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

Abstract

There are three conserved N-linked glycosites, namely, Asn10, Asn23 and Asn286, in the stem region of haemagglutinin (HA) in H5N1 avian influenza viruses (AIVs). To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns of stem glycans on the HA of A/Mallard/ Huadong/S/2005. The results indicated that these three N-glycans were dispensable for the generation of replication-competent influenza viruses. However, when N-glycans at Asn10 plus either Asn23 or Asn268 were removed, the cleavability of HA was almost completely blocked, leading to a significant decrease of the growth rates of the mutant viruses in MDCK and CEF cells in comparison with that of the WT virus. Moreover, the mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn10, revealed a significant decrease in thermostability and pH stability compared with the WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing antibodies against the WT virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the WT virus. Taken together, these data strongly suggest that the HA stem glycans play a critical role in HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs.

Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design

“…6). These results are in agreement with a recent study using mutant viruses with the loss of N-glycans in the globular head region with lowered immunogenicity in mice (12). In contrast, others have reported that removal of N-glycans in the globular head region resulted in increased virulence in mice (35,36) and improved live-attenuated vaccine immunity in ferrets (37).…”

“…All experiments were performed at least two times each. (11,12), we found four potential H5HA N-glycosylation sites, with three located in the HA1 stem region (positions N10, N11, and N23) and one in the HA2 stem region (N484) (Fig. 1A).…”

“…No dose-dependent neutralization activities were shown for the antisera obtained from the PBS-immunized groups. Additionally, we also obtained the H5 and H1 mutant proteins by the removal of three potential N-linked glycosylation sites located in the globular head regions (H5HA residues 154, 165, and 286 and pH1N1 residues 109, 298, and 309), whereas only the N-glycan removal of H5HA residue 286 was recently reported (12). Antisera obtained from immunizations using the globular head mutant proteins H5 N154A, H5 N165A, H5 N286A, H1 N109A, H1 N298A, and H1 N309A were also measured against the H5N1, pH1N1, and H3N2 viruses.…”

“…However, while N-linked glycosylation sites on the globular head are variable among different strains and different subtypes (9), N-linked glycosylation sites in the stem region are mostly well conserved among various influenza virus strains (10). To date, several reports indicate that N-glycans in the HA1 stem regions of H7N1 and H5N1 viruses can affect the structural stability of less efficient HA cleavage, virus fusion, and virus replication (11,12). It remains unclear whether N-glycans in the HA stem region affect anti-influenza virus immune responses, especially in terms of eliciting broadly neutralizing antibodies (bNAbs) and increasing protective immunity.…”

The Length of N-Glycans of Recombinant H5N1 Hemagglutinin Influences the Oligomerization and Immunogenicity of Vaccine Antigen

“…Indeed, oligosaccharides attached to the globular head of HA were shown to modulate virus antigenic properties (8–10) and its receptor binding (11, 12). The oligosaccharides attached to the stem region were suggested to play a critical role in HA cleavage, replication, and pH stability (13–15). The other research groups also showed that the N -glycosylation is required for the efficient folding and oligomerization of HA protein (7, 16–20).…”