The shape of pleomorphic virions determines resistance to cell-entry pressure.

Many enveloped animal viruses produce a variety of particle shapes, ranging from small spherical to long filamentous types. Characterization of how the shape of the virion affects infectivity has been difficult because the shape is only partially genetically encoded, and most pleomorphic virus structures have no selective advantage in vitro. Here, we apply virus fractionation using low-force sedimentation, as well as antibody neutralization coupled with RNAScope, single-particle membrane fusion experiments and stochastic simulations to evaluate the effects of differently shaped influenza A viruses and influenza viruses pseudotyped with Ebola glycoprotein on the infection of cells. Our results reveal that the shape of the virus particles determines the probability of both virus attachment and membrane fusion when viral glycoprotein activity is compromised. The larger contact interface between a cell and a larger particle offers a greater probability that several active glycoproteins are adjacent to each other and can cooperate to induce membrane merger. Particles with a length of tens of micrometres can fuse even when 95% of the glycoproteins are inactivated. We hypothesize that non-genetically encoded variable particle shapes enable pleomorphic viruses to overcome selective pressure and may enable adaptation to infection of cells by emerging viruses such as Ebola. Our results suggest that therapeutics targeting filamentous virus particles could overcome antiviral drug resistance and immune evasion in pleomorphic viruses.

Conformational triggers associated with influenza matrix protein 1 polymerization.

A central role for the influenza matrix protein 1 (M1) is to form a polymeric coat on the inner leaflet of the host membrane that ultimately provides shape and stability to the virion. M1 polymerizes upon binding membranes, but triggers for conversion of M1 from a water-soluble component of the nucleus and cytosol into an oligomer at the membrane surface are unknown. While full-length M1 is required for virus viability, the N-terminal domain (M1NT) retains membrane binding and pH-dependent oligomerization. We studied the structural plasticity and oligomerization of M1NT in solution using NMR spectroscopy. We show that the isolated domain can be induced by sterol-containing compounds to undergo a conformational change and self-associate in a pH-dependent manner consistent with the stacked dimer oligomeric interface. Surface-exposed residues at one of the stacked dimer interfaces are most sensitive to sterols. Several perturbed residues are at the interface between the N-terminal subdomains and are also perturbed by changes in pH. The effects of sterols appear to be indirect and most likely mediated by reduction in water activity. The local changes are centered on strictly conserved residues and consistent with a priming of the N-terminal domain for polymerization. We hypothesize that M1NT is sensitive to changes in the aqueous environment and that this sensitivity is part of a mechanism for restricting polymerization to the membrane surface. Structural models combined with information from chemical shift perturbations indicate mechanisms by which conformational changes can be transmitted from one polymerization interface to the other.

Comparative Analyses of the Antiviral Activities of IgG and IgA Antibodies to Influenza A Virus M2 Protein.

The influenza A virus (IAV) matrix-2 (M2) protein is an antigenically conserved viral envelope protein that plays an important role in virus budding together with another envelope protein, hemagglutinin (HA). An M2-specific mouse monoclonal IgG antibody, rM2ss23, which binds to the ectodomain of the M2 protein, has been shown to be a non-neutralizing antibody, but inhibits plaque formation of IAV strains. In this study, we generated chimeric rM2ss23 (ch-rM2ss23) IgG and IgA antibodies with the same variable region and compared their antiviral activities. Using gel chromatography, ch-rM2ss23 IgA were divided into three antibody subsets: monomeric IgA (m-IgA), dimeric IgA (d-IgA), and trimeric and tetrameric IgA (t/q-IgA). We found that t/q-IgA had a significantly higher capacity to reduce the plaque size of IAVs than IgG and m-IgA, most likely due to the decreased number of progeny virus particles produced from infected cells. Interestingly, HA-M2 colocalization was remarkably reduced on the infected cell surface in the presence of ch-rM2ss23 antibodies. These results indicate that anti-M2 polymeric IgA restricts IAV budding more efficiently than IgG and suggest a role of anti-M2 IgA in cross-protective immunity to IAVs.

