Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription

Abstract

Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1͞P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1͞P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat͞TAR axis in viral transcription is to permit the recruitment of CycT1͞P-TEFb.The Tat protein encoded by HIV type 1 (HIV-1) is a potent activator of the viral long terminal repeat (LTR) promoter and is required for virus replication (reviewed by refs. 1, 2). Properties that distinguish Tat from other transcription factors include that it acts predominantly at the level of transcription elongation (3-6), rather than initiation, and that the target sequence for Tat is an RNA stem-loop structure, termed TAR, located immediately 3Ј to the LTR transcription start site. The human cyclin T1 (hCycT1) component of positive transcription elongation factor b (P-TEFb) rescues Tat function in otherwise nonpermissive murine cells and binds to TAR cooperatively with Tat (7-10). Recruitment of hCycT1͞P-TEFb to TAR may enhance transcription elongation by allowing the CDK9 component of P-TEFb to phosphorylate the carboxyl-terminal domain (CTD) of initiated RNA polymerase II (PolII) complexes (11-18), a modification thought to be required for efficient elongation by PolII (19). Although hCycT1͞P-TEFb is therefore clearly an essential Tat cofactor, it has remained uncertain whether hCycT1͞P-TEFb recruitment to the LTR is the only activity of Tat or, instead, one of two or more critical functions.In addition to hCycT1, a number of other potential Tat interacting proteins and͞or cofactors have been proposed including MSS1, HT2A, CA150, TFIID, Tat-SF1, TIP30, and PolII itself (20-26), and other groups have reported interactions between Tat and the coactivator proteins p300 and CREB-binding protein (CBP) (27)(28)(29) or between Tat and the transcription factor...

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

“…Maximal activities obtained with the Hex⅐Tat48 or LaRP7⅐Tat48 chimeras were somewhat lower (5-to 8-fold) than those obtained with other systems, such as Rev and SLIIB (20-to 30-fold) (34,35,37,44). This difference could reflect the ability of Rev to form higher-order oligomers that recruit more activators to the elongation complex.…”

“…In these systems, the TAR in the HIV LTR is replaced by an RNA sequence of interest (34), and RNA-binding proteins that interact with it are used as fusion partners of specific effectors. One well studied plasmid target contains the stem-loop IIB (SLIIB) from the HIV Rev response element and Rev proteins fused to activators of transcription elongation (35,37,44). In particular, CycT1 from P-TEFb is a strong activator when fused to Rev, as are the transcriptional activation domain of Tat (residues 1-48, TAD48) and RelA, a subunit of nuclear factor B (44, 45).…”

TAR RNA loop: A scaffold for the assembly of a regulatory switch in HIV replication

“…Under similar conditions, CycT1-Tat bound all three s U TAR RNA sequences with varying efficiencies of RNA-protein complex formation: 75% with s U31 TAR, 60% with s U33 TAR, and 30% with A30-s U34 TAR. We used CycT1(1-303) in these RNA gel-shift assays instead of P-TEFb for three reasons: (i) this region of CycT1 is sufficient to form stable complexes with Tat-TAR and CDK9 (8,(10)(11)(12)(13)(14)(19)(20)(21), (ii) the kinase subunit CDK9 does not interfere with CycT1 specificity for Tat-TAR binding (8,(10)(11)(12)(13)(14)(19)(20)(21), and (iii) detection of the CycT1(1-303)-Tat-TAR complex by native gel electrophoresis methods is more straightforward and reliable compared with P-TEFb-Tat-TAR or CycT1(1-726)-Tat-TAR complexes. These results indicate that all 4-thiouridine-containing RNA sequences can form complexes with CycT1-Tat.…”

“…Recruitment of P-TEFb to TAR has been proposed to be both necessary and sufficient for activating transcription elongation from the HIV-1 long terminal repeat promoter (10). Neither CycT1 nor the P-TEFb complex bind TAR RNA in the absence of Tat; thus TAR binding is highly cooperative for both Tat and P-TEFb (7,9).…”

Discovery of a Small Molecule Tat-trans-Activation-responsive RNA Antagonist That Potently Inhibits Human Immunodeficiency Virus-1 Replication

“…Tat interacts with the cyclin T1 (CycT1) subunit of P-TEFb and recruits the kinase complex to TAR RNA. Recruitment of P-TEFb to TAR has been proposed to be both necessary and sufficient to activate transcription elongation from the HIV-1 long terminal repeat promoter (13). Recent fluorescence resonance energy transfer studies, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein, showed that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA, and that TAR RNA further enhances interaction between Tat and CycT1 (14).…”