Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

Abstract

Protein-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.

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

“…Detected MAY-induced hemifusion diaphragms had diameters consistent with hemifusion diaphragms found in wild-type HA (9 Ϯ 2 nm)-and G1S mutant (9 Ϯ 3 nm)-driven fusion published in our recent study (20). Some of the fusion events were similar to those described by Calder and Rosenthal as fusion pores in their recent study (49) or similar to those described by Gui et al as target membrane-pinching deformations at liposome-virus contact sites (50). However, due to the presence of an electrondense material between the MAYNAM1M2 VLPs and liposomes, we classified these events as hemifusion diaphragms/stalks, as in our previous study (20).…”

“…Isolated MAYNAM1M2 particles form complete fusion products with liposomes. Cryo-ET was recently used to structurally characterize the influenza A VLPdriven hemifusion intermediates (20) and the influenza A virus-driven fusion intermediates (49)(50)(51). To investigate the role of HA acylation in HA-mediated membrane fusion in a system that more closely mimics influenza virus fusion, we used cryo-ET.…”

Monomeric Intermediates Formed by Vesiculovirus Glycoprotein during Its Low-pH-induced Structural Transition

“…It is worth noting that EM images and cryo-ET have also provided several views of the membrane remodeling events and some structural snapshots of HA during the fusion process [81][82][83][84]. They are consistent with the initial formation of flexible extended intermediates which interact with the target membrane and refold into a post-fusion-like structure which is perpendicular to the axis of the fusion pore (i.e., parallel to the fusing membranes).…”

“…Cryo-EM and cryo-ET enhanced by direct electron detection devices, improved microscopes with more stable optics, and advances in image processing software [88][89][90][91] should make it possible to visualize the conformation of the glycoproteins while they interact with a target membrane. Indeed, as mentioned above, some progresses have already been made for both class I [81][82][83][84]92] and class II fusion glycoproteins [67][68][69]; however, a higher resolution will be required to get a reliable quasi-atomic model of those intermediates. [74] and in two monomeric HA structures [74,77].…”

Structure and organization of paramyxovirus particles

“…Multiple studies investigating different type I viral membrane fusion proteins have concluded that a concerted action of several fusion protein complexes is required to induce sufficient negative curvature in the viral envelope and cellular membrane to trigger local lipid disarray in the outer leaflets of the approaching bilayers, allowing merger of the disordered monolayers at the fusion tip and ultimately opening of a fusion pore [85,86,87,88,89,90,91]. If individual paramyxovirus glycoprotein homo-oligomers interact with each other for membrane fusion, each attachment protein tetramer would be sterically able to contact two F protein trimers in parallel.…”