as in is a of the from the membrane multiple that be HIV and that the of CD4 the in at and the of in the membrane that be by virus and we similar structures to be the of full the products of multiple of virus and fusion. of HIV fusion intermediates are in were with a cell that viral particles with of HIV A. L. L. J. J. of on HIV-1 Virol. PubMed Scopus Google but we not as by with produced from subsequent were with HIV produced from In using and blebs as a target membrane to HIV membrane fusion by cryoET, we have intermediates of HIV membrane fusion. TIRF microscopy and cryoET to fusion of HIV was a in and HIV pseudovirus particles binding to and as with that not incorporate but for However, of the particles that and of viral a similar fusion to as by M. A. M. G.B. HIV-1 fusion pore by functional of envelope Biol. Chem. 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The restriction HIV-1 without the lipid and of viral Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. the of the host to HIV-1 Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. of is into HIV-1 Virol. PubMed Scopus Google Scholar, J. R. P. H. S. B. HIV-1 restriction by of the Virol. PubMed Scopus (46) Google Scholar, S. A. H. S. E. J. Schwartz of as Virol. 2019; PubMed Scopus Google Scholar). the of particles visualized by cryoET at of pseudovirus and blebs was smaller in of and and blebs at the when to fusion products we visualized hemifusion and fusion in with Serinc3 and Serinc5 of similar to the fusion products with at the same in of we only fusion of the fusion products in Serinc3 and and Serinc5 and a of and in the viral can be the not the the snapshots of fusion with cryoET and the information on the process of HIV pseudovirus fusion to from TIRF microscopy that Serinc3 and Serinc5 the of observing hemifusion and fusion products and the opening of fusion for of To the at Serinc3 and Serinc5 HIV we a into the membrane of HIV membrane have been to the and of viral fusion R.J. and in fusion J. 2019; Full Text Full Text PDF PubMed Scopus Google Scholar), we a to a R.J. with lipid PubMed Scopus Google that have of an on and we the same fusion for or not were with not a lipid R. Viard M. 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