Resveratrol inhibited Tat-induced HIV-1 LTR transactivation via NAD+-dependent SIRT1 activity

Resveratrol inhibited Tat-induced HIV-1 LTR transactivation via NAD+-dependent SIRT1 activity

Life Sciences 85 (2009) 484–489 Contents lists available at ScienceDirect Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m...

316KB Sizes 1 Downloads 9 Views

Life Sciences 85 (2009) 484–489

Contents lists available at ScienceDirect

Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Resveratrol inhibited Tat-induced HIV-1 LTR transactivation via NAD+-dependent SIRT1 activity Hong-Sheng Zhang ⁎, Yue Zhou, Meng-Ran Wu, Hong-Sen Zhou, Fei Xu Department of Virology and Pharmacology, College of Life Science and Bioengineering, Beijing University of Technology, Pingleyuan 100#, District of Chaoyang, Beijing, 100124, China

a r t i c l e

i n f o

Article history: Received 12 January 2009 Accepted 4 July 2009 Keywords: SIRT1 Tat Resveratrol HIV-1

a b s t r a c t Aims: Tat protein plays a pivotal role in both the human immunodeficiency virus type 1 (HIV-1) replication cycle and the pathogenesis of HIV-1 infection. Sirtuins 1 (SIRT1) is a possible candidate for redox modulation because its activity is regulated by nicotinamide adenine dinucleotide (NAD+) or NAD+/NADH ratio. The aim of the present study was to determine whether the redox status and SIRT1 expression are related to HIV-1 Tat protein-induced transactivation. Main methods: HeLa-CD4-long terminal repeat (LTR)-β-gal (MAGI) cells were transfected with Tat plasmid. Tat-induced HIV-1 LTR transactivation was determined by MAGI cell assay. The NAD+ or NADH levels and SIRT1 activity were measured. In addition, the protein expression of SIRT1 was assayed by western blotting. Key findings: Pretreatment with resveratrol increased intracellular NAD+ levels and SIRT1 protein expression after Tat plasmid transfection in a concentration-dependent manner. Pretreatment with resveratrol attenuated Tat-induced HIV-1 transactivation in MAGI cells. These effects of resveratrol were largely abolished by knockdown of SIRT1 by short interfering RNA (siRNA). Pretreatment with nicotinamide, a SIRT1 inhibitor, potentiated Tat-induced HIV-1 transactivation in MAGI cells, and overexpression of SIRT1 attenuated Tat-induced HIV-1 transcription in MAGI cells. Significance: Inhibition of SIRT1 activity by Tat is considered a critical step of Tat transactivation. Resveratrol and related compounds represent potential candidates for novel anti-HIV therapeutics. © 2009 Elsevier Inc. All rights reserved.

Introduction Human immunodeficiency virus type 1 (HIV-1) transactivator Tat protein plays a pivotal role in both the HIV-1 replication cycle and the pathogenesis of HIV-1 infection. HIV-1 Tat modulates the expression of several cellular genes and triggers the activation of certain signal transduction pathways and transcription factors, suggesting a complex role in HIV-1 infection (Harrich et al. 2006; Stevens et al. 2006; Richter and Palu 2006; Karn 1999). Replication of HIV-1 is controlled by a variety of viral and host proteins. The viral protein Tat acts in concert with host cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR) through a specific interaction with a 59-residue stem-loop RNA known as the transactivation responsive element (TAR) (Karn 1999). Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. The highly dynamic nature of Tat posttranslational modifications is illustrated by the fact that Tat binds deacetylases as well as acetyltransferases (Ott et al. 2004). Deacetylases remove the acetyl moieties from lysines. Lysine 50 in Tat is deacetylated by the class III

