Asymmetrical Biantennary Glycans Prepared by a Stop-and-Go Strategy Reveal Receptor Binding Evolution of Human Influenza A Viruses

Abstract

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biologically relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical biantennary N-glycans having various numbers of N-acetyl lactosamine (LacNAc) repeating units capped by α2,3or α2,6-sialosides resembling structures found in airway tissues. It exploits that the branching enzymes MGAT1 and MGAT2 can utilize unnatural UDP-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNTFA) as donor. The TFA moiety of the resulting glycans can be hydrolyzed to give GlcNH 2 at one of the antennae, which temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of the evolutionary distinct human A(H3N2) and A(H1N1)pdm09 viruses. It was found that not only the sialoside type but also the length of the LacNAc chain and presentation at the α1,3-antenna of N-glycans are critical for binding. Early A(H3N2) viruses bound to 2,6-sialosides at a single LacNAc moiety at the α1,3-antenna whereas later viruses required the sialoside to be presented at a tri-LacNAc moiety. Surprisingly, most of the A(H3N2) viruses that appeared after 2021 regained binding capacity to sialosides presented at a di-LacNAc moiety. As a result, these viruses again agglutinate erythrocytes, commonly employed for antigenic characterization of influenza viruses. Human A(H1N1)pdm09 viruses have similar receptor binding properties as recent A(H3N2) viruses. The data indicate that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.

Construction of an IFNAR1 knockout MDBK cell line using CRISPR/Cas9 and its effect on bovine virus replication

“…Therefore, the upregulation of SLC2A1 after IFNAR1 knockout may synergistically promote BVDV replication ( Figure 4E ). Conversely, the significant downregulation of MGAT2 may reduce the n-acetylglucosamine modification on the cell surface, thereby affecting the interaction between the virus and host cells, and inhibiting virus replication ( Figure 4F ) ( 29 ). Although the specific roles of some DEGs in antiviral defense are unclear, the upregulation and downregulation of immune system-related DEGs, including SEMA3A ( 30 ), PADI1 ( 31 ), and MAP2K6 ( 32 ), as well as cell survival-related DEGs like STC1 ( 33 ), HSPA6 ( 34 ), and FGF17 ( 35 ), reflect the complex adaptability of BVDV-infected IFNAR1 knockout cells in antiviral defense.…”