S Acylation of the Hemagglutinin of Influenza Viruses: Mass Spectrometry Reveals Site-Specific Attachment of Stearic Acid to a Transmembrane Cysteine

Kordyukova, Larisa V.; Serebryakova, Marina V.; Baratova, Ludmila A.; Veit, Michael

doi:10.1128/jvi.00704-08

Cited by 95 publications

(80 citation statements)

References 20 publications

(29 reference statements)

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“…Two cysteines present within the cytoplasmic tail (CT) sequence and one cysteine in the transmembrane domain (TM) sequence of HA are S acylated by two palmitates (CT cysteines) and one stearic or palmitic acid (TM cysteine), respectively (34,35). The role of HA acylation has been studied extensively since these cysteines are conserved at their respective positions to a greater extent than other residues among all otherwise highly variable HA subtypes.…”

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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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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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“…3B). To prove the specificity of HA-M2 clustering, we used an HA mutant in which the raft- targeting features-S acylation at three cysteine residues (12) and the sequence VIL in the exoplasmic half of the transmembrane domain (19,26)-had been replaced by serines and alanines, respectively (6). A similar mutant of HA has been shown by electron microscopy to be uniformly distributed on the plasma membrane, in contrast to the patched appearance of wild-type HA (26).…”

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Intrinsic Cytoskeleton-Dependent Clustering of Influenza Virus M2 Protein with Hemagglutinin Assessed by FLIM-FRET

Thaa

Herrmann

Veit

2010

J Virol

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The hemagglutinin (HA) of influenza virus organizes the virus bud zone, a domain of the plasma membrane enriched in raft lipids. Using fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET), a technique that detects close colocalization of fluorescent proteins in transfected cells, we show that the viral proton channel M2 clusters with HA but not with a marker for inner leaflet rafts. The FRET signal between M2 and HA depends on the raft-targeting signals in HA and on an intact actin cytoskeleton. We conclude that M2 contains an intrinsic signal that targets the protein to the viral bud zone, which is organized by raft-associated HA and by cortical actin.

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“…Although the function of heterogenous S-acylation of proteins with different fatty acids was not known, it was shown that the length and composition of the transmembrane seuqences as well as the cytoplasmic sequences can influence the choice of fatty acids for acylation [64][65][66]. Identification and quantification of acyl moieties are essential for understanding the detailed mechanisms of protein palmitoylation.…”

Section: Identification and Quantification Of Acyl Moietiesmentioning

confidence: 99%

Chemical and genetic probes for analysis of protein palmitoylation

Dong

Xia

et al. 2011

Journal of Chromatography B

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S Acylation of the Hemagglutinin of Influenza Viruses: Mass Spectrometry Reveals Site-Specific Attachment of Stearic Acid to a Transmembrane Cysteine

Cited by 95 publications

References 20 publications

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

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

Intrinsic Cytoskeleton-Dependent Clustering of Influenza Virus M2 Protein with Hemagglutinin Assessed by FLIM-FRET

Chemical and genetic probes for analysis of protein palmitoylation

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