Spiky Nanostructures with Geometry-matching Topography for Virus Inhibition.

Geometry-matching has been known to benefit the formation of stable biological interactions in natural systems. Herein, we report that the spiky nanostructures with matched topography to the influenza A virus (IAV) virions could be used to design next-generation advanced virus inhibitors. We demonstrated that nanostructures with spikes between 5 and 10 nm bind significantly better to virions than smooth nanoparticles, due to the short spikes inserting into the gaps of glycoproteins of the IAV virion. Furthermore, an erythrocyte membrane (EM) was coated to target the IAV, and the obtained EM-coated nanostructures could efficiently prevent IAV virion binding to the cells and inhibit subsequent infection. In a postinfection study, the EM-coated nanostructures reduced >99.9% virus replication at the cellular nontoxic dosage. We predict that such a combination of geometry-matching topography and cellular membrane coating will also push forward the development of nanoinhibitors for other virus strains, including SARS-CoV-2.

Amphipathic Helices of Cellular Proteins Can Replace the Helix in M2 of Influenza A Virus with Only Small Effects on Virus Replication.

M2 of influenza virus functions as a proton channel during virus entry. In addition, an amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the assembly site where it inserts into the inner membrane leaflet to induce curvature that causes virus scission. Since vesicularization of membranes can be performed by a variety of amphiphilic peptides, we used reverse genetics to investigate whether the peptides can substitute for M2's helix. Virus could not be generated if M2's helix was deleted or replaced by a peptide predicted not to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was exchanged by helices known to induce membrane curvature. Infectious virus titers were marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor from the Epsin N-terminal homology domain or the nonnatural membrane inducer RW16. Transmission electron microscopy of infected cells did not reveal unequivocal evidence that virus budding or membrane scission was disturbed in any of the mutants. Instead, individual virus mutants exhibit other defects in M2, such as reduced surface expression, incorporation into virus particles, and ion channel activity. The protein composition and specific infectivity were also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication but that other helices can replace its basic (curvature-inducing) function.IMPORTANCE Influenza virus is unique among enveloped viruses since it does not rely on the cellular ESCRT machinery for budding. Instead, viruses encode their own scission machine, the M2 protein. M2 is targeted to the edge of the viral assembly site, where it inserts an amphiphilic helix into the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins with only small effects on virus replication. No evidence was obtained that budding is disturbed, but individual mutants exhibit other defects in M2 that explain the reduced virus titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic helix to induce membrane curvature, but its biophysical properties are more important than the amino acid sequence.

Frustration and fidelity in influenza genome assembly.

The genome of the influenza virus consists of eight distinct single-stranded RNA segments, each encoding proteins essential for the viral life cycle. When the virus infects a host cell, these segments must be replicated and packaged into new budding virions. The viral genome is assembled with remarkably high fidelity: experiments reveal that most virions contain precisely one copy of each of the eight RNA segments. Cell-biological studies suggest that genome assembly is mediated by specific reversible and irreversible interactions between the RNA segments and their associated proteins. However, the precise inter-segment interaction network remains unresolved. Here, we computationally predict that tree-like irreversible interaction networks guarantee high-fidelity genome assembly, while cyclic interaction networks lead to futile or frustrated off-pathway products. We test our prediction against multiple experimental datasets. We find that tree-like networks capture the nearest-neighbour statistics of RNA segments in packaged virions, as observed by electron tomography. Just eight tree-like networks (of a possible 262 144) optimally capture both the nearest-neighbour data and independently measured RNA-RNA binding and co-localization propensities. These eight do not include the previously proposed hub-and-spoke and linear networks. Rather, each predicted network combines hub-like and linear features, consistent with evolutionary models of interaction gain and loss.

A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages.

