Influenza Virus Hemagglutinins H2, H5, H6, and H11 are not Targets of Pulmonary Surfactant Protein D: T-glycan subtypes in host-pathogen interactions

Abstract

Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low pathogenicity A type influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortmant and emergence in human populations. Since the presence of head region high mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here we investigate the activities of two recombinant human SP-D forms against representative LPAIV including H2N1, H5N1, H6N1, H11N9, an avian H3N8 and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPIAV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite "N165" (H3 numbering) is occupied by high mannose glycans in H3 HA but by complex glycans in all LVIAV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high mannose glycan on the head region our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three dimensional structural analysis.Importance Low pathogenicity A type influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations as was seen in the 1957 pandemic, where an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first line innate immunity defence, removes IAV through interaction with hemagglutinin (HA) head region high mannose glycan(s). While it is known that both H1 and H3 HAs, have a key high mannose glycosite(s) in the head region, little is known about such glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high mannose glycans, do not interact with SP-D, and that sequence analysis can predict glycan subtype thus predicting presence or absence of this virulence marker.