Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal … (original) (raw)
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The HIV-1 trans-activator protein, tat, is an RNA binding protein with a high affinity for a U-rich bulge near the tip of the stem in the RNA stem-loop structure encoded by the tans-activation responsive region (TAR). A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with BiIV-1TAR RNA with a dissociation constant of Kd = 12 nM. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6-to 8-fold lower affinity than wild-type TAR. Introduction of a point mutation expected to destabilize base pairing in nearby residues of the TAR stem-loop structure reduced tat binding 10-fold. In contrast, mutations that alter the sequence of the six nucleotide long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs carrying TAR sequences encoding any of the mutations known to produce transcripts which bind tat weakly, are not stimulated efficiently by tat in vivo.
Proceedings of The National Academy of Sciences, 1989
tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunopripitation assays. tat does not bind detectably to anfisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-oop structures, or TAR DNA.
Journal of Virology, 1992
Protein kinase C (PKC) is involved in the mitogenic stimulation of cell proliferation and has recently been reported to be essential for Tat-mediated trans activation. We have determined that RNA binding of a cellular factor which specifically interacts with the trans-activation response region (TAR) is blocked in cells depleted of PKC activity by chronic phorbol myristate acetate stimulation. We also show that nuclear extracts can be depleted of the cellular TAR-binding factor by in vitro treatment with purified protein phosphatase 2A. Furthermore, TAR RNA-binding activity can be partially restored to depleted nuclear extracts in vitro by addition of PKC. Chimeric constructs in which the Tat protein is artificially tethered to viral RNA show PKC independence for Tat-mediated trans activation. Specific mutations in the TAR RNA stem region which cause reduced binding of host cell factor in vitro also cause reduced Tat-mediated trans activation in vivo. Together, these results suggest that phosphorylation-dependent binding of a cellular cofactor to TAR RNA is an essential step in Tat-mediated trans activation. Deciphering the regulation of Tat-mediated trans activation by phosphorylation will be critical in fully understanding the regulation of human immunodeficiency virus type 1 activation.
Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II
Proceedings of the National Academy of Sciences, 1996
The Tat-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein Tat. Recently, we demonstrated by gel-retardation analysis that RNA polymerase II binds to TAR RNA and that Tat prevents this binding even when Tat does not bind to TAR RNA. These results suggested that direct interactions between Tat and RNA polymerase II may prevent RNA polymerase II pausing and lead to Tat-mediated increases in transcriptional elongation. To test this possibility, we performed protein interaction studies with RNA polymerase II and both the HIV-1 and the closely related HIV-2 Tat protein. These studies indicated that both the HIV-1 and HIV-2 Tat proteins could specifically interact with RNA polymerase II. Mutagenesis of both HIV-1 and HIV-2 Tat demonstrated that the basic domains of both the HIV-1 and HIV-2 Tat proteins were required for this in...
TAR RNA loop: A scaffold for the assembly of a regulatory switch in HIV replication
Proceedings of the National Academy of Sciences, 2002
Replication of HIV requires the Tat protein, which activates elongation of RNA polymerase II transcription at the HIV-1 promoter by interacting with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex b (P-TEFb). The transactivation domain of Tat binds directly to the CycT1 subunit of P-TEFb and induces loop sequence-specific binding of P-TEFb onto nascent HIV-1 trans-activation responsive region (TAR) RNA. We used systematic RNA–protein photocross-linking, Western blot analysis, and protein footprinting to show that residues 252–260 of CycT1 interact with one side of the TAR RNA loop and enhance interaction of Tat residue K50 to the other side of the loop. Our results show that TAR RNA provides a scaffold for two protein partners to bind and assemble a regulatory switch in HIV replication. RNA-mediated assembly of RNA–protein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cel...
Virology, 1997
Tat stimulates HIV-1 gene expression during transcription initiation and elongation. Tat functions primarily through specific interactions with TAR RNA and several putative cellular cofactors to increase the processivity of RNA polymerase II complexes during HIV-1 transcription elongation. Although HIV-1 transactivation by Tat in most cell types requires intact TAR sequences, previous reports demonstrate that Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression in several central nervous system-derived astrocytic/glial cell lines in the absence of TAR. Within this study, transient expression assays performed in the astrocytic/glial cell line, U87-MG, confirm that kB elements within the HIV-1 LTR mediate TAR-independent transactivation by Tat and demonstrate additionally that distinct amino acid residues within the cysteine-rich activation domain of Tat are required for TAR-independent versus TAR-dependent transactivation. Established U87-MG cell lines expressing a transdominant negative mutant of IkBa, IkBaDN, fail to support TAR-independent transactivation by Tat, suggesting that binding of NF-kB to kB enhancer elements within the HIV-1 LTR is necessary for Tat-mediated transactivation in the absence of TAR. Ribonucleic acid protection analyses of promoter-proximal and-distal transcripts derived from TAR-deleted and TARcontaining HIV-1 LTR reporter constructs in U87-MG cells indicate that the predominant effect of Tat during TAR-independent transactivation occurs at the level of transcription initiation, whereas a prominent elongation effect of Tat is observed in the presence of TAR. These data suggest an alternative regulatory pathway for Tat transactivation in specific cells derived from the central nervous system that is independent of TAR and that requires direct or indirect interaction of Tat with NF-kBbinding sites in the HIV-1 LTR.
