Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans

Abstract

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemagglutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for α2,6 sialylated branched N-glycans with at least three Nacetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs.

Evolution of human H3N2 influenza virus receptor specificity has substantially expanded the receptor-binding domain site

“…A similar preference of H1N1 viruses for extended glycans has also been noted, but appears less extreme as for H3N2 viruses, where binding has been reported to α2–6 sialylated glycans with two LacNAc repeats. 55 , 56 These observations suggest that extended glycan receptors are present on cells within the human airway. In this regard, extended N-linked glycans have been documented on a human epithelial cell line, 56 in lung tissues, 22 in the glycome of whole lung, 23 and within ferret respiratory tissues 57 .…”

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding

“…While on hCK cells binding was only observed for HA A/PR/8/34, A/HK/1/68 and A/SG/16. Glycan binding specificities differ between the selected strains and define whether binding occurs on MDCK and hCK cells: A/PR/8/34 (α2‐6‐linked SA), A/VN/1203/04 (α2‐3‐linked SA), A/HK/1/68 (α2‐6‐linked SA), A/SG/16 (α2‐6‐linked SA) and A/duck/UA/63 (α2‐3‐linked SA) (Stevens et al, 2006 ; Nemanichvili et al, 2019 ; Canales et al, 2023 ; Broszeit et al, 2019 ; Peng et al, 2017 , 2018 ). Following Vibrio cholerae neuraminidase treatment (VCNA, sialidase), binding was abrogated for all mOrange2‐HA‐fusions, demonstrating that the measured binding interactions are SA‐dependent.…”

Sialic acids in infection and their potential use in detection and protection against pathogens

“…While the scalable enzymatic synthesis of sialic acid [155][156][157][158] and sialylated glycans [159][160][161] has been achieved, the correct presentation of this key sugar recognition element is critical to achieving physiologically or therapeutically relevant biological recognition. For instance, the valency of NeuAc presentation 97 as well as secondary interactions from the glycan chain to which it is attached 140 can have a profound impact on target engagement. In addition, the efficiency of glycan recognition is context dependent, in that monovalent affinity does not directly correlate with polyvalent avidity.…”

“…For instance, H3N2 viruses have specificity for a-2,6-sialylated branched N-glycans with at least three N-acetyllactosamine units (tri-LacNAc); the length of the glycan chain can be used to target enhance discrimination between virus strains. 140 Virucidal sialic acid materials Precise presentation of sialic acid is an important factor in achieving inhibition of influenza virus infection. Decorating a b-cyclodextrin scaffold with three copies of sialic acid derivatives (Fig.…”

“…For instance, H3N2 viruses have specificity for α-2,6-sialylated branched N -glycans with at least three N -acetyllactosamine units (tri-LacNAc); the length of the glycan chain can be used to target enhance discrimination between virus strains. 140 …”