Biophysical studies of viral membrane fusion proteins

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SUMMARY Hemagglutinin (HA) is an envelope glycoprotein that resides on the surface of influenza A virus (IAV) particles. HA mediates attachment of IAV virions to the cell surface through interaction with sialic acid (SA) moieties. Following internalization by endocytosis, the acidic environment of the late endosome triggers conversion of HA from a metastable prefusion conformation to a highly stable postfusion coiled coil. This cascade of conformational changes in HA results in fusion of the viral and endosomal membranes. Importantly, exposure of IAV to acidic pH prior to attachment to a cellular membrane prematurely converts HA to the postfusion conformation, which renders IAV noninfectious. Therefore, ensuring proper timing of HA conformational changes is critical to the life cycle of IAV. While endpoints of the HA conformational changes during membrane fusion are well characterized, recent structural data is beginning to identify intermediate states. However, the HA conformational trajectory that leads from prefusion to postfusion is not yet validated, nor has the timing of membrane fusion with respect to HA conformation been determined. The funded project leverages the combined strengths of the Munro and Somasundaran laboratories in cutting-edge single-molecule Förster resonance energy transfer (smFRET) imaging, single-virion fusion assays, cryoelectron tomography (cryoET), and computational approaches to address these questions in viral fusion. The funded project relies heavily on the application of advanced, quantitative fluorescence microscopy within the Munro laboratory. With institutional support from UMass Chan Medical School, the Munro laboratory has custom built a microscope specifically designed to conduct the proposed experiments. The requested Administrative Supplement for Equipment Purchases will provide upgrades to the existing microscope. These upgrades will address shortcomings of the microscope, which limit the ability to successfully complete the proposed Aims, and which were not foreseen at the time of application.