Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.

Abstract

Based on nucleotide sequence analysis of the hemagglutinin (HA) gene from the virulent and avirulent A/chicken/Pennsylvania/83 influeza viruses, it was previously postulated that acquisition of virulence was associated with a point mutation that resulted in loss of a glycosylation site. Since there are two potential glycosylation sites in this region of the HA molecule and since all Asn-Xaa-Thr/Ser sequences in the HAs of different strains are not necessarily glycosylated, the question remained open as to whether either one of these sites was glycosylated. We now provide direct evidence that a site-specific glycosylation affects cleavage of the influenza virus HA and thus virulence. We have identified the glycosylation sites on the HAl subunit from the virulent and avirulent strains by direct structural analysis of the isolated proteins. Our results show that the only difference in glycosylation between the HAls of the virulent and avirulent strains is the lack of an asparagine-linked carbohydrate on the virulent HAl polypeptide at residue 11. Further, we show that the HAls of both the avirulent and virulent viruses are not glycosylated at one potential site, while all other sites contain carbohydrate. Amino acid sequence analysis of the HAl of an avirulent revertant of the virulent strain confirmed these findings.In April 1983, an avirulent (avir) influenza virus, A/chicken/Pennsylvania/1/83 (Chick/Penn) (HSN2), appeared in chickens of eastern Pennsylvania (1). By October 1983, this virus had become virulent (vir), causing up to 80% mortality in domestic poultry (1, 2). Gene reassortment studies established that the difference between the viruses was a mutation of the hemagglutinin (HA) (3), a major surface glycoprotein responsible for viral attachment to, and penetration of, host cells. Nucleotide sequencing analysis suggested that the critical mutation eliminated a possible site for the attachment of an N-linked oligosaccharide at Asn-11 of HAl of the avirulent virus by altering a sequence for glycosylation. Preliminary evidence based on molecular weight differences in HAl and growth in the presence of tunicamycin suggested loss of a carbohydrate (4). However, since not all Asn-XaaSer/Thr sites are glycosylated in different influenza virus HAs, we do not know how many of the potential glycosylation sites contain carbohydrate, and since all of the influenza virus HAs studied to date contain a carbohydrate at this location in the HA molecule (5, 6), the question of glycosylation at this site remained unresolved.The HA protein of influenza viruses is post-translationally modified by cleavage at a connecting peptide region into subunits HA1 and HA2 (7). This cleavage is a prerequisite for viral infectivity (8), and a correlation has been drawn between HA cleavage and virus production in tissue culture and the virulence of the strain (9-11). Although exogenously added trypsin can cleave the HA and permit infectivity in vitro, the mechanism by which HA is processed and cleaved in vivo is unknown.To determin...

Kex2p: a model for cellular endoprotease processing human immunodeficiency virus type 1 envelope glycoprotein precursor

“…Another possibility may be the masking effect of carbohydrate chains of the N-glycosylation site. This has been shown for the uncleaved hemagglutinin of the avirulent strain of H5N2 influenza virus which differs from the correctly cleaved hemagglutinin of the virulent strains by the presence of an additional N-glycosylation site at Asnll [44]. On the other hand, Dedera et al [45] recently reported the importance of two highly conserved gp41 cysteines residues in the processing of HIV-1 gp160.…”

Role of stem glycans attached to haemagglutinin in the biological characteristics of H5N1 avian influenza virus

“…Previous studies in MDCK cells and embryonated chicken eggs have revealed that the N-glycan at Asn12 is crucial for virus replication (Wagner et al, 2002). It has been proven that the HA sequence at positions 10-12, namely Asn-Asn-Ser, in either avirulent or virulent strains of H5N2 AIV is not a glycosylation site and that the HA protein in an avirulent H5N2 AIV is glycosylated at residue Asn11 (Deshpande et al, 1987). In the present study, the H5N1 AIV A/Mallard/ Huadong/S/2005 strain possesses the same sequence, AsnAsn-Ser-Thr, at positions 10-13.…”

“…It has been previously reported that a single point mutation in the HA of the Ck/Penn (H5N2) virus resulted in the loss of N-linked glycans in the stalk region at residue 11, leading to virulence (Deshpande et al, 1987). Moreover, it has been demonstrated that glycans in the stalk and the length of the connecting peptide determine the cleavability of the H5 influenza virus HA (Kawaoka & Webster, 1989).…”

N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

“…These data suggest that changes in the E2 protein due to the mutation N185A in N3v, unlike the N116A mutation in N1v, are not sufficient to alter virus range within the host. Cleavage and glycosylation patterns of the hemagglutinin gene of H5 avian influenza viruses have been shown to affect pathogenicity in chickens (4,11). More recently it has been shown that glycosylation patterns of the neuraminidase gene of highly pathogenic H5N1 avian flu viruses are important for increased virulence in chickens (13).…”