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Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1)  [PDF]
Naoki Sakane,Hye-Sook Kwon,Sara Pagans,Katrin Kaehlcke,Yasuhiro Mizusawa,Masafumi Kamada,Kara G. Lassen,Jonathan Chan,Warner C. Greene,Martina Schnoelzer,Melanie Ott
PLOS Pathogens , 2011, DOI: 10.1371/journal.ppat.1002184
Abstract: The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear. We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells. Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.
SIRT1 Regulates HIV Transcription via Tat Deacetylation  [PDF]
Sara Pagans,Angelika Pedal,Brian J. North,Katrin Kaehlcke,Brett L. Marshall,Alexander Dorr,Claudia Hetzer-Egger,Peter Henklein,Roy Frye,Michael W. McBurney,Henning Hruby,Manfred Jung,Eric Verdin,Melanie Ott
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.0030041
Abstract: The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.
SIRT1 Regulates HIV Transcription via Tat Deacetylation  [PDF]
Sara Pagans,Angelika Pedal,Brian J North,Katrin Kaehlcke,Brett L Marshall,Alexander Dorr,Claudia Hetzer-Egger,Peter Henklein,Roy Frye,Michael W McBurney,Henning Hruby,Manfred Jung,Eric Verdin,Melanie Ott
PLOS Biology , 2005, DOI: 10.1371/journal.pbio.0030041
Abstract: The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.
Dominant negative mutant Cyclin T1 proteins inhibit HIV transcription by specifically degrading Tat
Julie K Jadlowsky, Masanori Nojima, Antje Schulte, Matthias Geyer, Takashi Okamoto, Koh Fujinaga
Retrovirology , 2008, DOI: 10.1186/1742-4690-5-63
Abstract: 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.The transcription of human immunodeficiency virus type 1 (HIV-1) is a highly regulated process in which several host cellular co-factors and the viral transactivator protein Tat are involved [1,2]. Tat stimulates the elongation of transcription with the aid of the positive transcription elongation factor b (P-TEFb), a heterodimer comprised of cyclin T1 (CycT1) and cyclin dependent kinase 9 (Cdk9). Tat and CycT1 bind to the transactivation response element (TAR), an RNA stem loop structure located at the 5'-end (+1 to +59) of all viral transcripts [3-5]. This interaction results in the recruitment of Cdk9 and the subsequent stimulation of its kinase activity by Tat [6]. Among three distinct P-TEFb complexes (CycT1/Cdk9, CycT2/Cdk9, and CycK/Cdk9), only the CycT1/Cdk9 complex can support Tat transactivation [7-9].The interaction between Tat, TAR, and CycT1 has been extensively studied [2-5,8,10]. Tat binds to the bulge region (+23 to +25) of TAR and the CycT1 subunit of P-TEFb through i
Effect of SWI/SNF chromatin remodeling complex on HIV-1 Tat activated transcription
Emmanuel Agbottah, Longwen Deng, Luke O Dannenberg, Anne Pumfery, Fatah Kashanchi
Retrovirology , 2006, DOI: 10.1186/1742-4690-3-48
Abstract: Here, we describe the effect of Tat activated transcription at the G1/S border of the cell cycle and analyze the interaction of modified Tat with the chromatin remodeling complex, SWI/SNF. HIV-1 LTR DNA reconstituted into nucleosomes can be activated in vitro using various Tat expressing extracts. Optimally activated transcription was observed at the G1/S border of the cell cycle both in vitro and in vivo, where chromatin remodeling complex, SWI/SNF, was present on the immobilized LTR DNA. Using a number of in vitro binding as well as in vivo chromatin immunoprecipitation (ChIP) assays, we detected the presence of both BRG1 and acetylated Tat in the same complex. Finally, we demonstrate that activated transcription resulted in partial or complete removal of the nucleosome from the start site of the LTR as evidenced by a restriction enzyme accessibility assay.We propose a model where unmodified Tat is involved in binding to the CBP/p300 and cdk9/cyclin T1 complexes facilitating transcription initiation. Acetylated Tat dissociates from the TAR RNA structure and recruits bromodomain-binding chromatin modifying complexes such as p/CAF and SWI/SNF to possibly facilitate transcription elongation.Human immunodeficiency virus (HIV) is the etiological agent of AIDS. The pathogenesis of HIV-induced disease is complex and multifactorial [1]. Following infection, reverse transcriptase complexes synthesize a double stranded DNA molecule that is then incorporated into the host genome. A robust cellular and humoral immune response inhibits viral production within weeks. However, a chronic persistent infection in lymphoid tissue persists throughout the life (median period of 10–20 years) of the infected individual. Several key HIV-1 and cellular proteins have been determined to be necessary for this course of infection, including the trans-activator Tat. Viral clones deficient in Tat do not effectively replicate in vitro or in vivo. Furthermore, infected T cells quiescent at the G0
Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein
Michael Y Mi, Jiying Zhang, Yukai He
Retrovirology , 2005, DOI: 10.1186/1742-4690-2-71
