Influenza A Virus Infection: Thriving on Receptor Diversity

Abstract

To enter a cell, a virus must bind to a receptor. Influenza A virus (IAV) attaches to sialic acids (Sia) on glycan chains. IAV binding requires multivalent binding due to the low binding affinity of protein-glycan interactions. Sialoglycans, a diverse group of sugars, are distributed over the cell surface and display structural diversity. Two well-established characteristics of sialoglycans are of major importance for IAV binding. (1) Due to the mostly low binding affinity that has generally been observed for protein-glycan interactions (see Chapter 4), the tight virus binding that is observably necessary for infecting a cell requires the virus to bind by multiple interactions, i.e., multivalent binding. (2) Sialoglycans are a highly diverse group of sugars that are heterogeneously distributed over the cell surface and display a large structural diversity depending on cell type, tissue and host species. Collectively, these properties enforce an IAV particle to bind by multiple, rapidly alternating, interactions with different sialoglycan receptors. This highly dynamic multivalent binding mode challenges the virus with the necessity to handle (or exploit) kinetic parameters that fall within a range. Hemagglutinin (HA) binds sialoglycans and establishes fusion of the viral envelope with the endosomal membrane whereas neuraminidase (NA) cleaves Sias from a sialoglycan. NA activity is essential to avoid being trapped by decoy receptors that prevent cell entry (reviewed in Chapter 2) and to assist release of newly formed particles by preventing virus particle aggregation and sticking to the originating cell. In Chapter 3 , we analyzed the consequence of multivalent binding to heterogeneous receptor surfaces. The results showed that the ability to simultaneously interact with sialoglycans of higher and lower affinity reduces the required threshold density for a higher affinity receptor provided the presence of sufficient lower affinity interactions. These findings nuance the canonical dichotomy in receptor utilization between human IAVs, binding to α2-6 linked Sias (2-6Sia), and avian IAVs, binding to 2-3Sias). In Chapter 4, we determined, by a novel biolayer interferometry-based method, the affinities of individual HA-Sia interactions of different IAV strains for several receptors. The results showed that gradual adaptation of H3N2 to human-type 2-6Sia receptors took a decade and was dependent on changes in association rate whereas the koff for 2-3Sia and 2-6Sia remained constant. In Chapter 5, we further developed a cell-based glycan array to study the ability of specific glycan assemblies displayed on the surface of genetically modified HEK293 cells to support IAV entry and to directly correlate entry to virus binding to the cells. We showed that three different glycoconjugate groups (N-glycans, O-glycans or glycosphingolipids) could support infection at high efficiency depending on the specific sialyltransferase that was used to install Sias. In Chapter 6, we analyzed whether different sialoglycans, attached to specific glycoconjugate groups, affect the specific endocytic entry routes that can be utilized. To assist dissection of dynamin-dependent entry routes we identified LFM-A13 as a specific inhibitor of a dynamin-dependent IAV entry route that was (partially) redundant to IAV entry by clathrin-mediated endocytosis.