Decoding the Structural Determinants of Hemagglutinin Mediated Influenza Entry and Antigenicity
Garcia, Natalie K.
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The fusion glycoproteins found on the surface of enveloped viruses enable the delivery of the viral genome into the host cell by facilitating the merger of the viral membrane with the host cell membrane, an essential step for a productive infection. The thermodynamically favorable but kinetically slow process of spontaneous membrane fusion is permitted by such complex viral molecular machines, which work by releasing enough conformational energy to surmount the activation barrier associated with fusion between two separate membranes. The trimeric influenza viral fusion glycoprotein hemagglutinin (HA) binds cell surface sialic acid moieties to trigger endocytosis of the virion into the host cell. During endosomal maturation, the drop in pH induces structural rearrangements within HA, to drive membrane fusion. While the pre-fusion and fragmentary post-fusion states of HA have been crystallized, the intermediates that promote fusion are too dynamic and transient for characterization through classical methodologies. HA is also the major target for neutralizing antibodies that bind to either prevent receptor binding or membrane fusion. Understanding the fusion mechanism and antigenic profile of HA is a sought after goal for the rational design of universal therapeutics and vaccines that can prevent influenza infection. This dissertation aims to discover how HA is triggered to undergo pH-dependent membrane fusion, and how the process is inhibited by neutralizing antibodies, therapeutic proteins, and small molecules. Using a combination of solution-phase structural techniques such as hydrogen-deuterium exchange mass spectrometry, small angle X-ray scattering, and light scattering, we find the structural elements that carry out membrane fusion are isolate specific. We also find that while neutralizing antibodies prevent fusion by stabilizing the pre-fusion state, other therapeutic proteins and small molecules stabilize some structural features but also allosterically destabilize portions of the trimer. In the efforts to find a broad-spectrum inhibitor, and characterize the mechanism of acid-activation during membrane fusion, it is imperative to study multiple isolates and subtypes of HA to comprehensively survey the structural and consequently mechanistic differences between variants.
- Medicinal chemistry