Influenza Virus Budding Research Articles

Imaging Sphingomyelin- and Cholesterol-Enriched Domains in the Plasma Membrane Using a Novel Probe and Super-Resolution Microscopy.

In this chapter, we show the visualization of lipid domains using a specific lipid-binding protein and super-resolution microscopy. Lipid rafts are plasma membrane domains enriched in both sphingolipids and sterols that play key roles in various physiological events. We identified a novel protein that specifically binds to a complex of sphingomyelin (SM) and cholesterol (Chol). The isolated protein, nakanori, labels the SM/Chol complex at the outer leaflet of the plasma membrane in mammalian cells. Structured illumination microscopic images suggested that the influenza virus buds from the edges of the SM/Chol domains in MDCK cells. Furthermore, a photoactivated localization microscopy analysis indicated that the SM/Chol complex forms domains in the outer leaflet, just above the phosphatidylinositol 4,5-bisphosphate domains in the inner leaflet. These observations provide significant insight into the structure and function of lipid rafts.

Cholesterol Alters the Orientation and Activity of the Influenza Virus M2 Amphipathic Helix in the Membrane.

The influenza virus M2 amphipathic helix (M2AH) alters membrane curvature in a cholesterol-dependent manner, mediating viral membrane scission during influenza virus budding. Here, we have investigated the biophysical effects of cholesterol on the ability of an M2AH peptide to manipulate membrane properties. We see that the ability of the M2AH to interact with membranes and form an α-helix is independent of membrane cholesterol concentration; however, cholesterol affects the angle of the M2AH peptide within the membrane. This change in membrane orientation affects the ability of the M2AH to alter lipid order. In low-cholesterol membranes, the M2AH is inserted near the level of the lipid head groups, increasing lipid order, which may contribute to generation of the membrane curvature. As the cholesterol content increases, the M2AH insertion becomes flatter and slightly deeper in the membrane below the lipid headgroups, where the polar face can continue to interact with the headgroups while the hydrophobic face binds cholesterol. This changed orientation minimizes lipid packing defects and lipid order changes, likely reducing the generation of membrane curvature. Thus, cholesterol regulates M2 membrane scission by precisely modulating M2AH positioning within the membrane. This has implications for the understanding of many of amphipathic-helix-driven cellular budding processes that occur in specific lipid environments.

Direct Imaging of the Cholesterol and Sphingolipid Abundance at the Site of Influenza Virus Assembly with High‐Resolution SIMS

The influenza virus is hypothesized to assemble and bud from plasma membrane domains that are enriched with cholesterol and sphingolipids. This hypothesis is consistent with the reported enrichment of the influenza viral envelope with cholesterol and sphingolipids, the reduction in influenza virus protein clustering in the plasma membrane induced by cholesterol depletion, and the sensitivity of influenza virus formation to cellular cholesterol levels. Nonetheless, the levels of cholesterol and sphingolipids in the plasma membrane at the site of influenza virus budding have never been directly measured. The main challenge has been a lack of analytical methods with the sensitivity and lateral resolution that is required to measure the relative cholesterol and sphingolipid levels in the plasma membrane on the 100‐nm‐length scales that are relevant to influenza virus budding. We recently overcame this challenge by using a secondary ion mass spectrometry (SIMS) technique with high spatial resolution and sensitivity to image the distributions of stable isotope‐labeled cholesterol, sphingolipids, and immunolabeled influenza hemagglutinin in the plasma membranes of virus‐infected MDCK cells. Our results show that the plasma membranes of both influenza‐infected and uninfected MDCK cells contained regions that were enriched with sphingolipids, but cholesterol appeared to be uniformly distributed within the membrane. Examination of the influenza‐infected MDCK cells revealed the hemagglutinin‐rich sites of influenza virus assembly in the plasma membrane were not enriched with cholesterol, and seldom enriched with sphingolipids. These direct imaging results demonstrate that the influenza virus does not assemble within plasma membrane domains that are enriched with cholesterol and sphingolipids.Support or Funding InformationThis research was supported by the U.S. National Science Foundation under CHE 15‐08662.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influenza A M2 Channel Clustering at High Protein/Lipid Ratios: Viral Budding Implications

