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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 binds to a region of 7SK resembling TAR. (A) Analysis of the sequence of 7SK reveals three AUCUG Tat consensus-binding sites in the first 100 nt. Several structured and unstructured RNA oligos with or without this consensus sequence were designed and chemically synthesized with most coming from the native 7SK sequences indicated. 7SK (10–48 M) has one insertion and one deletion in an otherwise wild-type 7SK (10–48) sequence. (B) Predicted structures of all oligos with sufficient stability to be the predominate form at room temperature. (C) Competition EMSA analysis of Tat–7SK complex formation. Tat, 32P-labeled 7SK, and the indicated cold RNA oligos were pre-incubated and the resulting complexes were resolved by gel electrophoresis on a native gel, followed by autoradiography to visualize the 7SK shift. The dsRNA was a 25-bp double-stranded RNA unrelated to 7SK sequence described previously (53). (D) Competition EMSA comparison between 7SK (10–48) and 7SK (10–48 M). Note in this experiment the 7SK was of higher specific activity than in (C) so less Tat was needed to achieve a higher fraction of Tat–7SK complex.
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Figure 5: HIV Tat binds to a region of 7SK resembling TAR. (A) Analysis of the sequence of 7SK reveals three AUCUG Tat consensus-binding sites in the first 100 nt. Several structured and unstructured RNA oligos with or without this consensus sequence were designed and chemically synthesized with most coming from the native 7SK sequences indicated. 7SK (10–48 M) has one insertion and one deletion in an otherwise wild-type 7SK (10–48) sequence. (B) Predicted structures of all oligos with sufficient stability to be the predominate form at room temperature. (C) Competition EMSA analysis of Tat–7SK complex formation. Tat, 32P-labeled 7SK, and the indicated cold RNA oligos were pre-incubated and the resulting complexes were resolved by gel electrophoresis on a native gel, followed by autoradiography to visualize the 7SK shift. The dsRNA was a 25-bp double-stranded RNA unrelated to 7SK sequence described previously (53). (D) Competition EMSA comparison between 7SK (10–48) and 7SK (10–48 M). Note in this experiment the 7SK was of higher specific activity than in (C) so less Tat was needed to achieve a higher fraction of Tat–7SK complex.

Mentions: To gain insight into what sequence Tat recognized in 7SK, the first 100 nt of 7SK were compared to HIV TAR, and a set of oligos were designed and tested for their ability to block Tat binding to 7SK. The target for Tat binding in TAR consists of an AUCUG forming a bulge in the apical region of the TAR stem-loop and this binding of Tat changes the structure of TAR so that a pocket forms between U23 and G26 (54–56). When the sequence of 7SK was analyzed, three AUCUG motifs were found (Figure 5A). This sequence would be expected to occur randomly once every 45 bp, or about 1 in 1000 nt, and so it may be significant that it is found three times in the first 100 nt of 7SK. A set of oligos were designed that contained AUCUG sequences in the context of sequences that would be unstructured or that could form secondary structure with the AUCUG in loops or in bulges. Other regions of 7SK were also sampled as controls (Figure 5A). mFold suggested that 7SK (10–48) would form a stem-loop with one AUCUG in a bulge and one in the loop and is shown compared to TAR in Figure 5B. HEXIM1 has been demonstrated to bind to a region of in the 5′ end of 7SK (57) and to this specific oligo as well as it binds to intact 7SK (53). Unlabeled oligos were then used as competitors in EMSAs with labeled full-length 7SK and Tat. No competition was seen for any of the oligos except for 7SK (10–48) (Figure 5C and D). This strongly suggests that Tat associated with the residues from 10 to 48 of 7SK. Because no competition was seen with oligos comprised of 11–34 or 20–36, Tat may require the secondary structure shown in Figure 5B to be able to interact with 7SK.Figure 5.


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 binds to a region of 7SK resembling TAR. (A) Analysis of the sequence of 7SK reveals three AUCUG Tat consensus-binding sites in the first 100 nt. Several structured and unstructured RNA oligos with or without this consensus sequence were designed and chemically synthesized with most coming from the native 7SK sequences indicated. 7SK (10–48 M) has one insertion and one deletion in an otherwise wild-type 7SK (10–48) sequence. (B) Predicted structures of all oligos with sufficient stability to be the predominate form at room temperature. (C) Competition EMSA analysis of Tat–7SK complex formation. Tat, 32P-labeled 7SK, and the indicated cold RNA oligos were pre-incubated and the resulting complexes were resolved by gel electrophoresis on a native gel, followed by autoradiography to visualize the 7SK shift. The dsRNA was a 25-bp double-stranded RNA unrelated to 7SK sequence described previously (53). (D) Competition EMSA comparison between 7SK (10–48) and 7SK (10–48 M). Note in this experiment the 7SK was of higher specific activity than in (C) so less Tat was needed to achieve a higher fraction of Tat–7SK complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1935001&req=5

Figure 5: HIV Tat binds to a region of 7SK resembling TAR. (A) Analysis of the sequence of 7SK reveals three AUCUG Tat consensus-binding sites in the first 100 nt. Several structured and unstructured RNA oligos with or without this consensus sequence were designed and chemically synthesized with most coming from the native 7SK sequences indicated. 7SK (10–48 M) has one insertion and one deletion in an otherwise wild-type 7SK (10–48) sequence. (B) Predicted structures of all oligos with sufficient stability to be the predominate form at room temperature. (C) Competition EMSA analysis of Tat–7SK complex formation. Tat, 32P-labeled 7SK, and the indicated cold RNA oligos were pre-incubated and the resulting complexes were resolved by gel electrophoresis on a native gel, followed by autoradiography to visualize the 7SK shift. The dsRNA was a 25-bp double-stranded RNA unrelated to 7SK sequence described previously (53). (D) Competition EMSA comparison between 7SK (10–48) and 7SK (10–48 M). Note in this experiment the 7SK was of higher specific activity than in (C) so less Tat was needed to achieve a higher fraction of Tat–7SK complex.
Mentions: To gain insight into what sequence Tat recognized in 7SK, the first 100 nt of 7SK were compared to HIV TAR, and a set of oligos were designed and tested for their ability to block Tat binding to 7SK. The target for Tat binding in TAR consists of an AUCUG forming a bulge in the apical region of the TAR stem-loop and this binding of Tat changes the structure of TAR so that a pocket forms between U23 and G26 (54–56). When the sequence of 7SK was analyzed, three AUCUG motifs were found (Figure 5A). This sequence would be expected to occur randomly once every 45 bp, or about 1 in 1000 nt, and so it may be significant that it is found three times in the first 100 nt of 7SK. A set of oligos were designed that contained AUCUG sequences in the context of sequences that would be unstructured or that could form secondary structure with the AUCUG in loops or in bulges. Other regions of 7SK were also sampled as controls (Figure 5A). mFold suggested that 7SK (10–48) would form a stem-loop with one AUCUG in a bulge and one in the loop and is shown compared to TAR in Figure 5B. HEXIM1 has been demonstrated to bind to a region of in the 5′ end of 7SK (57) and to this specific oligo as well as it binds to intact 7SK (53). Unlabeled oligos were then used as competitors in EMSAs with labeled full-length 7SK and Tat. No competition was seen for any of the oligos except for 7SK (10–48) (Figure 5C and D). This strongly suggests that Tat associated with the residues from 10 to 48 of 7SK. Because no competition was seen with oligos comprised of 11–34 or 20–36, Tat may require the secondary structure shown in Figure 5B to be able to interact with 7SK.Figure 5.

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