⁎ Corresponding author. Tel.: +86 10 67396212 83; fax: +86 10 67392780. E-mail address: [email protected] (H.-S. Zhang). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.07.014

deacetylase sirtuin 1 (SIRT1) (Pagans et al. 2005). Sirtuins, unlike other deacetylases, require nicotinamide adenine dinucleotide (NAD+) as a cofactor, inducing the hydrolysis of the cofactor to acetyl-ADP ribose and nicotinamide. It is demonstrated that Tat transactivating activity regulated by SIRT1 connects HIV transcription with the metabolic state of the cell (Kwon et al. 2008; Pagans et al. 2005). NAD+ and its reduced form (NADH) serve as cofactors in many metabolic and stress reactions involving oxidation and reduction (Yang and Sauve 2006). Changes in the cellular NAD+/NADH ratio may, therefore, regulate Tat activity through deacetylation by SIRT1. Because of the important role of nonacetylated lysine 50 in this initial step, Tat acetylation and deacetylation are important for Tat transactivation. The possibility of exploiting Tat posttranslational modifications for therapeutic purposes is especially exciting. Since it becomes apparent that a highly regulated balance between acetylated and nonacetylated Tat regulates HIV transcription, “locking” Tat in one of these modified states could render Tat inactive during HIV infection (Robinson 2007; van Lint et al. 1996; Weinberger and Shenk 2007; Yang and Sauve 2006). Resveratrol, the SIRT1 activator, is a natural product induced in plants after a variety of stresses and shows potential cancer chemoprotective properties (Athar et al. 2007). Activation of SIRT1 may be a useful chemopreventative agent in age-related cancers and HIV-1 infection (Fulda and Debatin 2006). In this study, HeLa-CD4-βgal cells, HeLa cells that express CD4 and are stably transfected with

H.-S. Zhang et al. / Life Sciences 85 (2009) 484–489

HIV-1 LTR-β-galactosidase reporter DNA were selected. It was demonstrated that HIV-1 Tat depleted intracellular NAD+ levels, caused a decline in NAD+/NADH ratio and inhibited SIRT1 protein expression. SIRT1 activation by resveratrol attenuated Tat-induced HIV-1 LTR transactivation through modulating NAD+ levels or NAD+/ NADH ratio. This indicates the SIRT1 modulated by redox status as a novel target of HIV Tat and provides evidence of a primary molecular response in a potential target cell of this important regulator of transcription activation.

485

Materials and methods

for 20 min at room temperature and the reactions were stopped by adding 50 μl of a developer solution supplemented with 1 μM TSA and 10 mM NAM (final concentrations). The plate was read after 10 min incubation at room temperature using a multi-well fluorometer (excitation 360 nm, emission 460 nm). A standard curve was generated using a deacetylated substrate Fluor-de-Lys (ranging from 1–40 μM). SIRT1 activity was determined as NAM-inhibitable, TSA-independent ability of cell extracts to deacetylate the specific fluorometric substrate. Experimental values are presented as pmol deacetylated product/μg protein/min. The negative controls (10 mM NAM) were subtracted from each treatment to give the final values.

Materials

Western blotting analysis

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and trypsin/EDTA were obtained from Hyclone (Logan, UT). Nicotinamide, bovine serum albumin (BSA), reduced NAD, resveratrol, and MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies against SIRT1, β-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence (ECL) kit was obtained from Pierce Biotechnology (Rockford, IL). Trizol, LipofectAMINE 2000 was from Gibco-BRL/Life Technologies. (Grand Island, NY). The SIRT1 and SIRT1 H363Y (Langley et al. 2002) expression plasmids were a gift from Dr. Kouzarides (Cancer Research UK Gurdon Institute, Cambridge). Tat plasmid (Sun et al. 2006) was provided by Prof. Ping-Kun Zhou (Beijing Institute of Radiation Medicine, Beijing). All other chemicals were of the highest commercial grade available.

MAGI cells were washed with phosphate-buffered saline (PBS), harvested and lysed with radioimmune precipitation assay (RIPA) buffer. Protein concentrations were determined by Bio-Rad Protein Assay (Bio-Rad) according to the manufacturer's instructions. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and blotted to a nitrocellulose membrane. SIRT1 and β-actin antibodies were used at a dilution of 1:500 followed by anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (Amersham) at a dilution of 1:5000. Detection of HRP-conjugated antibodies was performed with ECL Plus Blotting Reagent using a Quality One documentation system (Bio-Rad) (Zhang and Wang 2006a).

