In order to replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host. Although the virus-encoded components of the DNA integration system are a promising target for antiretroviral therapy, no clinically useful inhibitors have yet been developed. The studies proposed here are designed to reveal critical aspects of the molecular mechanism of DNA integration that will potentially guide the design of such inhibitors. These studies focus on the virus-encoded integrase protein, which covalently links the viral cDNA to host DNA, and its role in the subviral """"""""preintegration complex"""""""" that carries out DNA integration in vivo. The integrase acts at the ends of the unintegrated linear viral DNA; the parts of the integrase that direct specific DNA recognition will be mapped by assaying the activities in vitro of hybrid integrases containing potential recognition domains from integrases of different retroviruses. Since integrase oligomerization is also probably required for function, sequences capable of oligomerization will be identified using genetic assays with fusion proteins in vivo, and binding assays in vitro. Mutant derivatives of integrase generated in these studies will be used in collaborative attempts to solve the structure of integrase by X-ray crystallography or NMR. To examine the higher order assembly of the preintegration complex in vivo, improved methods will be developed for purifying such complexes from freshly infected cells, and the protein composition and stoichiometry will be determined. The interactions between proteins and the viral DNA in the preintegration complex will be mapped in DNA footprinting experiments using site specific methylases and dimethyl sulfate as probes. A panel of anti-integrase monoclonal antibodies is presently being developed. The binding sites of these antibodies on integrase will be mapped, and the arrangement of integrase monomers in the preintegration complex will be analyzed by monitoring the accessibility of particular integrase epitopes using immunoprecipitation. Guided by results from these studies, experiments will be undertaken to assemble active preintegration complexes from purified components. Ultimately, these studies taken together will provide a high-resolution picture of the organization and mechanism of action of the preintegration complex.
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