The long-term persistence of HIV in a latent state in memory T cells in patients treated with HAART prevents the eradication of HIV and forces patients to remain on HAART for their whole life. While the transcriptional regulation of HIV has been extensively studied in transformed cell lines, our understanding of how latent HIV infection occurs in primary memory CD4 T lymphocytes is rudimentary. The purpose of this application is to develop new single cell technology to examine the transcriptional status of HIV in single primary lymphoid cells over time after an infection in vitro. These studies will bridge the two research fields of human immunology and HIV molecular virology. Understanding HIV latency in primary lymphocytes may lead to the identification of cellular proteins that control the entry of HIV in latency, the maintenance of latency or its reactivation. Such cellular targets could represent new avenues for the treatment of HIV/AIDS among drug abusers and possibly lead to the eradication of infection. I propose to use a novel live cell, time-lapse fluorescence microscopy combined with cell trapping via microfluidic chips and the use of HIV expressing recombinant fluorescent protein (destabilized GFP) to study the kinetics of HIV transcription at the single cell level. This novel technique will allow the fate of HIV expression to be followed in live individual cells over time. Human lymphoid cells will be activated in vitro, infected with an HIV expressing a fluorescent protein, activation signals will be removed and HIV transcription will be followed over time. We anticipate that HIV transcription will be restricted in a subset of cells returning to quiescence. The time separating removal of activation signal to infection is likely to be critical in allowing infection to proceed until HIV integration while restricting HIV transcriptional activation. The nature of the activation signal could also prove critical. Experiment will eventu

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
NIH Director’s Pioneer Award (NDPA) (DP1)
Project #
5DP1DA031126-03
Application #
8312649
Study Section
Special Emphasis Panel (ZDA1-NXR-B (15))
Program Officer
Satterlee, John S
Project Start
2010-09-30
Project End
2015-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
3
Fiscal Year
2012
Total Cost
$936,050
Indirect Cost
$451,050
Name
J. David Gladstone Institutes
Department
Type
DUNS #
099992430
City
San Francisco
State
CA
Country
United States
Zip Code
94158
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Calvanese, Vincenzo; Chavez, Leonard; Laurent, Timothy et al. (2013) Dual-color HIV reporters trace a population of latently infected cells and enable their purification. Virology 446:283-92
Shirakawa, Kotaro; Chavez, Leonard; Hakre, Shweta et al. (2013) Reactivation of latent HIV by histone deacetylase inhibitors. Trends Microbiol 21:277-85
Hakre, Shweta; Chavez, Leonard; Shirakawa, Kotaro et al. (2012) HIV latency: experimental systems and molecular models. FEMS Microbiol Rev 36:706-16
Rafati, Haleh; Parra, Maribel; Hakre, Shweta et al. (2011) Repressive LTR nucleosome positioning by the BAF complex is required for HIV latency. PLoS Biol 9:e1001206