Role of Cyclin T1 acetylation in HIV transcription

Sungyoo Cho, J. David Gladstone Institutes
Molecular Biology

The viral Tat protein and its cellular interaction partners are necessary for the transcriptional activity of the HIV promoter. Tat binds to an RNA stem-loop structure called TAR and recruits the positive elongation factor b (P-TEFb) to the 5' extremities of HIV-1 transcripts. P-TEFb is composed of the regulatory subunit cyclin T1 and the cyclin-dependent kinase 9 (CDK9) and phosphorylates the C-terminal domain of the RNA polymerase II (RNAPII) to enhance processivity of the polymerase complex. Tat also recruits the histone acetyltransferase activity of p300 to the HIV promoter and is itself acetylated by p300 at a single lysine (K50) located in the RNA-binding region of Tat. We have previously shown that Tat acetylation by p300 disrupts the formation of the Tat-TAR-P-TEFb complex and transfers acetylated Tat directly to the elongating RNAPII, where it can interact with the transcriptional coactivator PCAF via the PCAF bromodomain. We now show that cyclin T1 is also acetylated by p300. By using mass spectrometry and site-directed mutagenesis, we have mapped the site of acetylation to four lysines. Cyclin T1 acetylation activates P-TEFb transcriptional activity by dissociation of ribonucleoprotein complexes containing Hexim 1. Several reports demonstrated that the activity of the core P-TEFb complex (a heterodimer of cyclin T1, T2a or T2b and CDK9) is regulated by the association with different factors. The active "small" P-TEFb complex is composed of the core complex and can associate with the bromodomain protein Brd4. The inactive "large" complex consists of the core complex together with 7SK small nuclear (sn) RNA, and Hexim 1. The relative partitioning of P-TEFb into active and inactive complexes changes rapidly by several stress-inducing agents.

We propose to analyze the effect of cyclin T1 acetylation on Tat function and HIV infection. To test whether acetylated cyclin T1 associates with large, inactive or small, active P-TEFb complexes we will generate antibodies specific for acetylated cyclin T1. We will perform glycerol gradient sedimentations to fractionate large and small P-TEFb and examine the presence of endogenous acetylated cyclin T1 in the different fractions. Since cyclin T1 binds cooperatively with Tat to TAR RNA we will analyze in in vitro RNA bandshift assays if the affinity of cyclin T1 to Tat and TAR RNA is altered by acetylation. We will also determine in chromatin immunoprecipitation experiments whether acetylated cyclin T1 associates with the HIV promoter in vivo. To examine the role of cyclin T1 acetylation in the HIV life cycle we will test the ability of an acetylation-deficient mutant of cyclin T1 to suppress HIV-1 replication in a dominant-negative manner. We will also express the acetylation-deficient cyclin T1 mutant in murine cells (naturally lacking Tat-binding cyclin T1) together with Tat and analyze its ability to transactivate the HIV promoter. Finally, we will generate a fusion protein composed of the HIV Rev protein and the acetylation-deficient cyclin T1 mutant and test its ability to transactivate an HIV promoter construct expressing the rev response element (RRE) instead of TAR. These experiments should characterize the role of cyclin T1 acetylation in P-TEFb biology and demonstrate whether cyclin T1 acetylation regulates HIV infection.