Journal of Biological Chemistry, 1997
We demonstrate that the interferon-induced, doublestranded (ds) RNA-activated kinase, PKR, is able to bind to and phosphorylate the human immunodeficiency virus type 1 (HIV-1) trans-activating protein, Tat. Furthermore, Tat can inhibit the activation and activity of the kinase. Phosphorylation of Tat by PKR is dependent on the prior activation of PKR by dsRNA and occurs on serine and threonine residues adjacent to the basic region important for TAR RNA binding and Tat function. Activated PKR efficiently phosphorylates both the twoexon form of Tat (Tat-86) and the single exon form (Tat-72). Mutagenesis indicates that the interaction between PKR and Tat requires the RNA-binding region of Tat. Tat competes with eukaryotic initiation factor 2, a wellcharacterized substrate of PKR, for phosphorylation by activated PKR. Tat also inhibits the autophosphorylation of PKR by dsRNA. This biochemical evidence of an intimate relationship between Tat, an important regulator of HIV transcription, and PKR, a pleiotropic cellular regulator, may provide insights into HIV-1 pathogenesis and, more generally, virus/host interactions.
HIV1 Tat Directly Interacts with the Interferon-Induced, Double-Stranded RNA-Dependent Kinase, PKR
Virology, 1995
We present evidence that the HIV-1 Tat protein and the RNA-dependent cellular protein kinase, PKR, interact with each other both in vitro and in vivo. Using GST fusion chromatography, we demonstrate that PKR, interacts directly with the HIV-1 Tat protein. The region in Tat sufficient for binding PKR maps within amino acids 20 to 72. In in vitro assays, the two-exon form of Tat (Tat 86) was phosphorylated by PKR, while the one exon form of Tat (Tat 72) inhibited PKR autophosphorylation and substrate phosphorylation. The ability of Tat to interact with PKR was demonstrated in both yeast and mammalian cells. Expression of PKR in yeast results in a growth suppressor phenotype which was reversed by coexpression of a one exon form of Tat. Expression of Tat 72 in HeLa cells resulted in direct interaction with PKR as detected by coimmunprecipitation with a Tat antibody. Tat and PKR also form a coimmunoprecipitable complex in cell-free extracts prepared from productively infected T lymphocytes. The interaction of Tat with PKR provides a potential mechanism by which HIV could suppress the interferon system. ᭧ 1995 Academic Press, Inc.
Journal of virology, 1997
Tat protein mediates transactivation of human immunodeficiency virus type 1 (HIV-1), which results in more-efficient transcript elongation. Since phosphorylation of C-terminal domain (CTD) of RNA polymerase II correlates with its enhanced processivity, we studied the properties of a Tat-associated CTD kinase derived from mitogenically stimulated human primary T lymphocytes (TTK). TTK binds to full-length Tat and specifically phosphorylates CTD and CDK2. This dual kinase activity is characteristic of CDK-activating kinase (CAK). The CTD kinase activity is induced upon mitogenic stimulation of primary T lymphocytes. Fractionation of T-cell lysate demonstrates that Tat-associated CTD kinase activity elutes in two peaks. About 60% of Tat-associated CTD kinase copurifies with CDK2 kinase activity and contains the CAK components CDK7 and cyclin H. The rest of Tat-associated kinase is free of CDK2 kinase activity and the CAK components and thus may represent a novel CTD kinase. The kinase activities of TTK are blocked by the adenosine analog 5,6-dichloro-1--D-ribofuranosyl-benzimidazole (DRB) as well as by the kinase inhibitor H8 at concentrations known to block transcript elongation. Importantly, the Tat-associated kinase markedly induced CAK. We suggest that the mechanism of Tat-mediated processive transcription of the HIV-1 promoter includes a Tat-associated CAK activator.