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Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR.

Sedore SC, Byers SA, Biglione S, Price JP, Maury WJ, Price DH - Nucleic Acids Res. (2007)

Bottom Line: P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2.Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat.In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa, Iowa City, IA, USA.

ABSTRACT
Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb-HEXIM1-7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb-HEXIM1-7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.

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HIV Tat competes with HEXIM1 for binding to 7SK and inhibits the formation of the P-TEFb–HEXIM1–7SK complex (A) The binding of 10 ng of HEXIM1 or the indicated amounts of Tat recombinant proteins to in vitro transcribed, radiolabeled 7SK was evaluated by electrophoretic mobility shift assay (EMSA) under equilibrium conditions as described in Materials and Methods. The proteins were added to the radiolabeled 7SK individually or HEXIM1 (H1) was added 10 min before the indicated amounts of Tat (HEXIM1/Tat) was added for an additional 10 min. Reactions were also carried out with the indicated amounts of Tat added to 7SK before 10 ng of HEXIM1 (Tat/HEXIM1) was added. Complexes were resolved by gel electrophoresis on a native gel and visualized by autoradiography. (B) The ability to form a P-TEFb–HEXIM1–7SK complex or inhibit formation of the complex was evaluated by titrating the indicated amounts of P-TEFb and/or Tat onto a preformed HEXIM1–7SK complex containing 3 ng of HEXIM1 and resolving complexes as in (A).
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Figure 3: HIV Tat competes with HEXIM1 for binding to 7SK and inhibits the formation of the P-TEFb–HEXIM1–7SK complex (A) The binding of 10 ng of HEXIM1 or the indicated amounts of Tat recombinant proteins to in vitro transcribed, radiolabeled 7SK was evaluated by electrophoretic mobility shift assay (EMSA) under equilibrium conditions as described in Materials and Methods. The proteins were added to the radiolabeled 7SK individually or HEXIM1 (H1) was added 10 min before the indicated amounts of Tat (HEXIM1/Tat) was added for an additional 10 min. Reactions were also carried out with the indicated amounts of Tat added to 7SK before 10 ng of HEXIM1 (Tat/HEXIM1) was added. Complexes were resolved by gel electrophoresis on a native gel and visualized by autoradiography. (B) The ability to form a P-TEFb–HEXIM1–7SK complex or inhibit formation of the complex was evaluated by titrating the indicated amounts of P-TEFb and/or Tat onto a preformed HEXIM1–7SK complex containing 3 ng of HEXIM1 and resolving complexes as in (A).

Mentions: The in vivo studies just presented indicate that the expression of Tat leads to the release of P-TEFb from the P-TEFb–HEXIM1–7SK complex and to the formation of a P-TEFb complex. However, due to the ambiguity of the in vivo system, it is not clear whether this release is due to a direct effect of Tat on the complex or more a more indirect effect, such as Tat association with P-TEFb complex leading to lower levels of P-TEFb activity and less general transcription that might trigger the cellular mechanism that releases P-TEFb from the large form. Because of this, a defined in vitro system was used to investigate the biochemical mechanism of this release. EMSAs were carried out under equilibrium binding conditions with protein in vast excess over 32P-labeled in vitro transcribed full-length 7SK. Two-hundred nanograms of highly structured tRNA (>1000-fold excess over 7SK) was used in each reaction as a non-specific competitor instead of poly (rI):poly (rC) that was used previously (19,21,25,52) because we recently found that HEXIM1 binds tightly to dsRNA (53). Dimeric HEXIM1 (25) formed its characteristic specific complex with 7SK (Figure 3A, H1–7SK). Somewhat surprisingly, as increasing amounts of Tat were added to 7SK a more slowly migrating complex formed, demonstrating that Tat can bind to 7SK forming a Tat–7SK complex. A portion of the 7SK was also shifted into the well and this could be due to association of 7SK with oxidized aggregates of Tat. When HEXIM1 was pre-incubated with 7SK to form the HEXIM1–7SK complex and then increasing amounts of Tat were added before being loaded onto the native gel, the HEXIM1–7SK complex was gradually eliminated and was replaced by a Tat–7SK complex. When the Tat–7SK complex was preformed and then HEXIM1 was added, the competition was even stronger. The amounts of the Tat–7SK complex formed were virtually identical with or without HEXIM1 being present. The fact that larger molar amounts of Tat were required is likely due to the presence of a large fraction of inactive Tat in the preparation used. We conclude that Tat can compete strongly with HEXIM1 for binding to 7SK, and that even when the relatively stable HEXIM1–7SK complex is preformed, Tat can disrupt it. There was no evidence for a significant amount of a Tat–HEXIM1–7SK complex, indicating that Tat and HEXIM1 bind to an identical or overlapping region of 7SK or that Tat and HEXIM1 bind to different mutually exclusive conformations of 7SK.Figure 3.


Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR.

