Inhibition of Tat activity by the HEXIM1 protein.
BACKGROUND
The positive transcription elongation factor b (P-TEFb) composed by CDK9/CyclinT1 subunits is a dedicated co-factor of HIV transcriptional transactivator Tat protein. Transcription driven by the long terminal repeat (LTR) of HIV involves formation of a quaternary complex between P-TEFb, Tat and the TAR element. This recruitment is necessary to enhance the processivity of RNA Pol II from the HIV-1 5' LTR promoter. The activity of P-TEFb is regulated in vivo and in vitro by the HEXIM1/7SK snRNA ribonucleic-protein complex.


RESULTS
Here we report that Tat transactivation is effectively inhibited by co-expression of HEXIM1 or its paralog HEXIM2. HEXIM1 expression specifically represses transcription mediated by the direct activation of P-TEFb through artificial recruitment of GAL4-CycT1. Using appropriate HEXIM1 mutants we determined that effective Tat-inhibition entails the 7SK snRNA basic recognition motif as well as the C-terminus region required for interaction with cyclin T1. Enhanced expression of HEXIM1 protein modestly affects P-TEFb activity, suggesting that HEXIM1-mediated repression of Tat activity is not due to a global inhibition of cellular transcription.


CONCLUSIONS
These results point to a pivotal role of P-TEFb for Tat's optimal transcription activity and suggest that cellular proteins that regulate P-TEFb activity might exert profound effects on Tat function in vivo.
Fraldi
Alessandro
A
Department of Structural and Functional Biology, University of Naples Federico II, Naples, Italy. fraldi@tigem.it
Varrone
Francesca
F
Napolitano
Giuliana
G
Michels
Annemieke A
AA
Majello
Barbara
B
Bensaude
Olivier
O
Lania
Luigi
L
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D042925:Positive Transcriptional Elongation Factor B / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012342:RNA, Small Nuclear / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
D015696:Gene Products, tat; C476123:HEXIM1 protein, human; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D042925:Positive Transcriptional Elongation Factor B