Influenza A virus (IAV) propagates efficiently in epithelial cells, its primary target in the respiratory tract. In contrast, productive infection of most IAV strains is either blocked or highly inefficient in macrophages. The exact nature of the defect in IAV replication in human macrophages remains unknown. In this study, we showed that even compared to a monocytic cell line differentiated to macrophage-like cells, primary human monocyte-derived macrophages (MDM) are inefficient in IAV production, despite comparable levels of expression of viral glycoproteins at the plasma membrane. Correlative fluorescence scanning electron microscopy revealed that formation of budding structures at the cell surface is inefficient in MDM even though clustering of a viral glycoprotein, hemagglutinin (HA), is observed, suggesting that a step in IAV particle assembly is blocked in MDM. Using an in situ proximity ligation assay, we further determined that HA associates with neuraminidase (NA) but fails to associate with another viral transmembrane protein, M2, at the MDM plasma membrane. Notably, the defects in HA-M2 association and particle assembly in MDM were reversed upon cytochalasin D treatment that inhibits actin polymerization. These results suggest that HA-M2 association on the plasma membrane is a discrete step in IAV production, which is susceptible to suppression by actin cytoskeleton in MDM. Virus release remained inefficient in MDM upon cytochalasin D treatment, suggesting the presence of an additional defect(s) in virus release in this cell type. Overall, our study revealed the presence of multiple cell-type-specific mechanisms negatively regulating IAV production at the plasma membrane in MDM.IMPORTANCE Identification of host cell determinants promoting or suppressing replication of viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM, which are not permissive to IAV replication, fail to support virus particle formation. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association and particle budding, but not virus release, in MDM are rescued by disruption of actin cytoskeleton, revealing a previously unknown, negative role for actin, which specifically targets an early step in the multistep IAV production. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.

Correlative Light and Electron Microscopy of Influenza Virus Entry and Budding.

Influenza A virus (IAV) entry is a stepwise process regulated by viral and cellular cues, facilitating cellular functions. Virus entry begins by attachment of hemagglutinin to cell surface sialic acids, followed by endocytic uptake, vesicular transport along microtubules, low-pH-mediated viral membrane fusion with the late endosomal membrane, capsid uncoating, viral ribonucleoprotein (vRNP) release, and nuclear import of vRNPs. Here we show a basic methodology to visualize incoming and egressing IAV particles by correlative light and electron microscopy (CLEM). We combine fluorescence microscopy of virus-infected human lung carcinoma A549 cells with high-pressure freezing (HPF) and in-resin fluorescence CLEM and the Tokuyasu CLEM method. This approach forms a basis to study the virus life cycle and virus-host interactions at the ultrastructural level.

Metal-Tagging Transmission Electron Microscopy and Immunogold Labeling on Tokuyasu Cryosections to Image Influenza A Virus Ribonucleoprotein Transport and Packaging.

Transmission electron microscopy (TEM) has been instrumental for studying viral infections. In particular, methods for labeling macromolecules at the ultrastructural level, by integrating biochemistry, molecular biology, and morphology, have allowed to study the functions of viral macromolecular complexes within the cellular context. Here, we describe a strategy for imaging influenza virus ribonucleoproteins in infected cells with two complementary labeling methods, metal-tagging transmission electron microscopy or METTEM, a highly sensitive technique based on the use of a metal-binding protein as a clonable tag, and immunogold labeling on thawed cryosections, a very specific labeling method that allows to study the distribution of different proteins simultaneously. The combination of both labeling methods offers new possibilities for TEM analysis of viral components in cells.

Selection of influenza virus resistant to the novel camphor-based antiviral camphecene results in loss of pathogenicity.

Due to the ability of influenza virus to develop drug resistance, the search for novel antivirals is an important goal of medical science and health care systems. We assessed the ability of the influenza virus to develop resistance to the hemagglutinin inhibitor camphecene and characterized laboratory-selected resistant strains. We showed by electron microscopy that camphecene decreases the number of virions fusing their envelopes with endosomal membranes. A 160-fold decrease in virus susceptibility was observed after six passages in cells. This was associated with the emergence of a V458L mutation in the HA2 subunit of HA and with a decrease in viral pathogenicity. Molecular modeling predicts that this substitution results in a more stable HA molecule compared to wild-type HA; and an altered camphecene-binding site. Therefore, despite the relatively rapid development of resistance, camphecene remains promising as a potential antiviral due to the low pathogenicity of resistant viruses that may arise.

