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

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.

Genetic Analysis of the Structure and Function of 7SK Small Nuclear Ribonucleoprotein (snRNP) in Cells

“…This region contains the PYNT motif (from positions 203-206) and the phenylalanine at position 208, which also help to inhibit the kinase activity of CDK9 (26, 29, 30). These RNA-protein interactions cause a conformational change to create a combinatorial binding surface for CycT1, which resembles those between Tat, TAR, and CycT1 (23,31) where the central loop of TAR is required for binding to CycT1 (46). Our results provide a genetic and func- Fig.…”

“…3A). In addition, because Tat can recruit P-TEFb from 7SK snRNP by competing with HEXIM1 for binding to 7SK and to P-TEFb, we wondered whether the full-length Tat protein also activates M3 (21)(22)(23). Indeed, the Tat, Hex(150 -220)⅐Tat48, and Hex(150 -177) chimeras activated M3 to similar levels (Fig.…”

“…Because Tat can compete with HEXIM1 for binding to CycT1 (22,23), P-TEFb recruited via Tat48 is also not inhibited by HEXIM1 in the context of the Hex⅐Tat48 chimera. However, HEXIM1 alone inhibited M3 (Fig.…”

Bromodomain and Extra-terminal (BET) Bromodomain Inhibition Activate Transcription via Transient Release of Positive Transcription Elongation Factor b (P-TEFb) from 7SK Small Nuclear Ribonucleoprotein

“…Consequently, the increased synthesis of HEXIM1 led to the reassembly of the 7SK snRNP and thus the inactivation of P-TEFb. Importantly, HIV transcription was activated by JQ1 and then transactivated by Tat, which can not only utilize P-TEFb from its active and inactive complexes but also compete with BRD4 for its binding (9,10,13).…”

“…Active P-TEFb can be recruited to its target genes via interaction with various DNA-bound activators such as NF-B, c-Myc, the class II transactivator, and steroid hormone receptors, which otherwise function poorly when levels of free P-TEFb are low (1,3). On the other hand, the HIV transcriptional transactivator Tat can utilize P-TEFb directly from the 7SK snRNP (9,10). BRD4 can also compete with HEXIM1 for binding to P-TEFb (11,12), and the BRD4⅐P-TEFb complex is important for the activation of primary response genes (13,14).…”