Architecture of a nascent viral fusion pore
2010; Springer Nature; Volume: 29; Issue: 7 Linguagem: Inglês
10.1038/emboj.2010.13
ISSN1460-2075
Autores Tópico(s)Bacteriophages and microbial interactions
ResumoArticle18 February 2010Open Access Architecture of a nascent viral fusion pore Kelly K Lee Corresponding Author Kelly K Lee Department of Medicinal Chemistry, University of Washington, Seattle, WA, USA Search for more papers by this author Kelly K Lee Corresponding Author Kelly K Lee Department of Medicinal Chemistry, University of Washington, Seattle, WA, USA Search for more papers by this author Author Information Kelly K Lee 1 1Department of Medicinal Chemistry, University of Washington, Seattle, WA, USA *Corresponding author. Department of Medicinal Chemistry, University of Washington, PO Box 357610, Seattle, WA 98195-7610, USA. Tel.: +1 206 616 3972; Fax: +1 206 685 3252; E-mail: [email protected] The EMBO Journal (2010)29:1299-1311https://doi.org/10.1038/emboj.2010.13 There is a Have you seen ...? (April 2010) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Enveloped viruses use specialized protein machinery to fuse the viral membrane with that of the host cell during cell invasion. In influenza virus, hundreds of copies of the haemagglutinin (HA) fusion glycoprotein project from the virus surface. Despite intensive study of HA and its fusion activity, the protein's modus operandi in manipulating viral and target membranes to catalyse their fusion is poorly understood. Here, the three-dimensional architecture of influenza virus–liposome complexes at pH 5.5 was investigated by electron cryo-tomography. Tomographic reconstructions show that early stages of membrane remodeling take place in a target membrane-centric manner, progressing from punctate dimples, to the formation of a pinched liposomal funnel that may impinge on the apparently unperturbed viral envelope. The results suggest that the M1 matrix layer serves as an endoskeleton for the virus and a foundation for HA during membrane fusion. Fluorescence spectroscopy monitoring fusion between liposomes and virions shows that leakage of liposome contents takes place more rapidly than lipid mixing at pH 5.5. The relation of ‘leaky’ fusion to the observed prefusion structures is discussed. Introduction Membrane fusion is a fundamental biological process that lies at the heart of enveloped virus infection, synaptic signaling, intracellular vesicle trafficking, gamete fertilization, and cell–cell fusion. Despite intensive study, we have a limited mechanistic understanding of how fusion protein machinery manipulates lipid membranes to induce their fusion. The haemagglutinin (HA) envelope glycoprotein mediates influenza virus membrane fusion and hence has a key function in host cell invasion by this major human pathogen. It is the archetypal class-I fusion protein and shares core architectural elements with fusogens in retroviruses (e.g. HIV), filoviruses (e.g. ebola virus), coronaviruses (e.g. SARS virus), and paramyxoviruses (e.g. respiratory syncytial virus, measles virus) (Hughson, 1997; Lamb and Jardetzky, 2007). HA ‘spikes’ project outward from the viral envelope and become fusion active when exposed to the acidic pH found in endosomes. Activated proteins grab hold of the host membrane, and in concert with an energy-releasing conformational change, are able to draw the host (target) and viral membranes into apposition and induce them to fuse. To date, the organization of lipid and protein in the virus–target membrane complexes during fusion has eluded structural characterization. Numerous models of membrane deformation leading to fusion have been proposed (reviewed, for example, in Chernomordik and Kozlov, 2003, 2008; Tamm et al, 2003; Martens and McMahon, 2008). The majority of models describing HA-mediated membrane fusion focus on the properties of a hemifusion stalk in which the outer leaflets of the two previously distinct lipid bilayers have joined, the inner leaflets remain separate, and aqueous contents have not mixed. The existence of a hemifused state is supported by fluorescence and electrophysiology assays (reviewed in Chernomordik and Kozlov, 2003, 2005). The events leading up to and following the formation of the hemifusion stalk are less understood. It has been suggested that HA induces the formation of highly curved dimples in the target membrane to initiate the fusion process (Kuzmin et al, 2001; Efrat et al, 2007). Alternatively, some have proposed that the fusion peptides act primarily on the viral membrane, generating local bending and protrusions directed towards the target membrane (Kozlov and Chernomordik, 1998). Computational simulations have yet to resolve pre-stalk fusion intermediates including dimples. In the absence of structural characterization, our understanding of the mechanisms of membrane fusion is likely to remain nebulous. The stoichiometry and