Localization of the influenza virus nucleoprotein: cell-associated and extracellular non-virion forms.

Both the supernatant of influenza virus-infected chick embryo cells and allantoic fluid containing influenza virus were shown to contain non-virion nucleoprotein (NP), which reacted readily with anti-NP monoclonal antibodies. Adsorption onto erythrocytes and centrifugation at 70,000 g for 2 h resulted in the removal of about 20% of the extracellular NP, whereas centrifugation at 100,000 g for 4 h eliminated about 50%, and practically all [3H]uridine-labelled virions. These results suggest that of the extracellular NP about 30% exists in the form of ribonucleoprotein, about 20% is precipitated with virions and about 50% occurs as free molecules. Comparative analysis of the kinetics of the accumulation of NP in the supernatant of infected cells, on the cell surface and inside the cells in relation to virus production, showed that there is a significant correlation between them.

Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds.

The oligomeric structure of the influenza A virus M2 integral membrane protein was determined. On SDS-polyacrylamide gels under nonreducing conditions, the influenza A/Udorn/72 virus M2 forms disulfide-linked dimers (30 kDa) and tetramers (60 kDa). Sucrose gradient analysis and chemical cross-linking analysis indicated that the oligomeric form of M2 is a tetramer consisting of either a pair of disulfide-linked dimers or disulfide-linked tetramers. In addition, a small amount of a cross-linked species of 150-180,000 kDa, which the available data suggest contains only M2 polypeptides, was observed. The role of M2 cysteine residues in disulfide bond formation and their role in forming oligomers were examined by converting each of the two extracellular and single cytoplasmic cysteine residues to serine residues and expressing the altered M2 proteins in eukaryotic cells. Removal of either one of the N-terminal cysteines at residues 17 or 19 indicated that tetramers formed that consisted of a pair of noncovalently associated disulfide-linked dimers, suggesting that each of the cysteine residues is equally competent for forming disulfide bonds. When both cysteine residues were removed from the M2 N-terminal domain, no disulfide-linked forms were observed. When solubilized in detergent this double-cysteine mutant lost reactivity with a M2-specific mAb and exhibited an altered sedimentation pattern on sucrose gradients. However, chemical cross-linking of this double-cysteine mutant in membranes indicated that it can form tetramers. Taken together, these data suggest that disulfide bond formation, although not essential for oligomeric assembly, stabilizes the M2 tetramer from disruption by detergent solubilization.

Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses.

We determined the sequences of 7 serotypes (H4, H6, H8, H9, H11, H12, and H13) of hemagglutinin (HA) genes, which have not been reported so far. The coding regions consisted of 1692 nucleotides in H4, 1698 in H6, 1695 in H8, 1680 in H9, 1695 in H11, 1692 in H12, and 1698 in H13, and specified 564, 566, 565, 560, 565, 564, and 566 amino acids, respectively. By comparison of amino acid sequences, 13 HA serotypes could be divided into two families, i.e., an H1 group (H1, H2, H5, H6, H8, H9, H11, H12, and H13) and an H3 group (H3, H4, H7, and H10). The relationship was essentially similar to that reported by Air from the comparison of 80 amino-terminal amino acid sequence of 12 HA serotypes (G.M. Air, 1981, Proc. Natl. Acad. Sci. USA 78, 7639-7643). Though a considerable amino acid sequence difference exists between certain HA serotypes, several amino acid residues in fusion peptides (HA2(1-11)) and receptor-binding sites (HA1(98), -134, -138, -153, -183, and -195) were shown to be conserved among the 13 HA serotypes. Human H1 and avian H3, H4, H8, and H10 viruses preferentially bound NeuAc alpha 2,3Gal sequences, whereas human H2 and H3 and avian H6 and H9 viruses bound NeuAc alpha 2,6Gal sequences, although the amino acid residues at position 226 of human H2 and avian H6 and H9 serotype HAs are glutamine. These results show that the amino acid residue at position 226 is not necessarily a determinant of receptor specificity for all serotypes.

[The anomalous isoelectric properties of influenza virus matrix protein M1]. / Anomal'nye izoélektricheskie svoistva matriksnogo belka M1 virusov grippa.

