HIV-1 tat transcriptional activity is regulated by acetylation.
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The human immunodeficiency virus (HIV) trans- activator protein, Tat, stimulates transcription from the viral long-terminal repeats (LTR) through an RNA hairpin element, trans-activation responsive region (TAR). We and others have shown that trans-activator protein (Tat)-associated histone acetyltransferases (TAHs), p300 and p300/CBP-associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV-1 LTR. Here, we show that p300 and PCAF also directly acetylate Tat. We defined two sites of acetylation located in different functional domains of Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV-1 LTR. Synergism was TAR-dependent and required the intact presence of both Lys28 and Lys50. Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat-associated kinase, CDK9/P-TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation. These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD-kinase and release of Tat from TAR RNA. (+info)
Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription.
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Tat activation of HIV-1 transcription is mediated by human transcription elongation factor P-TEFb, which interacts with Tat and phosphorylates the C-terminal domain of RNA polymerase II. The catalytic subunit of the P-TEFb complex, Cdk9, has been shown to interact with cyclin T and several other proteins of unknown identity. Consequently, the exact subunit composition of active P-TEFb has not been determined. Here we report the affinity purification and identification of the Cdk9-associated proteins. In addition to forming a heterodimer with cyclin T1, Cdk9 interacted with the molecular chaperone Hsp70 or a kinase-specific chaperone complex, Hsp90/Cdc37, to form two separate chaperone-Cdk9 complexes. Although the Cdk9/cyclin T1 dimer was exceptionally stable and produced slowly in the cell, free and unprotected Cdk9 appeared to be degraded rapidly. Several lines of evidence indicate the heterodimer of Cdk9/cyclin T1 to be the mature, active form of P-TEFb responsible for phosphorylation of the C-terminal domain of RNA polymerase II interaction with the Tat activation domain, and mediation of Tat activation of HIV-1 transcription. Pharmacological inactivation of Hsp90/Cdc37 function by geldanamycin revealed an essential role for the chaperone-Cdk9 complexes in generation of Cdk9/cyclin T1. Our data suggest a previously unrecognized chaperone-dependent pathway involving the sequential actions of Hsp70 and Hsp90/Cdc37 in the stabilization/folding of Cdk9 as well as the assembly of an active Cdk9/cyclin T1 complex responsible for P-TEFb-mediated Tat transactivation. (+info)
Interactions between equine cyclin T1, Tat, and TAR are disrupted by a leucine-to-valine substitution found in human cyclin T1.
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Transcriptional transactivators (Tat) from human immunodeficiency and equine infectious anemia viruses (HIV and EIAV) interact with their transactivation response elements (TAR) to increase the rates of viral transcription. Whereas the human cyclin T1 is required for the binding of Tat to TAR from HIV, it is unknown how Tat from EIAV interacts with its TAR. Furthermore, Tat from EIAV functions in equine and canine cells but not in human cells. In this study, we present sequences of cyclins T1 from horse and dog and demonstrate that their N-terminal 300 residues rescue the transactivation of Tat from EIAV in human cells. Although human and equine cyclins T1 bind to this Tat, only the equine cyclin T1 supports the binding of Tat to TAR from EIAV. Finally, a reciprocal exchange of the valine for the leucine at position 29 in human and equine cyclins T1, respectively, renders the human cyclin T1 active and the equine cyclin T1 inactive for Tat transactivation from EIAV. Thus, the collaboration between a specific cyclin T1 and Tat for their high-affinity interaction with TAR is a common theme of lentiviral transactivation. (+info)
Physical interaction between CDK9 and B-Myb results in suppression of B-Myb gene autoregulation.