organization of HA spikes at fusion loci is likewise unresolved. Several studies of inter-HA spike cooperativity have indirectly estimated the number of fusion proteins required to induce fusion, with figures ranging from one spike to several (Ellens et al, 1990; Clague et al, 1991; Blumenthal et al, 1996; Danieli et al, 1996; Gunther-Ausborn et al, 2000; Mittal et al, 2002; Imai et al, 2006); however, fusion proteins involved in virus–target membrane contact sites have not been directly imaged. Despite significant gaps in our understanding of the mechanics of protein-mediated membrane fusion, much is known about the fusion proteins themselves (Skehel and Wiley, 2000; Eckert and Kim, 2001). Influenza HA is expressed as a single precursor polypeptide called HA0. Each 225 kDa HA spike is a homotrimer of HA0 subunits. Host proteases cleave the HA0 into two disulfide-bonded subunits, HA1 and HA2 (reviewed in Steinhauer, 1999). Cleavage is necessary for rendering HA fusion capable and thus for infectivity (Klenk et al, 1975; Lazarowitz and Choppin, 1975). HA1 contains the sialic acid receptor-binding site as well as the majority of antigenic features for influenza, whereas HA2 carries out membrane fusion. HA is bound to the viral envelope by a transmembrane anchor on the C-terminal tail of HA2; the N-terminus of HA2 generated by proteolysis of HA0 is the fusion peptide itself. Between 300 and 500 trimeric, HA spikes are arrayed on the surface of each virus (Inglis et al, 1976; Ruigrok et al, 1984; Harris et al, 2006). After attachment to the cell surface, influenza virus enters the cell by endocytosis (Figure 1). Endosomal maturation and the resulting decrease in endocytic pH trigger a multi-step, ‘spring-loaded’ conformational change of HA to an extended coiled-coil conformation that grapples the viral and target membranes together and induces them to fuse (Skehel et al, 1982; Carr and Kim, 1993; Gruenke et al, 2002). X-ray crystal structures of influenza HA have elucidated the protein machinery's detailed structure in the neutral pH and post-fusion conformations, and in the precursor HA0 form (Wilson et al, 1981; Bullough et al, 1994; Chen et al, 1998). Fusion between the viral and endosomal membranes takes place optimally at pH 5 in late endosomes (Maeda and Ohnishi, 1980; Matlin et al, 1981; White et al, 1982; Puri et al, 1990; Krumbiegel et al, 1994; Korte et al, 1999; Lakadamyali et al, 2003); however, prefusion intermediates are populated at the pH values between 5.5 and 6.0 found in early and maturing endosomes (Doms et al, 1985; White and Wilson, 1987; Stegmann et al, 1990; Korte et al, 1999). Figure 1.Influenza A virus, an enveloped, negative-sense RNA virus, enters cells by endocytosis. Endocytic maturation exposes the virus to stages of acidification. Fusion of viral and endosomal membranes occurs most rapidly at pH 5; however, studies have shown that the HA fusion protein becomes active at pH 5.5–6 to begin the process of membrane remodeling that ultimately leads to fusion. Download figure Download PowerPoint The earliest stage of conformational change at intermediate endosomal pH values involves release of the amphipathic, membrane-active fusion peptide from its sequestered position in the interior of the HA spike at neutral pH (White and Wilson, 1987; Stegmann et al, 1990). HA1 globular head domain dissociation is not required for fusion-peptide deployment, however, it is required for the refolding of HA2 into the TBHA2 extended coiled-coil conformation (Godley et al, 1992). The early conformational intermediates may dock the target membrane and be responsible for priming the membranes for fusion by inducing critical initial deformations. Influenza virus entry and ultrastructure have been investigated by electron microscopy (EM) in a number of seminal studies (Matlin et al, 1981; Ruigrok et al, 1986; Fujiyoshi et al, 1994; Wharton et al, 1995; Kanaseki et al, 1997; Shangguan et al, 1998; Harris et al, 2006; Noda et al, 2006). These have provided a glimpse of the virus's detailed construction, the general nature of changes exhibited by the virus in response to fusion activation, and its trafficking into cells. This study is the first, however, to provide a detailed three-dimensional characterization of fusion intermediates in this intensively studied system. Here, electron cryo-tomography (ECT) was used to image the three-dimensional architecture of loci formed between authentic X31 influenza virions and liposomal membranes under fusogenic conditions. Flash freezing of unstained biological specimens enables transient intermediates to be trapped, and the organization of protein and membranes in fusion intermediates to be determined. ECT builds a three-dimensional image of unfixed biological structures preserved in vitreous ice by imaging the specimen over a range of axial angular tilts (Lucic et al, 2005). Conditions were chosen to recreate