BIOPHYSICAL STUDIES OF HIV ASSEMBLY AND MATURATION

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Description (Adapted from Applicant's abstract): The objective of this proposal is to characterize the interaction between the structural domains of the Gag polyprotein involved in the assembly and maturation of HIV. The structure of the individual matrix, capsid, and nucleocapsid domains have been determined, but it has proven difficult to obtain structural information about these domains in the context of the polyprotein or virion. The pleomorphic, enveloped, non-icosahedral nature of the virion makes it likely that this information will be obtained by traditional structural means. A novel approach to determining hydrogen/deuterium exchange protection profiles has been developed and, in combination with spectroscopic approaches, will be applied to this problem. These techniques make it possible to identify inter-subunit interfaces and determine their stability and dynamics. After refining proteolytic digestion conditions, and assigning a library of peptide fragments as required for the mass spectrometry, the applicant will determine the alterations in exchange protection profiles of the Gag polyprotein and its domain which accompany polymerization. The applicant will then characterize the stability of capsid protein polymerized into spherical and cylindrical polymers. These species are thought to be structurally analogous to the spherical and cylindrical capsid core found in the immature and mature virion. Once the applicant has characterized these in vitro structures, he will characterize the inter-subunit interactions within immature and mature particles budded from cells. These studies will lead to a refined understanding of the inter-relationship between the structural domains of the Gag polyprotein in the HIV lifecycle. This refined understanding will prove invaluable in the discovery and design of antivirals targeted at assembly