Preparative method of isolating phosphoinositide fractions from brain tissue]

A simple technique for preparative isolation of chromatographically homogenous fractions of mono-, di- and triphosphoinositides from ox brain tissue is described. Podyphosphoinositides fractions were obtained after chromatography of lipid extract on DEAE cellulose, phosphomonoinositides fraction--after chromatography of polyphosphoinositide-free material on aluminium hydroxide column. Bivalent metal ions were eliminated from lipid extract using chromatography on Dowex-50 H+. Ammonium acetate was removed after precipitation of lipids in water: methanol (1:1) in the presence of 4 M this salt. The average yield of mono-, di- and triphosphates was 40, 22 and 58 mg respectively per 1 kg of brain tissue as callulated for lipid phosphorus.     

Functional interactions of the influenza virus glycoproteins

Hemagglutinin (HA) and neuraminidase (NA) are functionally related coat glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reasortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. A loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, HA affinity is reduced and aggregation lost owing to a mechanism common for different HA antigenic subtypes (H2, H3, H4, and H13). This is an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with generation of Flu variants having a pandemic potential.     

Preparative isolation of basic structural proteins of the influenza virus]

The schemes for preparative electrophoretic isolation and purification of major proteins from influenza virus are described. The viral envelope protein, hemagglutinin, two of its subunits, internal M and NP proteins of influenza viruses A/FPV/Rostock (H7N1), A/PR/8/34 (H1N1) and X-31 (H3N2) were obtained in preparative amounts and characterized by amino acid and N-terminus analyses.     

Purified glycoproteins of influenza virus stimulate cell-mediated cytotoxicity in vivo

Previously, we reported that purified surface influenza viral glycoproteins can induce cell-mediated cytotoxicity (CMC) in vitro. Both neuraminidase (NA) and hemagglutinin (HA) were equally good stimulators, on an equimolar basis. In order to broaden the scope of these observations, we examined whether these glycoproteins stimulate natural killer (NK) activity in vivo. Biologically active preparations of glycoproteins NA and HA were purified from virus A/USSR/90/77 (H1N1) and recombinant virus A/USSR/92/77 (H1) x A/Prague/1/56 (N7), respectively. The studies were carried out using the optimal doses of NA and HA. In a 4-hour NK assay, using NK-sensitive YAC-1 cells as targets, both viral glycoproteins stimulated the NK activity of splenocytes of BALB/c and C3H mice. This stimulation was independent of the route of administration (intravenous or intraperitoneal) of the antigen. The observed NK activity was viral antigen-specific and could be modulated to levels comparable to those observed with the standard stimulator, polyinosinic acid-polycytidylic acid, by the use of an appropriate synthetic adjuvant, stearyl tyrosinate. Direct and indirect evidences suggest that the enhanced CMC is due to NK cells. These observations imply that enhancement of NK activity is the intrinsic property of influenza NA and HA.     

Membrane fusion activity of reconstituted vesicles of influenza virus hemagglutinin glycoproteins

Reconstituted vesicles of hemagglutinin glycoproteins into egg yolk phosphatidylcholine/spin-labeled phosphatidylcholine/cholesterol (molar ratio 1.6:0.4:1) were prepared by dialysis. Preparations at appropriate protein-to-lipid ratios (1:44 and 1:105 mol/mol) contained vesicles with a diameter of 100-300 nm and a high density of spikes on the surface. These vesicles showed low pH-induced membrane fusion activity. At pH 5.2 and 37 degrees C, fusion with erythrocyte membranes took place very rapidly within 1-2 min and reached a plateau at 63-66% fusion. The fusion was negligibly small at neutral pH and was induced to occur at pH values lower than 6.0. The reconstituted vesicles caused hemolysis and fusion of human erythrocyte cells in the same pH range as that of the fusion with erythrocyte membranes. The low pH-induced fusion activity of the reconstituted vesicles is essentially the same as that of the parent virus. These vesicles can be used to deliver some reagents or drugs into target cell cytoplasm via fusion at lysosomes.     

Basis for selective incorporation of glycoproteins into the influenza virus envelope.

