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Biochemistry (Moscow) 86(2), 230 - 240 (2021). doi:10.1134/S0006297921020115, Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVA��32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVA��32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55��C was also different. The data of transmission electron microscopy showed that the PVA��32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen., Published by Springer Science + Business Media B.V, Dordrecht [u.a.]

Influenza A/H1N1 virus hemagglutinin (HA) is an integral type I glycoprotein that contains a large glycosylated ectodomain, a transmembrane domain, and a cytoplasmic tail (CT) of 10-14 amino acid residues. There are absolutely no data on the secondary or tertiary structure of the HA CT, which is important for virus pathogenesis. Three highly conserved cysteines are post-translationally modified by the attachment of fatty acid residues that pin the CT to the lipid membrane inside the virion. We applied circular dichroism (CD) and fluorescence spectroscopy analysis to examine four synthetic peptides corresponding to 14-15 C-terminal residues of H1 subtype HA (NH

Influenza A virus is a serious human pathogen that assembles enveloped virions on the plasma membrane of the host cell. The pleiomorphic morphology of influenza A virus, represented by spherical, elongated, or filamentous particles, is important for the spread of the virus in nature. Using fixative protocols for sample preparation and negative staining electron microscopy, we found that the recombinant A/WSN/33 (H1N1) (rWSN) virus, a strain considered to be strictly spherical, may produce filamentous particles when amplified in the allantoic cavity of chicken embryos. In contrast, the laboratory WSN strain and the rWSN virus amplified in Madin–Darby canine kidney cells exhibited a spherical morphology. Next-generation sequencing (NGS) suggested a rare Ser126Cys substitution in the M1 protein of rWSN, which was confirmed by the mass spectrometric analysis. No structurally relevant substitutions were found by NGS in other proteins of rWSN. Bioinformatics algorithms predicted a neutral structural effect of the Ser126Cys mutation. The mrWSN_M1_126S virus generated after the introduction of the reverse Cys126Ser substitution exhibited a similar host-dependent partially filamentous phenotype. We hypothesize that a shortage of some as-yet-undefined cellular components involved in virion budding and membrane scission may result in the appearance of filamentous particles in the case of usually “nonfilamentous” virus strains.

Post-translational modifications often regulate protein functioning. Covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage (known as protein palmitoylation or S-acylation) affects protein trafficking, protein-protein and protein-membrane interactions. This post-translational modification is coupled to membrane fusion or virus assembly and may affect viral replication in vitro and thus also virus pathogenesis in vivo. In this review we outline modern methods to study S-acylation of viral proteins and to characterize palmitoylproteomes of virus infected cells. The palmitoylation site predictor CSS-palm is critically tested against the Class I enveloped virus proteins. We further focus on identifying the S-acylation sites directly within acyl-peptides and the specific fatty acid (e.g, palmitate, stearate) bound to them using MALDI-TOF MS-based approaches. The fatty acid heterogeneity/ selectivity issue attracts now more attention since the recently published 3D-structures of two DHHC-acyl-transferases gave a hint how this might be achieved.

