Thesis defense Xuesheng Wu

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Sialoglycans, ubiquitous carbohydrate structures found on cells and within the mucus layer, serve as an entry receptors for many viruses. Sialoglycan-binding viruses therefore often also possess receptor-destroying activity to avoid being trapped by decoy receptors. The balance between receptor binding and cleavage is presumed to be an important determinant of host tropism and pathogenicity. Sialoglycan-binding paramyxoviruses (PMVs), including important pathogens such as human parainfluenza virus type 1 and 3 and Newcastle disease virus (NDV), depend on the hemagglutinin-neuraminidase (HN) glycoprotein, which mediates both sialic acid (Sia) binding and cleavage. By employing biolayer interferometry (BLI), we analyzed the dynamics of virion-receptor interactions for several animal and human PMVs, revealing, among other findings, sialidase-driven motility of virus particles on receptor-coated surfaces, resembling a "lawnmower" mechanism, which likely allows them to escape the mucus layer and find the functional receptors on the cell surface. One of the challenges in studying virus-receptor interactions is the requirement for high virus titers due to the low binding affinity of individual virion-receptor interaction. We therefore developed viromimic nanoparticles that multivalently display recombinant tetrameric HN proteins. These nanoparticles successfully replicated the receptor-interaction dynamics of live viruses and allowed us to study how substitutions in HN affect virus production. This approach led to the discovery of long-range effects between the primary and secondary Sia-binding sites of the NDV HN protein. Finally, we also investigated specific sialoglycans involved in PMV infection. While (human) PMVs are generally known to prefer α2-3-linked Sia (2-3Sia), in contrast to human influenza A viruses that prefer to bind to 2-6Sia, the precise sialoglycoconjugate responsible for infection so far remained unclear. Using cell-based glycan arrays, we demonstrated that parainfluenza virus infection strictly required 2-3Sia, whereas NDV could infect cells lacking both 2-3 and 2-6Sia, although less efficiently. For all PMVs studied, the presence of 2-6Sia alongside low levels of 2-3Sia significantly enhanced infection, indicating heteromultivalent binding, which will inevitably also occur in vivo. Our findings show that the terminal sialoglycan structure, particularly the identity of the final three monosaccharides, is the key determinant of PMV binding and infection, rather than the type of glycoconjugate (N-glycan, O-glycan, or glycolipid). Collectively, these findings provide novel insights into the receptor-binding dynamics and infection requirements of sialoglycan-binding PMVs, paving the way for further exploration of their roles in host tropism (changes) and pathogenicity.