Sedore SC, Byers SA, Biglione S, Price JP, Maury WJ, Price DH - Nucleic Acids Res. (2007)

HIV Tat competes with HEXIM1 for binding to 7SK and inhibits the formation of the P-TEFb–HEXIM1–7SK complex (A) The binding of 10 ng of HEXIM1 or the indicated amounts of Tat recombinant proteins to in vitro transcribed, radiolabeled 7SK was evaluated by electrophoretic mobility shift assay (EMSA) under equilibrium conditions as described in Materials and Methods. The proteins were added to the radiolabeled 7SK individually or HEXIM1 (H1) was added 10 min before the indicated amounts of Tat (HEXIM1/Tat) was added for an additional 10 min. Reactions were also carried out with the indicated amounts of Tat added to 7SK before 10 ng of HEXIM1 (Tat/HEXIM1) was added. Complexes were resolved by gel electrophoresis on a native gel and visualized by autoradiography. (B) The ability to form a P-TEFb–HEXIM1–7SK complex or inhibit formation of the complex was evaluated by titrating the indicated amounts of P-TEFb and/or Tat onto a preformed HEXIM1–7SK complex containing 3 ng of HEXIM1 and resolving complexes as in (A).
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Figure 3: HIV Tat competes with HEXIM1 for binding to 7SK and inhibits the formation of the P-TEFb–HEXIM1–7SK complex (A) The binding of 10 ng of HEXIM1 or the indicated amounts of Tat recombinant proteins to in vitro transcribed, radiolabeled 7SK was evaluated by electrophoretic mobility shift assay (EMSA) under equilibrium conditions as described in Materials and Methods. The proteins were added to the radiolabeled 7SK individually or HEXIM1 (H1) was added 10 min before the indicated amounts of Tat (HEXIM1/Tat) was added for an additional 10 min. Reactions were also carried out with the indicated amounts of Tat added to 7SK before 10 ng of HEXIM1 (Tat/HEXIM1) was added. Complexes were resolved by gel electrophoresis on a native gel and visualized by autoradiography. (B) The ability to form a P-TEFb–HEXIM1–7SK complex or inhibit formation of the complex was evaluated by titrating the indicated amounts of P-TEFb and/or Tat onto a preformed HEXIM1–7SK complex containing 3 ng of HEXIM1 and resolving complexes as in (A).
Mentions: The in vivo studies just presented indicate that the expression of Tat leads to the release of P-TEFb from the P-TEFb–HEXIM1–7SK complex and to the formation of a P-TEFb complex. However, due to the ambiguity of the in vivo system, it is not clear whether this release is due to a direct effect of Tat on the complex or more a more indirect effect, such as Tat association with P-TEFb complex leading to lower levels of P-TEFb activity and less general transcription that might trigger the cellular mechanism that releases P-TEFb from the large form. Because of this, a defined in vitro system was used to investigate the biochemical mechanism of this release. EMSAs were carried out under equilibrium binding conditions with protein in vast excess over 32P-labeled in vitro transcribed full-length 7SK. Two-hundred nanograms of highly structured tRNA (>1000-fold excess over 7SK) was used in each reaction as a non-specific competitor instead of poly (rI):poly (rC) that was used previously (19,21,25,52) because we recently found that HEXIM1 binds tightly to dsRNA (53). Dimeric HEXIM1 (25) formed its characteristic specific complex with 7SK (Figure 3A, H1–7SK). Somewhat surprisingly, as increasing amounts of Tat were added to 7SK a more slowly migrating complex formed, demonstrating that Tat can bind to 7SK forming a Tat–7SK complex. A portion of the 7SK was also shifted into the well and this could be due to association of 7SK with oxidized aggregates of Tat. When HEXIM1 was pre-incubated with 7SK to form the HEXIM1–7SK complex and then increasing amounts of Tat were added before being loaded onto the native gel, the HEXIM1–7SK complex was gradually eliminated and was replaced by a Tat–7SK complex. When the Tat–7SK complex was preformed and then HEXIM1 was added, the competition was even stronger. The amounts of the Tat–7SK complex formed were virtually identical with or without HEXIM1 being present. The fact that larger molar amounts of Tat were required is likely due to the presence of a large fraction of inactive Tat in the preparation used. We conclude that Tat can compete strongly with HEXIM1 for binding to 7SK, and that even when the relatively stable HEXIM1–7SK complex is preformed, Tat can disrupt it. There was no evidence for a significant amount of a Tat–HEXIM1–7SK complex, indicating that Tat and HEXIM1 bind to an identical or overlapping region of 7SK or that Tat and HEXIM1 bind to different mutually exclusive conformations of 7SK.Figure 3.

Bottom Line: P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2.Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat.In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa, Iowa City, IA, USA.

ABSTRACT
Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb-HEXIM1-7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb-HEXIM1-7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.

Show MeSH
Related in: MedlinePlus