Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro

Abstract

P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

Figure 1

The small subunit of Drosophila P-TEFb is similar to PITALRE. (Dm) Small subunit of Drosophila P-TEFb; (Hu) human PITALRE. Black reverse shading indicates identity.

Figure 2

Human P-TEFb is required for the generation of DRB-sensitive runoff transcripts. (A) Immunodepletion. HNE was passed over two successive protein A columns containing either affinity-purified PITALRE–CT IgG or affinity-purified rabbit anti-goat IgG (control) as diagramed. (B) Western blot of HNE depleted with indicated antibodies probed with PITALRE–CT antibodies. (Marker) Bacterially expressed PITALRE. (C) Transcriptional activity of human P-TEFb. The pulse–chase protocol is described in Materials and Methods. (DmP-TEFb) Drosophila P-TEFb; (tRNA) tRNA labeled with [³²P]CTP during transcription reaction (recovery control).

Figure 3

Immunoprecipitation of human P-TEFb. (A) CTD kinase assay. Purified Drosophila RNA polymerase II (Price et al. 1987; Marshall and Price 1995) was used as substrate. (DmP-TEFb) Drosophila P-TEFb; (Control) beads after depletion of HNE by affinity-purified rabbit anti-goat IgG; (HuP-TEFb) beads after depletion of HNE by PITALRE–CT IgG. Products were analyzed by 6%–15% gradient SDS-PAGE. Selected size standards from the 10-kD ladder are indicated and apply to all gels. (B) Silver-stained SDS-PAGE of proteins bound to PITALRE–CT IgG beads after washing with a buffer containing 20 mm HEPES (pH 7.6), 0.5% NP-40, 1% Triton X-100, and 5 mm DTT and the indicated amount of NaCl. Sizes of proteins remaining after high salt wash are indicated. Bands marked with an asterisk (*) are from IgG. (C) Kinase assay with only endogenous substrates of the fractions analyzed in B either without or with 50 μm DRB, as indicated.

Figure 4

DRB and H-8 inhibition of transcription and human P-TEFb activity. (A,B) Transcription of HIV LTR template (633-nucleotide runoff); (C,D) CTD kinase assay using immunoprecipitated human P-TEFb and Drosophila RNA polymerase II as substrate, as in Fig. 3A. (B,D) Plot of radioactivity in runoff or polymerase IIo after quantitation using a Packard InstantImager and normalization to the starting amount (100).

Figure 5

PITALRE associates with the activation domain of HIV-1 Tat. (A) TAK activity assay using CTD3 peptide as substrate. The indicated GST Tat or Tat mutant proteins were used. Both Tat72 and Tat48Δ have intact transactivation domains, whereas other constructs have mutations that abolish transactivation. (Tat72) HIV-1 Tat containing residues 1–72; (Tat48Δ) the activation domain of HIV-1 Tat containing residues 1–48; (Cys22) mutant Tat containing Gly instead of Cys at position 22; (Pro18IS) Tat mutant containing Glu and Phe insertion after Pro-18. (B) Western blot of the same samples using antibodies against PITALRE–CT. (C) TAK activity assays using GST–Tat48Δ were performed with whole or depleted (depHNE) HNE. In the kinase assay CTD3 peptide was used as substrate. (−) No kinase added; (*) no CTD3 peptide added. Human P-TEFb (HuP-TEFb) was immunoprecipitated from the equivalent of 1 μl of HNE.

Figure 6

PITALRE coelutes with TAK activity. (A–C) Heparin column fractions; (D–F) SP column fractions. TAK activity was assayed with GST–Tat48Δ across both columns using CTD3 as the kinase substrate (A,D); PITALRE was detected in Western blots using antibodies against whole PITALRE (B,E) or PITALRE–CT, (C,F).

Figure 7

P-TEFb is required for Tat transactivation in vitro. (A) In vitro Tat transactivation using a continuous labeling protocol with the indicated templates and extracts. Runoff transcripts were analyzed in a 6% TBE/urea gel. (B) Similar reaction mixtures as in A were subjected to a 2-min pulse, and the short transcripts generated (<30 nucleotides) were analyzed in an 18% TBE/urea gel. (C) Quantitation data for A and B. The amount of runoff or short transcripts for each template/extract combination was normalized to the corresponding lane with no DRB or Tat added.