Protein dynamics in crowded environments is important for understanding protein functions in vivo and is especially relevant for membrane proteins because of the roles of protein-protein interactions in membrane protein functions and their regulation. Here, using solid-state NMR spectroscopy in combination with coarse-grained molecular dynamics simulations, we report that the rotational correlation time for the transmembrane domain of the influenza A M2 proton channel in lipid bilayers increases dramatically at an elevated protein/lipid ratio. This increase is attributable to persistent protein-protein interactions, thus revealing for the first time, to the best of our knowledge, extensive cluster formation of the M2 tetrameric channel. Such clustering appears to have direct biological relevance during budding of the nascent influenza virus, which does not use the endosomal sorting complexes required for transport machinery. Indeed, initial coarse-grained molecular dynamics simulations of the longer M2 construct known as the conductance domain suggest clustering-induced membrane curvature formation.

Mutations in Influenza A Virus Neuraminidase and Hemagglutinin Confer Resistance against a Broadly Neutralizing Hemagglutinin Stem Antibody.

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

Probing the lipid composition at the site of influenza virus assembly and budding with high‐resolution SIMS

The formation of infectious influenza virions is sensitive to the cholesterol levels in the host cell. The findings that the influenza viral envelope is enriched with cholesterol and sphingolipids and cholesterol depletion alters the clustering of influenza virus proteins in the plasma membrane, which is detrimental to virus assembly, suggests that the influenza virus assembles and bud from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane. Although this hypothesis has been around for decades, whether the region of the plasma membrane from which the influenza virus buds is enriched with cholesterol and sphingolipids is still unknown. To answer this question, we have used high‐resolution secondary ion mass spectrometry (SIMS) to image the distributions of stable isotope‐labeled cholesterol and immunolabeled influenza virus protein in the plasma membrane during virus budding with ~100 nm lateral resolution. This work involved metabolically incorporating distinct stable isotopes into the cholesterol and lipids of interest in living MDCK cells. After infecting these MDCK cells with influenza virus for 24 h, we immunolabeled the influenza envelope protein, hemagglutinin. Then we imaged the isotope‐labeled cholesterol, lipids, and immunolabeled influenza hemagglutinin on the surfaces of the virus infected cells with ~100 nm lateral resolution. The resulting images clearly show that the influenza virus does bud from cholesterol‐enriched domains in the membranes of MDCK cells.Support or Funding InformationThis research was supported by the U.S. National Science Foundation under CHE 15‐08662.

Is the Site of Influenza Virus Assembly and Budding Enriched with Cholesterol and Sphingolipids?

The observation that the influenza viral envelope is enriched with cholesterol and sphingolipids contributed to the hypothesis that the influenza virus assembles and bud from cholesterol- and sphingolipid-enriched domains in the plasma membrane. The formation of infectious influenza virions and the clustering of influenza virus proteins in the plasma membrane during virus assembly are reportedly reduced by depletion of cellular cholesterol. Though this cholesterol sensitivity certainly suggests that cholesterol plays a role in influenza virus infection, whether the plasma membrane domain from which the influenza virus buds is enriched with cholesterol and sphingolipids has not been decisively determined. We have addressed this question by using high-resolution secondary ion mass spectrometry (SIMS) to image the distributions of stable isotope-labeled lipids and immunolabeled influenza virus proteins in the plasma membranes of virus-infected cells with ∼100 nm lateral resolution. In these experiments, we metabolically incorporated the distinct stable isotopes 15N and 18O into the sphingolipids and cholesterol, respectively, in living MDCK cells. We infected these MDCK cells with an H3N2 strain of the influenza virus for 24 h, and immunolabeled the influenza envelope protein, hemagglutinin, to permit the detection of budding virus. Finally, we imaged the metabolically incorporated 15N-sphingolipids and 18O-cholesterol and immunolabeled influenza hemagglutinin on the surfaces of the influenza-infected MDCK cells, and assessed co-localization between these components. The results of these experiments and there implications will be presented.

