Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro

Abstract

The HIV Tat protein is a potent transactivator of HIV transcription, increasing both RNA initiation and elongation. We now demonstrate that purified, full-length 86 amino acid Tat protein specifically transactivates the HIV LTR in vitro to a high level (25- to 60-fold). Tat transactivation was specifically blocked by anti-Tat serum, but not preimmune serum. Tat did not transactivate transcription from the control adenovirus major late promoter (AdMLP). HIV transcription was blocked at various functional steps during initiation and elongation complex formation. Similar to the control AdMLP, HIV basal initiation complex assembly was sensitive to the addition of 0.015% sarkosyl prior to the addition of nucleoside triphosphates. Resistance to 0.05% sarkosyl required the addition of G, C, and U, which constitute the first 13 bases of the HIV RNA transcript. The addition of Tat to the in vitro transcription relieved the 0.015% sarkosyl block. These Tat-induced complexes were sensitive to 0.05% sarkosyl, suggesting that transcriptional initiation had not occurred. Consistent with this hypothesis, the addition of G, C, and U to the Tat-induced transcription complexes allowed the rapid conversion to transcription initiation complexes. Tat also facilitated the formation of 0.015% sarkosyl-resistant complexes in a reconstituted transcription system containing partially purified transcription factors and polymerase II. Following the formation of stable initiation complexes, Tat increased the rate and efficiency of transcription elongation on the HIV but not the AdML template. Kinetic analysis of Tat transactivation suggests that approximately 30% of the Tat initiation complexes are converted to elongation complexes. We conclude that Tat, in addition to its demonstrated role in RNA elongation, facilitates transcription initiation in vitro.

Figure 1

Tat-specific transactivation of HIV-1 transcription in vitro. A. Silver stain of an SDS-PAGE gel of purified Tat. Lanes 1 and 4, molecular weight markers; lane 2, 0.5 μg Tat; lane 3, 5 μg Tat. B. Tat concentration dependence for maximal transactivation in vitro. The CD12 HIV template (75 ng) was incubated with HeLa whole-cell extract in the absence (lane 1) or presence of 0.04 μM (lane 2), 0.4 μM (lane 3), or 4.0 μM (lane 4) Tat. C. Inhibition of Tat transactivation by anti-Tat antibodies in vitro. The CD12 HIV or AdML DNA template was incubated in the absence (lanes 1 and 5) or presence (lanes 2–4 and 6–8) of 0.4 μM Tat. Rabbit anti-Tat polyclonal antibody (1:10 dilution; lanes 3 and 7) or rabbit preimmune serum (1:10 dilution; lanes 4 and 8) were added to in vitro transcription reactions containing the HIV (75 ng) or AdML (250 ng) templates in the presence of 0.4 μM Tat. D. Titration of AdMLP in the presence and absence of Tat. 75 ng (lanes 3 and 6), 150 ng (lanes 4 and 7), or 350 ng (lanes 5 and 8) of the AdMLP were transcribed in the presence or absence of 0.4 μM Tat as indicated. 75 ng of the HIV CD12 template was incubated in the absence (lane 1) or presence (lane 2) of 0.4 μM Tat. E. Cotranscription of HIV-1 wild-type and TAR mutant DNA templates. Wild-type HIV templates CD12 (75 ng) and LM2 (75 ng) or CD12 (75 ng) and TAR mutant template (TM25) were coincubated in HeLa whole-cell extracts in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 0.4 μM Tat. Full-length transcripts from CD12, LM2, and TM25 HIV-1 LTR DNA templates were 382, 313, and 309 nt, respectively.

Figure 2

Tat-specific transactivation of transcription from 5′-deletion and TAR-deletion mutant HIV-1 LTR DNA templates in vitro. A. Diagram of HIV-1 LTR DNA templates used for in vitro transcription. B. Tat trans-activation of wild-type and mutant HIV-1 LTR transcription in vitro. Promoter templates (75 ng) were added to in vitro transcription reactions containing either Tat storage buffer (−) or 0.4 μM Tat (+). C. Effect of SP1 on Tat transactivation. In vitro transactivation was performed with approximately 100 ng of either the wild-type HIV-1 LTR or an internal SP1 deletion mutant (SPE-CAT) in the presence or absence of Tat (0.4 μM). Bar graphs indicate fold transactivation as measured by ³²P cpm determination of RNA products.

Figure 3

Schematic diagram of discrete functional steps during transcription initiation and their differential sensitivities to sarkosyl in vitro. Adapted from Cai and Luse, 1987; Reinberg and Roeder, 1987; Hawley and Roeder, 1987; Buratowski et al., 1989; Saltzman and Weinmann, 1989.

