S acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine

Abstract

S acylation of cysteines located in the transmembrane and/or cytoplasmic region of influenza virus hemagglutinins (HA) contributes to the membrane fusion and assembly of virions. Our results from using mass spectrometry (MS) show that influenza B virus HA possessing two cytoplasmic cysteines contains palmitate, whereas HA-esterase-fusion glycoprotein of influenza C virus having one transmembrane cysteine is stearoylated. HAs of influenza A virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate. MS analysis of recombinant viruses with deletions of individual cysteines, as well as tandem-MS sequencing, revealed the surprising result that stearate is exclusively attached to the cysteine positioned in the transmembrane region of HA.

FIG. 1.

Results of MALDI-TOF MS analysis of fatty acid species attached to influenza B virus HA and influenza C virus HEF. (A) Sequences of the C terminus of HA from B/Hong Kong/8/73 (Flu B) and HEF from C/Johannesburg/1/66 (Flu C) indicating sites of cleavage by bromelain (arrows) and the TMR (underlined). Bromelain preferentially cleaves HEF after aspartic acid 615 (large arrow); in additional experiments, cleavage after glutamine 621 and serine 622 (small arrows) was also noticed. (B and C) Results of MALDI-TOF MS analysis. Mass spectra of indicated peptides plus palmitate and/or stearate obtained in reflector mode with pointed average masses are represented. (B) HA from influenza B virus. Additional pair of peaks were identified which matched to triply palmitoylated (m/z 5629.7; two asterisks) and doubly palmitoylated/monostearoylated (m/z 5656.7; one asterisk) peptides, which may indicate substoichiometric O-acylation of a serine located at the boundary between the TMR and CT or in the CT. (C) HEF from influenza C virus. Pal, palmitate; Str, stearate; a.u., arbitrary unit. Italic position numbers mark the numbers of acylated cysteine residues, and nonitalic position numbers mark the starting and ending numbers of the isolated peptides.

FIG. 2.

Results of MALDI-TOF MS analysis of fatty acid species attached to influenza A virus HA (FPV/Rostock/34, H7N1) and to HA mutants from recombinant virus with replacement of individual acylation sites. (A) Sequence of the C terminus of wild-type (wt) HA indicating the two sites of cleavage by bromelain (arrows), the TMR (underlined), and nomenclature of acylation mutants Ac1, Ac2, and Ac3 generated by exchanging cysteines for serines at positions 551, 559, and 562, respectively (19). (B) Results of MALDI-TOF MS analysis. Mass spectra of indicated peptides plus palmitate and/or stearate obtained in reflector mode with pointed average masses are represented. The m/z value of a parent ion subjected to MS-MS fragmentation analysis (see C) is boxed. Bromelain cleaves this HA at two distinct sites, preferentially after lysine 519 (large arrows) but also after serine 521 (small arrows). Thus, two different peptides were observed, a major peptide spanning amino acid 520 to the end of HA at amino acid 563 but also a smaller peptide starting at amino acid 522. The peptides generated from the cysteine mutants of HA have lower m/z values due to the exchange of serine for cysteine and the concomitant lack of one acyl chain. Italic position numbers mark the numbers of acylated cysteine residues, and nonitalic position numbers mark the starting and ending numbers of the isolated peptides. (C) Results of MS-MS sequencing of the FPV/Rostock/34 (wild type) HA C-terminal anchoring peptide. Shown is a part of the MALDI-TOF-TOF mass spectrum of the parent ion from peptide [HA 520-563 + 2Pal/1Str] (m/z 5564.0; boxed in panel B). A series of detected average masses and corresponding C-terminal daughter y ions are designated. Their matching to the amino acid sequence is depicted above the mass spectrum. Pal, palmitate; Str, stearate; a.u., arbitrary unit.

FIG. 3.

Summary of the MS data. The putative TMRs (embedded in lipids) and CTs of HA of influenza A and B virus (Flu A and B) and HEF of influenza C virus (Flu C), with palmitate (Pal) and stearate (Stear) attached to individual cysteine residues, are shown.