The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different fatty acids.

We present evidence that the hemagglutinin (HA) of influenza B virus and the glycoprotein of influenza C virus (HEF) are acylated. The fatty acid linkage is sensitive to treatment with hydroxylamine and mercaptoethanol, which points to a labile thioester-type linkage. The HA of influenza B virus contains mainly palmitic acid, whereas the HEF glycoprotein of influenza C virus is acylated with stearic acid which has not been observed before as the prevailing fatty acid in viral or cellular acyl proteins.

[The structure of carbohydrate chains of hemagglutinin and neuraminidase of influenza virus B/Leningrad/179/86]. / Struktura uglevodnykh tsepei gemaggliutinina i neiraminidazy virusa grippa B/Leningrad/179/86.

The main surface glycoprotein, hemagglutinin (HA), was obtained by treatment of influenza virus B/Leningrad/179/86 with bromelain. Amino acid and monosaccharide compositions of HA and neuraminidase (NA, earlier isolated from the same virus) were determined, thus showing HA and NA to contain 8-10 and 2 carbohydrate chains, respectively. The carbohydrate fragments were cleaved off by the alkaline LiBH4 treatment, the oligosaccharides released were reduced with NaB3H4 and fractionated by two-step HPLC on Ultrasphere-C18 and Zorbax-NH2 columns. Some higher mannose and complex oligosaccharides were identified in both cases by comparison with nonlabelled oligosaccharides of the known structure. The data obtained show that surface glycoproteins of influenza virus A and B are rather similar with regard to structure and heterogeneity of their carbohydrate chains.

The phosphorylation of the integral membrane (M1) protein of influenza virus.

The phosphorylation of the internal and integral membrane (M1) protein of influenza virus was studied. Four points can be made based on the data: (1) The M1 contains at least two moles of phosphate per mole of M1. (2) Phosphorylation of M1 is conserved between influenza A, B and C viruses. Other characteristics of the M1 are also conserved, such as solubility in organic solvent, heterogeneity and ability to partition into lipid vesicles. (3) M1 is phosphorylated in cells infected with a vaccinia recombinant (vP273) containing only the gene of M1, either as a result of a vaccinia virus associated kinase or a cellular one. (4) The phosphate is located within or in close proximity to the major stretch of neutral and hydrophobic amino acids found in M1, as determined by analyzing cyanogen bromide fragments.

Protein and nucleic acid analysis of influenza B viruses isolated in Italy in 1984.

Genomic and phenotypic analysis of 30 influenza B viruses, isolated in Italy in 1984, antigenically closely related to the B/USSR/100/83 prototype virus, was carried out using T1-oligonucleotide fingerprinting of total RNA and one-dimensional peptide mapping. The results of fingerprinting analysis indicated an oligonucleotide spot homology of 90-96%, corresponding to a nucleotide sequence variation of only 0.75-0.3%. All the strains appear to belong to the same evolutionary line. Nevertheless, heterogeneity was found at the structural and antigenic level, when the viruses were compared by peptide mapping and monoclonal antibody analysis. No correlation between the biochemical variability of the viruses and the epidemiological characteristics of the different strains was established, which is consistent with the hypothesis that distinct variants, arisen at different times from a parental strain, co-circulate during an epidemic, although the additional occurrence of random mutations during the evolution of the epidemic cannot be excluded.

Functional and structural analysis of the ribonucleoprotein complexes of different human influenza virus strains.

Ribonucleoprotein (RNP) cores were prepared from various strains of human influenza virus by treating the purified or spikeless virus particles with non-ionic detergents such as Nonidet P-40 and centrifugation in continuous linear glycerol gradients. In addition to RNA, the purified complexes contained viral nucleoprotein (NP) and the three P proteins (PA, PB1, PB2) as determined by polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. Contaminations with other viral polypeptides, especially HA1, HA2 and M were below 1%. All RNP complexes were transcriptionally active in vitro. Comparison of the polymerase activity of purified complexes revealed considerable differences depending not only on the content of polymerase proteins. The activity of RNP complexes was enhanced for all strains tested by adding of ApG.

[Identification of the antigenic components of the host cell in isolated glycoproteins of the influenza virus]. / Identifikatsiia antigennykh komponentov kletki khoziaina v izolirovannykh glikoproteidakh virusa grippa.

Immunological analysis performed by solid-phase enzyme immunoassay and complement fixation test revealed the presence of host cell antigenic components in purified intact influenza virions and isolated hemagglutinins and glycoproteins. Three antigens inherent to chick embryo cells were identified in virus preparations: the species-specific antigen, heterogenetic Forssman antigen, and one similar to human group A antigen. Antisera to purified whole-virion influenza vaccines, to glycoproteins and to hemagglutinin isolated from them were found to contain antibody populations which were conducive to broad cross reactions between them and heterotypic virus antigens. To avoid erroneous results, these antibody populations must be removed from the sera by pre-adsorption with host cell antigens.

Monoclonal antibodies detect M-protein epitopes on the surface of influenza virions.

