Role of overlapping glycosylation sequons in antigenic properties, intracellular transport and biological activities of influenza A/H2N2 virus haemagglutinin
The haemagglutinin (HA) protein of influenza A/H2N2 virus possesses five oligosaccharide attachment sites, two of which have overlapping glycosylation sequons at positions 20–23 (NNST) and 169–172 (NNTS). Here, the role of these two oligosaccharide attachment sites is investigated with regard to antigenic property, intracellular transport and biological activity of the HA protein. Glycosylation-site HA mutants with mutation(s) in their overlapping glycosylated sequons, each of which had one or two oligosaccharide attachment sites removed, were constructed. Comparison of electrophoretic mobility between the wt and mutant HA proteins showed that both Asn residues 20 and 21 and Asn residues 169 and 170 could be used for glycosylation. Analysis of reactivity of the mutants with anti-HA monoclonal antibodies suggested that amino acid changes at these two positions result in a conformational change of the HA molecule. Even if oligosaccharide chains linked to Asn 20 or 21 and Asn 169 or 170 are eliminated, the antigenic properties, intracellular transport and biological activities are not influenced strongly. Thus it is reasonable to conclude that the two overlapping glycosylation sequons at positions 20–23 and 169–172 are conserved among all of the HAs of influenza A/H2N2 viruses because conservation of the amino acid sequence itself rather than that of N-glycosylation is essential for the formation of the proper conformation, intracellular transport and biological activities of the H2 subtype HA.
BauseE.,
LeglerG.1981; The role of the hydroxy amino acid in the triplet sequence Asn–Xaa–Thr(Ser) for the N -glycosylation step during glycoprotein biosynthesis. Biochemical Journal 195:639–644
BrownL.,
WardC.,
WhiteD.,
JacksonD.1981; Antigenic carbohydrate determinants on influenza hemagglutinin. In Genetic Variation Among Influenza Viruses pp 233–252 Edited by
NayakD. P.
London: Academic Press;
DeshpandeK. L.,
FriedV. A.,
AndoM.,
WebsterR. G.1987; Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proceedings of the National Academy of Sciences, USA 84:36–40
GallagherP. J.,
HenneberryJ. M.,
SambrookJ. F.,
GethingM.-J.1992; Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus. Journal of Virology 66:7136–7145
HongoS.,
SugawaraK.,
MurakiY.,
KitameF.,
NakamuraK.1997; Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus. Journal of Virology 71:2786–2792
InksterM. D.,
HinshawV. S.,
SchulzeI. T.1993; The hemagglutinins of duck and human H1 influenza viruses differ in sequence conservation and in glycosylation. Journal of Virology 67:7436–7443
MatrosovichM.,
ZhouN.,
KawaokaY.,
WebsterR.1999; The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. Journal of Virology 73:1146–1155
MurakiY.,
HongoS.,
SugawaraK.,
MatsuzakiY.,
TakashitaE.,
KitameF.,
NakamuraK.1999; Location of a linear epitope recognized by monoclonal antibody S16 on the hemagglutinin–esterase glycoprotein of influenza C virus. Virus Research 61:53–61
NobusawaE.,
AoyamaT.,
KatoH.,
SuzukiY.,
TatenoY.,
NakajimaK.1991; Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology 182:475–485
OhuchiM.,
OrlichM.,
OhuchiR.,
SimpsonB. E.,
GartenW.,
KlenkH.-D.,
RottR.1989; Mutations at the cleavage site of the hemagglutinin alter the pathogenicity of influenza virus A/chick/Penn/83 (H5N2). Virology 168:274–280
OhuchiM.,
FeldmannA.,
OhuchiR.,
KlenkH.-D.1995; Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity. Virology 212:77–83
OhuchiR.,
OhuchiM.,
GartenW.,
KlenkH.-D.1997; Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity. Journal of Virology 71:3719–3725
RobertsP. C.,
GartenW.,
KlenkH.-D.1993; Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin. Journal of Virology 67:3048–3060
SkehelJ. J.,
StevensD. J.,
DanielsR. S.,
DouglasA. R.,
KnossowM.,
WilsonI. A.,
WileyD. C.1984; A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proceedings of the National Academy of Sciences, USA 81:1779–1783
SugawaraK.,
NishimuraH.,
KitameF.,
NakamuraK.1986; Antigenic variation among human strains of influenza C virus detected with monoclonal antibodies to gp88 glycoprotein. Virus Research 6:27–32
TakebeY.,
SeikiM.,
FujisawaJ.,
HoyP.,
YokotaK.,
AraiK.,
YoshidaM.,
AraiN.1988; SR α promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Molecular and Cellular Biology 8:466–472
TarentinoA. L.,
MaleyF.1974; Purification and properties of an endo- β- N -acetylglucosaminidase from Streptomyces griseus. Journal of Biological Chemistry 249:811–817
TsuchiyaE.,
SugawaraK.,
HongoS.,
MatsuzakiY.,
MurakiY.,
LiZ.-N.,
NakamuraK.2001; Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus. Journal of General Virology 82:2475–2484
TsuchiyaE.,
SugawaraK.,
HongoS.,
MatsuzakiY.,
MurakiY.,
LiZ.-N.,
NakamuraK.2002; Effect of addition of new oligosaccharide chains to the globular head of influenza A/H2N2 virus haemagglutinin on the intracellular transport and biological activities of the molecule. Journal of General Virology 83:1137–1146
WaterfieldsM. D.,
GethingM. J.,
ScraceG.,
SkehelJ. J.1980; The carbohydrate side chains and disulphide bonds of the haemagglutinin of the influenza virus A/Japan 305/57 (H2N1). In Structure and Variation in Influenza Virus pp 11–20 Edited by
LaverG.,
AirG.
Amsterdam: Elsevier;
WilsonI. A.,
SkehelJ. J.,
WileyD. C.1981; Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289:366–373
Role of overlapping glycosylation sequons in antigenic properties, intracellular transport and biological activities of influenza A/H2N2 virus haemagglutinin