Cell culture and transfection The HeLa-CD4-LTR-β-galactosidase indicator cell line is MAGI (multinuclear activation of galactosidase indicator) cell (Kimpton and Emerman 1992). MAGI cells were maintained in DMEM supplemented with 10% FCS, 200 μg/ml G418, 100 μg/ml hygromycin B, at 37 °C in 5% CO2 and 95% air in humidified atmosphere. For all transfections, MAGI cells were used at a confluency of 50–60%. Typically, cells were transfected with 50 nM of Tat plasmid per well in a 96-well dish by using LipofectAMINE 2000 transfection reagent (Invitrogen). Cell extracts were prepared 48 h after transfection. Cell viability was examined using MTT-based assays according to the manufacturer's instructions. NAD+ and NADH assay The NAD+ or NADH levels were measured using a BioChain NAD+/ NADH assay kit according to the manufacturer's instructions (BioChain, Hayward, CA). NAD+ and NADH levels were calculated according to the equivalent protein quantity (μg protein) per data point. NAD+ or NADH standards were used to quantify NAD+ or NADH samples and were normalized to total protein as measured by Bio-Rad Protein Assay (Bio-Rad) according to the manufacturer's instructions. SIRT1 activity assay SIRT1 activity was determined using a BIOMOL assay (Plymouth Meeting, PA) used according to the manufacturer's instructions with slight modifications. Briefly, 1 μM of Trichostatin A (TSA) was added to the cell cultures 1 h prior to harvesting to block the class I, II and IV histone deacetylases (HDACs). Whole cell extracts were obtained from cells cultured with the addition of 1 μM TSA. The extracts were added (15 μl) to an opaque multi-well plate with 1 μM TSA and 1 mM of acetylated Fluor-de-Lys substrate. As a negative control, 10 mM of NAM was also added to some extracts. The extracts were then swirled

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis Total RNA was isolated from MAGI cells using TRIzol reagent according to the manufacturer's instructions. The purity and quantity of the RNA were determined by A260/280 ratios and A260, respectively. For RT-PCR, first-strand cDNA was synthesized using random primers. Briefly, reverse transcriptional reaction (RT) was carried out in the reaction system of 20 μl reaction volume containing total RNA 2.0 μl, random primer 0.5 μl, 5× RT buffer 5.0 μl, 10 mmol/L dNTPs 2.0 μl, Rnase inhibitor 0.5 μl (20 U), and DEPC·H2O 14.0 μl. The temperature cycling conditions were as follows: incubation at 30 °C for 10 min, at 42 °C for 45 min and then at 90 °C for 5 min. PCR was carried out sequentially. The volume of PCR reaction system is 50.0 μl, containing cDNA 1.0 μl, 10 pmol/μl of sense primer 2.0 μl, 10 pmol/μl of antisense primer 2.0 μl, rTaq enzyme 0.5 μl (2.5 U), 10× Taq enzyme buffer (added with Mg2+) 5.0 μl, 10 mmol/L dNTPs 1.0 μl, and ddH2O 38.5 μl. The temperature cycling conditions were as follows: predenaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s and a final extension at 72 °C for 10 min. As an internal control, a housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also amplified. In this experiment, 2 μg of total RNA was reverse transcribed in a 50 μl reaction volume. A 5 μl aliquot of each PCR amplified product was resolved by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and photographed under ultraviolet light. For quantification, photographs showing PCR products were scanned by HVE50 and analyzed by using Quality One Image software. For the respective samples, the PCR product values were normalized to the GAPDH PCR product values (Zhang and Wang 2006b). Gene silencing experiments The siRNA sequence targeting SIRT1 was designed and synthesized by Santa Cruz Biotechnology. As a control for unspecific effects, a nonsilencing (NS) siRNA sequence was used (negative control). Cells were transfected with 50 nM siRNA against SIRT1 or control siRNA using LipofectAMINE 2000. After 24 h incubation, fresh medium was added and the cells were cultured for another 48 h. Analysis of the

486

H.-S. Zhang et al. / Life Sciences 85 (2009) 484–489

silencing efficiency was performed by measuring the mRNA and protein levels of SIRT1 by RT-PCR or immunoblotting, respectively (Zhang and Wang 2007).

enzymes potentiated Tat-induced NAD+ depletion in MAGI cells. There was no difference in cell viability after the above treatment (data not shown).

