Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR.
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.
Sedore
Stanley C
SC
Department of Biochemistry, University of Iowa, Iowa City, IA, USA.
Byers
Sarah A
SA
Biglione
Sebastian
S
Price
Jason P
JP
Maury
Wendy J
WJ
Price
David H
DH
D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D042925:Positive Transcriptional Elongation Factor B / Q000737:chemistry / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; C476123:HEXIM1 protein, human; D012342:RNA, Small Nuclear; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D042925:Positive Transcriptional Elongation Factor B