Controlling Cellular P-TEFb Activity by the HIV-1 Transcriptional Transactivator Tat

Abstract

The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5′-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5′-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

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

“…No other predicted secondary structures were found to bind to HEXIM1. This region also binds to Tat, which is consistent with previous studies in vitro (21). Although those binding studies demonstrated the importance of the U40-U41 bulge and the GUAC motif for the interaction between HEXIM1 and 7SK (43), the importance of the central loop for assembling the 7SK snRNP had not been appreciated.…”

“…This finding and the previous data on residues from positions 178 -220 of HEXIM1, which contribute to its binding specificity (Fig. 3), support the model where RNA-binding residues from positions 150 -177 and those from positions 178 -220 of HEXIM1 form a tripartite HEXIM1-CycT1-7SK complex that resembles the Tat-CycT1-TAR complex (21,47,48).…”

“…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.…”

“…In addition, there is another bulge (from positions 75-77) containing a uridine residue that could interact with HEXIM1. Finally, 7SK contains a central loop (from positions 50 -59) that resembles the central loop of TAR where CycT1 binds to form a stable complex between Tat, P-TEFb, and TAR (21,47,48). Therefore, we constructed a series of mutant M3 plasmid targets to identify other residues critical for these interactions.…”

Transcription control by long non-coding RNAs

“…87 Tat also binds to the 5' stem-loop of 7SK, apparently triggering a conformational change in the RNA, but it is unclear if RNA-binding is required for P-TEFb displacement or if it occurs after P-TEFb and Hexim1 are removed from the 7SK snRNP. 83,[88][89][90][91] A prominent model is that Tat captures active P-TEFb from a large pool of inactive 7SK-bound P-TEFb complexes via this competition mechanism and then delivers it to the HIV promoter by binding to TAR. 87 Recent ChIP analyses on an integrated HIV promoter indicate that the 7SK snRNP proteins Larp7 and Hexim1, along with cyclin T1 and Cdk9, the formation of DNA-DNA-RNA triple helical structures as in the case of DHFR and pRNA, or by contacting factors already bound to chromatin.…”