Superinduction of Tat activity in CLS fibroblasts.

<p>(A) Western blot analysis of cellular extracts of fibroblasts from a patient with CLS and control human fibroblasts. (B) Nuclear microinjection of CLS fibroblasts with synthetic Tat (amino acids 1–72), the HIV LTR luciferase reporter, a CMV-GFP expression plasmid, and either the empty vector, an RSK2 expression construct, or a plasmid expressing kinase-deficient RSK2. Values are means±SEM of five experiments. (C) Coinjection of the 5xUAS luciferase reporter, a plasmid expressing the Gal4-VP16 transactivator and CMV-GFP with either the RSK2-expressing plasmid or the vector alone. Values are means±SEM of three experiments.</p

Role of histone kinases in Tat transactivation.

<p>(A) Chromatin immunoprecipitation analysis of Jurkat T cells containing an integrated HIV promoter in the absence or presence of Tat. Immunoprecipitations were performed with α-phospho-histone H3 antibodies (serine 10) followed by radioactive PCR with primers specific for the HIV LTR, the c-fos, or the β-globin genes. (B) Jurkat 1G5 cells containing an integrated HIV LTR luciferase construct were transiently transfected with Tat/FLAG (25 ng) and kinase-deficient (KD) kinase expression vectors (200 ng). (C) Western blot analysis of cellular lysates from 293 cells cotransfected with the indicated expression plasmids. (D) Transfection of CMV luciferase (25 ng) with the KD RSK2 expression plasmid (200 ng) in Jurkat cells. (E) Transfection of 5xUAS luciferase and Gal4-CDK9 (20 ng) with the KD RSK2 expression plasmid (200 ng) in Jurkat cells. Values are means±SEM of three experiments.</p

Activation of RSK2 by Tat.

<p>(A) Autoradiography of radioactive <i>in vitro</i> synthesized RSK2 proteins before (Input) and after binding to biotinylated synthetic Tat (amino acids 1–72) or to beads alone. Increasing amounts of <i>in vitro</i> translated RSK2 were included in the binding reaction. (B) Kinase assay of endogenous RSK2 immunoprecipitated from Cos7 cells transfected with wild type Tat/FLAG, TatF38A/FLAG, or empty vector. Values are means±SEM of four experiments. (C) Western blotting of nuclear extracts isolated from Cos7 cells cotransfected with RSK2/HA and Tat/FLAG or with RSK2/HA and Tat F38A/FLAG constructs. Densitometric quantification of the phospho-S227-specific bands was performed using the NIH Image software. (D) Chromatin immunoprecipitation analysis of the Jurkat T cell line A2, latently infected with an HIV-based lentiviral vector expressing Tat/FLAG from the HIV LTR after treatment with TNF-α. At indicated time points, cells were harvested and immunoprecipitations were performed in duplicate with α-phospho-S227 antibodies followed by PCR with primers specific for the HIV LTR or the c-fos gene.</p

In Vitro Tat Deacetylation by Human SIRT Proteins

<div><p>(A) Scheme of Tat deacetylation assay with immunoprecipitated SIRT1–7 proteins. Expression vectors for FLAG-tagged SIRT proteins were transfected into HEK 293 cells, immunoprecipitated, and incubated with synthetic Tat (72 amino acids) carrying an N-terminal biotin label and an acetyl group at position 50 (AcTat) in the presence of NAD⁺. Immunoprecipitated material was also analyzed in a radioactive (³H) histone deacetylase assay using an H3 peptide as a substrate.</p> <p>(B) WB analysis of deacetylation reactions with antibodies specific for acetylated lysine 50 in Tat (α-AcTat), with SA-HRP, or with α-FLAG antibodies.</p> <p>(C) WB of Tat deacetylation by immunoprecipitated SIRT1 in the presence or absence of NAD⁺, TSA, or nicotinamide (Nic).</p></div

Inhibition of HIV Gene Expression by a Small Molecule Inhibitor of SIRT1

<div><p>(A) In vitro histone deacetylation assays including recombinant SIRT1, radioactively labeled histone H3 peptide, and indicated concentrations of splitomicin or HR73. The average (± SEM) of two experiments performed in duplicate is shown for each point.</p> <p>(B) Chemical structures of splitomicin and its derivative HR73.</p> <p>(C) Inhibition of Tat transactivation by HR73. RSV-Tat (0, 20, and 200 ng) and HIV LTR luciferase (200 ng) or RSV-luciferase (200 ng) vectors were transfected into HeLa cells. Transfected cells were treated with indicated concentrations of HR73 or DMSO for 8 h.</p> <p>(D) Inhibition of HIV gene expression by HR73. GFP expression in Jurkat T cells infected with HIV_NL4–3 containing the GFP open reading frame in place of the viral <i>nef</i> gene or with an HIV-based lentiviral vector expressing GFP from the heterologous EF-1α promoter. Treatment with HR73 (1 μM in DMSO) or DMSO was performed for 36 h after overnight infection. The average (± SEM) of four experiments is shown.</p></div