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B-Myb is a transcription factor belonging to the myb family, whose activity has been associated with augmented DNA synthesis and cell cycle progression. We showed recently that B-Myb autoregulates its own expression through promoter transactivation. We report in this study that CDK9, the cyclin T associated kinase, which phosphorylates and activates RNA-Polymerase II, suppresses B-Myb autoregulation through direct interaction with the carboxyl-terminus of the B-Myb protein. Down-regulation of the transactivating ability of B-Myb is independent of the kinase activity of CDK9, because a kinase deficient mutant (dn-CDK9) also represses B-myb gene autoregulation. Overexpression of CDK9 did not result in suppression of p53-dependent transactivation or inhibition of the basal activity of the promoters tested so far, demonstrating that CDK9 is a B-Myb-specific repressor. Rather, transfection of the dominant negative dn-CDK9 construct inhibited the basal activity of the reporter genes, confirming an essential role for CDK9 in gene transcription. In addition, Cyclin T1 restores B-Myb transactivating activity when co-transfected along with CDK9, suggesting that the down-regulatory effect observed on B-Myb is specifically due to CDK9 alone. Thus, our data suggest that CDK9 is involved in the negative regulation of activated transcription mediated by certain transcription factors, such as B-Myb. This may indicate the existence of a feedback loop, mediated by the different activities of CDK9, which links basal with activated transcription. (+info)
Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection.
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AIDS and the bare lymphocyte syndrome (BLS) are severe combined immunodeficiencies. BLS results from mutations in genes that regulate the expression of class II major histocompatibility (MHC II) determinants. One of these is the class II transactivator (CIITA). HIV and its transcriptional transactivator (Tat) also block the expression of MHC II genes. By binding to the same surface in the cyclin T1, which together with CDK9 forms the positive transcription elongation factor b (P-TEFb) complex, Tat inhibits CIITA. CIITA can also activate transcription when tethered artificially to RNA. Moreover, a dominant-negative CDK9 protein inhibits the activity of MHC II promoters. Thus, CIITA is a novel cellular coactivator that binds to P-TEFb for the expression of its target genes. (+info)
Canine cyclin T1 rescues equine infectious anemia virus tat trans-activation in human cells.
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Human immunodeficiency virus-1 Tat protein and human Cyclin T1 mediate transcriptional activation by enhancing the elongation efficiency of RNA polymerase II. Activation of transcription of the related equine infectious anemia virus (EIAV) requires a similar protein known as eTat, which does not function in human cells. Expression of equine Cyclin T1 in human cells rescues eTat function, suggesting a general mechanism of transcription activation among lentiviruses. Here we present the cloning of Cyclin T1 from canine D17 osteosarcoma cells, which support EIAV transactivation, and show that canine Cyclin T1 confers eTat transactivation to human cells. A two-amino-acid change, from 79-proline-glycine-80 to 79-histidine-arginine-80, confers on the human Cyclin T1 the ability to cooperate with eTat in transcriptional activation. These findings suggested that the regions of Cyclin T1 that interact with lentiviral Tat proteins and TAR RNA elements form an extended domain, which very likely has a conserved fold. (+info)
A block to human immunodeficiency virus type 1 assembly in murine cells.
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Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed. (+info)
P-TEFb kinase recruitment and function at heat shock loci.
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P-TEFb, a heterodimer of the kinase Cdk9 and cyclin T, was isolated as a factor that stimulates formation of productive transcription elongation complexes in vitro. Here, we show that P-TEFb is located at >200 distinct sites on Drosophila polytene chromosomes. Upon heat shock, P-TEFb, like the regulatory factor HSF, is rapidly recruited to heat shock loci, and this recruitment is blocked in an HSF mutant. Yet, HSF binding to DNA is not sufficient to recruit P-TEFb in vivo, and HSF and P-TEFb immunostainings within a heat shock locus are not coincident. Insight to the function of P-TEFb is offered by experiments showing that the direct recruitment of a Gal4-binding domain P-TEFb hybrid to an hsp70 promoter in Drosophila cells is sufficient to activate transcription in the absence of heat shock. Analyses of point mutants show this P-TEFb stimulation is dependent on Cdk9 kinase activity and on Cdk9's interaction with cyclin T. These results, coupled with the frequent colocalization of P-TEFb and the hypophosphorylated form of RNA polymerase II (Pol II) found at promoter-pause sites, support a model in which P-TEFb acts to stimulate promoter-paused Pol II to enter into productive elongation. (+info)