An Improved Inactivated Influenza Vaccine with Enhanced Cross Protection.

Current inactivated influenza vaccines are strain-specific and poorly effective against variant or mismatched viruses. They are standardized based on their hemagglutinin (HA) or ability to induce strain-specific hemagglutination inhibition (HAI) antibodies. The HA is known to undergo major conformational changes when exposed to the low pH environment of endosomes (pH 5.0 and 37°C), which are required for membrane fusion during virus cell entry. In an effort to improve these vaccines, influenza antigens treated under various low pH conditions were evaluated for increased cross-reactive antibody response and cross protection. It was found that a full range of structural and antigenic changes in HA could be induced by varying low pH treatment conditions from the mild (low pH at ≤25°C) to the strong (low pH at ≥37°C) as determined by analysis of potency, HA morphology, protease sensitivity, and reactivity with an anti-HA2 domain (CD) antibody. Inactivated antigens of both H1N1 and H3N2 strains treated at mild low pH conditions (0-25°C) exhibited only moderate HA structural and antigenic changes and markedly increased antibody response against HA2, the highly conserved part of HA, and cross protection against heterologous challenge in mice by up to 30% in survival. By contrast, antigen treated with low pH at 37°C showed more extensive structural and antigenic changes, and induced much less of an increase in antibody response against HA2, but a greater increase with response against HA1, and did not provide any increased cross protection. These results suggest that the increased response against HA2 obtained with the mild low pH treatment is associated with the increased cross protection. These antigens treated at the mild low pH conditions remained capable of inducing a high level of strain-specific HAI antibodies. Thus, they could readily be formulated as an inactivated influenza vaccine which not only provides the same strain-specific protection but also an increased cross protection against heterologous viruses. Such a vaccine could be particularly beneficial in cases of vaccine mismatch.

Influenza C and D Viruses Package Eight Organized Ribonucleoprotein Complexes.

Influenza A and B viruses have eight-segmented, single-stranded, negative-sense RNA genomes, whereas influenza C and D viruses have seven-segmented genomes. Each genomic RNA segment exists in the form of a ribonucleoprotein complex (RNP) in association with nucleoproteins and an RNA-dependent RNA polymerase in virions. Influenza D virus was recently isolated from swine and cattle, but its morphology is not fully studied. Here, we examined the morphological characteristics of D/bovine/Yamagata/10710/2016 (D/Yamagata) and C/Ann Arbor/50 (C/AA), focusing on RNPs packaged within the virions. By scanning transmission electron microscopic tomography, we found that more than 70% of D/Yamagata and C/AA virions packaged eight RNPs arranged in the "1+7" pattern as observed in influenza A and B viruses, even though type C and D virus genomes are segmented into only seven segments. These results imply that influenza viruses generally package eight RNPs arranged in the "1+7" pattern regardless of the number of RNA segments in their genome.IMPORTANCE The genomes of influenza A and B viruses are segmented into eight segments of negative-sense RNA, and those of influenza C and D viruses are segmented into seven segments. For progeny virions to be infectious, each virion needs to package all of their genomic segments. Several studies support the conclusion that influenza A and B viruses selectively package eight distinct genomic RNA segments; however, the packaging of influenza C and D viruses, which possess seven segmented genomes, is less understood. By using electron microscopy, we showed that influenza C and D viruses package eight RNA segments just as influenza A and B viruses do. These results suggest that influenza viruses prefer to package eight RNA segments within virions independent of the number of genome segments.

Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR.

The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon-fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid 31P spectra show an isotropic peak indicative of high membrane curvature. This M2-cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol's effects on membrane protein function.

Biophysical characterization of influenza A virions.

