Membrane fusion-inhibiting peptides do not inhibit influenza virus fusion or the Ca(2+)-induced fusion of negatively charged vesicles.

ABSTRACTProtein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved.IMPORTANCEEnveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.

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Precise Triggering and Chemical Control of Single-Virus Fusion within Endosomes

Journal of Virology ◽

10.1128/jvi.01982-20 ◽

2020 ◽

Vol 95 (1) ◽

Author(s):

Sourav Haldar ◽

Kenta Okamoto ◽

Rebecca A. Dunning ◽

Peter M. Kasson

Keyword(s):

Influenza Virus ◽

Membrane Fusion ◽

Influenza A ◽

Living Cells ◽

Membrane Curvature ◽

Endosomal Trafficking ◽

Viral Fusion ◽

Enveloped Viruses ◽

Endosomal Membrane ◽

Virus Fusion

ABSTRACT Many enveloped viruses infect cells within endocytic compartments. The pH drop that accompanies endosomal maturation, often in conjunction with proteolysis, triggers viral proteins to insert into the endosomal membrane and drive fusion. Fusion dynamics have been studied by tracking viruses within living cells, which limits the precision with which fusion can be synchronized and controlled, and reconstituting viral fusion to synthetic membranes, which introduces nonphysiological membrane curvature and composition. To overcome these limitations, we report chemically controllable triggering of single-virus fusion within endosomes. We isolated influenza (A/Aichi/68; H3N2) virus:endosome conjugates from cells, immobilized them in a microfluidic flow cell, and rapidly and controllably triggered fusion. Observed lipid-mixing kinetics were surprisingly similar to those of influenza virus fusion with model membranes of opposite curvature: 80% of single-virus events had indistinguishable kinetics. This result suggests that endosomal membrane curvature is not a key permissive feature for viral entry, at least lipid mixing. The assay preserved endosomal membrane asymmetry and protein composition, providing a platform to test how cellular restriction factors and altered endosomal trafficking affect viral membrane fusion. IMPORTANCE Many enveloped viruses infect cells via fusion to endosomes, but controlling this process within living cells has been challenging. We studied the fusion of influenza virus virions to endosomes in a chemically controllable manner. Extracting virus:endosome conjugates from cells and exogenously triggering fusion permits precise study of virus:endosome fusion kinetics. Surprisingly, endosomal curvature does not grossly alter fusion kinetics, although membrane deformability does. This supports a model for influenza virus entry where cells restrict or permit membrane fusion by changing deformability, for instance, using interferon-induced proteins.

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Faculty Opinions recommendation of Third helical domain of the nipah virus fusion glycoprotein modulates both early and late steps in the membrane fusion cascade.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738343089.793577097 ◽

2020 ◽

Author(s):

Rebecca Dutch

Keyword(s):

Membrane Fusion ◽

Nipah Virus ◽

Helical Domain ◽

Virus Fusion ◽

Fusion Glycoprotein

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Small Molecule Inhibitors of Influenza Virus Entry

Pharmaceuticals ◽

10.3390/ph14060587 ◽

2021 ◽

Vol 14 (6) ◽

pp. 587

Author(s):

Zhaoyu Chen ◽

Qinghua Cui ◽

Michael Caffrey ◽

Lijun Rong ◽

Ruikun Du

Keyword(s):

Influenza Virus ◽

Membrane Fusion ◽

Small Molecule ◽

Drug Target ◽

Small Molecule Inhibitors ◽

Virus Entry ◽

Critical Role ◽

Structural Basis ◽

Future Directions ◽

The Past

Hemagglutinin (HA) plays a critical role during influenza virus receptor binding and subsequent membrane fusion process, thus HA has become a promising drug target. For the past several decades, we and other researchers have discovered a series of HA inhibitors mainly targeting its fusion machinery. In this review, we summarize the advances in HA-targeted development of small molecule inhibitors. Moreover, we discuss the structural basis and mode of action of these inhibitors, and speculate upon future directions toward more potent inhibitors of membrane fusion and potential anti-influenza drugs.

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Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)45442-7 ◽

1987 ◽

Vol 262 (36) ◽

pp. 17744-17749 ◽

Cited By ~ 2

Author(s):

T Stegmann ◽

F P Booy ◽

J Wilschut

Keyword(s):

Influenza Virus ◽

Membrane Fusion ◽

Low Ph

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Role of Hemagglutinin Surface Density in the Initial Stages of Influenza Virus Fusion: Lack of Evidence for Cooperativity

Journal of Virology ◽

10.1128/jvi.74.6.2714-2720.2000 ◽

2000 ◽

Vol 74 (6) ◽

pp. 2714-2720 ◽

Cited By ~ 34

Author(s):

Susanne Günther-Ausborn ◽

Pieter Schoen ◽

Ingrid Bartoldus ◽

Jan Wilschut ◽

Toon Stegmann

Keyword(s):

Influenza Virus ◽

Surface Density ◽

Initial Rate ◽

Fusion Activity ◽

Cooperative Action ◽

Lipid Ratio ◽

Virus Fusion ◽

Influenza Virus Hemagglutinin ◽

Optimal Fusion

ABSTRACT Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.

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