Membrane Perturbation and Fusion Pore Formation in Influenza Hemagglutinin-mediated Membrane Fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".

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The Pathway of Membrane Fusion Catalyzed by Influenza Hemagglutinin: Restriction of Lipids, Hemifusion, and Lipidic Fusion Pore Formation

The Journal of Cell Biology ◽

10.1083/jcb.140.6.1369 ◽

1998 ◽

Vol 140 (6) ◽

pp. 1369-1382 ◽

Cited By ~ 273

Author(s):

Leonid V. Chernomordik ◽

Vadim A. Frolov ◽

Eugenia Leikina ◽

Peter Bronk ◽

Joshua Zimmerberg

Keyword(s):

Membrane Fusion ◽

Surface Density ◽

Pore Formation ◽

Membrane Lipid ◽

Low Ph ◽

Fusion Pore ◽

Present Evidence ◽

Membrane Lipid Composition ◽

Influenza Hemagglutinin ◽

Pore Opening

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell–cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.

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Mechanics of membrane fusion/pore formation

Chemistry and Physics of Lipids ◽

10.1016/j.chemphyslip.2014.07.010 ◽

2015 ◽

Vol 185 ◽

pp. 109-128 ◽

Cited By ~ 30

Author(s):

Marc Fuhrmans ◽

Giovanni Marelli ◽

Yuliya G. Smirnova ◽

Marcus Müller

Keyword(s):

Membrane Fusion ◽

Pore Formation ◽

Fusion Pore

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Cholesterol Promotes Hemifusion and Pore Widening in Membrane Fusion Induced by Influenza Hemagglutinin

The Journal of General Physiology ◽

10.1085/jgp.200709932 ◽

2008 ◽

Vol 131 (5) ◽

pp. 503-513 ◽

Cited By ~ 47

Author(s):

Subrata Biswas ◽

Shu-Rong Yin ◽

Paul S. Blank ◽

Joshua Zimmerberg

Keyword(s):

Membrane Fusion ◽

Membrane Lipid ◽

Fusion Pore ◽

Dye Transfer ◽

Influenza Hemagglutinin ◽

Human Red Blood Cells ◽

Cholesterol Levels ◽

Pore Opening ◽

Two Stages ◽

Pore Expansion

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (<4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.

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Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

The Journal of Cell Biology ◽

10.1083/jcb.136.5.995 ◽

1997 ◽

Vol 136 (5) ◽

pp. 995-1005 ◽

Cited By ~ 131

Author(s):

Grigory B. Melikyan ◽

Sofya A. Brener ◽

Dong C. Ok ◽

Fredric S. Cohen

Keyword(s):

Outer Membrane ◽

Negative Curvature ◽

Pore Formation ◽

Transmembrane Domain ◽

Positive Curvature ◽

Fusion Pore ◽

Spontaneous Curvature ◽

Wild Type ◽

Influenza Hemagglutinin ◽

Fusion Pores

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.

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Multifaceted Sequence-Dependent and -Independent Roles for Reovirus FAST Protein Cytoplasmic Tails in Fusion Pore Formation and Syncytiogenesis

Journal of Virology ◽

10.1128/jvi.01667-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12185-12195 ◽

Cited By ~ 20

Author(s):

Christopher Barry ◽

Roy Duncan

Keyword(s):

Membrane Fusion ◽

Fusion Proteins ◽

Pore Formation ◽

Syncytium Formation ◽

Fusion Pore ◽

Protein Fusion ◽

Sequence Dependent ◽

Cytoplasmic Tails ◽

Fast Proteins ◽

Pore Expansion

ABSTRACT Fusogenic reoviruses utilize the FAST proteins, a novel family of nonstructural viral membrane fusion proteins, to induce cell-cell fusion and syncytium formation. Unlike the paradigmatic enveloped virus fusion proteins, the FAST proteins position the majority of their mass within and internal to the membrane in which they reside, resulting in extended C-terminal cytoplasmic tails (CTs). Using tail truncations, we demonstrate that the last 8 residues of the 36-residue CT of the avian reovirus p10 FAST protein and the last 20 residues of the 68-residue CT of the reptilian reovirus p14 FAST protein enhance, but are not required for, pore expansion and syncytium formation. Further truncations indicate that the membrane-distal 12 residues of the p10 and 47 residues of the p14 CTs are essential for pore formation and that a residual tail of 21 to 24 residues that includes a conserved, membrane-proximal polybasic region present in all FAST proteins is insufficient to maintain FAST protein fusion activity. Unexpectedly, a reextension of the tail-truncated, nonfusogenic p10 and p14 constructs with scrambled versions of the deleted sequences restored pore formation and syncytiogenesis, while reextensions with heterologous sequences partially restored pore formation but failed to rescue syncytiogenesis. The membrane-distal regions of the FAST protein CTs therefore exert multiple effects on the membrane fusion reaction, serving in both sequence-dependent and sequence-independent manners as positive effectors of pore formation, pore expansion, and syncytiogenesis.

