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 glycoprotein of influenza C virus is the haemagglutinin, esterase and fusion factor.

Of the biological activities of influenza C virus, haemagglutination, receptor inactivation and fusion, only the latter has been conclusively correlated with its surface glycoprotein (gp). We have purified the gp by octylglucoside treatment of influenza C virions followed by centrifugation into a sucrose gradient. Evidence was obtained that gp also represents the receptor-destroying enzyme of influenza C virus, which has been characterized as a neuraminate 9-O-acetylesterase: (i) it inactivated the receptors for influenza C virus on chicken erythrocytes; (ii) it had acetylesterase activity as indicated by the release of acetate from bovine submandibulary mucin; (iii) monoclonal antibodies directed against gp inhibited the acetylesterase activity of influenza C virus. Although purified gp was unable to agglutinate chicken red blood cells, it blocked haemagglutination by viruses. This finding as well as the haemagglutination inhibition activity of monoclonal anti-gp antibodies indicate that gp is also responsible for the haemagglutinating activity of influenza C virus. Thus, as the influenza C glycoprotein is the only myxovirus glycoprotein with three different activities, we propose the designation HEF in order to describe its function as a haemagglutinin (H), an esterase (E) and a fusion factor (F).

Nucleotide sequence of the gene encoding the fusion glycoprotein of Newcastle disease virus.

The nucleotide sequence of the gene encoding the fusion (F) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from cDNA clones obtained from virion RNA. The gene is 1792 nucleotides long, including mRNA start and polyadenylation signals typical of paramyxoviruses. The single open reading frame encodes a polypeptide of 553 amino acids, with a predicted molecular weight of 59042. The F polypeptide has three regions of high hydrophobicity: an N-terminal signal peptide, the N terminus of F1 (known from protein sequencing) and a C-terminal membrane-spanning region by which the F glycoprotein is anchored to the membrane. The cleavage site of F0 is located in a highly basic region of the F polypeptide. Five potential asparagine-linked glycosylation sites are present in the amino acid sequence, of which one is in F2 and the others in F1. Comparison of the NDV F amino acid sequence to those from other paramyxoviruses reveals homology to Sendai virus, simian virus 5 and human respiratory syncytial virus. There is also limited homology between the N terminus of F1 of NDV and the N termini of HA2 of influenza viruses. Post-translational modifications of the NDV F polypeptide are discussed in the light of information provided by the amino acid sequence.

Analogous amino acid sequences in myelin proteolipid and viral proteins.

Computer analysis of the intrinsic membrane protein, myelin proteolipid, shows strong sequence similarities between the putative extramembrane segments of the proteolipid protein and a number of viral proteins, several of which infect humans. These similarities are even more striking than those reported previously between viral proteins and the encephalitogenic myelin basic protein (MBP). These findings, along with other reports of molecular mimicry by viruses, suggest that immunological cross-reactions between virus-induced antibodies or T-cells and analogous antigenic determinants (epitopes) in myelin proteolipid could be involved in the pathophysiology of multiple sclerosis or post-infectious demyelinating syndromes.

[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.

Expression of influenza virus NS2 nonstructural protein in bacteria and localization of NS2 in infected eucaryotic cells.

The nonstructural NS2 protein of influenza A/PR/8/34 virus was efficiently expressed in bacteria, and monospecific antisera were prepared against the bacterially synthesized polypeptide. These antisera were cross-reactive among the NS2 proteins of various influenza A viruses. However, they did not react with the NS2 of influenza B/Lee/40 virus nor with other proteins of influenza A viruses such as NS1. Antisera against NS2 were used to determine that the NS2 protein is localized in the cell nucleus during influenza virus infection, as shown by immunofluorescence microscopy. Cells infected with simian virus 40 recombinants containing the influenza virus NS gene revealed that both the NS1 and NS2 proteins appeared in the nucleus, even in the absence of expression of other influenza virus-specific components.

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.

Low resolution structure of the influenza C glycoprotein determined by electron microscopy.

