Project 3: Investigation of capsid-host factor interactions using minimal, soluble capsid lattice assemblies

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P3. Abstract The mature HIV-1 capsid is an ordered protein shell that houses the viral genome and other viral or host factors necessary for infection of a target cell. Its structure and stability are critical to infection—it must stay at least partially assembled until it reaches the nuclear periphery, and a variety of host factors, either beneficial or inhibitory to the virus, recognize specific capsid patterns only present in assembled cores. Potentially hundreds of host factors bind the HIV-1 capsid, but our knowledge of host factor-capsid interactions at the molecular level has been limited, in part caused by a lack of soluble capsid constructs that recapitulate lattice interfaces in the assembled state and by challenges in purifying recombinant capsid-binding host factors. To overcome these obstacles, we propose to design and build a repertoire of soluble capsid assemblies that represent all unique capsid-lattice interfaces. This will be achieved by making use of previously published intra-hexamer engineered disulfides and by incorporating the SpyCatcher/SpyTag system to create stable isopeptide bonds between hexamers. With these new capsid assemblies in hand, we will use well-established biophysical and biochemical techniques to map binding modes and determine high-resolution structures of these capsid assemblies in complex with a range of host factors including TRIM5α, TRIMCyp, MxB, Fez1, and CypA, most of which we have optimized for expression and purification. The proposed work will allow us to overcome major experimental hurdles to advance mechanistic understandings of the capsid-related key steps of the HIV life cycle.