Unveiling Glycosylation features in the oseltamivir-resistant NA protein of the H1N1pdm09 strain

Influenza A virus remains a persistent health threat in our society, despite ongoing efforts to develop more effective vaccines. The virus's ability to infect hosts relies on the coordinated actions of its surface antigens, haemagglutinin (HA) and neuraminidase (NA), which facilitate viral binding and spreading respectively. Traditionally, HA has been the primary focus of vaccine development. However, following the emergence of the H1N1pdm09 strain, which exhibited resistance to the NA inhibitor Tamiflu® (oseltamivir, OC) due to a single amino acid mutation (H275Y), there is increasing support among scientists to include NA antigens in annual influenza vaccines to enhance their effectiveness and coverage.

NA is a glycosylated protein crucial for budding off newly synthesised virions and facilitating the effective viral spreading. Therefore, understanding how glycans mediate the above process might be crucial for future vaccine design. NA is a tetrameric protein with a cytoplasmic tail, a transmembrane domain, and a stalk that anchors the catalytic head in the viral membrane. Given that the enzymatic activity resides in the head, most studies on NA's glycosylation have concentrated on this region. However, there is growing evidence of the potential antigenic role of the stalk region, although information on the glycan structures at the corresponding sites remains limited.

In this study, we aimed to characterize the glycan variation and occupancy in both the stalk and head regions of the wild-type and oseltamivir-resistant recombinant NA (rNA) from the H1N1pdm09 strain. We conducted site-directed mutagenesis to introduce mutations associated with OC resistance (H275Y) and mutations restoring viral fitness (V241I and N369K) and expressed rNA in mammalian cells (HEK293). Purified proteins were subjected to proteolytic digestion, and the resulting glycopeptides were analysed using the ZenoTOF and cyclic SELECT mass spectrometers. Identification of glycan formulas was performed in Byonic, and all glycopeptide spectra were manually curated for the presence of structure-specific fragments. Analysis of ion mobility data was performed in DriftScope with the retention times associated with glycopeptides manually extracted. The above analysis resulted in the identification of all four glycosites in the head domain (N88, N146, N235 and N386) and all three conserved sites in the stalk (N50, N58, N63 and N68). The majority of the glycans identified were complex, with fucose being either at the core or antenna of the glycan structure. Neither sialylated- nor sulphated-glycopeptides were identified. To the best of our knowledge, this study represents one of the initial attempts to provide a comprehensive glycosylation profile of a pandemic-emerged NA, and therefore could offer valuable insights for future vaccine designs.