Protein–lipid interactions critical to replication of the influenza A virus

Chlanda, Petr; Zimmerberg, Joshua

doi:10.1002/1873-3468.12118

Cited by 39 publications

(35 citation statements)

References 108 publications

(157 reference statements)

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“…Our data from studies with model and cell membranes indicate that PS can form domains in the inner leaflet of the PM of noninfected cells. Furthermore, it is well established that M1 interacts with PS (5,6,8,53,54), although this was never observed directly in living cells. In order to monitor the localization of M1 in infected cells, we expressed fluorescent M1 (Card-M1) in HEK cells infected with the X-31 strain of IAV.…”

Section: Resultsmentioning

confidence: 99%

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

Bobone

Hilsch

Storm

et al. 2017

J Virol

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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.KEYWORDS influenza, assembly, confocal microscopy, fluorescence image analysis, lipid rafts, matrix protein, model membranes, phosphatidylserine, plasma membrane I nfluenza A virus (IAV) is a major burden to human health and remains a prominent cause of mortality worldwide (1, 2). Despite significant research efforts, detailed knowledge about the molecular mechanisms involved in IAV infection is still limited. A deeper understanding of the virus machinery and its interaction with the host is

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Section: Resultsmentioning

confidence: 99%

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

Bobone

Hilsch

Storm

et al. 2017

J Virol

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“…At least four influenza virus membrane-associated proteins, hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), and the matrix 2 (M2) ion channel, orchestrate the plasma membrane remodeling to assemble new virions (2). HA and NA, both serving as the main antigens, stud the virion surface.…”

mentioning

confidence: 99%

“…The resulting virus particles have pleomorphic geometry, with a predominance of spheroidal and filamentous shapes (8). Effects of glycoproteins on the viral envelope curvature have been reviewed recently (2). Spheroidal and filamentous virions are able to infect cells and enter via receptormediated clathrin-dependent endocytosis (14) and macropinocytosis (15), respectively.…”

mentioning

confidence: 99%

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

Chlanda

Mekhedov

Waters

et al. 2017

J Virol

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The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strainindependent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/ Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryoelectron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.KEYWORDS cryo-electron microscopy, electron microscopy, membrane fusion, palmitoylation, protein acylation

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“…HA organizes the viral assembly site, since it is not randomly distributed in the plasma membrane of transfected cells, but is present in (partly cholesterol-sensitive) clusters of various sizes as demonstrated by quantitative immunoelectron microscopy (6–8) and FPALM (fluorescence-photoactivation-localization microscopy) (9). The other integral membrane proteins of the virus, the neuraminidase (NA) and the ion channel M2 contain their own signals for targeting to the viral assembly site (10), where they are supposed to recruit the internal components of viral particles, the matrix protein M1 and the eight ribonucleoparticles (containing the viral genome segments complexed to the nucleoprotein (NP) and the three polymerase proteins PA, PB1 and PB2) into budding virions (11, 12).…”

Section: Introductionmentioning

confidence: 99%

Cholesterol binding to the transmembrane region of a group 2 HA of Influenza virus is essential for virus replication affecting both virus assembly and HA’s fusion activity

Hoefer

Thiele

et al. 2019

Preprint

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Hemagglutinin (HA) of Influenza virus is incorporated into cholesterol enriched, 1 nanodomains of the plasma membrane. Phylogenetic group 2 HAs contain the 2 conserved cholesterol consensus motif (CCM) YKLW in the transmembrane region. 3 We previously reported that mutations in the CCM retarded intracellular transport of 4 HA and decreased its nanodomain association. Here we analyzed whether cholesterol 5 interacts with the CCM. Incorporation of photocholesterol into HA was significantly 6 reduced if the whole CCM is replaced by alanine, both using immunoprecipitated HA 7 and when HA is embedded in the membrane. Next, we used reverse genetics to 8 investigate the significance of the CCM for virus replication. No virus was rescued if 9 the whole motif is exchanged (YKLW4A); single (LA) or double (YK2A and LW2A)

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Protein–lipid interactions critical to replication of the influenza A virus

Cited by 39 publications

References 108 publications

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

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

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

Cholesterol binding to the transmembrane region of a group 2 HA of Influenza virus is essential for virus replication affecting both virus assembly and HA’s fusion activity

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