Proteomic Tools to Uncover the Role of Chromatin Remodeling in HIV-1 Infection

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DESCRIPTION (provided by applicant): With a genetic capacity of only 104 bp, HIV has triggered the devastating acquired immunodeficiency syndrome (AIDS) epidemic. In large part, the refined mechanisms that control the fate of cells invaded by the pathogen remain unknown. To understand the disease at the molecular level and develop therapies we are developing new approaches to reveal the nature of these temporal and spatial protein interactions. These methods entail a rapid cryolysis-based strategy that uses a single fluorescent tag (GFP) to both visualize viral proteins in live cells and capture their interacting partners during the course of infection. Our preliminary work on HIV-1 host protein interactions demonstrates that chromatin remodeling complexes are among other cellular functions modulated by the virus. This finding suggests that viruses have developed mechanisms to control host gene expression by usurping these complexes. Indeed, recent reports have shown that histone deacetylases may mediate the HIV latency. We propose to initiate a comprehensive analysis of the known eleven histone deacetylases during the course of HIV-1 infection. To gain insights into the location, composition, structure, and function of these multi-protein complexes, we will merge novel proteomic approaches with molecular biology, virology, and biochemistry methodologies. Using human cell lines expressing HDAC fused to GFP, we will visualize and isolate these chimeras, first in uninfected cells, and then upon HIV-1 infection. The state-of-the-art mass spectrometric configuration in my laboratory is the first of its kind, combining speed of analysis with the robustness of MALDI (matrix assisted laser desorption ionization), the enrichment in the linear ion trap, and the high accuracy measurement in the Orbitrap. This configuration is perfectly suited for the analysis of macromolecules. This comprehensive multidisciplinary analysis of HDAC complexes will provide insights into the host and viral processes that determine the fate of a cell and the ultimate outcome of infection.