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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
NIH Director’s Pioneer Award (NDPA) (DP1)
Project #
5DP1DA026192-05
Application #
8305794
Study Section
Special Emphasis Panel (ZDA1-NXR-B (26))
Program Officer
Satterlee, John S
Project Start
2008-09-05
Project End
2013-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
5
Fiscal Year
2012
Total Cost
$757,890
Indirect Cost
$287,151
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
Luo, Yang; Jacobs, Erica Y; Greco, Todd M et al. (2016) HIV-host interactome revealed directly from infected cells. Nat Microbiol 1:16068
Castellana, Michele; Hsin-Jung Li, Sophia; Wingreen, Ned S (2016) Spatial organization of bacterial transcription and translation. Proc Natl Acad Sci U S A 113:9286-91
Guise, Amanda J; Cristea, Ileana M (2016) Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5). Methods Mol Biol 1436:47-84
Diner, Benjamin A; Lum, Krystal K; Javitt, Aaron et al. (2015) Interactions of the Antiviral Factor Interferon Gamma-Inducible Protein 16 (IFI16) Mediate Immune Signaling and Herpes Simplex Virus-1 Immunosuppression. Mol Cell Proteomics 14:2341-56
Cristea, Ileana M; Graham, David (2015) Virology meets Proteomics. Proteomics 15:1941-2
Orzalli, Megan H; Broekema, Nicole M; Diner, Benjamin A et al. (2015) cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection. Proc Natl Acad Sci U S A 112:E1773-81
Koyuncu, Orkide O; Song, Ren; Greco, Todd M et al. (2015) The number of alphaherpesvirus particles infecting axons and the axonal protein repertoire determines the outcome of neuronal infection. MBio 6:
Diner, Benjamin A; Lum, Krystal K; Cristea, Ileana M (2015) The emerging role of nuclear viral DNA sensors. J Biol Chem 290:26412-21
Lin, Kah-Wai; McDonald, Karin R; Guise, Amanda J et al. (2015) Proteomics of yeast telomerase identified Cdc48-Npl4-Ufd1 and Ufd4 as regulators of Est1 and telomere length. Nat Commun 6:8290
Diner, Benjamin A; Li, Tuo; Greco, Todd M et al. (2015) The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA. Mol Syst Biol 11:787

Showing the most recent 10 out of 63 publications