title: Lipid raft-dependent targeting of the influenza A virus nucleoprotein to the apical plasma membrane.
abstract: Influenza virus acquires a lipid raft-containing envelope by budding from the apical surface of epithelial cells. Polarised budding involves specific sorting of the viral membrane proteins, but little is known about trafficking of the internal virion components. We show that during the later stages of virus infection, influenza nucleoprotein (NP) and polymerase (the protein components of genomic ribonucleoproteins) localised to apical but not lateral or basolateral membranes, even in cell types where haemagglutinin was found on all external membranes. Other cytosolic components of the virion either distributed throughout the cytoplasm (NEP/NS2) or did not localise solely to the apical plasma membrane in all cell types (M1). NP localised specifically to the apical surface even when expressed alone, indicating intrinsic targeting. A similar proportion of NP associated with membrane fractions in flotation assays from virus-infected and plasmid-transfected cells. Detergent-resistant flotation at 4 degrees C suggested that these membranes were lipid raft microdomains. Confirming this, cholesterol depletion rendered NP detergent-soluble and furthermore, resulted in its partial redistribution throughout the cell. We conclude that NP is independently targeted to the apical plasma membrane through a mechanism involving lipid rafts and propose that this helps determine the polarity of influenza virus budding.
authors.affiliation: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: Marlene
authors.initials: M
authors.lastname: Carrasco
authors.forename: Maria Joao
authors.initials: MJ
authors.lastname: Amorim
authors.forename: Paul
authors.initials: P
authors.lastname: Digard
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism*; D009976:Orthomyxoviridae Infections / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D002784:Cholesterol