Various data obtained with activable hydrophobic probes, proteolytic treatments and anti M-protein polyclonal antibodies strongly suggest that M-protein of influenza A is an integral part of the lipid bilayer of native virions and somehow spans at the surface of the virions. Therefore we have looked for the presence of M-protein epitopes on the surface of influenza A virion by using four type A M-protein monoclonal antibodies. We developed a specific and sensitive competition ELISA where intact virions, dodecyl-sulfate disrupted virions and spikeless particles obtained after proteolytic treatment with caseinase C were used to test their ability to inhibit the reaction between these monoclonal antibodies and pure M-protein. Intact virions or SDS disrupted virions prevented three monoclonal antibodies from reacting with the M-protein. Spikeless particles also inhibited the specific binding of two of these antibodies, whereas the other fourth antibody was inhibited by contact with SDS disrupted particles only. Data presented show that at least three distinct M-protein epitopes were detected, of which at least two are exposed on the surface of intact virions. Of these two epitopes, one is inactivated by the proteolytic treatment. The third epitope could only react with its monoclonal antibody when the virus particles were solubilized with SDS. This work provides a clear demonstration that a substantial part of the M-protein spans the lipid bilayer and that the rest, protected by lipids, resists proteolytic enzymes and is prevented from binding with anti M-protein monoclonal antibodies.

Structure and composition of influenza virus. A small-angle neutron scattering study.

A detailed analysis is presented of the small-angle neutron scattering curves of homogeneous solutions of influenza B virus, both intact and after treatment with bromelain, which removes the external glycoprotein spikes. The two sets of data are consistent with the following low-resolution structure: the virus particles are spherical, about 1200 A in diameter and of Mr about 180 X 10(6). The lipid bilayer is centred at a radius of 425 A, is 40 A to 50 A thick and constitutes 25% to 28% of the virus mass. The surface glycoproteins, predominantly haemagglutinin, contribute 40% to 46% of the total mass. Surprisingly little protein is found in the interior of the virus. It is suggested that the reason for this is that many particles do not contain the full complement of ribonucleoprotein complexes. These results are in good agreement with recent scanning transmission electron microscopic measurements of molecular mass and cryo-electron microscopic observations of the same preparations. Appendix 1 describes a new method of deriving spherical shell models from contrast variation neutron scattering data on viruses, in which scattering curves from all measured contrasts are used simultaneously. There is also a discussion of the assumptions and limitations implicit in the structural interpretation of such models, with emphasis on viruses containing lipid bilayers. Appendix 2 examines the effect on the scattering curves of various arrangements of the surface glycoproteins.

[Mobility study of influenza C virus proteins in cellulose acetate electrophoresis]. / Izuchenie podvizhnosti belkov virusa grippa C v élektroforeze na atsetate tselliulozy.

Four strains of influenza C virus were studied by cellulose acetate electrophoresis. All of them were found to contain super-capsid proteins destroyed by electrophoresis apparently due to contact with sodium dodecylsulphate. According to Laver's classification, influenza C viruses may be placed with Group 3 viruses. Among major virion proteins of influenza C viruses matrix protein was found to be the most stable forming a separate protein band on cellulose acetate, and it may be readily identified and eluted from paper in preparative amounts.

[Demonstration and study of the internal proteins of the influenza B virus using solid-phase radioimmunological analysis]. / Indikatsiia i izuchenie vnutrennikh belkov virusa grippa B s pomoshch'iu tverdofaznogo radioimmunologicheskogo analiza.

Internal proteins of influenza B/USSR/14/80 virus retaining their antigenic and immunogenic activities were obtained by electrophoresis in 1% agarose. Antisera to eluted NP and M polypeptides were prepared. Study of influenza B viruses isolated in 1940-1984, performed by solid-phase radioimmunoassay, revealed no differences in the antigenic properties of nucleoprotein and matrix protein, except in the B/Yamagata/73 virus.

Reassortants of influenza B viruses for use in vaccines: an evaluation.

Three slightly different procedures for the preparation of influenza B virus reassortants from B/Lee/40 and B/Lyon/79, and B/Johannesburg/58 and B/Hong Kong/82 parental viruses are described. Following cloning procedures in eggs or allantois-on-shell cultures in the presence of antisera to B/Lee or B/Johannesburg, twenty-eight putative reassortants were obtained. Using SDS-PAGE to determine the migration rates of virion polypeptides, eighteen isolates from mixed infections of B/Lee with B/Lyon, and three isolates from mixed infections of B/Johannesburg with B/Hong Kong, were found to be reassortants. All reassortants possessed surface proteins derived from either B/Lyon or B/Hong Kong and one to three internal polypeptides from either B/Lee or B/Johannesburg. None of the reassortants showed a growth capacity in embryonated eggs as high as that of their B/Lee or B/Johannesburg parent viruses. The procedures used, and the influenza B reassortants thus generated, would appear to offer no major advantages over the existing methods and influenza B strains at present employed for the preparation of inactivated influenza virus vaccines.