Abstract COVID‑19, a new human respiratory disease that has killed nearly 3 million people in a year since the start of the pandemic, is a global public health challenge. Its infectious agent, SARS‑CoV‑2, differs from other coronaviruses in a number of structural features that make this virus more pathogenic and transmissible. In this review, we discuss some important characteristics of the main SARS‑CoV‑2 surface antigen, the spike (S) protein, such as (i) ability of the receptor-binding domain (RBD) to switch between the “standing-up” position (open pre-fusion conformation) for receptor binding and the “lying-down” position (closed pre-fusion conformation) for immune system evasion; (ii) advantage of a high binding affinity of the RBD open conformation to the human angiotensin-converting enzyme 2 (ACE2) receptor for efficient cell entry; and (iii) S protein preliminary activation by the intracellular furin-like proteases for facilitation of the virus spreading across different cell types. We describe interactions between the S protein and cellular receptors, co-receptors, and antagonists, as well as a hypothetical mechanism of the homotrimeric spike structure destabilization that triggers the fusion of the viral envelope with the cell membrane at physiological pH and mediates the viral nucleocapsid entry into the cytoplasm. The transition of the S protein pre-fusion conformation to the post-fusion one on the surface of virions after their treatment with some reagents, such as β-propiolactone, is essential, especially in relation to the vaccine production. We also compare the COVID‑19 pathogenesis with that of severe outbreaks of “avian” influenza caused by the A/H5 and A/H7 highly pathogenic viruses and discuss the structural similarities between the SARS‑CoV‑2 S protein and hemagglutinins of those highly pathogenic strains. Finally, we touch on the prospective and currently used COVID‑19 antiviral and anti-pathogenetic therapeutics, as well as recently approved conventional and innovative COVID‑19 vaccines and their molecular and immunological features.

Influenza A viruses contain two S-acylated proteins, the ion channel M2 and the glycoprotein hemagglutinin (HA). Acylation of the latter is essential for virus replication. Here we analyzed expression of each of the 23 members of the family of ZDHHC acyltransferases in human airway cells, the site of virus replication. RT-PCR revealed that every ZDHHC acyltransferase (except ZDHHC19) is expressed in A549 and Calu cells. Interestingly, expression of one ZDHHC, ZDHHC22, is upregulated in virus-infected cells; this effect is more pronounced after infection with an avian compared to a human virus strain. The viral protein NS1 triggers ZDHHC22 expression in transfected cells, whereas recombinant viruses lacking a functional NS1 gene did not cause ZDHHC22 upregulation. CRISPR/Cas9 technology was then used to knock-out the ZDHHC22 gene in A549 cells. However, acylation of M2 and HA was not reduced, as analyzed for intracellular HA and M2 and the stoichiometry of S-acylation of HA incorporated into virus particles did not change according to MALDI-TOF mass spectrometry analysis. Comparative mass spectrometry of palmitoylated proteins in wt and ΔZDHHC22 cells identified 25 potential substrates of ZDHHC22 which might be involved in virus replication. This article is protected by copyright. All rights reserved.

Membranes 11(10), 772 (2021). doi:10.3390/membranes11100772, Influenza A virus envelope contains lipid molecules of the host cell and three integral viral proteins: major hemagglutinin, neuraminidase, and minor M2 protein. Membrane-associated M1 matrix protein is thought to interact with the lipid bilayer and cytoplasmic domains of integral viral proteins to form infectious virus progeny. We used small-angle X-ray scattering (SAXS) and complementary techniques to analyze the interactions of different components of the viral envelope with M1 matrix protein. Small unilamellar liposomes composed of various mixtures of synthetic or ���native��� lipids extracted from Influenza A/Puerto Rico/8/34 (H1N1) virions as well as proteoliposomes built from the viral lipids and anchored peptides of integral viral proteins (mainly, hemagglutinin) were incubated with isolated M1 and measured using SAXS. The results imply that M1 interaction with phosphatidylserine leads to condensation of the lipid in the protein-contacting monolayer, thus resulting in formation of lipid tubules. This effect vanishes in the presence of the liquid-ordered (raft-forming) constituents (sphingomyelin and cholesterol) regardless of their proportion in the lipid bilayer. We also detected a specific role of the hemagglutinin anchoring peptides in ordering of viral lipid membrane into the raft-like one. These peptides stimulate the oligomerization of M1 on the membrane to form a viral scaffold for subsequent budding of the virion from the plasma membrane of the infected cell., Published by MDPI, Basel