The isoelectric point (pI) values of matrix protein M1 of influenza A, B, and C viruses, calculated theoretically on the basis of its primary structure, were found to be about 10.0. However, experimental pI determination by electrofocusing in ampholyte-containing polyacrylamide gel showed it to be 5.0 for M1 protein isolated from A/WSN/33 (H1N1) and A/Aichi/2/68 (H3N2) viruses by mild deproteinization with nonionic detergents. This marked discrepancy between experimental and theoretical pI values indicated that influenza virus matrix protein M1 possessed an unusual tertiary structure and/or intensive posttranslational addition of charged residues.

Heterogeneous forms of polymerase proteins exist in influenza A virus-infected cells.

In influenza virus-infected cells a virus coded polymerase that consists of three polypeptide subunits, namely PB1, PB2 and PA, mediates both transcription and replication. Radioimmunoprecipitation with monospecific antisera to each of the polymerase proteins revealed additional forms of PB1 and PA proteins in infected cells. PA antiserum detected two additional proteins of 62k and 60k and PB1 antiserum recognized two additional proteins of 85k and 70k. Further investigation was carried out on the 62k PA and 85k PB1 related proteins. Limited proteolysis peptide mapping showed that these proteins are subsets of their normal counter-parts. These new forms of polymerase proteins are designated as "b" forms (PAb and PB1b) to distinguish them from the previously recognized forms designated as "a" forms (PAa and PB1a). Both PAb and PB1b proteins were found in cells infected with all the influenza type A viruses tested indicating that they are evolutionarily conserved. Pulse chase experiments showed that the "b" forms are not derived from "a" forms. This suggested that "b" forms are translated independently. The "b" forms were not detected in purified virus but were found to be associated with intracellular RNP templates, suggesting a role for these proteins in intracellular virus replication events.

NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells.

Purified viral preparations of influenza A virus were examined for the presence of NS2 protein hitherto considered as a viral nonstructural protein that is present only in infected cells. Analysis of purified virus by radioimmunoprecipitation with monospecific antisera to NS2 revealed its presence in the virus particle suggesting that it is a viral structural protein. NS2 protein was also shown to be phosphorylated in infected cells in this study. This brings the number of influenza virus phosphoproteins to three which include NP, NS1, and NS2. These observations raise important questions about the role of NS2 in the replication of influenza virus.

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.

[Standardization of conditions for detecting the internal proteins of the influenza virus in studying their antigenic properties in solid-phase immunoenzyme analysis]. / Standartizatsiia uslovii vyiavleniia vnutrennikh belkov virusa grippa pri issledovanii ikh antigennykh svoistv v tverdofaznom immunofermentnom analize.

The influence of the conditions of adsorption and virion destruction by freezing-thawing and detergents on the detection of M1 and NP proteins of different influenza virus strains by solid-phase enzyme immunoassay with direct virion adsorption on polystyrene was studied. It was found that for the detection of M1 protein the optimal conditions included virion disruption with detergent and adsorption to polystyrene at 4 degrees C, and for NP protein disruption by freezing-thawing at adsorption to polystyrene at 37 degrees C. In the study of the antigenic properties of protein M1 of different influenza virus strains using monoclonal antibodies it was shown to be necessary, first, to achieve maximum detection of proteins and, second, to standardize the amount of the adsorbed antigen with polyclonal antibodies.

[The structure of oligosaccharide fragments of glycoproteins from influenza virus A/Krasnodar/101/59/(H2N2)--heavy and light chains of hemagglutinin and neuraminidase]. / Struktura oligosakharidnykh fragmentov glikoproteinov virusa grippa A/Krasnodar/101/59(H2N2)--tiazheloi i legkoi tsepei gemaggliutinina i neiraminidazy.

The structure and heterogeneity of carbohydrate chains of hemagglutinin (HA) and neuraminidase (NA), the surface glycoproteins of influenza virus A/Krasnodar/101/59 (H2N2), were investigated. Hemagglutinin was reduced with beta-mercaptoethanol and its heavy (HA1) and light (HA2) chains were separated by gel chromatography. Amino acid and sugar composition of HA1, HA2 and NA was elucidated. The carbohydrate chains of the glycoproteins were cleaved off by the alkaline LiBH4 treatment and oligosaccharides were reduced with NaB[3H]4. They were fractionated by subsequent two-step HPLC on Ultrasphere-C8 and Zorbax-NH2 columns with simultaneous identification using nonlabelled oligosaccharides of known structures. Some of the major oligosaccharides isolated from HA1, HA2 and NA were thus identified as high mannose chains, containing 5-9 mannose residues, and complex chains, first of all biantennary chains having or not having bisecting N-acetylglucosamine and/or fucose residues. The approach which has been developed enables one to study the structure and heterogeneity of carbohydrate chains starting from one nmole of a desialylated N-glycoprotein.