SIRT1 Is a Positive Cofactor for Tat Transactivation

<div><p>(A) Cotransfection of SIRT1 or the catalytically inactive SIRT1 mutant SIRT1H363Y with the HIV LTR luciferase construct and increasing amounts of a Tat expression vector (RSV-Tat: 0, 2, 20, and 200 ng), an HIV LTR luciferase construct containing mutated binding sites for the transcription factor NF-κB and RSV-Tat (20 ng), or with an RSV-luciferase construct (200 ng) in HeLa cells. The average of three experiments is shown (± standard error of the mean [SEM]).</p> <p>(B) WB analysis of HeLa cells 72 h after transfection of siRNAs directed against SIRT1 or GL3 control siRNAs.</p> <p>(C) Cotransfection of the HIV LTR luciferase construct with increasing amounts of CMV-Tat or CMV-TatK50R (0, 50, 100, 200, 400, and 800 ng) 48 h after transfection of double-stranded siRNAs directed against SIRT1 or GL3 control siRNAs in HeLa cells. Luciferase activity was measured 24 h after plasmid transfection and 72 h after siRNA transfection. Note that all luciferase reporter vectors used in this study are based on the pGL2 luciferase vector, which is not affected by GL3-specific siRNAs [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030041#pbio-0030041-b36" target="_blank">36</a>]. The average of three experiments is shown (± SEM).</p> <p>(D) The transcriptional activity of increasing amounts of the CMV-luciferase reporter (0, 50, 100, 200, 400, and 800 ng) was similar in SIRT1 knockdown or GL3-treated control cells. The average of two experiments performed in duplicate is shown (± SEM).</p> <p>(E) WB of endogenous SIRT1 or actin 72 h after transfection of siRNA directed against SIRT1 or mutated SIRT1 siRNA.</p> <p>(F) Cotransfection of the HIV LTR luciferase with increasing amounts of CMV-Tat (0, 2, 20, and 200 ng) in HeLa cells pretransfected with wild-type or mutant SIRT1 siRNA oligonucleotides as described in (C). WT, wild-type.</p></div

Impaired Tat Transcriptional Activity in Murine SIRT1^−/− Cells

<div><p>(A) Nuclear microinjection of HIV LTR luciferase, RSV-Tat, and a human cyclinT1-expressing construct into MEFs derived from SIRT^+/+ or SIRT^−/− mice. In all experiments, a fixed amount of DNA was injected by adding the empty vector control. Cells were coinjected with CMV-GFP, and the luciferase activity per GFP-positive cell was calculated. An average of two injections is shown.</p> <p>(B) The HIV LTR luciferase construct together with RSV-Tat and the cyclinT1-expressing construct were coinjected into SIRT^−/− MEFs in the presence of increasing amounts of a plasmid expressing human SIRT1. The average of three experiments is shown (± SEM).</p> <p>(C) Coinjection of the human SIRT1 expression vector together with the 5xUAS luciferase construct containing five Gal4 binding sites upstream of the thymidine kinase promoter and a Gal4-VP16 expression plasmid into SIRT1^−/− MEFs. The average of three experiments is shown (± SEM).</p></div

Transcriptional Activity of AcTat Depends on Deacetylation by SIRT1

<div><p>(A) AcTat functions through TAR and cyclinT1 binding. Nuclear microinjection of increasing amounts of synthetic Tat or AcTat together with wild-type (wt TAR), TAR Δbulge, or TAR Δloop mutant HIV LTR luciferase constructs into HeLa cells. Cells were coinjected with CMV-GFP, and luciferase activity was calculated per GFP-positive cell. An average of three experiments is shown (± SEM).</p> <p>(B) AcTat transactivation requires CDK9. HeLa cells microinjected with Tat or AcTat (each 30 ng/μl) and the HIV LTR luciferase reporter were treated with increasing amounts of DRB, a known CDK9 inhibitor, for 4 h.</p> <p>(C) AcTat transcriptional activity is inhibited by nicotinamide, but not TSA. HeLa cells injected with HIV LTR luciferase and increasing amounts of AcTat were treated with TSA (400 nM) or nicotinamide (5 mM) for 4 h. The average of two experiments is shown.</p> <p>(D) SIRT1 is necessary for AcTat, but not Tat function. HeLa cells were transfected with siRNAs specific for SIRT1 or GL3 control siRNAs 48 h before microinjection of HIV LTR luciferase and Tat or AcTat (each 30 ng/μl). The average of three experiments is shown (± SEM).</p></div

Physical Interaction between Tat and SIRT1

<div><p>(A) Immunoprecipitation (IP) and WB of FLAG-tagged Tat (Tat-FLAG) and HA-tagged SIRT1 (SIRT1-HA) after transfection of corresponding expression vectors (+) or empty vector controls (−) into HEK 293 cells.</p> <p>(B) The same experiments as in (A) performed with T7-tagged Tat and FLAG-tagged SIRT1, SIRT2, and SIRT6.</p> <p>(C) Coimmunoprecipitation of FLAG-tagged Tat with endogenous SIRT1 in HEK 293 cells transfected with the Tat expression vector or the empty vector control. IPs were performed with or without rabbit α-SIRT1 antibodies.</p> <p>(D) WB of recombinant SIRT1 protein after pulldown with synthetic biotinylated Tat or AcTat. Tat proteins were detected with antibodies specific for acetylated lysine 50 in the Tat ARM (α-AcTat) or SA-HRP.</p> <p>(E) Immunoprecipitation/WB of FLAG-tagged Tat or TatK50R and HA-tagged SIRT1. WT, wild type.</p></div