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Andrew P. Rice - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Cyclin T1 during HIV replication and latency establishment in human memory CD4 T cells.
Virology journal, 2019Co-Authors: Jacob Couturier, Andrew P. Rice, Sona Budhiraja, Hongbing Liu, Aaron F. Orozco, Edward B. Siwak, Pramod N. Nehete, K. Jagannadha Sastry, Dorothy E. LewisAbstract:The regulatory Cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized. To better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes. In activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1- cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1- cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24- cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues. CycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1- cells during HIV replication.
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Cyclin T1 and cdk9 t loop phosphorylation are downregulated during establishment of hiv 1 latency in primary resting memory cd4 t cells
Journal of Virology, 2013Co-Authors: Sona Budhiraja, Marylinda Famiglietti, Alberto Bosque, Vicente Planelles, Andrew P. RiceAbstract:P-TEFb, a cellular kinase composed of Cyclin T1 and CDK9, is essential for processive HIV-1 transcription. P-TEFb activity is dependent on phosphorylation of Thr186 in the CDK9 T loop. In resting CD4+ T cells which are nonpermissive for HIV-1 replication, the levels of Cyclin T1 and T-loop-phosphorylated CDK9 are very low but increase significantly upon cellular activation. Little is known about how P-TEFb activity and expression are regulated in resting central memory CD4+ T cells, one of the main reservoirs of latent HIV-1. We used an in vitro primary cell model of HIV-1 latency to show that P-TEFb availability in resting memory CD4+ T cells is governed by the differential expression and phosphorylation of its subunits. This is in contrast to previous observations in dividing cells, where P-TEFb can be regulated by its sequestration in the 7SK RNP complex. We find that resting CD4+ T cells, whether naive or memory and independent of their infection status, have low levels of Cyclin T1 and T-loop-phosphorylated CDK9, which increase upon activation. We also show that the decrease in Cyclin T1 protein upon the acquisition of a memory phenotype is in part due to proteasome-mediated proteolysis and likely also to posttranscriptional downregulation by miR-150. We also found that HEXIM1 levels are very low in ex vivo- and in vitro-generated resting memory CD4+ T cells, thus limiting the sequestration of P-TEFb in the 7SK RNP complex, indicating that this mechanism is unlikely to be a driver of viral latency in this cell type.
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identification of novel cdk9 and Cyclin T1 associated protein complexes ccaps whose sirna depletion enhances hiv 1 tat function
Retrovirology, 2012Co-Authors: Rajesh Ramakrishnan, Hongbing Liu, Hart Donahue, Anna Malovannaya, Jun Qin, Andrew P. RiceAbstract:Background HIV-1 Tat activates RNA Polymerase II (RNAP II) elongation of the integrated provirus by recruiting a protein kinase known as P-TEFb to TAR RNA at the 5′ end of nascent viral transcripts. The catalytic core of P-TEFb contains CDK9 and Cyclin T1 (CCNT1). A human endogenous complexome has recently been described – the set of multi-protein complexes in HeLa cell nuclei. We mined this complexome data set and identified 12 distinct multi-protein complexes that contain both CDK9 and CCNT1. We have termed these complexes CCAPs for CDK9/CCNT1-associated protein complexes. Nine CCAPs are novel, while three were previously identified as Core P-TEFb, the 7SK snRNP, and the Super-Elongation Complex. We have investigated the role of five newly identified CCAPs in Tat function and viral gene expression.
