Tat activates human immunodeficiency virus type 1 transcriptional elongation independent of TFIIH kinase

Abstract

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of the human transcription elongation factor P-TEFb, consisting of Cdk9 and cyclin T1, to the HIV-1 promoter via cooperative binding to the nascent HIV-1 transactivation response RNA element. The Cdk9 kinase activity has been shown to be essential for P-TEFb to hyperphosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II and mediate Tat transactivation. Recent reports have shown that Tat can also interact with the multisubunit transcription factor TFIIH complex and increase the phosphorylation of CTD by the Cdk-activating kinase (CAK) complex associated with the core TFIIH. These observations have led to the proposal that TFIIH and P-TEFb may act sequentially and in a concerted manner to promote phosphorylation of CTD and increase polymerase processivity. Here, we show that under conditions in which a specific and efficient interaction between Tat and P-TEFb is observed, only a weak interaction between Tat and TFIIH that is independent of critical amino acid residues in the Tat transactivation domain can be detected. Furthermore, immunodepletion of CAK under high-salt conditions, which allow CAK to be dissociated from core-TFIIH, has no effect on either basal HIV-1 transcription or Tat activation of polymerase elongation in vitro. Therefore, unlike the P-TEFb kinase activity that is essential for Tat activation of HIV-1 transcriptional elongation, the CAK kinase associated with TFIIH appears to be dispensable for Tat function.

FIG. 1

Strong and specific binding of Tat to P-TEFb but not to TFIIH. (A) Binding of P-TEFb to Tat. Affinity-purified P-TEFb complex fraction (6 μl) was incubated with wild-type GST-Tat(1-48) and three GST-Tat(1-48) mutants (C22G, K41A, and H33A) bound to glutathione-Sepharose beads. After extensive washes, the bound proteins were analyzed by SDS-PAGE and Western blotting for the presence of cyclin T1 and Cdk9-HA with antibodies specific for cyclin T1 and HA (MAb 12CA5), respectively. One microliter of the input P-TEFb fraction was used in lane 1 as a reference. The lower panel is a Coomassie-stained SDS gel showing the relative amounts of wild-type and mutant GST-Tat(1-48) proteins bound to glutathione-Sepharose beads. (B) Binding of TFIIH to Tat. TFIIH fraction (2 μl) with associated Cdk7-HA at a level similar to that of Cdk9-HA in P-TEFb (6 μl) was tested for binding to Tat under the same conditions as for P-TEFb. After washes, Western blotting with 12CA5 was used to detect Cdk7-HA bound to the GST-Tat beads. (C) Five times more TFIIH fraction (10 μl) was tested for binding to GST, wild-type, or mutant GST-Tat(1-48) beads. Bound proteins were examined by Western blotting with antibodies specific for ERCC3, the HA tag of Cdk7-HA, and Mat1.

FIG. 2

Tat transactivation in HeLa nuclear extract depleted of Cdk7 or Cdk9. (A [left panel]) Transcription reactions containing both templates pHIV+TAR-G400 and pHIVΔTAR-G100 were performed in the absence (−) or presence (+) of Tat and mock-depleted HeLa nuclear extract (lanes 1 and 2) or HeLa nuclear extract immunodepleted of the Cdk9 subunit of P-TEFb (lanes 3 to 6). Affinity-purified P-TEFb complex was added to Cdk9-depleted reactions as indicated. Immunodepletion was performed in the presence of 0.8 M KCl. (Right panel) The depleted extracts were examined by Western blotting with Cdk9 antibodies. (B and C) Cdk7, a subunit of the CAK ternary complex, was removed from HeLa nuclear extract by immunodepletion with anti-Cdk7 antibodies under high-salt (0.8 M KCl [B]) or low-salt (0.1 M KCl [C]) conditions. Immobilized anti-Myc antibody was used in control depletion reactions. (Left halves of panels B and C) Transcription reactions containing depleted extracts and transcription templates were carried out in the absence (−) or presence (+) of Tat. (Right halves of panels B and C) The depleted extracts were analyzed by Western blotting with anti-Cdk7 antibodies.

FIG. 2

Tat transactivation in HeLa nuclear extract depleted of Cdk7 or Cdk9. (A [left panel]) Transcription reactions containing both templates pHIV+TAR-G400 and pHIVΔTAR-G100 were performed in the absence (−) or presence (+) of Tat and mock-depleted HeLa nuclear extract (lanes 1 and 2) or HeLa nuclear extract immunodepleted of the Cdk9 subunit of P-TEFb (lanes 3 to 6). Affinity-purified P-TEFb complex was added to Cdk9-depleted reactions as indicated. Immunodepletion was performed in the presence of 0.8 M KCl. (Right panel) The depleted extracts were examined by Western blotting with Cdk9 antibodies. (B and C) Cdk7, a subunit of the CAK ternary complex, was removed from HeLa nuclear extract by immunodepletion with anti-Cdk7 antibodies under high-salt (0.8 M KCl [B]) or low-salt (0.1 M KCl [C]) conditions. Immobilized anti-Myc antibody was used in control depletion reactions. (Left halves of panels B and C) Transcription reactions containing depleted extracts and transcription templates were carried out in the absence (−) or presence (+) of Tat. (Right halves of panels B and C) The depleted extracts were analyzed by Western blotting with anti-Cdk7 antibodies.

FIG. 3

Cdk7-containing complexes have different sizes at different salt concentrations. HeLa nuclear extracts were subjected to ultracentrifugation sedimentation through a 13 to 30% glycerol gradient containing either 0.1 M (B and D) or 0.8 M KCl (A and C). Gradient fractions were analyzed by Western blotting using antibodies specific for Cdk7 (A and B) or ERCC3 (C and D). A mixture of molecular mass marker proteins were sedimented in a parallel gradient. Their positions in the gradient were determined by SDS-PAGE and silver staining and are indicated in between panels B and C.

FIG. 4

Efficient removal of CAK from holo-TFIIH by anti-Cdk7 depletion in the presence of high salt concentrations. (A) CAK can be dissociated from holo-TFIIH by high salt concentrations. Human 293T cells transiently transfected with an HA-tagged Cdk7 (Cdk7-HA) construct were lysed with buffers containing either 500 mM or 150 mM NaCl. Immunoprecipitation with anti-HA tag MAb 12CA5 was carried out at the same salt concentrations as in the lysis buffers. The immunoprecipitated proteins were analyzed by Western blotting with antibodies directed against Cdk7, Mat1, cyclin H, and ERCC3 as indicated. Occasionally, Cdk7-HA can be seen as a doublet probably because it can be modified differently under different conditions. (B) Cdk7-CAK in highly purified TFIIH complex can be immunodepleted in the presence of high salt concentrations. Highly purified TFIIH preparation was incubated with immobilized Cdk7 antibodies in the presence of 0.8 M KCl. Western blotting with antibodies directed against Cdk7 and ERCC3 was carried out to examine the presence of these two proteins in the highly purified TFIIH preparation after immunodepletion.

FIG. 5

Immunodepletion of CAK from holo-TFIIH with anti-Mat1 antibodies does not affect Tat activation. Immobilized anti-Mat1 antibodies were used to immunodeplete Mat1 from HeLa nuclear extract containing 0.8 M KCl. Immobilized anti-Myc MAb was used in a control depletion reaction. (A) The depleted extracts were analyzed for their abilities to mediate Tat activation in transcription reactions as described in the legend for Fig. 1. (B) The removal of Mat1 and its associated Cdk7 in the CAK complex was confirmed by Western blotting with anti-Mat1 and anti-Cdk7 antibodies.