Structural and biochemical characterization of paramyxovirus fusion complexes

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DESCRIPTION (provided by applicant): The paramyxovirus family and viruses that undergo pH independent entry are responsible for major human morbidity and mortality. Successful entry of enveloped viruses requires the fusion of viral and cellular membranes. Viruses have evolved a variety of mechanisms to accomplish fusion including pH and protein-protein interaction induced conformational changes in the viral glycoprotein that ultimately lead to membrane fusion and pore formation. Although the broad mechanism of this process is understood for most enveloped viruses, the specific changes and conformational states have only been thoroughly elucidated for a handful of viruses. To better understand protein-mediated fusion at neutral pH, these experiments will investigate the structural rearrangements of an archetype of the paramyxovirus family, measles virus (MV). Specific Aim 1 - to biochemically define the pre- and post-fusion paramyxovirus glycoprotein hetero- oligomers and determine the effect of receptor binding on the pre-fusion complex using native- electrophoresis, carbohydrate shielding, site-directed mutagenesis, and functional complementation. Specific Aim 2 - to determine the geometry of paramyxovirus glycoproteins in situ by cryo-electron tomography (cryo-ET) of purified virions. Crystal structure data of the glycoproteins will be superimposed onto the electron densities identified from the cryo-ET to produce a high resolution hetero-oligomer pseudo-atomic structure. This work will further the understanding of the molecular mechanism of infection of a family of major human and animal pathogens. Understanding the molecular mechanism of fusion will provide a basis for structure-guided design of novel antiviral therapeutics. PUBLIC HEALTH RELEVANCE: Measles virus infections are on the rise in the US and around the world despite an effective vaccine. Understanding how the measles virus infects cells will lay the framework for the development of novel antiviral therapies against measles virus and other closely related human pathogens.