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We recently reported that members of group 1 influenza A virus (IAV) containing H2, H5, H6, and H11 hemagglutinins (HAs) are resistant to lung surfactant protein D (SP-D). H3 viruses, members of group 2 IAV, have high affinity for SP-D, which depends on the presence of high-mannose glycans at glycosite N165 on the head of HA. The low affinity of SP-D for the group 1 viruses is due to the presence of complex glycans at an analogous glycosite on the head of HA, and replacement with high-mannose glycan at this site evoked strong interaction with SP-D. Thus, if members of group 1 IAV were to make the zoonotic leap to humans, the pathogenicity of such strains could be problematic since SP-D, as a first-line innate immunity factor in respiratory tissues, could be ineffective as demonstrated in vitro. Here, we extend these studies to group 2 H4 viruses that are representative of those with specificity for avian or swine sialyl receptors, i.e., those with receptor-binding sites with either Q226 and G228 for avian or recent Q226L and G228S mutations that facilitate swine receptor specificity. The latter have increased pathogenicity potential in humans due to a switch from avian sialylα2,3 to sialylα2,6 glycan receptor preference. A better understanding of the potential action of SP-D against these strains will provide important information regarding the pandemic risk of such strains. Our glycomics and in vitro analyses of four H4 HAs reveal SP-D-favorable glycosylation patterns. Therefore, susceptibilities to this first-line innate immunity defense respiratory surfactant against such H4 viruses are high and align with H3 HA glycosylation.

The SARS-CoV-2 spike protein is heavily glycosylated, having 22 predicted N-glycosylation sites per monomer. It is also O-glycosylated, although the number of O-glycosites is less defined. Recent studies show that spike protein glycans play critical roles in viral entry and infection. The spike monomer has two subdomains, S1 and S2, and a receptor-binding domain (RBD) within the S1 domain. In this study, we have characterized the site-specific glycosylation patterns of the HEK293 recombinant spike RBD and S1 domains as well as the intact spike derived from the whole virus produced in Vero cells. The Vero cell-derived spike from the WA1 strain and a D614G variant was analyzed. All spike proteins, S1, and RBDs were analyzed using hydrophilic interaction chromatography (HILIC) and LC-MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer. N-glycans identified in HEK293-derived S1 were structurally diverse. Those found in the HEK293-derived RBD were highly similar to those in HEK293 S1 where N-glycosites were shared. Comparison of the whole cell-derived WA1 and D614G spike proteins revealed that N-glycosites local to the mutation site appeared to be more readily detected, hinting that these sites are more exposed to glycosylation machinery. Moreover, recombinant HEK293-derived S1 was occupied almost completely with complex glycan, while both WA1 and D614G derived from the Vero E6 cell whole virus were predominantly high-mannose glycans. This stands in stark contrast to glycosylation patterns seen in both CHO- and HEK cell-derived recombinant S1, S2, and the whole spike previously reported. Concerning O-glycosylation, our analyses revealed that HEK293 recombinant proteins possessed a range of O-glycosites with compositions consistent with Core type 1 and 2 glycans. The O-glycosites shared between the S1 and RBD constructs, sites T323 and T523, were occupied by a similar range of Core 1 and 2 type O-glycans. Overall, this study reveals that the sample nature and cell substrate used for production of these proteins can have a dramatic impact on the glycosylation profile. SARS-CoV-2 spike glycans are associated with host ACE2 receptor interaction efficiency. Therefore, understanding such differences will serve to better understand these host–pathogen interactions and inform the choice of cell substrates to suite downstream investigations.