BIOCHEMICAL MECHANISM OF HIV DNA INTEGRATION

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Human immunodeficiency virus type 1 (HIV-1) must integrate the cDNA copy of its RNA genome into an infected cell chromosome in order to efficiently replicate. The key viral players in integration are the trans-acting integrase (IN) protein, which enters the cell as a part of the virus, and the cis-acting DNA attachment (att) site, comprised of the ends of linear cDNA made by reverse transcription. In cells, integration is mediated through large subviral nucleoprotein preintegration complexes (PICs) that are derived from the cores of infecting virions. PICs isolated from infected cells can integrate their endogenous cDNA into an added target DNA in vitro. PICs were previously purified based on their large size. Sizing on its own is not a very efficient purification technique: it is inherently diluting, and large cellular assemblies copurify with PICs. Novel techniques based on affinity chromatography will be developed to increase the purity and yield of PICs approximately 100-fold over existing techniques. In addition to IN, host factors play essential roles in the structure and function of retroviral PICs. Two host proteins, HMG I(Y) and the barrier-to-autointegration factor (BAF), were previously implicated in HIV-1 integration, but it unclear which of these is physiologically-relevant. The stoichiometries of HMG I(Y) and BAF to IN and cDNA will be determined in purified PICs. Other viral proteins, including matrix, reverse transcriptase, Vpr, and nucleocapsid cofractionate with HIV-1 PICs. Although not needed for DNA recombination, matrix and Vpr may facilitate the nuclear import of PICs in nondividing cells. The stoichiometries of these viral proteins will be determined in purified samples, along with the folding topologies of IN, matrix and reverse transcriptase. Current models of protein-protein interactions important for PIC structure and function are not well defined, but predict the two cDNA ends are coupled for IN catalysis in vivo. Preliminary data supports a model where the cDNA ends are uncoupled for an initial catalytic step, and then come together through protein-protein interactions for integration. The contribution of viral and host factors to these interactions will be determined. A novel IN mutant was identified that in addition to normal integration promotes the integration of just one cDNA end at a time, a pathway that is deleterious to virus growth. These mutant PICs will be analyzed in detail to identify protein-protein and protein-DNA interactions required for normal PIC function. The results of these experiments will be used to formulate a detailed model of the structural and functional organization of HIV-1 PICs, which will aid the design of antiviral drugs targeted against HIV-1 integration.