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identification of novel cdk9 and Cyclin T1 associated protein complexes ccaps whose sirna depletion enhances hiv 1 tat function
Retrovirology, 2012Co-Authors: Rajesh Ramakrishnan, Hongbing Liu, Hart Donahue, Anna Malovannaya, Jun Qin, Andrew P. RiceAbstract:Background: HIV-1 Tat activates RNA Polymerase II (RNAP II) elongation of the integrated provirus by recruiting a protein kinase known as P-TEFb to TAR RNA at the 5 0 end of nascent viral transcripts. The catalytic core of P-TEFb contains CDK9 and Cyclin T1 (CCNT1). A human endogenous complexome has recently been described – the set of multi-protein complexes in HeLa cell nuclei. We mined this complexome data set and identified 12 distinct multi-protein complexes that contain both CDK9 and CCNT1. We have termed these complexes CCAPs for CDK9/ CCNT1-associated protein complexes. Nine CCAPs are novel, while three were previously identified as Core P-TEFb, the 7SK snRNP, and the Super-Elongation Complex. We have investigated the role of five newly identified CCAPs in Tat function and viral gene expression. Results: We examined five CCAPs that contain: 1) PPP1R10/TOX3/WDR82; 2) TTF2; 3) TPR; 4) WRNIP1; 5) FBXO11/ CUL1/SKP1. SiRNA depletions of protein subunits of the five CCAPs enhanced Tat activation of an integrated HIV-1 LTR-Luciferase reporter in TZM-bl cells. Using plasmid transfection assays in HeLa cells, we also found that siRNA depletions of TTF2, FBXO11, PPP1R10, WDR82, and TOX3 enhanced Tat activation of an HIV-1 LTR-luciferase reporter, but the depletions did not enhance expression of an NF-κB reporter plasmid with the exception of PPP1R10. We found no evidence that depletion of CCAPs perturbed the level of CDK9/CCNT1 in the 7SK snRNP. We also found that the combination of siRNA depletions of both TTF2 and FBXO11 sensitized a latent provirus in Jurkat cells to reactivation by sub-optimal amounts of αCD3/CD28 antibodies. Conclusions: Our results identified five novel CDK9/CCNT1 complexes that are capable of negative regulation of HIV-1 Tat function and viral gene expression. Because siRNA depletions of CCAPs enhance Tat function, it is possible that these complexes reduce the level of CDK9 and CCNT1 available for Tat, similar to the negative regulation of Tat by the 7SK snRNP. Our results highlight the complexity in the biological functions of CDK9 and CCNT1.
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Regulation of Cyclin T1 and HIV-1 Replication by MicroRNAs in Resting CD4+ T Lymphocytes
Journal of virology, 2011Co-Authors: Karen Chiang, Tzu-ling Sung, Andrew P. RiceAbstract:The replication of integrated human immunodeficiency virus type 1 (HIV-1) is dependent on the cellular cofactor Cyclin T1, which binds the viral Tat protein and activates the RNA polymerase II transcription of the integrated provirus. The activation of resting CD4(+) T cells upregulates Cyclin T1 protein levels independently of an increase in Cyclin T1 mRNA levels, suggesting a translational repression of Cyclin T1 in resting CD4(+) T cells. Hypothesizing that microRNAs (miRNAs) repress Cyclin T1 translation in resting CD4(+) T cells and that this inhibition is lifted upon cell activation, we used microarray expression analysis to identify miRNAs miR-27b, miR-29b, miR-150, and miR-223 as being significantly downregulated upon CD4(+) T cell activation. The overexpression of these miRNAs decreased endogenous Cyclin T1 protein levels, while treatment with the corresponding antagomiRs increased Cyclin T1 protein levels. An miR-27b binding site within the Cyclin T1 3' untranslated region (3'UTR) was identified and confirmed to be functional after the mutation of key resides abrogated the ability of miR-27b to decrease the expression of a luciferase reporter upstream of the Cyclin T1 3'UTR. Ago2 immunoprecipitation revealed an association with Cyclin T1 mRNA that was decreased following treatment with miR-27b and miR-29b antagomiRs. Cells overexpressing miR-27b showed decreased viral gene expression levels of the HIV-1 reporter virus and a decreased replication of strain NL4.3; a partial rescue of viral transcription could be seen following the transfection of Cyclin T1. These results implicate miR-27b as a novel regulator of Cyclin T1 protein levels and HIV-1 replication, while miR-29b, miR-223, and miR-150 may regulate Cyclin T1 indirectly.
Koh Fujinaga - One of the best experts on this subject based on the ideXlab platform.