A series of commercial inactivated influenza vaccines (IIVs) used in the Russian National Immunization Program were characterized to evaluate their protective properties on an animal model. Standard methods for quantifying immune response, such as hemagglutination inhibition (HAI) assay and virus neutralization (VN) assay, allowed us to distinguish the immunogenic effect of various IIVs from that of placebo. However, these standard approaches are not suitable to determine the role of various vaccine components in immune response maturation. The expanded methodological base including an enzyme-linked immunosorbent assay (ELISA) and a neuraminidase ELISA (NA-ELISA) helped us to get wider characteristics and identify the effectiveness of various commercial vaccines depending on the antigen content. Investigations conducted showed that among the IIVs tested, Ultrix&reg, Ultrix&reg, Quadri and VAXIGRIP&reg, elicit the most balanced immune response, including a good NA response. For Ultrix&reg, Quadri, and SOVIGRIPP&reg, (FORT LLC), the whole-virus specific antibody subclass IgG1, measured in ELISA, seriously prevailed over IgG2a, while, for VAXIGRIP&reg, and SOVIGRIPP&reg, (NPO Microgen JSC) preparations, the calculated IgG1/IgG2a ratio was close to 1. So, the immune response varied drastically across different commercial IIVs injected in mice.

Background: today&rsquo, s standard quality control methods used to control the protein composition of inactivated influenza vaccines only take into account a few key reference components. They do not allow for thorough characterization of protein compositions. As a result, observation of unpredictable variations in major viral constituents and admixtures of cellular proteins within manufactured vaccines that may seriously influence the immunogenicity and safety of such vaccines has become a pressing issue in vaccinology. This study aims at testing a more sophisticated approach for analysis of inactivated split influenza vaccines licensed in the Russian Federation. The formulations under study are the most available on the market and are included in the Russian National Immunization Program. Methods: liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, in combination with label-free protein quantitation via the intensity-based absolute-quantitation (iBAQ) algorithm, as well as a number of standard molecular analysis methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS), and negative-stain transmission electron microscopy (TEM) were applied. Results: the methods implemented were able to identify dozens of viral and host proteins and quantify their relative amounts within the final formulations of different commercially available inactivated split influenza vaccines. Investigation of molecular morphology of the vaccine preparations using TEM revealed typical rosettes of major surface proteins (hemagglutinin and neuraminidase). DLS was used to demonstrate a size distribution of the rosettes and to test the stability of vaccine preparations at increased temperatures. Conclusions: a holistic approach based on modern, highly productive analytical procedures was for the first time applied for a series of different commercially available inactivated split influenza vaccines licensed in Russia. The protocols probed may be suggested for the post-marketing quality control of vaccines. Comparison of different preparations revealed that the Ultrix&reg, and Ultrix&reg, Quadri vaccines produced by pharmaceutical plant FORT LLC and trivalent vaccine Vaxigrip&reg, produced by pharmaceutical company Sanofi Pasteur have well-organized antigen rosettes, they contain fewer admixture quantities of host cell proteins, and demonstrate good correlation among mostly abundant viral proteins detected by different methods.

The aim of this study was to construct a vaccine peptide candidate against pandemic Influenza H1N1 hemagglutinin and to test its structure. With the help of bioinformatic algorithms we showed that the sequence encoding the second polypeptide of pandemic Influenza H1N1 hemagglutinin (HA2) is protected from nonsynonymous mutations better than the sequence encoding its first polypeptide (HA1). With the help of secondary and ternary structure predicting algorithms we found the fragment of HA2 with the most reproducible secondary structure and synthesized the NY25 peptide corresponding to the residues Asn117 - Tyr141 of HA2. According to the circular dichroism spectra analysis, the peptide has short helix and beta hairpin. According to the analysis of differential fluorescence quenching results, two tyrosine residues are situated on a long distance from each other. These facts taken together with the positive results of affine chromatography with the serum of a person immunized by full-length hemagglutinin confirm that the structure of the fragment of viral full-length protein has been reproduced in the synthetic NY25 peptide. Amino acid sequence of the NY25 peptide (NLYEKVRSQLKNNAKEIGNGCFEFY) is relatively conserved in 18 subtypes of Influenza A virus hemagglutinin.

