Determinants of Receptor Binding in Influenza and Coronaviruses: Glycoconjugate Specificity and Spatial Distribution of Host Receptors

Abstract

Despite advances in glycan microarray technology, cryo-electron microscopy, and structural analysis, the relationships between host-specific glycan structures and viral tropism remain incompletely understood. This dissertation investigates the molecular mechanisms underlying receptor binding and viral infectivity, with a particular focus on protein glycosylation, multivalency, and glycan modifications such as O-acetylation. It further examines the spatial distribution of key entry factors ACE2 and TMPRSS2 in relevant animal models. Chapter 2 establishes a foundational toolset by engineering recombinant hemagglutinin (HA) and receptor-binding domain (RBD) proteins fused to fluorescent reporters, enabling visualization of influenza A and SARS-CoV-2 glycoprotein interactions with host receptors using advanced imaging approaches. Chapter 3 demonstrates that glycosylation state and trimeric architecture of the RBD substantially influence binding efficiency to ACE2, and that tissue-specific ACE2 expression patterns determine tropism. Fully glycosylated, trimeric RBDs outperform their unglycosylated or monomeric counterparts, underscoring the functional importance of native protein conformation. Chapter 4 shifts focus to antigenic drift in the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The Beta variant (501Y.V2) exhibits enhanced binding to sialic acids, particularly 9-O-acetylated forms, revealing how NTD evolution contributes to altered receptor engagement and potentially to changes in viral fitness and immune evasion. Chapter 5 broadens the scope to sialoglycan modifications across coronavirus species. Using multivalent ligand presentations, the study reveals that 9-O-acetylated sialic acids are recognized by multiple coronaviruses, suggesting a conserved receptor recognition strategy despite evolutionary divergence among CoV lineages. Chapter 6 addresses the in vivo dimension by mapping ACE2 and TMPRSS2 expression across lung lobes of SARS-CoV-2-infected Syrian hamsters. Expression levels vary by anatomical region and correlate with sites of active infection, highlighting the importance of receptor distribution in determining pathogenesis and informing the design of therapeutic interventions. Together, these chapters provide an integrated molecular and spatial framework for understanding how glycan biology shapes viral receptor binding, tissue tropism, and infection outcomes across host species.