Research Article: Phosphorylation of CDK9 at Ser175 Enhances HIV Transcription and Is a Marker of Activated P-TEFb in CD4+ T Lymphocytes

Date Published: May 2, 2013

Publisher: Public Library of Science

Author(s): Uri R. Mbonye, Giridharan Gokulrangan, Manish Datt, Curtis Dobrowolski, Maxwell Cooper, Mark R. Chance, Jonathan Karn, Michael Emerman.


The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.

Partial Text

HIV infections persist throughout the lifetimes of patients due to the creation of a latent viral reservoir that is refractory to both antiviral immune responses and antiretroviral therapy (ART) [1], [2], [3], [4], [5]. Genetic and biochemical evidence strongly suggests that the major latent viral reservoir comprises a small population of resting memory CD4+ T-cells (∼1 in 106 cells) [6], [7], [8] that are created when effector T-cells acquire a Go resting memory phenotype [9] or when resting memory T-cells become infected [10]. Interruption of ART invariably leads to a rebound of virus production, even in patients that have been suppressed to below detectable levels of viremia for decades [11], [12], [13], [14], [15], [16]. The need to develop novel therapeutic tools to attack the latently infected population is now a widely recognized goal [1], [5], but implementation of this will require both a more detailed understanding of mechanisms underlying proviral latency and the creation of improved analytical tools to monitor the state of the latent proviral reservoir [3], [4], [17].




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