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Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion.

ABSTRACT: Lipid raft microdomains are enriched in sphingomyelin and cholesterol and function as platforms for signal transduction and as the site of budding of several enveloped viruses, including influenza virus. The influenza virus hemagglutinin (HA) glycoprotein, which mediates both viral-cell attachment and membrane fusion, associates intrinsically with lipid rafts. Residues in the HA transmembrane (TM) domain are important for raft association as sequence substitutions in the HA TM domain ablate HA association with rafts (nonraft HA). Cells expressing either WT or nonraft HA cause complete fusion (lipid mixing and content mixing) over widely varying HA expression levels. However, the number of fusion events measured for nonraft HA mutant protein at all HA surface densities was reduced to approximately 55% of the events for WT HA protein. Mutant influenza viruses were generated that contain the nonraft HA TM domain alterations. Electron microscopy experiments showed that WT HA was distributed at the cell surface in clusters of 200-280 nm in diameter, whereas nonraft HA was distributed mostly randomly at the plasma membrane. Nonraft HA virus showed reduced budding, contained reduced amounts of HA protein, was greatly reduced in infectivity, and exhibited decreased virus-membrane fusion activity. Cholesterol depletion of virus did not affect the ability of virions to cause either virus-cell lipid mixing or virus-mediated hemolysis, a surrogate for content mixing. Taken together, the data suggest that HA clusters in rafts to provide a sufficient concentration of HA in budding virus to mediate efficient virus-cell fusion.

SUBMITTER: Takeda M

PROVIDER: S-EPMC299746 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion.

Takeda Makoto M Leser George P GP Russell Charles J CJ Lamb Robert A RA

Proceedings of the National Academy of Sciences of the United States of America 20031015 25

Lipid raft microdomains are enriched in sphingomyelin and cholesterol and function as platforms for signal transduction and as the site of budding of several enveloped viruses, including influenza virus. The influenza virus hemagglutinin (HA) glycoprotein, which mediates both viral-cell attachment and membrane fusion, associates intrinsically with lipid rafts. Residues in the HA transmembrane (TM) domain are important for raft association as sequence substitutions in the HA TM domain ablate HA a ...[more]

PMID: 14561897

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Project description:Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.

Dataset Information

Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion.

Publications

Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion.

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