Filamentous Influenza Viruses.

Influenza A virus is a pathogen of global medical importance causing significant health and socio-economic costs every year. Influenza virus is an unusual pathogen in that it is pleomorphic, capable of forming virions ranging in shape from spherical to filamentous. Despite decades of research on the influenza virus, much remains unknown about the formation of filamentous influenza viruses and their role in the viral replication cycle. Here, we discuss what is known about influenza virus assembly and budding, focusing on the viral and host factors that are involved in the determination of viral morphology. Whilst the biological function of the filamentous morphology remains unknown, recent results suggest a role in facilitating viral spread in vivo. We discuss these results and speculate on the consequences of viral morphology during influenza virus infection of the human respiratory tract.

Is the site of influenza virus assembly and budding enriched with cholesterol and sphingolipids?

The observation that the influenza viral envelope is enriched with cholesterol and sphingolipids contributed to the hypothesis that the influenza virus assembles and bud from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane. Protein imaging experiments have demonstrated that the clustering of influenza virus proteins in the plasma membrane is sensitive to depletion of cellular cholesterol. However, the precise distributions of cholesterol and sphingolipids in relation to the site of influenza budding remains speculative due to an inability to directly image lipids without the use of potentially perturbing labels. We have used high‐resolution secondary ion mass spectrometry (SIMS), which is performed on a Cameca NanoSIMS 50, to image the distributions of stable isotope‐labeled lipids in the plasma membranes of intact cells with ~100 nm lateral resolution. Here we report using this approach to directly test the long‐standing hypothesis that the influenza virus buds from plasma membrane domains that are enriched with cholesterol and sphingolipids. We imaged metabolically incorporated 15N‐sphingolipids and 18O‐cholesterol and immunolabeled influenza virus envelope proteins on the surfaces of influenza‐infected MDCK cells with high‐resolution SIMS. The sites of virus assembly and budding in the plasma membrane were identified as the regions on the MDCK cell that were enriched with the immunolabeled influenza virus envelope protein, hemagglutinin. High‐resolution SIMS imaging revealed the sites of influenza virus assembly and budding were not enriched with cholesterol and sphingolipids. Although sphingolipid domains were detected in the plasma membranes of these MDCK cells, they had little co‐localization with the hemagglutinin‐rich membrane patches. These findings argue against the hypothesis that the influenza virus buds from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane.Support or Funding InformationThis research was supported by the U.S. National Science Foundation under CHE 15‐08662.

Does the Influenza Virus Bud from Cholesterol- and Sphingolipid-Enriched Plasma membrane domains?

The segregation of different lipid and protein species into compositionally distinct membrane domains is undoubtedly essential for cellular function. Advances in protein imaging have vastly improved our knowledge of the distributions and stimuli-dependent clustering of various protein species within cellular membranes. However, much less is known about the distributions of distinct lipid species, and especially their proximity to proteins of interest, within the plasma membrane. We have shown that high-resolution secondary ion mass spectrometry (SIMS) performed on a Cameca NanoSIMS 50 enables imaging the distributions of stable isotope-labeled lipids in model and cellular membranes with ∼100 nm lateral resolution. Our previous NanoSIMS imaging of metabolically incorporated 15N-sphingolipids and 18O-cholesterol on the surfaces of fibroblast cells revealed the plasma membrane contains cytoskeleton-dependent sphingolipid domains that are not enriched with cholesterol. Unexpectedly, cholesterol was found to be uniformly distributed within the plasma membrane. Here we use this approach to test the long-standing hypothesis that the influenza virus buds from plasma membrane domains that are enriched with cholesterol and sphingolipids. Specifically, we have imaged metabolically incorporated 15N-sphingolipids and 18O-cholesterol and immunolabeled viral envelope proteins on the surfaces of influenza-infected MDCK cells, and assessed co-localization between these components. The results of these experiments and there implications will be presented.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Alterations of membrane curvature during influenza virus budding.