Influenza A virus, a member of the Orthomyxoviridae family of enveloped viruses, is one of the human and animal top killers, and its structure and components are therefore extensively studied during the last decades. The most abundant component, M1 matrix protein, forms a matrix layer (scaffold) under the viral lipid envelope, and the functional roles as well as structural peculiarities of the M1 protein are still under heavy debate. Despite multiple attempts of crystallization, no high resolution structure is available for the full length M1 of Influenza A virus. The likely reason for the difficulties lies in the intrinsic disorder of the M1 C-terminal part preventing diffraction quality crystals to be grown. Alternative structural methods including synchrotron small-angle X-ray scattering (SAXS), atomic force microscopy, cryo-electron microscopy/tomography are therefore widely applied to understand the structure of M1, its self-association and interactions with the lipid membrane and the viral nucleocapsid. These methods reveal striking similarities in the behavior of M1 and matrix proteins of other enveloped RNA viruses, with the differences accompanied by the specific features of the viral lifecycles, thus suggesting common interaction principles and, possibly, common evolutional ancestors. The structural information on the Influenza A virus M1 protein obtained to the date strongly suggests that the intrinsic disorder in the C-terminal domain has important functional implications.

Palmitoylation or S-acylation is the posttranslational attachment of fatty acids to cysteine residues and is common among integral and peripheral membrane proteins. Palmitoylated proteins have been found in every eukaryotic cell type examined (yeast, insect, and vertebrate cells), as well as in viruses grown in these cells. The exact functions of protein palmitoylation are not well understood. Intrinsically hydrophilic proteins, especially signaling molecules, are anchored by long-chain fatty acids to the cytoplasmic face of the plasma membrane. Palmitoylation may also promote targeting to membrane subdomains enriched in glycosphingolipids and cholesterol or affect protein-protein interactions.This chapter describes (1) a standard protocol for metabolic labeling of palmitoylated proteins and also the procedures to prove a covalent and ester-type linkage of the fatty acids, (2) a simple method to analyze the fatty acid content of S-acylated proteins, (3) two methods to analyze dynamic palmitoylation for a given protein, and (4) protocols to study cell-free palmitoylation of proteins.

Investigations of short peptides that can be used in the next phase of synthetic HIV1 vaccine development are an urgent goal, as well as investigations of peptides that can be used in immunological tests with the aim to check the titer of antibodies against the alpha helix 1 from the first conserved region of HIV1 gp120 that are known to cause antibody-dependent cellular cytotoxicity (ADCC). The aim of this work was to study the structure of the NQ21 peptide corresponding to the less mutable part of the first conserved region of HIV1 gp120 (residues 94–114). The NQ21 peptide and its conjugate with biotin (biotin-NQ21) are absolutely alpha-helical in phosphate buffer solutions at pH = 6.8, 7.4 and 8.0, as well as in the dried form, according to the results of surface-enhanced Raman scattering (SERS) spectroscopy. Results of the native gel electrophoresis and thermal analysis under the control of spectrofluorometer and near UV circular dichroism (CD) showed that the peptide exists in form of octamers and tetramers at pH = 7.4, that is important information for further vaccine development. Strong signal of interacting Trp residues in oligomers in the far UV CD obscures the signal from secondary structure, but becomes less intensive during the heating.

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.