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Functional characterization of a human Cyclin T1 mutant reveals a different binding surface for Tat and HEXIM1
Virology, 2012Co-Authors: Alona Kuzmina, Koh Fujinaga, Uzi Hadad, Ran TaubeAbstract:HIV transcription is regulated at the step of elongation by the viral Tat protein and the cellular positive transcription elongation factor b (P-TEFb; Cdk9/Cyclin T1). Herein, a human Cyclin T1 mutant, Cyclin T1-U7, which contains four substitutions and one deletion in the N-terminal Cyclin box, was stably expressed in HeLa cells. HIV transcription was efficiently inhibited in HeLa-HA-CycT1-U7 stable cells. Cyclin T1-U7 bound Tat but did not modulate its expression levels, which remained high. Importantly Cyclin T1-U7 failed to interact with Cdk9 or HEXIM1 and did not interfere with endogenous P-TEFb activity to stimulate MEF2C or NFkB mediated transcription. In a T cell line and primary CD4+ cells, Cyclin T1-U7 also inhibited HIV transcription. We conclude that Cyclin T1-U7 sequesters Tat from P-TEFb and inhibits HIV transcription. Importantly, N-terminal residues in Cyclin T1 are specifically involved in the binding of Cyclin T1 to HEXIM1 but not to Tat.
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Cyclin T1 stabilizes expression levels of HIV-1 Tat in cells.
The FEBS journal, 2009Co-Authors: Kenichi Imai, Koh Fujinaga, Kaori Asamitsu, Ann Florence B. Victoriano, Marni E. Cueno, Takashi OkamotoAbstract:Transcription from HIV-1 proviral DNA is a rate-determining step for HIV-1 replication. Interaction between the Cyclin T1 (CycT1) subunit of positive transcription elongation factor b (P-TEFb) and the Tat transactivator protein of HIV-1 is crucial for viral transcription. CycT1 also interacts directly with the transactivation-responsive element (TAR) located on the 5'end of viral mRNA, as well as with Tat through the Tat-TAR recognition motif (TRM). These molecular interactions represent a critical step for stimulation of HIV transcription. Thus, Tat and CycT1 are considered to be feasible targets for the development of novel anti-HIV therapies. In this study, we demonstrate that CycT1 is positively involved in the Tat protein stability. Selective degradation of CycT1 by small interfering RNA (siRNA) culminated in proteasome-mediated degradation of Tat and eventual inhibition of HIV-1 gene expression. We noted that the siRNA-mediated knockdown of CycT1 could inhibit HIV-1 transcription without affecting cell viability and Tat mRNA levels. These findings clearly indicate that CycT1 is a feasible therapeutic target, and inactivation or depletion of CycT1 should effectively inhibit HIV replication by destabilizing Tat and suppressing Tat-mediated HIV transcription.
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Dominant negative mutant Cyclin T1 proteins that inhibit HIV transcription by forming a kinase inactive complex with Tat.
Journal of General Virology, 2008Co-Authors: Julie K Jadlowsky, Masanori Nojima, Takashi Okamoto, Koh FujinagaAbstract:Transcription of the human immunodeficiency virus type 1 (HIV) requires the interaction of the Cyclin T1 (CycT1) subunit of a host cellular factor, the positive transcription elongation factor b (P-TEFb), with the viral Tat protein, at the transactivation response element (TAR) of nascent transcripts. Because of this virus-specific interaction, CycT1 may potentially serve as a target for the development of anti-HIV therapies. Here we report the development of a mutant CycT1 protein, containing three threonine-to-alanine substitutions in the linker region between two of the Cyclin boxes, which displays a potent dominant negative effect on HIV transcription. Investigation into the inhibitory mechanism revealed that this mutant CycT1 interacted with Tat and the Cyclin-dependent kinase 9 (Cdk9) subunit of P-TEFb, but failed to stimulate the Cdk9 kinase activity critical for elongation. This mutant CycT1 protein may represent a novel class of specific inhibitors of HIV transcription which could lead to development of new antiviral therapies.
