title: Site-specific S-acylation of influenza virus hemagglutinin: the location of the acylation site relative to the membrane border is the decisive factor for attachment of stearate.
abstract: S-Acylation of hemagglutinin (HA), the main glycoprotein of influenza viruses, is an essential modification required for virus replication. Using mass spectrometry, we have previously demonstrated specific attachment of acyl chains to individual acylation sites. Whereas the two cysteines in the cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to a cysteine positioned at the end of the transmembrane region (TMR). Here we analyzed recombinant viruses containing HA with exchange of conserved amino acids adjacent to acylation sites or with a TMR cysteine shifted to a cytoplasmic location to identify the molecular signal that determines preferential attachment of stearate. We first developed a new protocol for sample preparation that requires less material and might thus also be suitable to analyze cellular proteins. We observed cell type-specific differences in the fatty acid pattern of HA: more stearate was attached if human viruses were grown in mammalian compared with avian cells. No underacylated peptides were detected in the mass spectra, and even mutations that prevented generation of infectious virus particles did not abolish acylation of expressed HA as demonstrated by metabolic labeling experiments with [(3)H]palmitate. Exchange of conserved amino acids in the vicinity of an acylation site had a moderate effect on the stearate content. In contrast, shifting the TMR cysteine to a cytoplasmic location virtually eliminated attachment of stearate. Thus, the location of an acylation site relative to the transmembrane span is the main signal for stearate attachment, but the sequence context and the cell type modulate the fatty acid pattern.
authors.affiliation: From the Institut für Virologie, Free University Berlin, 14163 Berlin, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Brett
authors.affiliation: A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia.
authors.forename: Larisa V
authors.initials: LV
authors.lastname: Kordyukova
authors.affiliation: A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Serebryakova
authors.affiliation: A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia, I. I. Mechnikov Research Institute of Vaccines and Sera, Russian Academy of Medical Sciences, 105064 Moscow, Russia, and.
authors.forename: Ramil R
authors.initials: RR
authors.lastname: Mintaev
authors.affiliation: A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia, Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia.
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Alexeevski
authors.affiliation: From the Institut für Virologie, Free University Berlin, 14163 Berlin, Germany, mveit@zedat.fu-berlin.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Veit
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism / Q000821:virology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000378:metabolism* / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D013228:Stearates / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D013228:Stearates
keywords: Fatty Acid; Glycoprotein; Hemagglutinin; Influenza Virus; Mass Spectrometry (MS); Palmitate; Protein Acylation; Protein Palmitoylation; Stearate; Transmembrane Domain