Differential phosphorylation of the nucleoprotein of influenza A viruses.

An analysis of the nucleoprotein (NP) of 29 different influenza A viruses by phosphopeptide fingerprinting revealed three prototype patterns. The first, which was a complex pattern consisting of six to seven phosphopeptides, another which was relatively simple consisted of two or three phosphopeptides, and a third one which was complex but was missing the main phosphopeptide shared by the two other patterns. Phosphoserine was the only labelled phosphamino acid detected. A tentative deduction of two of the phosphate attachment sites (serine residues at positions 3 and 473) could be made by comparison of the known amino acid sequences of the NPs of 25 strains. No correlation was found between species specificity or subtype or year of isolation of the strains. During the infectious cycle the fingerprint underwent significant changes, indicating subtle phosphorylation and dephosphorylation of the NP at various stages during viral multiplication. Most of the phosphopeptides were metabolically stable; however one major phosphopeptide, which was not found in the NP of mature virions, exhibited a high turnover (presumably serine at position 3). The phosphopeptide fingerprint could be significantly influenced in vivo by the specific stimulation of cellular protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate or by its inhibition with the isoquinoline sulphonamide H7.H7 specifically inhibited the replication of influenza A viruses by deregulation of viral protein synthesis without interfering with the multiplication of a parainfluenza virus (Newcastle disease virus), an alphavirus (Semliki Forest virus) or a flavivirus (West Nile). Therefore the correct phosphorylation of the NP of influenza viruses appears to be essential for influenza virus replication.

Fluorescence anisotropy of UV-irradiated viruses.

The steady-state fluorescence anisotropy measurements on influenza and parainfluenza viruses, showed no changes in the microviscosity of the viral membranes after exposure to UV-irradiation, when a fluorescent probe was used, but the intrinsic fluorescence of viral proteins presented, under the same experimental conditions, a significant difference of anisotropy behaviour in the two viruses used.

[The spatial interrelationships between influenza virus hemagglutinin and the lipid phase in the liposomal membrane]. / Issledovanie prostranstvennykh vzaimootnoshenii mezhdu gemaggliutinom virusa grippa i lipidnoi fazoi v membrane liposom.

A scheme has been proposed demonstrating the location of tryptophan residues of hemagglutinin molecule in relation to the middle of the lipid layer 1.2 nm thick with a fluorescent probe pyrene. In the immediate proximity to it, one tryptophanyl of protein molecule is located in a hydrophobic "pocket". At a distance of 2.85 nm from the middle of the lipid zone 3 tryptophanyls are located and the remaining five at a distance over 3.6 nm. After treatment with proteolytic enzyme bromelin of the liposomes with hemagglutinin incorporated into their bilayer, the hydrophobic "anchor" of protein molecule contains one tryptophanyl which is raised by 0.3 nm and its hydrophobic environment is changed.

[The structure of complex carbohydrate chains of hemagglutinin from influenza viruses A/Kiev/59/79 (H1N1), A/Chile/1/83/25(H1N1) and X/79(H3N2)]. / Struktura uglevodnykh tsepei kompleksnogo tipa v gemaggliutinine virusov grippa A/Kiev/59/79(H1N1), A/Chili/1/83/25(H1N1) i X/79(H3N2).

An earlier developed method of identification of oligosaccharides by HPLC was used for studying the carbohydrate chains of three hemagglutinins from various influenza virus strains. The structures of main oligosaccharides of the complex type were elucidated on the basis of their chromatographic characteristics and monosaccharide composition. Oligosaccharide patterns varied in the above hemagglutinin samples but in all cases the major complex chains were fucosylated and nonfucosylated biatennary chains; bisected and triantennary chains were also found.

[An optical sensor for analyzing the flow of biological particles in chromatographic processes]. / Opticheskii datchik dlia analiza potoka biologicheskikh chastits v protsessakh khromatografii.