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dominant negative mutant Cyclin T1 proteins inhibit hiv transcription by specifically degrading tat
Retrovirology, 2008Co-Authors: Julie K Jadlowsky, Antje Schulte, Masanori Nojima, Takashi Okamoto, Koh FujinagaAbstract:Background The positive transcription elongation factor b (P-TEFb) is an essential cellular co-factor for the transcription of the human immunodeficiency virus type 1 (HIV-1). The Cyclin T1 (CycT1) subunit of P-TEFb associates with a viral protein, Tat, at the transactivation response element (TAR). This represents a critical and necessary step for the stimulation of transcriptional elongation. Therefore, CycT1 may serve as a potential target for the development of anti-HIV therapies.
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Dominant negative mutant Cyclin T1 proteins inhibit HIV transcription by specifically degrading Tat
Retrovirology, 2008Co-Authors: Julie K Jadlowsky, Antje Schulte, Masanori Nojima, Takashi Okamoto, Koh FujinagaAbstract:The positive transcription elongation factor b (P-TEFb) is an essential cellular co-factor for the transcription of the human immunodeficiency virus type 1 (HIV-1). The Cyclin T1 (CycT1) subunit of P-TEFb associates with a viral protein, Tat, at the transactivation response element (TAR). This represents a critical and necessary step for the stimulation of transcriptional elongation. Therefore, CycT1 may serve as a potential target for the development of anti-HIV therapies. To create effective inhibitors of HIV transcription, mutant CycT1 proteins were constructed based upon sequence similarities between CycT1 and other Cyclin molecules, as well as the defined crystal structure of CycT1. One of these mutants, termed CycT1-U7, showed a potent dominant negative effect on Tat-dependent HIV transcription despite a remarkably low steady-state expression level. Surprisingly, the expression levels of Tat proteins co-expressed with CycT1-U7 were significantly lower than Tat co-expressed with wild type CycT1. However, the expression levels of CycT1-U7 and Tat were restored by treatment with proteasome inhibitors. Concomitantly, the dominant negative effect of CycT1-U7 was abolished by these inhibitors. These results suggest that CycT1-U7 inhibits HIV transcription by promoting a rapid degradation of Tat. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription that could potentially be used in the design of anti-viral therapy.
Tariq M. Rana - One of the best experts on this subject based on the ideXlab platform.
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specific hiv 1 tar rna loop sequence and functional groups are required for human Cyclin T1 tat tar ternary complex formation
Biochemistry, 2002Co-Authors: Sara N Richter, Hong Cao, Tariq M. RanaAbstract:Replication of human immunodeficiency virus requires Tat protein which activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the Cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex (P-TEFb). Tat binds directly through its transactivation domain to the CycT1 subunit of the P-TEFb and induces loop sequence specific binding of the P-TEFb onto nascent HIV-1 TAR RNA. By using a gel electrophoresis method and a comprehensive set of TAR loop mutants, we have identified the sequence and structural determinants for high-affinity CycT1-Tat-TAR ternary complex formation. Our results show that CycT1 and Tat binding to TAR RNA is highly cooperative, and a capacity of 85%, a Hill coefficient of 2.7, and a dissociation constant (K(D)) of 2.45 nM were observed. These results indicate that there are three binding sites on TAR RNA. CycT1 does not bind TAR RNA in the absence of Tat, and Tat binding to TAR, while detectable, is very inefficient in the absence of CycT1. It is conceivable that the CycT1-Tat heterodimer directly binds to TAR RNA in the U-rich RNA bulge region and this binding facilitates the interactions of the CycT1-Tat heterodimer at the other two sites in the RNA loop region. On the basis of our results, we suggest a model where CycT1 interacts with Tat protein and positions the protein complex to make contacts with the G34 region of the loop sequence; G34 is critical for CycT1-Tat binding and forms a C30.G34 base pair. Two functional groups, O6 and N7, at nucleotide positions 32 and 34 in the TAR loop are essential for CycT1-Tat interactions with TAR RNA. The identity of two nucleotides, U31 and G33, is not critical, but they contribute to the stabilization of the RNA-protein complex. The presence of a single-nucleotide bulge of A35 or C35 is essential for distortion of the backbone RNA structure as well as the accessibility of functional groups in the major groove of the double-helical region. CycT1-Tat interaction with TAR RNA represents another example of the flexibility and complexity of RNA structure involved in protein recognition.
