P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro - PubMed (original) (raw)

P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro

H S Mancebo et al. Genes Dev. 1997.

Abstract

To identify novel inhibitors of transcriptional activation by the HIV Tat protein, we used a combination of in vitro and in vivo Tat-dependent transcription assays to screen >100,000 compounds. All compounds identified blocked Tat-dependent stimulation of transcriptional elongation. Analysis of a panel of structurally diverse inhibitors indicated that their target is the human homolog of Drosophila positive transcription elongation factor b (P-TEFb). Loss of Tat transactivation in extracts depleted of the kinase subunit of human P-TEFb, PITALRE, was reversed by addition of partially purified human P-TEFb. Transfection experiments with wild-type or kinase knockout PITALRE demonstrated that P-TEFb is required for Tat function. Our results suggest that P-TEFb represents an attractive target for the development of novel HIV therapeutics.

PubMed Disclaimer

Figures

Figure 1

Tat-dependent in vitro transcription assay. Transcription reactions were as described in Materials and Methods and were reconstituted with purified Pol II, basal factors (GF mix), and a small amount of nuclear extract. Reaction products were quantitated with a Fuji PhosphorImager. The (−) and (+) signs indicate reactions without or with Tat protein (25 n

), respectively. (A) The effect of increasing concentrations of nuclear extract on Tat transactivation. The fold activation for each reaction set (bracketed) is indicated at the bottom of the panel. The amount (μg) of HeLa nuclear extract (Ne) added to the reactions is also indicated. (B) Optimal Tat transactivation requires an intact TAR element and HIV LTR promoter sequences from −80 to −40, relative to the start site of transcription. Brackets group sets of reactions minus (−) and plus (+) Tat according to the LTR promoter derivative used as the DNA template. (wt) Wild-type TAR sequences; (ΔTAR) deletion of TAR; (Lm) loop mutant; (Bm) bulge mutant. (C) The effect of increasing Tat concentrations on Tat-dependent transcription from either a wild-type or TAR-deleted −80 LTR promoter. Tat was added at 0.7, 2.8, 7, 28, and 66 n

Figure 2

Selective inhibition of Tat activation in vitro by various types of kinase inhibitors. (A) A schematic representation of the DNA templates used to measure transcriptional activation by HCMV IE2, BZLF, and GAL4–VP16 proteins is shown. The fold induction attained in response to these proteins is indicated by the bar graph. (B) The effects of TRB, T276339, and H7 on various transcription assays. Transcription reactions were as described Materials and Methods and were quantitated with a PhosphorImager. For each reaction type, the amount of product obtained in the absence of drug is defined as 100% activity. (•) Tat; (□) IE2; (▪) BZLF; (♦) GAL4–VP16; (▴) LTR–basal.

Figure 3

Selective inhibition of Tat-mediated potentiation of transcription elongation in vitro and in vivo by various types of kinase inhibitors. Lanes labeled (−) and (+) refer to experiments performed in the presence or absence of Tat, respectively. (A) Use of RNase protection to assess the effects of kinase inhibitors on the production of short and long mRNA transcripts. RNase protection and transcription reactions were performed as described in Materials and Methods. Transcription reactions were reconstituted with the HIV LTR promoter fused to a luciferase reporter gene so that the same probes could be used to measure reaction products from in vitro and in vivo experiments. A schematic representation of the DNA template and the RNA probes is shown. The thin line in the 5′ probe denotes LTR promoter sequences that are not protected by accurately initiated transcripts. TRB (1 μ

), T276339 (10 μ

), H7 (10 μ

), or DMSO solvent were added to reactions as indicated at the top. The mobility of products protected by the 5′ and 3′ probes in 6% polyacrylamide sequencing gels is indicated at the right. (B) Quantitation of products generated in the experiments in A. Values obtained in the absence of drug are defined as 100% activity. (▪) 5′ transcript; (□) 3′ transcript.

Figure 4

Structures of compounds analyzed in Tables 1 and 2. Compounds are grouped according to their structural class. The chemical name and source of each compound are described in Materials and Methods.

