Abstract
Enveloped viruses deliver their genomes into host cells by undergoing fusion between the viral lipid envelope and a cellular membrane. For influenza A virus (IAV) and Ebola virus (EBOV), fusion occurs with the endosomal membrane after endocytic uptake. Engagement of receptors on the cellular membrane by viral glycoproteins facilitates adsorption to cellular membranes and endocytosis but is not generally thought of as a direct regulator of membrane fusion. In this dissertation, I demonstrate that receptor binding by the IAV fusion protein hemagglutinin (HA) regulates IAV membrane fusion and present preliminary observations suggesting that receptor engagement by the EBOV glycoprotein (GP) also modulates membrane fusion. I developed two key methodological advances to enable the presented experiments. To define how receptor binding regulates IAV membrane fusion, I developed a flow-cytometry-based assay which measures lipid mixing of individual virion-target pairs (spCALM). The high experimental throughput of this approach relative to previous single-particle-resolution approaches enabled analysis of lipid mixing in highly attenuated regimes such as low receptor density membranes. I combined this approach with a novel, programmable DNA-mediated receptor-mimic display approach to achieve precise control over the receptor environments displayed on liposome targets, allowing receptor density and structure to be controlled without altering membrane composition. These approaches revealed that HA-receptor engagement promotes the efficiency of lipid mixing. A suite of experimental parameters which influence receptor binding likewise influence lipid mixing. Mechanistic analysis suggests that receptor binding promotes the formation of fusion-competent clusters of target-membrane-engaged HAs but does not directly prompt initiation of conformational changes leading to membrane engagement. The functional balance between HA and the viral receptor-destroying enzyme, NA, has been appreciated at the level of virion motility and progeny release; our results show that HA-NA balance influences the efficiency of membrane fusion as well. The conformational changes of EBOV GP and the triggers that initiate them are less thoroughly characterized than for IAV HA. To study EBOV, I established a reconstituted single-particle TIRF microscopy-based assay which probes lipid mixing between fluorescently labeled EBOV virus-like particles (VLPs) and NPC1-C-bearing supported planar bilayers (SPBs). Attachment of EBOV VLPs to SPBs required both proteolytic priming of the EBOV glycoprotein GP and the presence of NPC1-C, validating the specificity of the assay. NPC1-C engagement alone did not trigger lipid mixing at neutral pH. Exposure to acidic pH, however, induced lipid mixing in a subset of attached particles. Together with experiments testing receptor elution and pH re-neutralization, these preliminary results suggest a model in which acidic pH promotes a long-lived GP intermediate conformation which is competent for membrane engagement and can later mediate lipid mixing. Receptor disengagement may collapse this intermediate, leading to lipid mixing. The IAV and EBOV studies, taken together, suggest that receptor binding can modulate viral lipid mixing activity beyond serving as an attachment factor. Furthermore, this dissertation details several novel single-particle-resolution approaches to assess the determinants of lipid mixing activity for IAV and EBOV.