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specific hiv 1 tar rna loop sequence and functional groups are required for human Cyclin T1 tat tar ternary complex formation
Biochemistry, 2002Co-Authors: Sara N Richter, Hong Cao, Tariq M. RanaAbstract:Replication of human immunodeficiency virus requires Tat protein which activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the Cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex (P-TEFb). Tat binds directly through its transactivation domain to the CycT1 subunit of the P-TEFb and induces loop sequence specific binding of the P-TEFb onto nascent HIV-1 TAR RNA. By using a gel electrophoresis method and a comprehensive set of TAR loop mutants, we have identified the sequence and structural determinants for high-affinity CycT1−Tat−TAR ternary complex formation. Our results show that CycT1 and Tat binding to TAR RNA is highly cooperative, and a capacity of 85%, a Hill coefficient of 2.7, and a dissociation constant (KD) of 2.45 nM were observed. These results indicate that there are three binding sites on TAR RNA. CycT1 does not bind TAR RNA in the absence of Tat, and Tat binding to TAR, while detectable, is very inefficient in t...
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HIV-1 TAR RNA enhances the interaction between Tat and Cyclin T1.
The Journal of biological chemistry, 2000Co-Authors: Jing Zhang, Natarajan Tamilarasu, Seongwoo Hwang, Mitchell E Garber, Ikramul Huq, Katherine A. Jones, Tariq M. RanaAbstract:Human immunodeficiency virus, type 1 (HIV-1), Tat activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the Cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex, P-TEFb. Binding of Tat to CycT1 induces cooperative binding of the P-TEFb complex onto nascent HIV-1 TAR RNA. Here the specific interaction between Tat protein, human Cyclin T1, and HIV-1 TAR RNA was analyzed by fluorescence resonance energy transfer, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein synthesized through solid-phase chemistry. We find that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA and that TAR RNA further enhances the interaction between Tat and CycT1. We conclude that TAR RNA nucleates the formation of the Tat.P-TEFb complex through an induced fit mechanism.
Ran Taube - One of the best experts on this subject based on the ideXlab platform.
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A single point mutation in Cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription
Retrovirology, 2014Co-Authors: Alona Kuzmina, Nina Verstraete, Sigal Galker, Maayan Maatook, Olivier Bensaude, Ran TaubeAbstract:BackgroundHuman immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II.ResultsTo map residues in the Cyclin box of Cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal Cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in Cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique Cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus.ConclusionsThis study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of Cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription.
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A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat
Retrovirology, 2014Co-Authors: Nina Verstraete, Alona Kuzmina, Olivier Bensaude, Ran Taube, Van Trung Nguyen, Gaelle Diribarne, Lydia Kobbi, Monika LudanyiAbstract:Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding.
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A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat
Retrovirology, 2014Co-Authors: Nina Verstraete, Alona Kuzmina, Ran Taube, Van Trung Nguyen, Gaelle Diribarne, Lydia Kobbi, Monika Ludanyi, Olivier BensaudeAbstract:The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two Cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription.
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Functional characterization of a human Cyclin T1 mutant reveals a different binding surface for Tat and HEXIM1
Virology, 2012Co-Authors: Alona Kuzmina, Koh Fujinaga, Uzi Hadad, Ran TaubeAbstract:HIV transcription is regulated at the step of elongation by the viral Tat protein and the cellular positive transcription elongation factor b (P-TEFb; Cdk9/Cyclin T1). Herein, a human Cyclin T1 mutant, Cyclin T1-U7, which contains four substitutions and one deletion in the N-terminal Cyclin box, was stably expressed in HeLa cells. HIV transcription was efficiently inhibited in HeLa-HA-CycT1-U7 stable cells. Cyclin T1-U7 bound Tat but did not modulate its expression levels, which remained high. Importantly Cyclin T1-U7 failed to interact with Cdk9 or HEXIM1 and did not interfere with endogenous P-TEFb activity to stimulate MEF2C or NFkB mediated transcription. In a T cell line and primary CD4+ cells, Cyclin T1-U7 also inhibited HIV transcription. We conclude that Cyclin T1-U7 sequesters Tat from P-TEFb and inhibits HIV transcription. Importantly, N-terminal residues in Cyclin T1 are specifically involved in the binding of Cyclin T1 to HEXIM1 but not to Tat.