The ability of mutant or chimeric A/Japan hemagglutinins (HAs) to compete for space in the envelope of A/WSN influenza viruses was investigated with monkey kidney fibroblasts that were infected with recombinant simian virus 40 vectors expressing the Japan proteins and superinfected with A/WSN influenza virus. Wild-type Japan HA assembled into virions as well as WSN HA did. Japan HA lacking its cytoplasmic sequences, HAtail-, was incorporated into influenza virions at half the efficiency of wild-type Japan HA. Chimeric HAs containing the 11 cytoplasmic amino acids of the herpes simplex virus type 1gC glycoprotein or the 29 cytoplasmic amino acids of the vesicular stomatitis virus G protein were incorporated into virions at less than 1% the efficiency of HAtail-. Thus, the cytoplasmic domain of HA was not required for the selection process; however, foreign cytoplasmic sequences, even short ones, were excluded. A chimeric HA having the gC transmembrane domain and the HA cytoplasmic domain (HgCH) was incorporated at 4% the efficiency of HAtail-. When expressed from simian virus 40 recombinants in this system, vesicular stomatitis virus G protein with or without (Gtail-) its cytoplasmic domain was essentially excluded from influenza virions. Taken together, these data indicate that the HA transmembrane domain is required for incorporation of HA into influenza virions. The slightly more efficient incorporation of HgCH than G or Gtail- could indicate that the region important for assembling HA into virions extends into part of the cytoplasmic domain.     

Computational investigation of interaction mechanisms between juglone and influenza virus surface glycoproteins

Surface glycoproteins of influenza virus [haemagglutinin (HA) and neuraminidase (NA)] are vital target proteins in current rational drug designs. Here, the molecular recognitions between juglone and A/H5N1 influenza virus membrane glycoproteins were studied through flexible docking and molecular dynamic simulations. The results revealed that juglone has the binding specificity to HA (H5) and NA (N1), especially the spatial match of 2-cyclohexene-1,4-dione ring. N1 rather than H5 protein is responsible for the binding, with the interaction energies of ? 72.48 and ? 41.91 kcal mol^? 1, respectively. The residues Arg152, Arg156, Glu276, Glu277 and Arg292 of N1 protein had important roles during the binding process. Compared with other NA inhibitors, juglone is a potential source of anti-influenza ingredients, with better interaction energy and relatively smaller size. In addition, this work also pointed out how to effectively modify the functional groups of juglone. We hope that the results will aid our understanding of recognitions involving influenza surface glycoproteins with the phenolic compounds and warrant the experimental aspects to design novel anti-influenza drugs.     

Sequence determination of the Sendai virus HN gene and its comparison to the influenza virus glycoproteins

The nucleotide sequence of the Sendai virus (SV) HN (hemagglutinin-neuraminidase) gene was determined. The deduced primary structure of the protein showed only one hydrophobic domain likely to represent the transmembrane region, but at its N terminus. Since the SV F protein is anchored in the membrane at its C terminus, the two SV glycoproteins are thus membrane-anchored in opposite orientations, similar to the two influenza virus (FLU) glycoproteins. Amino acid sequence comparisons of the SV HN and the FLU HA and NA proteins revealed homologies between 100 amino acids of the hemagglutinin region of the FLU HA protein and the C terminus of the SV HN, and between 200 amino acids of the neuraminidase region of the FLU NA and the central region of SV HN. Alignment of the neuraminidase, hemagglutinin, and fusion regions shared by these glycoproteins suggest the structure of a possible ancestral gene.     

Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.     

Interplay between lipids and viral glycoproteins during hemolysis and fusion by influenza virus

Since mixtures of lipids alone are known to elicit membrane fusion without participation of fusion proteins, the role of viral lipids in the so-called virus-induced hemolysis and cell fusion has been investigated, using as a model the fowl plague virus (influenza A/FPV/Rostock/H7N1). The experiments were planned in a way that allowed quantitative modification of viral lipids without changing envelope glycoproteins. Under the conditions employed, cholesterol oxidase of Nocardia erythropolis and phospholipase C of Bacillus cereus were shown to completely modify their substrates in the virus without altering virus-associated hemagglutinating and neuraminidase activities. It was found with such enzyme treatment that virus-induced hemolysis and cell fusion are greatly influenced by cholesterol and phospholipids of the envelope. It became clear, that hemolysis and fusion are differently dependent on the nature of lipid components even though mediated by the same viral glycoproteins.     

In vitro enhancement of human natural cell-mediated cytotoxicity by purified influenza virus glycoproteins.

The role of the glycoproteins of influenza virus, hemagglutinin (HA), and neuraminidase (NA) in the in vitro stimulation of natural cell-mediated cytotoxicity (NCMC) or natural killer activity of human peripheral blood lymphocytes was evaluated with radiolabeled K562 cells as target cells in an overnight chromium release assay. Three different approaches were used. (i) Purified viral proteins were obtained by extraction with Nonidet P-40, separation on a sucrose gradient, and further purification by affinity chromatography. Ficoll-Hypaque-purified peripheral blood lymphocytes exposed to HA or NA individually or to a mixture of both significantly increased NCMC (32 to 50%). (ii) Treatment of HA and NA with their respective homologous antisera or F(ab')2 antibody abrogated the stimulation of NCMC by these glycoproteins. (iii) Virions treated with proteolytic enzymes resulted in viral cores lacking either HA or NA or both activities. Compared to whole virions, viral cores devoid of HA activity only induced a 50% increase in NCMC, whereas viral cores lacking HA activity and with traces of NA activity stimulated only 10% of the NCMC. These results suggest that influenza virus-induced cell-mediated cytotoxicity is largely due to its glycoproteins.     