Figure 4

Effects of sarkosyl on in vitro transcription from the adenovirus major late promoter (AdMLP). The indicated concentrations of sarkosyl were added before (lanes 1–8) or after (lanes 9–15) a 30-minute preincubation in the absence of exogenous nucleoside triphosphates to in vitro transcription reactions containing the AdML template (300 ng).

Figure 5

Facilitation of preinitiation complex formation on the HIV-1 LTR promoter in the presence of Tat. A. Resistance of HIV-1 transcription to 0.015% sarkosyl added at the start of preincubation in the presence of Tat. 75 ng of the CD12 template was transcribed in HeLa whole-cell extracts containing 0.0% to 0.25% sarkosyl during the preincubation period. Reactions were carried out in the absence (lanes 1–5) or presence (lanes 6–10) of 0.4 μM Tat. B. Effect of sarkosyl addition to in vitro transcription reactions after preincubation.

Figure 6

Ability of Tat to facilitate transcription preinitiation complex formation in the absence of nascent RNA synthesis. A. Effect of preincubation of HIV template and Tat in the presence of various nucleoside triphosphates. Different combinations of nucleoside triphosphates were added to in vitro transcription reactions containing the HIV CD12 promoter template (75 ng) without (lanes 1–3) or with (lanes 4–6) 0.4 μM Tat. After a 30-minute preincubation, 0.05% sarkosyl and then a full complement of nucleoside triphosphates were added to the reactions. B. Effect of limiting exogenous nucleoside triphosphates on HIV-1 transcription in vitro. Different combinations of nucleoside triphosphates were added to in vitro transcription reactions containing 300 ng of either AdMLP or HIV-1 LTR DNA templates. Transcription reactions were analyzed after a 30-minute incubation.

Figure 7

Stimulation of preinitiation complex formation by Tat in an in vitro transcription system reconstituted with partially purified transcription factors and RNA polymerase II. A. Kinetics of Tat transactivation in an in vitro transcription system reconstituted with purified transcription factors (TF) and RNA polymerase II. Transcription reactions containing a HIV-1 LTR DNA (CD12R1) template in the absence (lanes 1 and 3) or presence (lanes 2, 4, and 5) of 0.4 μM Tat were reconstituted with partially purified TFIIA, TFIIB, TFIID, TFIIE/F, Spl, and RNA polymerase II. Sarkosyl (0.015%) was added 7.5 minutes (lanes 1 and 2) or 15 minutes (lanes 3 and 4) after the start of incubation. In a separate reaction, 0.05% sarkosyl was added 5 minutes before addition of exogenous nucleoside triphosphates (lane 5). B. Tat transactivation of HIV-1 transcription initiation in an in vitro reconstituted system. Purified TFIIA, TFIIB, TFIID, TFIIE/F, Spl, and RNA polymerase II were preincubated with an HIV-1 LTR DNA template (75 ng) and 0.015% sarkosyl in the absence (lanes 1 and 3) or presence (lanes 2, 4, and 5) of 0.4 μM Tat. Sarkosyl concentration was either maintained at 0.015% throughout the 90-minute incubation (lanes 1 and 2) or raised to 0.05% 5 minutes before (lane 5) or 45 seconds after (lanes 3 and 4) exogenous nucleoside triphosphate addition.

Figure 8

Stimulation of transcription elongation on the HIV-1 LTR promoter in the presence of Tat. Wild-type (CD12R1) and TAR mutant (CD23dlS) HIV-1 LTR DNA templates (A) or AdML template (B) in HeLa whole-cell extracts were permitted to form preinitiation complexes for 30 minutes. Subsequent addition of nucleoside triphosphates (500 μM) and [α-³²P]UTP began productive initiation. Transcription was allowed to proceed for 10 minutes prior to the addition of purified Tat (0.4 μM). Following mock or Tat addition, assays were terminated at 45 minutes.

Figure 9

Kinetics of Tat transactivation in unfractionated HeLa cell extracts. Sarkosyl (0.015% or 0.05%) was added (5, 10, or 30 minutes) to complete in vitro transcription reactions containing the HIV CD12 template (75 ng), HeLa whole-cell extract, and exogenous nucleoside triphosphates (300 μM) in the absence (lanes 1, 3, 5, 7, 9, 11) or presence (lanes 2, 4, 6, 8, 10, 12) of 0.4 μM Tat.