Two enveloped virus families, Orthomyxoviridae and Paramyxoviridae, comprise a large number of dangerous pathogens that enter the host cell via fusion of their envelope with a target cell membrane at acidic or neutral pH. The Class I prototypic glycoproteins responsible for this reaction are the Influenza virus haemagglutinin (HA) protein or paramyxovirus fusion (F) protein. X-ray crystallography and cryoelectron microscopy data are available for the HA and F ectodomains in pre- and post-fusion conformations, revealing similar spiky architectures, albeit with clear differences in the details. In contrast, their anchoring segments, which possess a linker region, transmembrane domain and cytoplasmic tail that is specifically modified with long fatty acids (highly conserved in HA and occasional in F), are not resolved. Recent experimental, bioinformatics and molecular modelling data showing the primary, secondary and quaternary organization of the HA and F anchoring segments are summarized in this review. Some amino acid patterns that are crucial for protein thermal stability or lipid membrane order/cholesterol binding are addressed, and new achievements in vaccine practice using HA transmembrane domain chimaeras are discussed. The oligomerization properties of the transmembrane domains are considered in the context of Group-1 and Group-2 antigenic HA subtypes and various genera/subfamilies of paramyxoviruses. A specific focus is the late steps of fusion that are reportedly facilitated by (1) β-sheet-promoting β-branched amino acids (valine and isoleucine) that are enriched in the transmembrane domain of paramyxovirus F or (2) a post-translational modification of C-terminal cysteines with palmitate/stearate (differential S-acylation) that is highly conserved in Influenza virus HA.

Hemagglutinin (HA) of influenza virus is S-acylated with stearate at a transmembrane cysteine and with palmitate at two cytoplasmic cysteines. The amount of stearate varies from 35 (in avian strains) to 12% (in human strains), although the acylation region exhibits only minor or even no amino acid differences between HAs. To address whether matrix proteins and neuraminidase affect stearoylation of HA, we used mass spectrometry to analyze laboratory reassortants containing avian virus HA and the internal proteins from a human virus. Only minor fluctuations in the amount of stearate were observed, implying that other viral proteins do not affect acylation of HA.

This review considers principles of the use of mass spectrometry for the study of biological macromolecules. Some examples of protein identification, virion proteomics, testing vaccine preparations, and strain surveillance are represented. Possibilities of structural characterization of viral proteins and their posttranslational modifications are shown. The authors’ studies by MALDI-MS on S-acylation of glycoproteins from various families of enveloped viruses and on oligomerization of the influenza virus hemagglutinin transmembrane domains are summarized.

Interactions between model enzymes and the influenza virus hemagglutinin (HA) homotrimeric spike were addressed. We digested influenza virions (naturally occurring strains and laboratory reassortants) with bromelain or subtilisin Carlsberg and analyzed by MALDI-TOF mass spectrometry the resulting HA2 C-terminal segments. All cleavage sites, together with (minor) sites detected in undigested HAs, were situated in the linker region that connects the transmembrane domain to the ectodomain. In addition to cleavage at highly favorable amino acids, various alternative enzyme preferences were found that strongly depended on the HA subtype/type. We also evaluated the surface electrostatic potentials, binding cleft topographies and spatial dimensions of stem bromelain (homologically modeled) and subtilisin Carlsberg (X-ray resolved). The results show that the enzymes (∼45Å(3)) would hardly fit into the small (∼18-20Å) linker region of the HA-spike. However, the HA membrane proximal ectodomain region was predicted to be intrinsically disordered. We propose that its motions allow steric adjustment of the enzymes' active sites to the neck of the HA spike. The subtype/type-specific architectures in this region also influenced significantly the cleavage preferences of the enzymes.

Many glycoproteins of enveloped viruses are known to be “palmitoylated” at cysteines located either in the transmembrane region or in the cytoplasmic tail. Although it was recognized early on that “palmitoylation” is not specific for this carbon chain, the exact fatty acid composition of S-acylated proteins has been difficult to determine. Advancements in mass-spectrometry (MS) now allow one to quantify the fatty acids linked to single acylation sites. We report that G of Vesicular Stomatitis virus contains palmitate at a cytoplasmic cysteine, whereas F of Newcastle Disease virus and E1 of Semliki Forest virus (SFV) are stoichiometrically acylated with stearate at a transmembrane cysteine. E2 of SFV contains three molecules of palmitate and one molecule of stearate, the latter probably attached to a transmembrane cysteine. Thus, site-specific attachment of palmitate or stearate, previously described only for HA of influenza virus, is a common feature of viral spike proteins.