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A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells
Journal of virology, 2002Co-Authors: Koh Fujinaga, Ran Taube, Dan Irwin, Fan Zhang, B. Matija PeterlinAbstract:The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human Cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5' end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn(2+)-dependent manner via the cysteine at position 261, which is a tyrosine in murine Cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn(2+)-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex.
Matthias Geyer - One of the best experts on this subject based on the ideXlab platform.
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specificity of hexim1 and hexim2 complex formation with Cyclin T1 t2 importin α and 7sk snrna
Journal of Molecular Biology, 2010Co-Authors: Nadine Czudnochowski, Friederike Vollmuth, Sascha Baumann, Karin Vogelbachmayr, Matthias GeyerAbstract:Positive transcription elongation factor b (P-TEFb) stimulates the transition from transcription initiation to productive elongation by phosphorylation of the C-terminal domain of RNA polymerase II. P-TEFb consists of the Cyclin-dependent kinase Cdk9 and a T-type Cyclin and is regulated by the small nuclear RNA 7SK and the coupling protein Hexim1 or Hexim2. In this study, we analyzed the tripartite protein-RNA complex formation between Hexim, Cyclin T and 7SK snRNA. Using isothermal titration calorimetry, we observed higher affinities for Cyclin T1-Hexim1 and Cyclin T2-Hexim2 complex formations compared with the interactions in reverse. Importin alpha, which is part of the Ran-mediated nuclear import pathway, bound Hexim1 and Hexim2 with dissociation constants of 2.0 and 0.5 muM, respectively. Furthermore, tripartite complex formations between Cyclin T, Hexim and Importin alpha showed the suitability of a collaborative nuclear import pathway for Cyclin T. Electrophoretic mobility shift assays using radioactively labelled full-length 7SK snRNA revealed a tight association of the RNA to Cyclin T1-Hexim1 with dissociation constants lower than 0.3 muM. Similar binding affinities were recorded for both Hexim orthologues to a 66-mer double-stranded 5' hairpin loop encompassing nucleotides 23-88 of 7SK, while a 39-mer fragment, resulting from different RNA folding predictions, did not bind as tightly. These results provide the molecular basis for the generation of a core complex for the inhibition of P-TEFb.
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structural insights into the Cyclin T1 tat tar rna transcription activation complex from eiav
Nature Structural & Molecular Biology, 2008Co-Authors: Kanchan Anand, Antje Schulte, Klaus Scheffzek, Karin Vogelbachmayr, Matthias GeyerAbstract:The replication of many retroviruses is mediated by a transcriptional activator protein, Tat, which activates RNA polymerase II at the level of transcription elongation. Tat interacts with Cyclin T1 of the positive transcription-elongation factor P-TEFb to recruit the transactivation-response TAR RNA, which acts as a promoter element in the transcribed 5' end of the viral long terminal repeat. Here we present the structure of the Cyclin box domain of Cyclin T1 in complex with the Tat protein from the equine infectious anemia virus and its corresponding TAR RNA. The basic RNA-recognition motif of Tat adopts a helical structure whose flanking regions interact with a Cyclin T-specific loop in the first Cyclin box repeat. Together, both proteins coordinate the stem-loop structure of TAR. Our findings show that Tat binds to a surface on Cyclin T1 similar to where recognition motifs from substrate and inhibitor peptides were previously found to interact within Cdk-Cyclin pairs.