Abstract: We synthesized a short peptide named Tat-P, which contained the TAR RNA binding and PTD domains to examine whether the peptide has the potential of inhibiting TAT dependent HIV replication. We investigated the inhibiting effects of Tat-P in vitro using a HIV derived lentiviral vector model. We found that the TAT PTD domain not only efficiently transduced test cells, but also effectively inhibited the production of lentiviral particles in a TAT dependent manner. These results were also supported by data derived from the TAT activated LTR-luciferase expression model and RNA binding assays.Tat-P may become part of a category of anti-HIV drugs that competes with full length TAT proteins to inhibit HIV replication. In addition, this study indicates that the HIV derived lentiviral vector system is a safe and reliable screening method for anti-HIV drugs, especially for those targeting the interaction of TAT and TAR RNAs.The HIV TAT protein is a key regulator of viral replication [1]. Binding of the TAT protein to the TAR element, a 59 nt sequence at the 5' end of nascent RNA, is the first critical step for producing full length HIV RNA. The transcription of HIV RNA from both integrated and non-integrated HIV genome is dependent on TAT protein [2]. Thus, interruption of this TAT-TAR interaction has been considered as a possible way to inhibit HIV replication [3]. TAR RNA decoys have been shown to be able to interfere with the binding of TAT proteins to native TAR elements, thus inhibiting HIV replication [4-6]. However, delivery of oligonucleotides in vivo is not trivial. Conversely, small synthetic substances, or short TAT peptides mimicking the TAT and TAR RNA binding domains have been shown to be promising inhibitors of HIV replication [7,8]. Furthermore, a different fragment of the TAT protein could compete for the binding site of the CXCR4 receptor on T cells and inhibit HIV entry [9]. Recently, several research groups have identified the TAR RNA binding domain of the
Tat gets the "green" light on transcription initiation
John Brady, Fatah Kashanchi
Retrovirology , 2005, DOI: 10.1186/1742-4690-2-69
Abstract: Transcription of the HIV-1 provirus is characterized by an early, Tat-independent and a late, Tat-dependent phase. Transcription from the HIV-1 LTR is increased several hundred-fold in the presence of Tat and the ability of Tat to activate transcription is essential for virus replication. Tat is an unusual transcription factor because it interacts with a cis acting RNA enhancer element, TAR, present at the 5' end of all viral transcripts (nt +1 to +59) [1-4]. In fact, TAR was the first demonstration of a RNA enhancer element. Unlike other eukaryotic enhancers, however, the TAR element was only functional when it was placed 3' to the HIV promoter and in the correct orientation and position [5]. The location of the TAR in transcribed regions was surprising, and to many, inconsistent with a role for TAR in transcription initiation. In fact, the uniqueness of the RNA enhancer element drove many investigators to search for unique pathways in HIV Tat transactivation. When Kao et al. [6] reported that in the absence of Tat the majority of RNA polymerases initiating transcription stall near the promoter, and later Laspia et al. [7] reported a small effect of Tat on transcription initiation but a large effect on transcription elongation, the initiation model quickly lost support. The observation that Tat binds specifically to the TAR RNA [8] and could function as an RNA binding protein [9] gave further support for the elongation model, and it became quite well accepted that through interaction with TAR, Tat promotes the assembly of an active transcription elongation complex. The more recent finding that Tat promotes the binding of P-TEFb, a transcription elongation factor composed of cyclin T1 and cdk9 [10] and, more recently, Brd4 in the active nuclear complex [11] seemed consistent with the elongation model. In fact, it has been shown that the interaction of Tat with P-TEFb and TAR leads to hyperphosphorylation of the C-terminal domain (CTD) of RNA Pol II and increased pro
Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat
Qinmiao Sun, Hittu Matta, Preet M Chaudhary
Retrovirology , 2005, DOI: 10.1186/1742-4690-2-9
Abstract: We present evidence that vFLIP K13 activates HIV-1 LTR via the activation of the classical NF-κB pathway involving c-Rel, p65 and p50 subunits. K13-induced HIV-1 LTR transcriptional activation requires the cooperative interaction of all three components of the IKK complex and can be effectively blocked by inhibitors of the classical NF-κB pathway. K13 mutants that lacked the ability to activate the NF-κB pathway also failed to activate the HIV-1 LTR. K13 could effectively activate a HIV-1 LTR reporter construct lacking the Tat binding site but failed to activate a construct lacking the NF-κB binding sites. However, coexpression of HIV-1 Tat with K13 led to synergistic activation of HIV-1 LTR. Finally, K13 differentially activated HIV-1 LTRs derived from different strains of HIV-1, which correlated with their responsiveness to NF-κB pathway.Our results suggest that concomitant infection with KSHV/HHV8 may stimulate HIV-1 LTR via vFLIP K13-induced classical NF-κB pathway which cooperates with HIV-1 Tat protein.The human immunodeficiency virus type 1 (HIV-1) establishes latent infection following integration into the host genome [1]. The expression