The design of an optic flow sensor operating in the UV spectrum is described. The sensor is intended to monitor changes in the size and/or concentration of light-diffusing particles against the background of low-molecular admixtures, as well as for analysis of these admixtures. The sensor has been employed in gel chromatography and adsorption chromatography of virus-containing suspensions.

Mixed anionic detergent/aliphatic alcohol-polyacrylamide gel electrophoresis alters the separation of proteins relative to conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The order and relative mobility of proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is affected by unknown components that are differentially present in SDS preparations obtained from different sources [J.B. Swaney, G.F. Vande Woude, and H.L. Bachrach (1974) Anal. Biochem. 58, 337-346]. The modified separation capabilities of such SDS preparations are useful but the use of this phenomenon in a controlled manner requires that the components responsible for the altered separation be identified. Accordingly, this paper describes a polyacrylamide gel electrophoresis system [mixed alcohol/detergent-polyacrylamide gel electrophoresis (MAD-PAGE)] that employs a mixture of alcohol and detergent instead of SDS alone to modify and enhance protein separation relative to conventional SDS-PAGE. A defined mixture consisting of four sulfated alkyl detergents (dodecyl sulfate, tetradecyl sulfate, hexadecyl sulfate, octadecyl sulfate) as well as the four alcohols of corresponding aliphatic chain length was found to be effective at duplicating the electrophoretic effect of USP-grade SDS and thus changed the relative order and position of polypeptides on electrophoresis relative to conventional SDS-PAGE. This method serves as an adjunct to conventional SDS-PAGE by providing another means of resolving proteins that are not normally resolved by SDS-PAGE. Further, it was found that MAD-PAGE is capable of resolving the NS1 protein of influenza virus into three fractions, whereas conventional SDS-PAGE yields one electrophoretic species. Reelectrophoresis of these novel NS1 bands by conventional SDS-PAGE indicated that they were not modified during MAD-PAGE and probably represented distinct molecular forms present in infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a unique protein produced by influenza A virus recovered from a long-term persistent infection.

Virus isolated from a persistent infection initiated in BHK cells with influenza A/WSN/33 (H1N1) produced an unusual pattern of protein synthesis in productive infections of BHK cells: The levels of NP and M1 proteins were slightly reduced compared to an infection with wild-type WSN, while the other proteins (Pb1, Pb2, Pa, HA, NS1, and NS2) were synthesized at very low or undetectable levels. In addition, a new viral protein with an approximate molecular weight of 11 kDa (Pi protein) is made (Frielle et al., Virology 138, 103-117, 1984). When viral RNA was analyzed by the Northern blot technique, a deletion was found in the NS gene segment and in NS1 mRNA; all other RNAs were full-sized. Immunoprecipitation of in vitro translation products demonstrated that the Pi protein reacts specifically with anti-NS1 serum. In addition, the Pi protein, like the NS1 of the parental wild-type virus, accumulated in the nucleus of infected cells. These results indicate that the Pi protein is a mutated form of the NS1 protein encoded by a deleted NS segment and suggest that this mutation may be involved in the expression of the persistent virus phenotype.

The intracellular distribution of influenza virus matrix protein and nucleoprotein in infected cells and their relationship to haemagglutinin in the plasma membrane.

Pre- and post-embedding immune electron microscopy techniques employing ferritin and large and small gold markers to detect cell surface and intracellular antigens respectively, have been combined in a study of influenza virus-infected cells. This has permitted, for the first time, the simultaneous detection of intracellular virus matrix protein (M), nucleoprotein (NP) and membrane haemagglutinin (HA). The technique facilitated an investigation of the possible physical interrelationship between these three proteins both in the infected cell, and on the infected cell membrane. Electron-dense bodies uniformly labelled by antibody to M protein were observed in the nucleus and cytoplasm. Similarly, NP was detected in both the nucleus and cytoplasm. Approximately 50% of the nuclear NP was located in close proximity to the M protein-containing dense bodies but mainly on the perimeter of the structures. A similar relationship of NP to the M-containing dense bodies was observed in the cytoplasm. M protein and NP were readily detected in sections of budding virions. Labelling of these proteins was also observed on the cytoplasmic face of the plasma membrane but the density of labelling only occasionally approached that of newly formed virions. These findings suggest that budding occurs very quickly after the internal proteins arrive at the plasma membrane. Double labelling experiments on the cell surface indicate that NP and HA behave as independent molecules and do not form tight complexes with each other.

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.