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structure of the Cyclin t binding domain of hexim1 and molecular basis for its recognition of p tefb
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Sonja A. Dames, Antje Schulte, André Schönichen, Stephan Grzesiek, Matija B Peterlin, Matjaz Barboric, Matthias GeyerAbstract:Hexim1 is a cellular protein that associates with the positive transcription elongation factor b (P-TEFb) to regulate RNA polymerase II elongation of nascent mRNA transcripts. It directly binds to Cyclin T1 of P-TEFb and inhibits the kinase activity of Cdk9, leading to an arrest of transcription elongation. Here, we report the solution structure of the Cyclin T binding domain (TBD) of Hexim1 that forms a parallel coiled-coil homodimer composed of two segments and a preceding alpha helix that folds back onto the first coiled-coil unit. NMR titration, fluorescence, and immunoprecipitation experiments revealed the binding interface to Cyclin T1, which covers a large surface on the first coiled-coil segment. Electrostatic interactions between an acidic patch on Hexim1 and positively charged residues of Cyclin T1 drive the complex formation that is confirmed by mutagenesis data on Hexim1 mediated transcription regulation in cells. Thus, our studies provide structural insights how Hexim1 recognizes the Cyclin T1 subunit of P-TEFb, which is a key step toward the regulation of transcription elongation.
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identification of a Cyclin t binding domain in hexim1 and biochemical analysis of its binding competition with hiv 1 tat
Journal of Biological Chemistry, 2005Co-Authors: Antje Schulte, André Schönichen, Nadine Czudnochowski, Matija B Peterlin, Matjaz Barboric, Dalibor Blazek, Matthias GeyerAbstract:Abstract The active form of the positive transcription elongation factor b (P-TEFb) consists of Cyclin T and the kinase Cdk9. P-TEFb stimulates transcription by phosphorylating the C-terminal domain of RNA polymerase II. It becomes inactivated when associated in a tetrameric complex with the abundant 7SK small nuclear RNA and the recently identified protein Hexim1. In this study, we identified a stable and soluble C-terminal domain (residues 255–359) in Hexim1 of 12.5-kDa size that binds the Cyclin boxes of Cyclin T1. Functional assays in HeLa cells showed that this Cyclin T-binding domain (TBD) is required for the binding of Hexim1 to P-TEFb and inhibition of transcriptional activity in vivo. Analytical gel filtration and GST pull-down experiments revealed that both full-length Hexim1 and the TBD are homodimers. Isothermal titration calorimetry yielded a weak multimer for the TBD with a multimerization constant of 1.3 × 103 m. The binding affinity between the TBD and Cyclin T1 was analyzed with fluorescence spectroscopy methods, using a dansyl-based fluorescence label at position G257C. Equilibrium fluorescence titration and stopped flow fast kinetics yield a dissociation constant of 1.2 μm. Finally, we tested the effect of the HIV-1 Tat protein on the Cyclin T1-TBD complex formation. GST pull-down experiments and size exclusion chromatography exhibit a mutually exclusive binding of the two effectors to Cyclin T1. Our data suggest a model where HIV-1 Tat competes with Hexim1 for Cyclin T1 binding, thus releasing P-TEFb from the inactive complex to stimulate the transcription of HIV-1 gene expression.