Influenza A virus belongs to the Orthomyxoviridae family. It is an enveloped virus that contains a segmented and negative-sense RNA genome. Influenza A viruses cause annual epidemics and occasional major pandemics, are a major cause of morbidity and mortality worldwide, and have a significant financial impact on society. Assembly and budding of new viral particles are a complex and multi-step process involving several host and viral factors. Influenza viruses use lipid raft domains in the apical plasma membrane of polarized epithelial cells as sites of budding. Two viral glycoproteins, haemagglutinin and neuraminidase, concentrate in lipid rafts, causing alterations in membrane curvature and initiation of the budding process. Matrix protein 1 (M1), which forms the inner structure of the virion, is then recruited to the site followed by incorporation of the viral ribonucleoproteins and matrix protein 2 (M2). M1 can alter membrane curvature and progress budding, whereas lipid raft-associated M2 stabilizes the site of budding, allowing for proper assembly of the virion. In the later stages of budding, M2 is localized to the neck of the budding virion at the lipid phase boundary, where it causes negative membrane curvature, leading to scission and virion release.

Influenza A Matrix Protein M1 Multimerizes upon Binding to Lipid Membranes

The matrix protein M1 plays a pivotal role in the budding of influenza virus from the plasma membrane (PM) of infected cells. This protein interacts with viral genetic material and envelope proteins while binding to the inner leaflet of the PM. Its oligomerization is therefore closely connected to the assembly of viral components and the formation of new virions. Of interest, the molecular details of M1 interaction with lipids and other viral proteins are far from being understood, and it remains to be determined whether the multimerization of M1 is affected by its binding to the PM and interaction with its components. To clarify the connection between M1 oligomerization and binding to lipid membranes, we applied a combination of several quantitative microscopy approaches. First, we used number and brightness (N&B) microscopy to characterize protein multimerization upon interaction with the PM of living cells. Second, we used controlled biophysical models of the PM (i.e., supported bilayers) to delve into the details of M1-lipid and M1-M1 interactions by employing a combination of raster image correlation spectroscopy (RICS), fluorescence correlation spectroscopy (FCS), and atomic force microscopy (AFM). Our results show that M1 oligomer formation is strongly enhanced by membrane binding and does not necessarily require the presence of other viral proteins. Furthermore, we propose a specific model to explain M1 binding to the lipid bilayer and the formation of multimers.

Type I Interferon Imposes a TSG101/ISG15 Checkpoint at the Golgi for Glycoprotein Trafficking during Influenza Virus Infection

Several enveloped viruses exploit host pathways, such as the cellular endosomal sorting complex required for transport (ESCRT) machinery, for their assembly and release. The influenza A virus (IAV) matrix protein binds to the ESCRT-I complex, although the involvement of early ESCRT proteins such as Tsg101 in IAV trafficking remain to be established. We find that Tsg101 can facilitate IAV trafficking, but this is effectively restricted by the interferon (IFN)-stimulated protein ISG15. Cytosol from type I IFN-treated cells abolished IAV hemagglutinin (HA) transport to the cell surface in infected semi-intact cells. This inhibition required Tsg101 and could be relieved with deISGylases. Tsg101 is itself ISGylated in IFN-treated cells. Upon infection, intact Tsg101-deficient cells obtained by CRISPR-Cas9 genome editing were defective in the surface display of HA and for infectious virion release. These data support the IFN-induced generation of a Tsg101- and ISG15-dependent checkpoint in the secretory pathway that compromises influenza virus release.

Influenza Virus A M2 Protein Generates Negative Gaussian Membrane Curvature Necessary for Budding and Scission

