Larisa V. Kordyukova

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.

Mass spectrometric sequencing and acylation character analysis of the C-terminal anchoring segment from Influenza A hemagglutinin

Influenza A virus hemagglutinin (HA) is a major envelope glycoprotein mediating viral and cell membrane fusion. HA is anchored in the viral envelope by a light HA2 chain containing one transmembrane domain and a cytoplasmic tail. Three cysteine residues in the C-terminal region, one in the transmembrane domain and two in the cytoplasmic tail, are highly conserved and potentially palmitoylated in all HA subtypes. The HA2 C-terminal anchoring segments were extracted to organic phase from the bromelain-digested viruses (subviral particles) of three strains: A/X-31 (H3 subtype), A/Puerto Rico/8/34 (H1 subtype) and A/FPV/Weybridge/34 (H7 subtype). Their primary structures were assessed by matrix-assisted laser desorption/ionization time-of-flight time-of-flight mass spectrometry (MALDI-ToF-ToF MS). Trypsin-type protease-cleaved peptides prevailed over bromelain-cleaved ones in the peptide mixtures. All of them included transmembrane domains. Several distinctive features of the C-terminal HA2 peptides acylation character were discovered by MALDI-ToF MS: 1) the peptides isolated from the viruses, which were digested by bromelain in the absence of β-mercaptoethanol, were predominantly triply acylated; 2) the peptides were acylated not only by palmitic, but also by stearic acid residues; 3) the palmitate/stearate ratio was different for the three strains studied; 4) the A/FPV/Weybridge/34 strain has a priority to stearate binding. This fatty acid residue was discovered at the first of three conservative cysteine residues located in the transmembrane domain. It was found that presence of thiol reagent during preparation of subviral particles led to the appearence of the C-terminal HA2 peptides acylated to different degrees. Triply, doubly, mono-and even unacylated peptides were detected. It was demonstrated that the thioester bond in the isolated acylpeptides was extremely sensitive to thiol reagents.

Palmitoylation of influenza virus proteins

Influenza viruses contain two palmitoylated (S-acylated) proteins: the major spike protein HA (haemagglutinin) and the proton-channel M2. The present review describes the fundamental biochemistry of palmitoylation of HA: the location of palmitoylation sites and the fatty acid species bound to HA. Finally, the functional consequences of palmitoylation of HA and M2 are discussed regarding association with membrane rafts, entry of viruses into target cells by HA-mediated membrane fusion as well as the release of newly assembled virus particles from infected cells.

Determination of concentration and aggregate size in influenza virus preparations from true UV absorption spectra

Light scattering is known to make a considerable contribution to ultraviolet absorption spectra of influenza virus (Flu) preparations. We applied extrapolation to analysis of this contribution. Ultraviolet spectra were recorded and true extinction coefficients (A1 cm, 2800.1%) were determined in suspensions of intact virions of Flu strain Puerto Rico/8/34 and subviral particles obtained by bromelain digestion of the same strain (1.26 ± 0.17 and 0.96 ± 0.11 OD, respectively). This allowed simple and rapid measurement of virus concentration. It was shown that UV spectra allowed efficient monitoring of virion aggregation. The pH dependence of aggregation properties of influenza subviral particles was studied.

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

Background: S-Acylation of hemagglutinin with stearate and palmitate is essential for influenza virus replication. Results: Mass spectrometry showed that shifting a cysteine from the transmembrane region to a cytoplasmic position eliminates attachment of stearate. Conclusion: The location of the acylation site is the decisive factor for site-specific acylation. Significance: Similar differential acylation might occur in cellular transmembrane proteins. 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 [3H]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.

The In Situ Structural Characterization of the Influenza A Virus Matrix M1 Protein within a Virion

The first attempt has been made to suggest a model of influenza A virus matrix M1 protein spatial structure and molecule orientation within a virion on the basis of tritium planigraphy data and theoretical prediction results. Limited in situ proteolysis of the intact virions with bromelain and surface plasmon resonance spectroscopy study of the M1 protein interaction with lipid coated surfaces were used for independent confirmation of the proposed model.

INFLUENZA A HEMAGGLUTININ C-TERMINAL ANCHORING PEPTIDE: IDENTIFICATION AND MASS SPECTROMETRIC STUDY

MALDI-TOF MS and N-terminal amino acid sequencing allowed us to identify several fragments of the C-terminal peptide of Influenza A hemagglutinin (HA) containing transmembrane domains (TMD). These fragments were detected in the organic phase of chloroform-methanol extracts from bromelain-treated virus particles. Heterogeneous fatty acylation of the C-terminus was revealed. Tritium bombardment technique might open an opportunity for 3D structural investigation of the HA TMD in situ.

Influenza A virus M1 protein structure probed by in situ limited proteolysis with bromelain.

Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.

Structural investigation of influenza virus hemagglutinin membrane-anchoring peptide

Hemagglutinin (HA), the trimeric spike of influenza virus, catalyzes fusion of viral and cellular membranes. We have synthesized the anchoring peptide including the linker, transmembrane region and cytoplasmic tail (HA-TMR-CT) in a cell-free system. Furthermore, to mimic the palmitoylation of three conserved cysteines within the CT, we chemically alkylated HA-TMR-CT using hexadecyl-methanethiosulfonate. While the nuclear magnetic resonance spectroscopy showed pure and refolded peptides, the formation of multiple oligomers of higher order impeded further structural analysis. Circular dichroism spectroscopy of both alkylated and non-alkylated HA-TMR-CT revealed an α-helical secondary structure. No major impact of the fatty acids on the secondary structure was detected.

Studying liposomes by tritium bombardment

Bilayer liposomes from a mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPC:DPPE=8:2, molar ratio) or DPPC labeled with 14C-DPPC (DPPC:14C-DPPC) were bombarded with thermally activated tritium atoms. The tritiated liposomes were hydrolyzed by phospholipase C, and the tritium incorporation into different parts of the bilayer along its thickness was determined. The tritium flux attenuation coefficients were calculated for the headgroup (k1=0.176±0.032 Å−1) and acylglycerol residue (k2=0.046±0.004 Å−1) layers indicating a preferential attenuation of the tritium flux in the headgroup region and relative transparence of the membrane hydrophobic part. The finding is potentially important to apply tritium bombardment for investigation of spatial organization of transmembrane proteins in their native lipid environment.