Influenza A virus N-linked glycosylation sites with very low glycan occupancy: identification, evolutionary dynamics, and impact on virus biology
Introduction: Influenza A virus (IAV) is a common pathogen of the human respiratory tract that causes both seasonal endemics and intermittent pandemics. The IAV major surface protein and antigen, hemagglutinin (HA), is extensively glycosylated, with 12 N-linked glycosylation sites in A(H3N2)2013 IAV...
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| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-03-01
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| Series: | International Journal of Infectious Diseases |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1201971224007112 |
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| Summary: | Introduction: Influenza A virus (IAV) is a common pathogen of the human respiratory tract that causes both seasonal endemics and intermittent pandemics. The IAV major surface protein and antigen, hemagglutinin (HA), is extensively glycosylated, with 12 N-linked glycosylation sites in A(H3N2)2013 IAV. Here, we compare glycan site occupancy on HA from A(H3N2)2013 IAV produced either in embryonated chicken eggs, MDCK cells, or human lung carcinoma (A549) cells, and investigate the effects of low glycosylation site occupancy at residues N45 and N144 on generation of adaptive immune responses against the virus and on viral virulence. Methods: We constructed reverse-genetics IAV lacking a glycosylation sequon at residues N45 or N144 of 2013 A(H3N2) HA. Site-specific glycosylation of the wild-type virus (containing both N45 and N144 sites) and of these glycosylation mutant viruses was determined by mass spectrometry. The ability of concentrated, purified, inactivated wild-type or glycosylation mutant viruses to generate adaptive immune responses was assessed by immunization of naïve mice with two intramuscular injections of 10 µg of HA, followed by measurement of total, protective, and neutralizing serum antibody titers (by ELISA, hemagglutination inhibition, and microneutralization assays, respectively) at post-immunization days 35, 43, 49, and 67. Antibody responses generated by glycosylation mutant viruses were compared to those of the wild-type virus. In addition, receptor binding of viruses was determined using binding to 0.5% turkey red blood cells. Results: Site occupancy was similar in viruses grown in eggs, MDCK cells, or A549 cells. Nine of the 12 N-glycosylated sites had high site occupancy (near 100%). The other three sites, all located on the HA globular head, were either not detectably glycosylated (site N122) or glycosylated at levels below 10% (sites N45 and N144). Sites 122 and 144 first arose in the 1980s, and became ubiquitous in the early 2000s. Interestingly, around 2015 a drastic reduction of the frequency of site N144 coincided with a sharp rise in the frequency of site N45. The presence of a low level of glycosylation at either amino acid 45 or 144 reduces total homologous and heterologous serum antibody responses as well as titers of antibodies that correlate with protection against influenza disease and virus neutralization, and reduces receptor binding of A(H3N2)2013 IAV. Discussion: Adding or removing existing HA N-glycosylated sites has been shown to alter antibody-mediated neutralization of influenza viruses, but there is no information on how low N-glycosylation site occupancy affects antibody immune responses and virus virulence. Our data suggest that IAV uses sites with low glycan occupancy to reduce immune pressure from the host on antigenic sites and receptor-binding site of HA without compromising protein function. Conclusion: N-glycosylation sites with low glycan occupancy are important for sustained circulation of influenza virus in the human population. |
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| ISSN: | 1201-9712 |