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Updated: May 17, 2026
Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells
Published on: January 26, 2017
Identification of novel CDK9 and Cyclin T1-associated protein complexes (CCAPs) whose siRNA depletion enhances HIV-1
Rajesh Ramakrishnan1, Hongbing Liu, Hart Donahue
1Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas, USA.
Background:
HIV-1 Tat activates RNA Polymerase II (RNAP II) elongation of the integrated provirus by recruiting a protein kinase known as P-TEFb to TAR RNA at the 5' end of nascent viral transcripts. The catalytic core of P-TEFb contains CDK9 and Cyclin T1 (CCNT1). A human endogenous complexome has recently been described - the set of multi-protein complexes in HeLa cell nuclei. We mined this complexome data set and identified 12 distinct multi-protein complexes that contain both CDK9 and CCNT1. We have termed these complexes CCAPs for CDK9/CCNT1-associated protein complexes. Nine CCAPs are novel, while three were previously identified as Core P-TEFb, the 7SK snRNP, and the Super-Elongation Complex. We have investigated the role of five newly identified CCAPs in Tat function and viral gene expression.
Results:
We examined five CCAPs that contain: 1) PPP1R10/TOX3/WDR82; 2) TTF2; 3) TPR; 4) WRNIP1; 5) FBXO11/CUL1/SKP1. SiRNA depletions of protein subunits of the five CCAPs enhanced Tat activation of an integrated HIV-1 LTR-Luciferase reporter in TZM-bl cells. Using plasmid transfection assays in HeLa cells, we also found that siRNA depletions of TTF2, FBXO11, PPP1R10, WDR82, and TOX3 enhanced Tat activation of an HIV-1 LTR-luciferase reporter, but the depletions did not enhance expression of an NF-κB reporter plasmid with the exception of PPP1R10. We found no evidence that depletion of CCAPs perturbed the level of CDK9/CCNT1 in the 7SK snRNP. We also found that the combination of siRNA depletions of both TTF2 and FBXO11 sensitized a latent provirus in Jurkat cells to reactivation by sub-optimal amounts of αCD3/CD28 antibodies.
Conclusions:
Our results identified five novel CDK9/CCNT1 complexes that are capable of negative regulation of HIV-1 Tat function and viral gene expression. Because siRNA depletions of CCAPs enhance Tat function, it is possible that these complexes reduce the level of CDK9 and CCNT1 available for Tat, similar to the negative regulation of Tat by the 7SK snRNP. Our results highlight the complexity in the biological functions of CDK9 and CCNT1.
Insights
Researchers identified five novel CDK9/CCNT1 complexes (CCAPs) that negatively regulate HIV-1 Tat function. Depleting these CCAPs enhances viral gene expression, revealing new targets for therapeutic intervention.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- HIV-1 Tat protein is crucial for viral gene expression by recruiting P-TEFb (CDK9/Cyclin T1) to activate RNA Polymerase II elongation.
- The human endogenous complexome provides a resource for identifying novel protein complexes.
- Twelve distinct CDK9/CCNT1-containing complexes (CCAPs) were identified, including nine novel ones.
Purpose of the Study:
- To investigate the role of newly identified CCAPs in HIV-1 Tat function and viral gene expression.
- To determine if these novel complexes modulate Tat-activated transcription.
Main Methods:
- Mining the human endogenous complexome data for CDK9/CCNT1 complexes.
- Utilizing siRNA depletions to assess the impact of CCAP subunit loss on HIV-1 LTR-driven luciferase reporter expression in TZM-bl and HeLa cells.
- Evaluating the effect of CCAP depletion on NF-κB reporter activity.
- Assessing the reactivation of latent HIV-1 provirus in Jurkat cells.
Main Results:
- Depletion of subunits from five novel CCAPs enhanced Tat-activated HIV-1 transcription.
- Specific CCAP subunit depletions (TTF2, FBXO11, PPP1R10, WDR82, TOX3) boosted Tat activity, with PPP1R10 also affecting NF-κB activity.
- CCAP depletion did not alter CDK9/CCNT1 levels within the 7SK snRNP.
- Combined depletion of TTF2 and FBXO11 sensitized latent HIV-1 to reactivation.
Conclusions:
- Five novel CCAPs negatively regulate HIV-1 Tat function and viral gene expression.
- These CCAPs may reduce the availability of CDK9/CCNT1 for Tat-mediated activation, similar to the 7SK snRNP.
- The findings underscore the complex regulatory roles of CDK9 and CCNT1 in viral processes.