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the basic protein components of the virion environment: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently deleted HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA2 185-211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a basic site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM beta-mercaptoethanol than in the presence of 50 mM. However, the complete removal of exodomains of HA, HA, and low-activity enzyme by the HA high- and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein Ml1. The action of trans-epoxysuccinyl-L-leucylamido)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl2 on the hydrolysis of proteins HA and M1 by bromelain was investigated.

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 (A 1 cm, 280 0.1% ) 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.

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.

The Center of Influenza Ecology and Epidemiology (IEEC, Moscow) has shown the cocirculation of influenza A/H1N1, A/H3N2 and B viruses in humans during the 2002–2003 season. All strains were isolated in MDCK system, but A/H1N1 viruses still had the propensity to propagate in embryonated eggs. The epidemic A/H1N1 strains were related to the reference strain A/New Caledonia/20/99. The population of A/H3N2 strains included A/Moscow/10/99, A/Panama/2007/99 and a new reference strain A/Fujian/411/02-like. Reemergence of influenza B/Victoria/2/87 lineage viruses absent in Russia for more than 10 years were discovered. Hemagglutinin (HA) of influenza B strains was related to that of B/Hong Kong/22/01; neuraminidase (NA) was more related to that of either B/Victoria/2/87 or B/Yamagata/16/88-like viruses. Serological data confirmed the circulation of all isolated strains. Different sensitivities of influenza A and B viruses to treatment by proteolytic enzyme bromelain were revealed; the most sensitive was B virus, and less sensitive were A/H3N2 and A/H1N1 viruses. Specific activities of the two bromelain preparations used were tested in relation to the high specific chromogenic substrate for thiol proteinase assay ( l -pyroglutamyl- l -phenylalanyl- l -alanine- p -nitroanilide).

The density of glycoprotein (GP) distribution on the virion surface substantially influences the virus infectivity and pathogenicity. A method to quantitatively determine the area occupied by surface GP spikes was proposed for influenza virus (Flu) strain A/PR/8/34 on the basis of data of tritium bombardment and dynamic light scattering. The latter was used to measure the diameter of intact virions and subviral particles (Flu virions lacking GP spikes after bromelain digestion). Intact virions and subviral particles were bombarded with a hot tritium atom flux, and the specific radioactivity of the matrix M1 protein was analyzed. The tritium label was incorporated into the amino acid residues of a thin exposed protein layer and partly penetrated through the lipid bilayer of the viral envelope, labeling M1, located under the lipid bilayer. The tritium label distribution among different amino acid residues was the same in M1 isolated from subviral particles and M1 isolated from intact virions, demonstrating that the M1 spatial structure remained unchanged during proteolysis of GP spikes. The difference in specific radioactivity between the M1 proteins isolated from intact virions and subviral particles was used to calculate the GP-free portion of the viral surface. Approximating the Flu virion as a sphere, the GP-covered area was estimated at 1.4 × 104 nm2, about 40% of the total virion surface. This was consistent with the cryoelectron tomography data published for Flu strain A/X-31. The approach can be applied for other enveloped high pathogenic viruses, such as HIV and the Ebola virus.

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.

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.