the fusogenic interactions between virus and target membrane that are present at the early and maturing endosomal stage of cell invasion (pH∼5.5–6.0), with the goal of imaging nascent fusion loci at the earliest stages of membrane remodeling. In parallel with the ECT studies, time-resolved fluorescence measurements at pH 5.5 and 5.0 were performed to validate that the liposomes are a reasonable facsimile of a target membrane and verify that the virions were capable of fusing with the liposomes. Results Fluorescence spectroscopy of fusion between DOPC liposomes and virions at acidic pH A series of fluorescence measurements at pH 5.0 and 5.5 were performed using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes that contained the water-soluble fluorophore sulforhodamine-B (SRB) at self-quenching concentrations and the lipophilic fluorophore 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) incorporated into the bilayer, also at self-quenching concentrations. SRB and DiD have been used previously to monitor liposome permeabilization under acidic conditions (e.g. Lee et al, 1998; Odegard et al, 2009) and influenza fusion respectively (e.g. Lakadamyali et al, 2003). When the doubly labeled liposomes were mixed with unlabeled virus particles at acidic pH, dequenching of both SRB and DiD fluorescence was observed (Figure 2). At pH 5.5 DiD dequenching was a slow process, reaching only 9% after 60 min. Interestingly, SRB fluorescence dequenching took place significantly more rapidly compared with DiD fluorescence changes and reached 30% by 60 min. When the pH of the reaction was 5.0, changes in the two signals were nearly concomitant, and DiD dequenching was also more complete than at pH 5.5 reaching 17% by 60 min post-acidification. Figure 2.Fluorescence spectroscopy monitoring lipid exchange and liposomal content transfer and leakage. SRB/DiD-labeled DOPC liposomes were incubated with X31 influenza virions at pH 5.5 and 5.0. Fluorescence dequenching of the water-soluble (SRB, solid circles) and lipophilic (DiD, open circles) dyes was monitored as a function of time. The vertical scales for the two pH conditions are the same. Extent of dequenching was calculated as [F(t)−F(0)]/[FTX-100−F(0)], where FTX-100 was the fluorescence measured in the presence of 0.1% w/v TritonX-100 detergent. Fitted curves are only meant to guide the eye and are not intended to reflect a particular kinetic model. Download figure Download PowerPoint The dequenching observed for SRB fluorescence was significantly larger in magnitude than dequenching of the lipidic dye. A reasonable explanation for this is that though some of the dequenching signal may be due to transfer of some SRB to the viral lumen, additional dye leaks out of the liposomes and diffuses into the entire volume of the cuvette. This appears to precede lipid mixing. By contrast, as lipid mixing takes place between virus and liposome, the two-dimensional surface area coverage of DiD only increases modestly (∼50%) as ∼150 nm virus envelopes fuse with membrane of the 100 nm liposomes, leading to a relatively small dequenching signal. Fluorescence experiments were carried out with an excess of liposomes to disfavour disruption of liposomes by multiple virus particles. As a result, the total extent of dequenching of the dyes did not reach 100% (as determined by complete dequenching in the presence of 0.1% TritonX-100) over the time frame examined. No qualitatively different fluorescence kinetics was observed when the amount of DiD incorporated in the DOPC membrane was varied by a factor of 4. A complementary set of experiments were performed in which the DiD fluorophore was incorporated into the virus membrane, and liposomes contained only encapsulated SRB (Supplementary Figure S1). The changes and pH effects closely paralleled the changes observed when the lipophilic DiD dye was in the liposomal bilayer. These data confirm that DOPC liposomes and viruses are capable of merging their membranes, which occurs more efficiently at pH 5.0, and the HA-mediated process is ‘leaky’. Influenza A virus exhibits a complex envelope and glycoprotein coat In the electron cryo-tomograms of X31 influenza A virions at pH 5.5, HA spikes are generally well ordered on the surface of the particles and resemble the neutral pH HA structure (Figure 3), although it is not possible to distinguish between the neutral pH structure and a similar fusogenic conformation that has been reported (Wilson et al, 1981; Bottcher et al, 1999). As in a previous ECT study of influenza virus ultrastructure, the HA and neuraminidase surface glycoproteins can often be distinguished (Harris et al, 2006). In most virions a thick (∼8–12 nm) lipoprotein envelope lies beneath the glycoprotein coat and encapsulates the virus's ribonucleoprotein material (the individual segments are unresolved). The envelope's braided appearance results from the compositional