Figure 5

Requirement of P-TEFb for Tat transactivation in vitro. (A) Purification scheme for HeLa-derived P-TEFb. (B) CTD kinase assay (top) and PITALRE Western blot analysis (bottom) of Superdex 200 column fractions. Conditions for each assay were as described in Materials and Methods. (C) HeLa cell-purified P-TEFb restores Tat transactivation to in vitro transcription reactions reconstituted with nuclear extracts that had been immunodepleted with antibodies against PITALRE. The presence (+) or absence (−) of Tat and/or DRB are indicated at the top. Reactions are grouped with brackets according to the factors added to complement Tat transactivation. (GF) General factors and Pol II; (Ne) HeLa nuclear extracts (14 μg) depleted with control antibodies; (dNE) HeLa nuclear extracts (14 μg) depleted with antibodies to PITALRE; (P-TEFb) HeLa cell-purified P-TEFb. Lane numbers are indicated at the bottom. (D) Western blot analysis showing the depletion of PITALRE but not Cdk7 from HeLa nuclear extracts by the anti-PITALRE antibody.

Figure 5

Figure 6

Participation of P-TEFb in Tat transactivation in intact cells. (A) Inhibition of Tat-dependent transactivation by overexpression of mutated PITALRE in Jurkat cells. All transfections were performed with a wild-type LTR–luciferase reporter with (+) or without (−) a Tat expression vector and without (-) or with wild-type (wt) or mutated (mt) PITALRE expression vectors as indicated. Luciferase values were normalized with respect to the luciferase activity obtained with the reporter plasmid alone. In each case the luciferase values represent the average of three independent experiments. For each experiment, luciferase values were normalized with respect to those obtained upon transfection of the reporter plasmid alone. (B) Stimulation of Tat-dependent activation in HeLa cells by wild-type, but not mutated PITALRE. Transfection assays with (+) or without (−) a Tat expression vector are indicated.

Cited by

Transcriptional and posttranscriptional regulation of HIV-1 gene expression.
Karn J, Stoltzfus CM. Karn J, et al. Cold Spring Harb Perspect Med. 2012 Feb;2(2):a006916. doi: 10.1101/cshperspect.a006916. Cold Spring Harb Perspect Med. 2012. PMID: 22355797 Free PMC article. Review.
Bovine HEXIM1 inhibits bovine immunodeficiency virus replication through regulating BTat-mediated transactivation.
Guo HY, Ma YG, Gai YM, Liang ZB, Ma J, Su Y, Zhang QC, Chen QM, Tan J. Guo HY, et al. Vet Res. 2013 Mar 27;44(1):21. doi: 10.1186/1297-9716-44-21. Vet Res. 2013. PMID: 23537346 Free PMC article.
Understanding HIV latency: the road to an HIV cure.
Dahabieh MS, Battivelli E, Verdin E. Dahabieh MS, et al. Annu Rev Med. 2015;66:407-21. doi: 10.1146/annurev-med-092112-152941. Annu Rev Med. 2015. PMID: 25587657 Free PMC article. Review.
Inhibition of human immunodeficiency virus type 1 replication by RNA interference directed against human transcription elongation factor P-TEFb (CDK9/CyclinT1).
Chiu YL, Cao H, Jacque JM, Stevenson M, Rana TM. Chiu YL, et al. J Virol. 2004 Mar;78(5):2517-29. doi: 10.1128/jvi.78.5.2517-2529.2004. J Virol. 2004. PMID: 14963154 Free PMC article.
Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency.
Kim YK, Bourgeois CF, Pearson R, Tyagi M, West MJ, Wong J, Wu SY, Chiang CM, Karn J. Kim YK, et al. EMBO J. 2006 Aug 9;25(15):3596-604. doi: 10.1038/sj.emboj.7601248. Epub 2006 Jul 27. EMBO J. 2006. PMID: 16874302 Free PMC article.

References

1. Akoulitchev S, Makela TP, Weinberg RA, Reinberg D. Requirement for IIH kinase activity in transcription by RNA polymerase II. Nature. 1995;377:557–560. - PubMed
1. Ausubel F, Brent R, Kingston RE, Moore D, Seidman JG, Smith JA, Struhl K. Current protocols in molecular biology. Vol. 1. New York, NY: Greene-Wiley; 1995. RNase protection assay; pp. 4.7.1–4.7.8.
1. Blau J, Xiao H, McCracken S, O’Hare P, Greenblatt J, Bentley D. Three functional classes of transcriptional activation domains. Mol Cell Biol. 1996;16:2044–2055. - PMC - PubMed
1. Byrnes V, Hazuda D. A system to analyze and identify inhibitors of HIV-1 gene regulation using a defective integrated provirus. Methods Enzymol. 1996;275:348–361. - PubMed
1. Chang C, Kostrub CF, Burton ZF. RAP30/74 (transcription factor IIF) is required for promoter escape by RNA polymerase II. J Biol Chem. 1993;268:20482–20489. - PubMed

P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro - PubMed (original) (raw)