The influenza C glycoprotein is clearly seen to be a trimer in specimens prepared with uranyl stains. Three-dimensional reconstructions from naturally occurring hexagonal arrays show that at low resolution (approximately 30 A) the influenza C glycoprotein exhibits similar features to the haemagglutinin glycoprotein of influenza A. Both have a triangular stalk near the membrane. Further from the membrane, the stalk becomes broader and the monomers more separated, leaving an open centre. The molecule narrows at the top. The regions of greatest contact between adjacent trimers in the arrays are situated nearer the distal end of the molecule. These contact zones can be related to equivalent zones on the influenza A haemagglutinin. Differences between the structure of the influenza A haemagglutinin glycoprotein determined by X-ray analysis and reconstructions of the influenza C glycoprotein are greatest at either end of the molecule, where the reconstructions are least reliable. Ordered glycoprotein arrays have not been observed on influenza C virions incubated at low pH. The staining patterns of glycoproteins on intact virions are essentially determined by the pH at which the virus is incubated, and the stain type, but not the pH of the stain.

[Comparative characteristics of the biophysical properties of influenza A, B and C proteins]. / Sravnitel'naia kharakteristika biofizicheskikh svoistv belkov virusov grippa A, B i C.

A comparative analysis of electrophoregrams of influenza A, B, and C virus proteins in polyacrylamide gel and in agarose was made. Separation of proteins was similar in three influenza C virus strains tested and differed from that of influenza A and B virus proteins. The possibility of preparative isolation of supercapsid and internal proteins of influenza C virus with preservation of their antigenic and immunogenic properties was demonstrated. Antisera to internal proteins and hemagglutinin of influenza C virus were prepared. Both antisera reacted in the double immunodiffusion test and ELISA, and antiserum to hemagglutinin also in hemagglutination inhibition test.

Characterization of three highly purified influenza virus strains by electron microscopy.

Mass determinations on highly purified influenza virus preparations were performed using the technique of scanning transmission electron microscopy. The masses of the three strains, X49, B/Singapore/222/79 and B/Hong Kong/8/73 were determined. The average value was 174 X 10(6) daltons with only small differences between the three strains. The mass of virus particles after removal of the protruding spike proteins, haemagglutinin and neuraminidase by bromelain treatment was determined to be 86 X 10(6) daltons. From the mass difference and the known molecular weight of the spike proteins the number of spikes was estimated to lie in the range 400 to 500.

Comparison of nine strains of influenza C virus in growth characteristics and viral polypeptides. Brief report.

Nine strains of influenza C virus were compared in their growth characteristics and viral polypeptides using LLCMK2 cells. The results suggested that influenza C virus undergoes genetic changes like influenza A or B virus.

Comparative studies on influenza B virus. II. The effect of host cells on biological properties and polypeptide composition.

The reproduction patterns of various influenza B virus strains isolated in 1970-1976 using roller cultures of MDCK cells and chick embryos (CE) were compared. The cultural and allantoic virus populations did not differ in their sensitivity to non-specific inhibitors from mammalian sera and in their reactivity with specific haemagglutinins (HA). The content of infectious virus and HA in the harvested allantoic fluids as compared to medium fluids were 94- and 8-fold higher, respectively, even though the fluids did not differ in the titres of complement-fixing (CF) antigen. The mol. weight (MW) of HA1 polypeptide of the B/Len/75 virus prepared in culture was higher than that Of the allantoic virus (57.5 K and 55.5 K, respectively). Under reducing conditions, the HA of the virus from culture was represented mostly by the uncleaved HA0 polypeptide, while that of the allantoic virus by the HA1 and HA2 subunits. Under non-reducing conditions, the virus from medium fluid was found to contain glycopeptide D with MW of 90 K.

Comparative studies on influenza B virus. I. Biological properties and polypeptide composition of strains isolated in different years.

Influenza B viruses isolated in 1940, 1950 and 1970 belonged to antigenically remote subgroups according to the antigenic composition of haemagglutinin (HA). Antigenically related strains isolated in 1972-1976 differed by their reproduction capability, by their sensitivity to inhibitors in mammalian sera and by affinity to specific antibodies in human sera. Certain correlation between these properties was observed by strains of the Hong Kong variant. Polyacrylamide gel electrophoresis (PAGE) showed that the viruses under study differed in the mobility of the major HA chain (HA1). Molecular weights (MW) of HA1 of antigenically similar influenza B viruses isolated in 1972-1976 revealing different biological properties varied from 52.5 K to 58 K. Neuraminidase (NA) of influenza B/Leningrad/369/75 virus purified by electrophoresis in acetate cellulose consisted of 2 polypeptides with apparent MW of 62 K and 57 K. All viruses examined showed similar mobility of the NA polypeptide MW 57 K; the mobility of the former polypeptide was similar to that of nucleoprotein (NP).