Influenza virus hemagglutinin is a homotrimeric spike glycoprotein crucial for virions' attachment, membrane fusion, and assembly reactions. X-ray crystallography data are available for hemagglutinin ectodomains of various types/subtypes but not for anchoring segments. To get structural information for the linker and transmembrane regions of hemagglutinin, influenza A (H1–H16 subtypes except H8 and H15) and B viruses were digested with bromelain or subtilisin Carlsberg, either within virions or in non-ionic detergent micelles. Proteolytical fragments were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Within virions, hemagglutinins of most influenza A/Group-1 and type B virus strains were more susceptible to digestion with bromelain and/or subtilisin compared to A/Group-2 hemagglutinins. The cleavage sites were always located in the hemagglutinin linker sequence. In detergent, 1) bromelain cleaved hemagglutinin of every influenza A subtype in the linker region; 2) subtilisin cleaved Group-2 hemagglutinins in the linker region; 3) subtilisin cleaved Group-1 hemagglutinins in the transmembrane region; 4) both enzymes cleaved influenza B virus hemagglutinin in the transmembrane region. We propose that the A/Group-2 hemagglutinin linker and/or transmembrane regions are more tightly associated within trimers than type A/Group-1 and particularly type B ones. This hypothesis is underpinned by spatial trimeric structure modeling performed for transmembrane regions of both Group-1 and Group-2 hemagglutinin representatives. Differential S-acylation of the hemagglutinin C-terminal anchoring segment with palmitate/stearate residues possibly contributes to fine tuning of transmembrane trimer packing and stabilization since decreased stearate amount correlated with deeper digestion of influenza B and some A/Group-1 hemagglutinins.

Introduction Influenza A hemagglutinin ( ) is a major envelope glycoprotein having attachment and fusion functions to deliver viral nucleoprotein into the host cell. It is a spike homotrimer; each monomer consists of two disulfide-linked chains (HA1 and HA2) and is anchored in the membrane by the light HA2 chain C-terminal segment. Three conservative cysteine residues of the segment were shown to be palmitoylated via thioester bond [1] (Fig.1). Probably, fatty acids may serve to orient the TMD in the lipid bilayer and influence membrane fusion processes.

Introduction Influenza A virus hemagglutinin (HA) is a transmembrane glycoprotein of the viral envelope which plays a central role in the delivery of the viral nucleocapsid into the host cell. HA ectodomain cleaved by enzyme bromelain was solved by X-ray crystallography. On the other hand, for the C-terminal anchoring segment which includes a transmembrane domain (TM) and a cytoplasmic tail (Fig. 1a) there is no 3D-structural information available, and data about its primary structure is insufficient. Another transmembrane glycoprotein of the envelope, neuraminidase (NA) is anchored by its N-terminal domain (Fig. 1b).

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.

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.

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.

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.

The topography of bacteriorhodopsin (bR) in situ was earlier studied by using the tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95 (1998) 11673], we estimated the influence of membrane environment (lipid and protein) on tritium incorporation into amino acid residues forming transmembrane helices. We have determined the tritium flux attenuation coefficients for residues 10–29 of helix A. They turned out to be low (0.04±0.02 A −1 ) for residues adjacent to the lipid matrix, and almost fourfold higher (0.15±0.05 A −1 ) for those oriented to the neighboring transmembrane helices. We believe that tritium incorporation data could help modeling transmembrane segment arrangement in the membrane.

Interactions between integral membrane proteins hemagglutinin (HA), neuraminidase (NA), M2 and membrane-associated matrix protein M1 of influenza A virus are thought to be crucial for assembly of functionally competent virions. We hypothesized that the amino acid residues located at the interface of two different proteins are under physical constraints and thus probably co-evolve. To predict co-evolving residue pairs, the EvFold ( http://evfold.org ) program searching the (nontransitive) Direct Information scores was applied for large samplings of amino acid sequences from Influenza Research Database ( http://www.fludb.org/ ). Having focused on the HA, NA, and M2 cytoplasmic tails as well as C-terminal domain of M1 (being the less conserved among the protein domains) we captured six pairs of correlated positions. Among them, there were one, two, and three position pairs for HA–M2, HA–M1, and M2–M1 protein pairs, respectively. As expected, no co-varying positions were found for NA–HA, NA–M1, and NA–M2 pairs obviously due to high conservation of the NA cytoplasmic tail. The sum of frequencies calculated for two major amino acid patterns observed in pairs of correlated positions was up to 0.99 meaning their high to extreme evolutionary sustainability. Based on the predictions a hypothetical model of pair-wise protein interactions within the viral envelope was proposed.

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.