mixture of phospholipids, cholesterol, transmembrane protein anchors, M2 ion channels, and associated M1 matrix proteins. Three distinct layers of the envelope can be discerned in volume slices through many of the viruses (Supplementary Figure S2). The two outermost layers exhibit ∼4–6 nm separation, whereas the inner layer, presumably matrix protein, is ∼4–6 nm further below the central layer. Fujiyoshi et al (1994) have proposed that the inner portion of the envelope reflects a matrix protein–lipid composite, rather than a classical phospholipid bilayer leaflet with associated protein. Further study is required to elucidate the detailed construction and stratification of the envelope; however, for the purpose of this study, virus particles that exhibit thick envelopes are inferred to possess an intact matrix layer. Density that extends beneath some glycoprotein spikes and bores completely through the virus envelope to the matrix layer may correspond to the glycoproteins’ transmembrane anchors (Figure 3A). A direct association between glycoprotein anchors and matrix protein is consistent with previous reports that have described the importance of this interaction for virus morphology (Enami and Enami, 1996; Jin et al, 1997; Ali et al, 2000). The leaflets of the DOPC liposomes tend to be difficult to resolve under the imaging conditions, although in some tomograms, two leaflets with ∼4 nm spacing are evident (Supplementary Figure S2). Figure 3.Electron cryo-tomograms of influenza virions at pH 5.5. (A) 5.3 nm-thick section through the reconstructed tomographic density for an X31 influenza A virion at pH 5.5 after 5 min acidification. The glycoprotein fringe is evident on the exterior. Most particles bear an ∼8–12 nm-thick viral envelope. Density beneath the exterior spikes (red arrowheads), which bores through the viral envelope (white arrowheads), may be HA and NA transmembrane anchors. HA (green arrowhead) and NA (orange arrowhead) ectodomains are distinguishable. Inset: HA spike density, cropped from the tomogram is in good agreement with the HA ectodomain crystal structure; anchors are modeled into the structure (Wilson et al, 1981). The spike is tethered to three layers of the virus envelope (postulated strata: black arrowhead outer leaflet, white, inner leaflet, grey, M1 matrix protein layer). Microscope defocus 3 μm. Scalebar 50 nm. (B) At early timepoints, DOPC liposomes do not appear to be deformed by virions even when they are in close proximity. Sample acidified at pH 5.5 for 2.5 min. Microscope defocus 3 μm. Scalebar 50 nm. Download figure Download PowerPoint Summary of liposome–virion complexes To produce fusion-active complexes of influenza virus and target membrane for ECT, whole X31 strain virions were combined with DOPC liposomes at pH 5.5 and flash frozen. For samples that were acidified for 2.5 min, few changes in virus or liposome structure were observed (Figure 3B). In this case, virus particles and liposomes were sometimes found close to each other but with no apparent deformation of the liposome, suggesting that the majority of HA on the virus surface was still inactive. Between 5 and 15 min post-acidification, more significant numbers of virions and liposomes were observed to form a range of complexes, with the liposomal bilayer typically showing clear signs of perturbation (Figures 4, 5 and 6; Supplementary Movies 1–3). Identification of relative concentrations of virus and liposome that produce significant numbers of discrete pairings of the particle types and also vitreous ice suitable for low-dose ECT has been a challenge. The majority (n>100) of virions in the ECT specimens for this study were not participating in complexes. Of the 53 loci observed between virions and liposomes at pH 5.5, nine exhibited dimple-like features on the target membranes without apparent disruption of the liposomal bilayer density (Figure 4). 23 exhibited a funnel-shaped deformation of the target liposomal membrane with what appears to be a break in the target membrane (Figures 5 and 6). Two instances of intermediates further along the pathway were observed (Figure 7), the low count number being consistent with the relatively low efficiency of lipid mixing and fusion at pH 5.5 (Figure 2). The features are described further below. The remainder of contacts involved various types of flat contacts in which the target membrane runs parallel to the outer viral envelope and otherwise does not exhibit any detectable deformation. In some cases, these appear to be mediated by HA (Figure 4D), in others, the virus and liposome may simply have been brought into contact because of surface tension effects in the thin water film that spans the 2 μm grid hole. A limited number of putative post-fusion complexes were observed (Supplementary Figure S3), again consistent with the relatively low efficiency of lipid mixing and fusion at pH 5.5. These complexes