of integrated HIV-1 provirus in cells latently infected with this virus is controlled at the level of transcription by an interplay between distinct cellular and viral transcription factors which bind to the HIV-1 long terminal repeat (LTR) [1-4]. The HIV-1 LTR is divided into three regions: U3, R and U5, which contain four functional elements: transactivation response element (TAR), a basal or core promoter, a core enhancer, and a modulatory element [1,4]. The viral transactivator Tat is a key activator of HIV-1 LTR via its binding to the TAR region, while the core region contains three binding sites for Sp1 transcription factor and a TATA box [1]. The enhancer region of HIV-1 LTR contains two highly conserved consecutive copies of κB elements at nucleotides -104 to -81 that are critical for HIV-1 replication in T cells [1]
Proliferative activity of extracellular HIV-1 Tat protein in human epithelial cells: expression profile of pathogenetically relevant genes
Alessia A Bettaccini, Andreina Baj, Roberto S Accolla, Fulvio Basolo, Antonio Q Toniolo
BMC Microbiology , 2005, DOI: 10.1186/1471-2180-5-20
Abstract: small concentrations of Tat (100 ng/ml) stimulated cell proliferation. Tat antibodies neutralized the mitogenic Tat activity. Changes of gene expression in Tat-treated cells were evaluated by RT-PCR and gene-array methods. Within 4 hours of treatment, exposure to Tat is followed by up-regulation of some cell cycle-associated genes (transcription factors, cyclin/cdk complexes, genes of apoptotic pathways) and of genes relevant to HIV pathogenesis [chemokine receptors (CXCR4, CCR3), chemotactic cytokines (SDF-1, RANTES, SCYC1, SCYE1), IL6 family cytokines, inflammatory cytokines, factors of the TGF-beta family (TGFb, BMP-1, BMP-2)]. Up-regulation of anti-inflammatory cytokines (IL-10, IL-19, IL-20), a hallmark of other persistent viral infections, was a remarkable feature of Tat-treated epithelial cell lines.extracellular Tat is mitogenic for mammary and amniotic epithelial cells and stimulates the expression of genes of pathogenetic interest in HIV infection. These effects may favor virus replication and may facilitate the mother-to-child transmission of virus.Numerous extracellular roles for the HIV-1 transactivator protein (Tat) have emerged from experiments showing that Tat is taken up by cultured cells, enters the nucleus, and transactivates genes linked to the HIV LTR [1].Early experiments showing that recombinant Tat was able to inhibit antigen-induced, but not mitogen-induced, proliferation of peripheral blood mononuclear cells indicated Tat as a viral immunosuppressant [2]. This view has been confirmed by subsequent observations indicating that: a) Tat down-regulates HLA class I [3] and class II [4] expression in T-lymphocytes and macrophages, b) Tat represses transcription of the mannose receptor [5] a key molecule in the early response to invading pathogens, c) Tat suppresses CD26-dependent T cell proliferation [6], d) Tat down-regulates TCR/CD3 surface complexes [7], e) Tat induces suppressive levels of alpha interferon in T cells [8], and f) Tat causes hu
HIV-1 Tat protein alter the tight junction integrity and function of retinal pigment epithelium: an in vitro study
Ling Bai, Zhenping Zhang, Hui Zhang, Xiumei Li, Qiurong Yu, Haotian Lin, Wenhui Yang
BMC Infectious Diseases , 2008, DOI: 10.1186/1471-2334-8-77
Abstract: A human RPE cell line (D407) cultured on microporous filter-supports was used. After treating with HIV-1 Tat protein, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudins were determined by real-time polymerase chain reaction, immunofluorescence, and Western blot analysis. Activation of ERK1/2 was detected by Western blot analysis with specific antiphospho protein antibodies. NF-κB DNA binding activity was determined by transcription factor assay. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in barrier destruction of RPE cells exposed to HIV-1 Tat.Treating cultured human retinal pigment epithelial cells with 100 nM Tat for 24 hours increased the permeability and decreased the TER of the epithelial monolayer. HIV-1 Tat also disrupted and downregulated the tight-junction proteins claudin-1, claudin-3, and claudin-4 in these cells, whereas claudin-2 was upregulated, and the expression of occludin was unaffected. HIV-1 Tat protein also induced activation of ERK1/2 and NF-κB. HIV-1 Tat protein induced barrier destruction, changes in expression of TJs, and activation of ERK1/2 and NF-κB were abrogated by inhibitor of ERK1/2 and NF-κB.HIV-1 Tat protein causes increases in the paracellular permeability of RPE cells in vitro concomitant with changes in expression of certain transmembrane proteins associated with the tight junction. The effects of HIV-1 Tat on barrier function of the RPE may be mediated by ERK MAPK and NF-κB activation, which may represent potential targets for novel therapeutic approaches for the retinopathy induced by HIV infection.Serious ophthalmic diseases can cause blindness in the absence of prompt diagnosis and therapy. These diseases often result from opportunistic infections and are common in HIV-infected patients [1]. The exa
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