The M2 protein is a multifunctional protein, which plays several roles in the replication cycle of the influenza A virus. Here we focus on its ability to promote budding of the mature virus from the cell surface. Using high-resolution small-angle X-ray scattering we show that M2 can restructure lipid membranes into bicontinuous cubic phases which are rich in negative Gaussian curvature (NGC). The active generation of negative Gaussian membrane curvature by M2 is essential to influenza virus budding. M2 has been observed to colocalize with the region of high NGC at the neck of a bud. The structural requirements for scission are even more stringent than those for budding, as the neck must be considerably smaller than the virus during 'pinch off'. Consistent with this, the amount of NGC in the induced cubic phases suggests that M2 proteins can generate high curvatures comparable to those on a neck with size 10× smaller than a spherical influenza virus. Similar experiments on variant proteins containing different M2 domains show that the cytoplasmic amphipathic helix is necessary and sufficient for NGC generation. Mutations to the helix which reduce its amphiphilicity and are known to diminish budding attenuated NGC generation. An M2 construct comprising the membrane interactive domains, the transmembrane helix and the cytoplasmic helix, displayed enhanced ability to generate NGC, suggesting that other domains cooperatively promote membrane curvature. These studies establish the importance of M2-induced NGC during budding and suggest that antagonizing this curvature is a viable anti-influenza strategy.

Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells

The epithelium of conducting airways represents the main target for influenza virus in mammals. However, the peculiarities of virus interactions with differentiated airway epithelial cells remain largely unknown. Here, influenza virus budding was studied in differentiated cultures of human tracheobronchial epithelial cells using transmission electron microscopy. Budding of spherical and filamentous virions was observed on the apical surfaces of cells with no association with cilia and secretory granules. Quantitative analysis of the distribution of viral buds on the cell surface indicated that the tips of the microvilli represented a prominent site of influenza virus budding in the human airway epithelium. As the microvilli of differentiated cells are involved in many fundamental cell functions, these data will prompt further studies on the biological significance of microvilli-associated budding for virus replication, transmission and pathogenicity.

Illustrating the machinery of life: Viruses

Data from electron microscopy, X-ray crystallography, and biophysical analysis are used to create illustrations of viruses in their cellular context. This report describes the scientific data and artistic methods used to create three illustrations: a depiction of the poliovirus lifecycle, budding of influenza virus from a cell surface, and a mature HIV particle in blood serum.

F1Fo-ATPase, F-type proton-translocating ATPase, at the plasma membrane is critical for efficient influenza virus budding

The identification of host factors involved in virus replication is important to understand virus life cycles better. Accordingly, we sought host factors that interact with the influenza viral nonstructural protein 2 by using coimmunoprecipitation followed by mass spectrometry. Among proteins associating with nonstructural protein 2, we focused on the β subunit of the F1Fo-ATPase, which received a high probability score in our mass spectrometry analysis. The siRNA-mediated down-regulation of the β subunit of the F1Fo-ATPase reduced influenza virion formation and virus growth in cell culture. We further found that efficient influenza virion formation requires the ATPase activity of F1Fo-ATPase and that plasma membrane-associated, but not mitochondrial, F1Fo-ATPase is important for influenza virion formation and budding. Hence, our data identify plasma membrane-associated F1Fo-ATPase as a critical host factor for efficient influenza virus replication.

Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane

The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin-Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. The virus membrane exhibited a further enrichment of SPs and cholesterol compared with the donor membrane at the expense of phosphatidylcholines. Our data are consistent with and extend existing models of membrane raft-based biogenesis of the apical membrane and IFV envelope.

Hydrophobic Matching Between Phospholipid Bilayers and Helical Peptides Affects the Memrbane Affinity of Sterols

The lateral organization of cell membranes is important for several cellular functions including signaling and membrane trafficking. In addition pathogen uptake and influenza virus budding is associated with sphingolipid and cholesterol enriched lateral membrane domains. The lateral structure of membranes is in part controlled by lipid self-organization, but it is becoming increasingly clear that also membrane proteins can play an active role in the maintenance of the lateral structure. Cholesterol is thought to have an important role in lateral organization of eukaryotic cell membranes. As cholesterol also has been implicated to take part in the sorting of cellular transmembrane proteins it is a good starting point to determine how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of different transmembrane peptides, mimicking the transmembrane helices of proteins. By measuring the equilibrium partitioning of the fluorescent cholesterol analogue cholestatrienol (CTL) between large unilamellar vesicles and methyl-beta-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. The results showed that increasing positive mismatch led to higher affinity of the sterol for the bilayers. This suggests that hydrophobic matching could affect the lateral organization of cholesterol in cell membranes and have an important role especially in membrane trafficking.