tend to be large, on the order of 200–250 nm in diameter; they exhibit granular contents that are localized on primarily one side of the vesicle near concentrations of glycoprotein spikes; other regions on the vesicle surface are bare, with low density of associated spikes. Figure 4.Target membrane deformations in virion–liposome complexes observed by ECT. Samples had been acidified at pH 5.5 for 5–8 min. Microscope defocus was set at 3–5 μm; 5.3 nm-thick slices through reconstructed cryo-tomographic density are shown. Scalebars 50 nm. (A) At early stages of docking, the liposomal bilayer is pulled into ∼5 nm-wide dimples (black arrowheads). (B) Some liposomes make glancing contacts (arrowhead in slice 3) with the viral envelope. (C) Liposomes can form extended planar contacts with virus envelope. (D) The liposome in this complex is engaged by two virions, one above and one to the left hand side. The liposome in this case forms a nearly planar contact with the virus on the left and appears to be coordinated by HA. The boxed HA spike in slices 2 and 3 exhibits density in a ‘Y’ shape bridging the virus surface and liposome. This resembles features seen at virus membrane junctions in HSV-1 entry studies (Maurer et al, 2008). The interaction with the upper virus is more localized, with the liposomal membrane being pinched into an acute angle (arrowhead, slice 1). Download figure Download PowerPoint Figure 5.Electron cryo-tomograms of deformations in the liposomal membrane induced by interaction with virions at pH 5.5. Also see Supplementary Movies 1 and 2. (A) In this complex, the liposomal membrane appears to have been breached (white arrowhead, slice 1) at the contact zone where it is coordinated by HA spikes (black arrowheads, slice 1). Sample acidified for 5 min. Microscope defocus 3 μm. (B) In this tomogram, the liposome has been remodeled into a funnel that is apposed to the viral envelope; a putative opening at the funnel's mouth is indicated by the white arrowheads in slices 1 and 2. The virus envelope by contrast is intact. HA spikes (black arrowheads) ring the prefusion locus. Sample acidified for 8 min. Microscope defocus 5 μm; 5.3 nm virtual serial sections through the reconstructed tomograms are shown. Scalebars 50 nm. Download figure Download PowerPoint Figure 6.Electron cryo-tomograms of a funnel-shaped deformation in the liposomal membrane induced by interaction with virions at pH 5.5. Also see Supplementary Movie 3. (A) The virus has grabbed hold of a liposome and drawn its membrane towards the viral envelope. A putative opening at the funnel's mouth is indicated by the white arrowheads in slice 1. The viral envelope appears to be intact; 5.3 nm-thick serial sections are shown. Samples have been acidified to pH 5.5 for 5 min; 50 nm scalebar. (B) A cross-sectional density plot (path indicated by black arrowheads in (A), slice 1) maps the ∼5 nm-wide channel through the lipid funnel. The channel is lined by the liposomal bilayer with 3–4 nm leaflet spacings. (C) A 10 nm-thick volume section shows density that is interpreted to be HA spikes in a ‘Y’ shaped conformation (white arrowheads) coordinating the liposomal funnel. Density rendered using Chimera. Inset shows a schematic of the key density features. Download figure Download PowerPoint Figure 7.Constrained necks can join virions and liposomes at pH 5.5. (A) Serial 5.3 nm-thick sections through a liposome–virion complex; liposome marked with ‘L’ and virus with ‘F’ in slice 1. A neck with 10–15 nm-wide mouth is seen in slices 4–6. Liposomal membrane and viral envelope density appear continuous, although the strong liposomal density exhibits a pronounced boundary at the base of the neck. Sample acidified for 8 min. Microscope defocus 5 μm. Scalebar 50 nm. (B) In the 10.6 nm-thick section through another complex, an ∼15 nm-wide neck connects a virion and liposome. Sample acidified for 5 min. Microscope defocus 3 μm. Scalebar 50 nm. (C) A density plot along the channel axis (path indicated by black arrowheads in (B)) indicates that three ridges of density traverse the central section of the neck. The density may be due to an intact matrix layer or liposome and virus membrane leaflets. (D) Low-dose cryo-EM image of influenza X31 virions at pH 5.0 in the absence of liposomes suggests that the viral envelopes are more labile at pH 5.0 and give rise to envelope-derived lipidic vesicles (marked ‘V’); no comparable vesicles were observed in identical samples that were kept at pH 7.5. Two virions (marked ‘F’) seen fusing with a vesicle in the lower left quadrant appear to be undergoing expansive pore dilation; an alternative explanation for this feature is that the virions may be caught in the process of shedding or blebbing their membranes. Sample acidified at pH 5.0 for 10 min. Microscope defocus 3 μm. Scalebar 100 nm. Download figure Download PowerPoint A number of ∼20 nm diameter