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Minimal Aggregate Size and Minimal Fusion Unit for the First Fusion Pore of Influenza Hemagglutinin-Mediated Membrane Fusion

Biophysical Journal ◽

10.1016/s0006-3495(00)76587-8 ◽

2000 ◽

Vol 78 (1) ◽

pp. 227-245 ◽

Cited By ~ 73

Author(s):

Joe Bentz

Keyword(s):

Membrane Fusion ◽

Aggregate Size ◽

Fusion Pore ◽

Influenza Hemagglutinin

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Molecular mechanisms of membrane fusion: Steps during phospholipid and exocytotic membrane fusion

Bioscience Reports ◽

10.1007/bf01121447 ◽

1987 ◽

Vol 7 (4) ◽

pp. 251-268 ◽

Cited By ~ 30

Author(s):

Joshua Zimmerberg

Keyword(s):

Membrane Fusion ◽

Pore Formation ◽

Molecular Mechanisms ◽

Fusion Pore ◽

Pore Widening

Exocytosis is considered as four separate steps: adhesion, fusion/pore formation, pore widening, and content discharge. Experiments on both synthetic and natural membranes are presented to show each of these steps. Major differences are seen in the two fusing systems. These differences are discussed in terms of molecular mechanisms of fusion.

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The block to membrane fusion differs with the site of ligand insertion in modified retroviral envelope proteins

Journal of General Virology ◽

10.1099/vir.0.83445-0 ◽

2008 ◽

Vol 89 (4) ◽

pp. 1049-1058 ◽

Cited By ~ 4

Author(s):

Byoung Y. Ryu ◽

Tatiana Zavorotinskaya ◽

Bernadette Trentin ◽

Lorraine M. Albritton

Keyword(s):

Membrane Fusion ◽

Pore Formation ◽

Leukemia Virus ◽

Point Mutations ◽

Retroviral Vectors ◽

Membrane Lipid ◽

Fusion Pore ◽

Subsequent Step ◽

Cell Type Specific ◽

Proline Rich Region

Efforts to achieve cell type-specific transduction of retroviral vectors for gene therapy have centred on modification of the envelope protein (Env). Typically, addition of a ligand to Env gives binding to the new or target receptor, but little or no infection, and affects the subunit association of the modified Env. We previously discovered two point mutations that increase targeted infection by over 1000-fold when added to an Env modified by N-terminal insertion of the receptor-binding domain from amphotropic murine leukemia virus Env. Here, we asked whether these mutations would similarly increase transduction by Env modified with a clinically relevant ligand, human interleukin-13 (IL-13L). Addition of the point mutations stabilized the weak subunit association observed in some IL-13L-modified Env proteins, but infection via the target IL-13 receptor still did not occur. Fluorescence-based cell–cell fusion assays and studies with a membrane-curving agent revealed that defects in membrane fusion differed with the site of ligand insertion. When IL-13 was inserted into the N terminus of Env, membrane fusion was blocked prior to membrane-lipid mixing, regardless of whether flanking flexible linkers were added. Unexpectedly, insertion of IL-13 in the proline-rich region showed evidence of initiation of fusion and fusion-peptide exposure, but fusion was blocked at a subsequent step prior to fusion-pore formation. Thus, the site of ligand insertion influenced initiation of membrane fusion and its progression. These observations suggest that a novel site for ligand insertion must be identified before clinically useful targeted transduction will be achieved.

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Single-virus content mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency

10.1101/2021.04.26.441491 ◽

2021 ◽

Author(s):

Katherine N Liu ◽

Steven G. Boxer

Keyword(s):

Membrane Fusion ◽

Influenza A ◽

Pore Formation ◽

Fluorescent Labeling ◽

Fusion Pore ◽

Viral Fusion ◽

Enveloped Viruses ◽

Virus Assay ◽

Virus Content ◽

Target Membrane

In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses.

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