The HIV-1 Tat transactivator interacts with P-TEFb (Cyclin T1:CDK9) and the AFF4:AFF1:AF9:ENL:ELL2 complex to stimulate elongation at the HIV-1 promoter. It was recently shown that Tat also recruits the SKIP splicing/elongation factor and MLL1:Menin H3K4 methyltransferase, assembling a multicomponent complex similar to that used by leukemogenic MLL activators. Here, novel components of the Tat and SKIP complexes (Aim 1) are identified, and their function in HIV-1 elongation, mRNA splicing, and virus replication (Aim 2) assessed. Residues in SKIP critical for HIV-1 splicing and transcription will be defined, and the role of 3'splice site recognition factors in pausing and elongation at the HIV-1 promoter (Aim 3) will be examined. In the cell, SKIP is required for expression of the anti-apoptotic p21Cip1 protein, a potent inhibitor of HIV-1 replication. It will be assessed whether Tat blocks the normal anti-apoptotic function of SKIP, thereby downregulating p21Cip1 expression and inducing p53-mediated T cell apoptosis. Tat-induced apoptosis will be characterized in the wild-type and isogenic p53-/- HCT116 cell lines, a stable SKIP expressing cell line that strongly resists p53 apoptosis, and in primary CD4+ T cells (Aim 4). Together, these experiments will better define the Tat transactivation mechanism and its role in p21Cip1 downregulation and T cell apoptosis.

Public Health Relevance

We have analyzed and identified an mRNA splicing/elongation factor, SKIP, which associates with HIV-1 Tat and is a highly regulated protein critical for Tat transactivation of the HIV-1 promoter. New findings also indicate that SKIP and Tat:P-TEFb are carried along the viral genome in an RNAPII elongation complex that is assembled by the Spt6 and Iws1 factors, and that Tat may regulate alternative spicing and other steps downstream of elongation through this complex. Here we will analyze new proteins identified in Tat and SKIP complexes for their role in Tat transactivation and earlier steps in RNAPII pausing and corepressor exchange using novel models for HIV-1 latency with integrated pNL4-3 proviruses in Jurkat and primary T cells, and evaluate these proteins as important new targets for novel anti-viral inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI044615-13
Application #
8459374
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sharma, Opendra K
Project Start
1998-12-01
Project End
2017-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
13
Fiscal Year
2013
Total Cost
$451,200
Indirect Cost
$216,200
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
078731668
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Bres, Vanessa; Yoh, Sunnie M; Jones, Katherine A (2008) The multi-tasking P-TEFb complex. Curr Opin Cell Biol 20:334-40
Yoh, Sunnie M; Cho, Helen; Pickle, Loni et al. (2007) The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev 21:160-74
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