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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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HEXIM1–TAR inhibits the kinase activity of P-TEFb. (A) HEXIM1, TAR RNA, 7SK RNA or mixtures of HEXIM1 and TAR or 7SK found by EMSA to result in 1:1 HEXIM1–TAR or HEXIM1–7SK complexes were titrated into in vitro kinase assays containing purified P-TEFb and incorporation of 32P into the Spt5 subunit of DSIF was analyzed by SDS–PAGE followed by autoradiography. (B) Kinase activity of P-TEFb in the presence HEXIM1, TAR and 7SK as described in (A) was quantitated using an Instant Imager and plotted as a function of increasing HEXIM1.
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Figure 7: HEXIM1–TAR inhibits the kinase activity of P-TEFb. (A) HEXIM1, TAR RNA, 7SK RNA or mixtures of HEXIM1 and TAR or 7SK found by EMSA to result in 1:1 HEXIM1–TAR or HEXIM1–7SK complexes were titrated into in vitro kinase assays containing purified P-TEFb and incorporation of 32P into the Spt5 subunit of DSIF was analyzed by SDS–PAGE followed by autoradiography. (B) Kinase activity of P-TEFb in the presence HEXIM1, TAR and 7SK as described in (A) was quantitated using an Instant Imager and plotted as a function of increasing HEXIM1.

Mentions: While this result supports the hypothesis that HEXIM1 can bind to TAR, with subsequent recruitment and binding of P-TEFb, inhibition of P-TEFb requires that the RNA change the conformation of HEXIM1 to relieve the autoinhibitory properties of the protein. To determine whether HEXIM1–TAR can inhibit P-TEFb, in vitro kinase assays were performed using recombinant P-TEFb(1–290), HEXIM1 and TAR RNA as in the EMSAs to monitor γ-32P(ATP) incorporation into DSIF, and the complexes resolved by SDS–PAGE (Figure 7A). Compared to P-TEFb alone and with DRB, an inhibitor of P-TEFb, there was no qualitative effect of HEXIM1 alone on P-TEFb kinase activity. Similarly, TAR alone, or 7SK alone as a control, demonstrated no appreciable effect. However, when HEXIM1 and TAR are combined and titrated in a constant ratio determined by EMSA to give 1:1 HEXIM1–TAR binding, there was a dose-dependent inhibition of P-TEFb, similar to that observed with HEXIM1–7SK. When the assay was quantitated (Figure 7B), HEXIM1 and the RNAs separately had no significant effect; however, HEXIM1–TAR appeared to inhibit as effectively as equimolar amounts of HEXIM1–7SK. These findings support the hypothesis that HEXIM1 may actually be inhibiting P-TEFb at the HIV LTR.Figure 7.


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)

HEXIM1–TAR inhibits the kinase activity of P-TEFb. (A) HEXIM1, TAR RNA, 7SK RNA or mixtures of HEXIM1 and TAR or 7SK found by EMSA to result in 1:1 HEXIM1–TAR or HEXIM1–7SK complexes were titrated into in vitro kinase assays containing purified P-TEFb and incorporation of 32P into the Spt5 subunit of DSIF was analyzed by SDS–PAGE followed by autoradiography. (B) Kinase activity of P-TEFb in the presence HEXIM1, TAR and 7SK as described in (A) was quantitated using an Instant Imager and plotted as a function of increasing HEXIM1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: HEXIM1–TAR inhibits the kinase activity of P-TEFb. (A) HEXIM1, TAR RNA, 7SK RNA or mixtures of HEXIM1 and TAR or 7SK found by EMSA to result in 1:1 HEXIM1–TAR or HEXIM1–7SK complexes were titrated into in vitro kinase assays containing purified P-TEFb and incorporation of 32P into the Spt5 subunit of DSIF was analyzed by SDS–PAGE followed by autoradiography. (B) Kinase activity of P-TEFb in the presence HEXIM1, TAR and 7SK as described in (A) was quantitated using an Instant Imager and plotted as a function of increasing HEXIM1.
Mentions: While this result supports the hypothesis that HEXIM1 can bind to TAR, with subsequent recruitment and binding of P-TEFb, inhibition of P-TEFb requires that the RNA change the conformation of HEXIM1 to relieve the autoinhibitory properties of the protein. To determine whether HEXIM1–TAR can inhibit P-TEFb, in vitro kinase assays were performed using recombinant P-TEFb(1–290), HEXIM1 and TAR RNA as in the EMSAs to monitor γ-32P(ATP) incorporation into DSIF, and the complexes resolved by SDS–PAGE (Figure 7A). Compared to P-TEFb alone and with DRB, an inhibitor of P-TEFb, there was no qualitative effect of HEXIM1 alone on P-TEFb kinase activity. Similarly, TAR alone, or 7SK alone as a control, demonstrated no appreciable effect. However, when HEXIM1 and TAR are combined and titrated in a constant ratio determined by EMSA to give 1:1 HEXIM1–TAR binding, there was a dose-dependent inhibition of P-TEFb, similar to that observed with HEXIM1–7SK. When the assay was quantitated (Figure 7B), HEXIM1 and the RNAs separately had no significant effect; however, HEXIM1–TAR appeared to inhibit as effectively as equimolar amounts of HEXIM1–7SK. These findings support the hypothesis that HEXIM1 may actually be inhibiting P-TEFb at the HIV LTR.Figure 7.

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