Antigenic drift of the influenza A virus requires that vaccine production is targeted to the strains circulating each year. Live-attenuated influenza A vaccine manufacturing is used to produce intact virions with the surface antigens of the circulating strains. Influenza A typically contains a large percentage (>90%) of non-infective virions. The ribonucleoprotein (RNP) content, virion structure, and aggregation are factors that are thought to have an impact on infectivity. However, these factors are difficult to study because of the intrinsic variability in virion size, shape and overall structural integrity. Negative stain TEM for total particle counts and cryoTEM for detailed size/structural analysis are established benchmark techniques for virus characterization. Other methods may be valuable for certain sample types or circumstances. The aim of this work is to establish a benchmark comparison between orthogonal biophysical techniques for particle counts, population size distribution, structural integrity, and aggregate levels. NTA and FFF-MALS rapidly provided total counts, size distribution, and aggregate/elongated virion content. CryoTEM with size analysis and fraction counting yielded detailed information about the pleomorphism of the sample. The structural integrity of virions was inferred from multi-signal AUC-SV and CryoTEM. The current work provides a comparative assessment and a baseline for the selection of biophysical tools for the determination of particle counts, aggregation and pleomorphic characteristics of influenza A virus.

Visualization of early influenza A virus trafficking in human dendritic cells using STED microscopy.

Influenza A viruses (IAV) primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs). Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED) microscopy, we visualized input IAV nucleoprotein (NP), early and late endosomal compartments (EEA1 and LAMP1 respectively), and HLA-DR (DC membrane/cytosol) by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP+ signals were also EEA1+. Peak association with late endosomes occurred at 15 min when 60% of NP+ signals were LAMP1+. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells.IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them.

Cryomicroscopy provides structural snapshots of influenza virus membrane fusion.

The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we used electron cryomicroscopy and cryotomography to image the fusion of influenza virus with target membranes at low pH. We visualized structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultrastructural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane-fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy.

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion.

UNLABELLED: 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. IMPORTANCE: Enveloped 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.

Cryo-electron Microscopy Structures of Chimeric Hemagglutinin Displayed on a Universal Influenza Vaccine Candidate.

UNLABELLED: Influenza viruses expressing chimeric hemagglutinins (HAs) are important tools in the quest for a universal vaccine. Using cryo-electron tomography, we have determined the structures of a chimeric HA variant that comprises an H1 stalk and an H5 globular head domain (cH5/1 HA) in native and antibody-bound states. We show that cH5/1 HA is structurally different from native HA, displaying a 60° rotation between the stalk and head groups, leading to a novel and unexpected "open" arrangement of HA trimers. cH5/1N1 viruses also display higher glycoprotein density than pH1N1 or H5N1 viruses, but despite these differences, antibodies that target either the stalk or head domains of hemagglutinins still bind to cH5/1 HA with the same consequences as those observed with native H1 or H5 HA. Our results show that a large range of structural plasticity can be tolerated in the chimeric spike scaffold without disrupting structural and geometric aspects of antibody binding. IMPORTANCE: Chimeric hemagglutinin proteins are set to undergo human clinical trials as a universal influenza vaccine candidate, yet no structural information for these proteins is available. Using cryo-electron tomography, we report the first three-dimensional (3D) visualization of chimeric hemagglutinin proteins displayed on the surface of the influenza virus. We show that, unexpectedly, the chimeric hemagglutinin structure differs from those of naturally occurring hemagglutinins by displaying a more open head domain and a dramatically twisted head/stalk arrangement. Despite this unusual spatial relationship between head and stalk regions, virus preparations expressing the chimeric hemagglutinin are fully infectious and display a high glycoprotein density, which likely helps induction of a broadly protective immune response.

Protein-lipid interactions critical to replication of the influenza A virus.

Influenza A virus (IAV) assembles on the plasma membrane where viral proteins localize to form a bud encompassing the viral genome, which ultimately pinches off to give rise to newly formed infectious virions. Upon entry, the virus faces the opposite task-fusion with the endosomal membrane and disassembly to deliver the viral genome to the cytoplasm. There are at least four influenza proteins-hemagglutinin (HA), neuraminidase (NA), matrix 1 protein (M1), and the M2 ion channel-that are known to directly interact with the cellular membrane and modify membrane curvature in order to both assemble and disassemble membrane-enveloped virions. Here, we summarize and discuss current knowledge of the interactions of lipids and membrane proteins involved in the IAV replication cycle.