Analyses of structural polypeptides of seven different isolates of influenza C virus.

The major structural polypeptides (gp88, NP and M) of seven different influenza C virus strains isolated between 1947 and 1981 in U.S.A. and Japan were compared by SDS-polyacrylamide gel electrophoresis and one-dimensional mapping of the peptide fragments produced after limited proteolysis with various proteases. Of the three polypeptides analysed, the membrane (M) protein appeared to be the most highly conserved since the electrophoretic mobility as well as the mapping pattern of this protein was found to be identical among all seven strains. The structure of nucleoprotein (NP) was also found to be highly conserved. The proteins of five isolates from 1964 to 1981 showed migration rates and mapping patterns indistinguishable from each other though they were slightly different in mapping patterns from the earlier isolates, C/Taylor/1233/47 and C/JJ/50. The similarities between influenza C strains were also evident with the surface glycoprotein, gp88. The gp88 proteins of the five strains isolated in 1947, 1950, 1971 and 1981 were virtually identical in migration rates as well as in mapping patterns, while the two isolates of 1964 and 1974 showed minor differences. These results strongly suggest that the surface glycoprotein of influenza C virus is structurally much more stable than the haemagglutinin and neuraminidase glycoproteins of influenza A and B viruses. Further, the findings that differences from the original influenza C strain, Taylor/1233/47 were detectable in the strains isolated in 1964 and 1974 but not in the strains isolated in 1971 and 1981 suggest that unlike the antigenic drift of types A and B influenza viruses, the structural variation of gp88 may not be a sequential event.

Nucleic acid-binding properties of adenovirus structural polypeptides.

The ability of adenovirus structural polypeptides to bind nucleic acids was assessed by separating the polypeptides on SDS-polyacrylamide gels, transferring them electrophoretically to nitrocellulose and probing with 32P-labelled nucleic acids. Polypeptides IVa2, V, VI and VII, as well as trace amounts of pVII and a polypeptide of apparent mol. wt. 40 X 10(3) were able to bind label under these conditions. Labelling was also detected with a smaller polypeptide, possibly related to the cleavage products of pVII and/or pVI. The binding of DNA to polypeptide VI appeared to be more sensitive to detergents than the others. No sequence specificity could be detected in the DNA binding.

[Abortive influenza virus infection of Ehrlich cells. Virion and subcellular fraction proteins]. / Abortivnaia infektsiia virusa grippa v kletkakh Erlikha. Belki virionov i subkletochnykh fraktsii.

Ehrlich ascitic carcinoma cells infected with the WSN strain of influenza virus produced noninfectious virus particles containing no M protein. Polypeptides P1, NP, and NS were synthesized in the infected ascites cells to the same concentration was in permissive cells (chick fibroblasts and MDCK cells); the synthesis of HA and M proteins was markedly disturbed, however. In the permissive cells, M protein was found in the fraction of the perinuclear martial and plasma membranes whereas in the ascitic cells M protein was detected only in perinuclear material but not in plasma membrane. Thus, nonpermissiveness of the ascitic cells appears to be due to disturbance of both synthesis and transport of M protein to the plasma membrane.

[Reaction of influenza viruses with thymus-dependent lymphocytes]. / Reaktionen von Influenzaviren mit thymusabhängigen Lymphozyten.

Apart from the humoral immune response against influenza viruses recent years much attention has focussed on problems of cell-mediated immunity. The most important cell-mediated immune reactions of humans and experimental animals against influenza viruses and the most used in-vitro-assays were reviewed in the present paper. Especially the specificity of these reactions against the surface antigens of influenza viruses, hemagglutinin and neuraminidase was presented. A lot of open questions in the field of the cellular immune defense against influenza viruses were also shown.