The role of proteolytic cleavage of viral glycoproteins in the pathogenesis of influenza virus infections

Infectivity, tropism, spread, and pathogenicity of influenza viruses are based on the interplay between the fusogenic glycoproteins and appropriate host endoproteases. The hemagglutinin (HA) of influenza A and B viruses and the HEF (hemagglutinating, esterase, fusion) glycoprotein of influenza C virus receive their full biological activity by proteolytic cleavage of a precursor molecule at a definite cleavage site. The amino acid motifs at the cleavage site and the availability of suitable proteases are critical for the clinical manifestation of the infection. Prototype cleavage proteases, including bacterial enzymes, are described.     

Fusion-mediated microinjection of liposome-enclosed DNA into cultured cells with the aid of influenza virus glycoproteins

Influenza viruses were able to mediate fusion of DNA-loaded liposomes with living cultured cells such as monkey COS-7 cells. This was inferred from the appearance of CAT activity in recipient cells incubated with the combination of influenza viruses and liposomes loaded with the plasmid pSV2CAT. Influenza virions were found to be as efficient as intact Sendai virions in mediating microinjection of foreign DNA into living cells. Also, reconstituted envelopes bearing either influenza glycoproteins or the combination of Sendai and influenza glycoproteins were highly efficient in promoting fusion of loaded liposomes with recipient cells. Introduction of DNA into cultured cells required the presence of an active influenza fusion protein; namely, an active HA glycoprotein. Very little or no CAT activity was observed in cells incubated with loaded liposomes and unfusogenic influenza viruses. The virus-induced fusion event probably occurs within intracellular organelles such as endosomes following receptor-mediated endocytosis of virus-liposome complexes. This is due to the fact that the viral fusion glycoprotein is activated only at acidic pH values such as those which characterize the intraendosomal environment. Results of the present work demonstrate for the first time microinjection of foreign DNA via fusion with membranes of intracellular organelles. The potential of the present system to serve as a biological carrier for in vivo use is discussed.     

Carbohydrates of influenza virus. III. Nature of oligosaccharide-protein linkage in viral glycoproteins.

Four different glycopeptides can be distinguished after pronase digestion of influenza A virus glycoproteins: Ia and Ib, containing N-acetylglucosamine, mannose, galactose, and fucose, and IIa and IIb, containing mannose and N-acetylglucosamine. All glycopeptides yielded N-acetylglucosaminyl-asparagine after mild acid hydrolysis. There was no evidence for O-glycosidic bonds. Thus, the carbohydrate complement is linked to the polypeptide exclusively by N-glycosidic linkages between N-acetylglucosamine and asparagine.     

Preparative isolation of myxovirus glycoproteins and the study of their immunogenic properties

Preparative isolation of glycoproteins from ortho- and paramyxoviruses is described. The purified concentrated virus has been treated with nonionic detergent MESK with subsequent removal of viral cores by centrifugation. Supernatant was sterilized by filtration through the nuclear filters and cleared from detergent by dialysis. Glycoproteins obtained have not contained contaminating cellular or core viral proteins or viral shell lipids. In the absence of detergent, glycoproteins have formed the peculiar mycelial complexes. Biological activity of glycoproteins was kept at high level. Glycoproteins output at isolation from different strains of influenza viruses A, B and Sendai virus varied from 75 to 98%. Immunogenetic study of the preparations obtained has demonstrated their capability to stimulate the formation of antibodies against both viral glycoproteins comparable with the capability of intact virus. The obtained level of immunity was enough to protect organism against homologous infection. Samples of glycoproteins obtained are up to standards for subunit vaccines, and the technique of their preparation is perspective as far as the production of vaccine preparations is concerned.     

The use of the Soviet detergents dezintegron-O and dezintegron-B for the solubilization of influenza virus glycoproteins]

Native detergents, desintegron-O-and desintegron-B, solubilize hemagglutinins of the influenza virus as intensively, as foreign drugs (mulgophen, zwittergent and sodium sarcozyl) do. This permits recommending desintegrons for quantitative determination of hemagglutinin of the influenza virus in virus-containing materials by means of the reaction of single radial immunodiffusion. Desintegron-O and desintegron-B permit extracting enzymatically active neuraminidase from influenza A viruses with 44.6-83.4% yield.