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optimized chimeras between kinase inactive mutant cdk9 and truncated Cyclin T1 proteins efficiently inhibit tat transactivation and human immunodeficiency virus gene expression
Journal of Virology, 2002Co-Authors: Koh Fujinaga, Matthias Geyer, Dan Irwin, Matija B PeterlinAbstract:The expression of human immunodeficiency virus type 1 (HIV) is regulated by the viral transcriptional transactivator Tat (22). Unlike other DNA-bound activators, Tat binds RNA and effects the elongation step of transcription (41). Without Tat, RNA polymerase II terminates prematurely, and short transcripts accumulate. By binding the transactivation response (TAR) element, which forms an RNA stem loop at the 5′ end of all viral transcripts, Tat relieves this transcriptional block. Tat also requires a cellular cofactor, the positive transcription elongation factor b (P-TEFb) (28, 29, 47). P-TEFb is a heterodimer composed of the Cyclin-dependent kinase 9 (Cdk9) and Cyclin T1, T2a, T2b, or K (38). Among these C-type Cyclins, only Cyclin T1 (CycT1) can support Tat transactivation (37, 45). CycT1 but not CycT2 or CycK binds Tat and the central loop of TAR, resulting in the recruitment of Cdk9 to the stalled RNA polymerase II. Cdk9 phosphorylates the C-terminal domain of RNA polymerase II, thus converting the initiating RNA polymerase IIa to its elongating form (RNA polymerase IIo). CycT1 (726 amino acids) contains two conserved Cyclin boxes (from positions 31 to 250), a coiled-coil sequence (from positions 379 to 430), a histidine-rich region (from positions 506 to 530), and a PEST sequence (from positions 709 to 726) (37, 45). N-terminal Cyclin boxes are important for binding and activating Cdk9. Early studies revealed that residues from positions 251 to 272 are essential for the binding between Tat and TAR (16). Since murine CycT1, which cannot support Tat transactivation in rodent cells, has a tyrosine at position 261, the cysteine at position 261 in human CycT1 (hCycT1) was found to be essential for this interaction (3, 14, 16, 21). Indeed, the change of this tyrosine to cysteine allows murine CycT1 to support Tat transactivation (14, 16, 21). This region also contains six positively charged residues that serve as an arginine-rich RNA binding motif and a nuclear localization signal. Thus, this sequence is called the Tat-TAR recognition motif. Although the C-terminal region of CycT1 might play a role in TAR binding and binds the C-terminal domain, the N-terminal 272 amino acids of CycT1 are sufficient for Tat transactivation in vitro and in vivo (3, 11, 14-16, 21, 25, 42; K. Fujinaga, D. Irwin, M. Geyer, R. Taube, and B. M. Peterlin, submitted for publication). The interaction between Tat and CycT1 is dependent on zinc (16). A Zn2+-mediated bond is formed between two cysteines and one histidine in the activation domain (from positions 1 to 48) in Tat and the cysteine at position 261 in hCycT1 (3, 14, 16, 21). No other cysteine and/or histidine is required for this interaction (Fujinaga et al., submitted). In addition, hCycT1 alone cannot bind TAR (45). Although the interaction between CycT1, Tat, and TAR can be detected in the absence of Cdk9 and Cdk9 alone cannot bind TAR, Cdk9 still plays a major role in stabilizing this RNA-protein complex (11, 15). Thus, adding Cdk9 to the electrophoretic mobility shift assay enhances binding between Tat, TAR, and hCycT1 in an ATP-dependent manner (11). Moreover, the autophosphorylation of Cdk9 in its C terminus is required for this binding (15). Mimicking the phosphorylation by mutating three serines (at positions 347, 353, and 357) and two threonines (at positions 350 and 354) to glutamates in the C terminus of Cdk9 (Cdk9-5E) restored this binding. These data imply that the negative charges in Cdk9 are important for the interaction between hCycT1, Tat, and TAR. Since P-TEFb is essential for Tat transactivation, it is a potential target for anti-HIV therapeutics. To date, a kinase-negative mutant Cdk9 protein [Cdk9(D167N)], an inhibitor that is specific for Cdk9 (flavopiridol), and compounds that bind TAR (phenothiazines) inhibit Tat transactivation (5, 10, 27, 44). However, since this kinase is important for the transcriptional elongation of other cellular genes, inhibiting P-TEFb might have pleiotropic effects. Thus, it is important to develop inactive mutant P-TEFb complexes that are highly specific for Tat. In this study, we constructed mutant hCycT1 proteins that were deficient in their interactions with Cdk9 or TAR and measured their inhibitory effects on HIV transcription. However, expression studies revealed that these mutant hCycT1 proteins had relatively modest effects, presumably because of the high stability of the endogenous P-TEFb. Stronger inhibitory effects were obtained when the mutant Cdk9 proteins were fused with hCycT1. The chimera between a kinase-inactive Cdk9 that contained glutamates rather than serines and threonines in its C terminus and hCycT1 [Cdk9(D167N,361-5E).hCycT280] especially inhibited Tat transactivation as well as HIV gene expression. Therefore, this fusion protein could provide an effective and specific anti-HIV therapy.