unilamellar vesicles were observed on the interior of nearly every liposomes that was in complex with a virus particle. These were comparatively less abundant in liposomes that had not been exposed to influenza virus, with ∼15% of virus-naive liposomes exhibiting similar features (Supplementary Figure S4). It is possible the small vesicles seen in virion–liposome complexes ‘blebbed’ off the parent liposome in response to the bending stresses on the liposomal membrane induced by interaction with HA. Similar vesicles have been observed by freeze-fracture EM, leading Kanaseki et al (1997) to postulate that the vesicles originate from a virion making multiple points of contact with its target membrane. SNARE protein-mediated vacuole fusion has also been shown to yield pinched off membrane fragments that end up inside the fused organelle (Wang et al, 2002). Target membrane remodeling by influenza virus Liposome membrane deformation is localized around points of contact between virus and liposome (Figure 4). For complexes at early stages of docking, individual, localized dimples on the liposomal bilayers were observed (Figure 4A and B). These resemble ‘dimples’ that have been proposed in physical modeling schemes (Efrat et al, 2007) and may correspond to similar punctate features observed by Kanaseki et al (1997) using freeze-fracture EM. Other liposomes are pinched by a small set of HA spikes as shown in Figure 5A (see also Supplementary Movie 1) and appear to be breached (white arrowhead, slice 1) at the point of contact with the HA spikes (black arrowheads, slice 1). A scenario in which target membrane scission is induced by fusogenic HA is consistent with the fluorescence observations that significant liposome content leakage precedes lipid mixing (Figure 2). Others have documented HA-induced content leakage through a range of target membranes and have found that the fusion peptide of HA can lead to significant changes in lipid order and membrane curvature (Epand and Epand, 1994, 2000; Shangguan et al, 1996; Jiricek et al, 1997; Longo et al, 1997; Bonnafous and Stegmann, 2000; Frolov et al, 2003; Lau et al, 2004; Ge and Freed, 2009). One of the most striking and highly populated (23 out of 53 loci) features observed in liposome–virion complexes is a state in which the target membrane has been drawn and pinched towards the viral envelope such that it is in close apposition to the envelope's face (Figures 5 and 6; Supplementary Movies 2 and 3). Nearly all such funnel-shaped target membrane structures show an apparent lack of density, ∼5 nm wide, at the point of closest approach to the virus particle, suggesting that the liposome may present an open mouth towards the virus envelope. The proximal virus membrane by comparison does not appear to exhibit similar breaks or deformations. Given the limitations in ECT resolution, partial information because of the missing wedge effect, as well as the potential for weak density to be obscured by contrast transfer function (CTF) artefacts (seen as white Fresnel fringes) around liposomal bilayers, one cannot definitively rule out the possibility that tenuous connectivity with the viral envelope has been established or that membrane in some non-bilayer form may be present in the funnel mouth. The putative membrane breaches are observed not only at higher microscope defocus settings (e.g. 5 μm in Figure 5B), but also closer to true focus as well (3 μm in Figure 6) where fringing is less pronounced and where individual liposomal leaflets (still associated as a bilayer) can be observed lining the channel through the liposome's pinched neck (see Figure 6A slice 1, and Figure 6B). An open-mouthed funnel would be consistent with observations of significant leakage of liposomal contents before lipid mixing. Once a hemifused state is reached, the joining of target membrane and viral lipids into a continuous barrier would inhibit further leakage of contents from the liposomal lumen. In Figure 7A, a liposome and virus are seen to be bridged by a neck that exhibits a 10–15 nm-wide central channel (indicated by the white arrowheads in slices 4–6). In this case, within resolution limitations, the liposomal membrane appears to exhibit density continuity with the viral envelope (particularly evident in slices 4–6), however the lipids may not have mixed extensively. Specifically, the high contrast DOPC liposomal membrane exhibits an abrupt boundary at the base of the neck. Previous studies of HA-mediated fusion have indicated that at early stages of membrane fusion, lipid transfer may be restrained (Zimmerberg et al, 1994; Chernomordik et al, 1998). The tomogram is not sharp enough to distinguish individual membrane leaflets, thus it is not possible to discern whether the complex exists as hemifused stalk-like structure or possibly as a constrained pore with joined lumens. Futu
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