MAGI cell assay

Effect of resveratrol on Tat-induced inhibition of SIRT1 protein expression and activity in MAGI cells

MAGI cell assays were performed according to the recommended protocol. Cells were exposed to HIV-IIIB virus or transfected with Tat plasmid for 48 h. Two days after treatment, the cells were fixed for 5 min with 0.2% glutaraldehyde, 1% formaldehyde in phosphatebuffered saline. Cells were washed twice with phosphate-buffered saline and then staining solution (4 mM ferrocyanide, 4 mM ferricyanide, 2 mM MgCl2, and 0.4 mg/ml 5-bromo-4-chloro-3-indolyl-Dgalactopyranoside (X-gal)) was added and incubated at 37 °C for 1 h. Blue cells were counted under microscopy. Statistical analysis Each data bar represents the mean values ± S.D. (standard deviations) of at least three independent experiments in all cases. Results were analyzed using SPSS for Windows. Differences between groups were analyzed by one-way analysis of variance (ANOVA). If the F values were significant, LSD post-hoc test was used to compare multiple groups. A P value of ≤ 0.05 was considered statistically significant in all cases. Results Effect of resveratrol on Tat-induced NAD+ depletion in MAGI cells Many of the biological processes regulated by SIRT1 result from the adaptation of complex gene-expression programs to the energetic state of the cell, sensed through NAD+ levels or an alteration in NAD+/NADH ratio. Therefore, intracellular NAD+ and NADH levels were measured using an enzymatic recycling assay as described in the Materials and methods section. As shown in Fig. 1, transfection with Tat plasmid causes a decline of intracellular NAD+ level and NAD+/NADH ratio in MAGI cells, whereas pretreatment with resveratrol suppressed Tatmediated NAD+ depletion and Tat-mediated a decline in NAD+/NADH ratio in a concentration-dependent manner. There was no significant difference in NADH concentration among all groups. However, pretreatment with nicotinamide, a natural byproduct of the sirtuin deacetylase reaction which functioned as a feedback inhibitor of these

Fig. 1. Effects of resveratrol and nicotinamide on Tat-mediated depletion of intracellular NAD+ levels in MAGI cells. MAGI cells were pretreated with 10, 25, and 50 μM resveratrol (Res) or 1, 2, and 5 mM nicotinamide (NAM) for 1 h, then transfected with Tat plasmid for 48 h. Intracellular NAD+ and NADH levels were assayed as described in the Materials and methods section. The empty expression vector was used in mock transfected controls. Intracellular NAD+ and NADH levels were 15 pg/μg protein and 8 pg/μg protein, respectively. The data shown are the means of six independent experiments. The error bars represent the standard error of the mean. Values represent means± standard deviation (n = 6). **P b 0.01 (compared to control). #Pb 0.05 (compared to Tat), ##P b 0.01 (compared to Tat).

As shown in Fig. 2, transfection with Tat plasmid inhibited SIRT1 expression and activity in MAGI cells. Pretreatment with SIRT1 activator resveratrol attenuated Tat-induced inhibition of SIRT1 protein expression and activity in a concentration-dependent manner in MAGI cells as shown in Fig. 2. Effect of resveratrol and nicotinamide on Tat-induced HIV-1 LTR transactivation in MAGI cells HeLa cells, that express CD4 and are stably transfected with HIV-1 long terminal repeat-β-galactosidase reporter DNA, were transfected with Tat for 24 h. The number of β-galactosidase-positive blue cells that were determined using light microscopy was scored to determine the effectivity of Tat-induced HIV-1 LTR transactivation. As shown in Fig. 4A, pretreatment with SIRT1 activator resveratrol significantly reduced the β-Gal positive cell level to about 73.1%, 63.3%, and 52.7% of the Tat alone level in a concentration-dependent manner (10, 25, and 50 μM); whereas, the β-Gal positive cell levels in nicotinamidepretreated cells significantly increased to about 132.4%, 146.9%, and 164.7% of the Tat treatment alone in a concentration-dependent manner (1, 2, and 5 mM). In the absence of Tat, no blue cells were detected after treatment with resveratrol or nicotinamide alone (data not shown). Similar results were found when cells were treated with HIV-IIIB virus infection instead of treated with transfection of Tat plasmid (Fig. 5A). Effect of overexpression of SIRT1 and siRNA SIRT1 on Tat-induced HIV-1 LTR transactivation in MAGI cells To confirm the participation of SIRT1 in the Tat-induced HIV-1 LTR transactivation, we performed overexpression with SIRT1 plasmid transfection and knockdown of SIRT1 by RNA silencing experiments (siSIRT1) in MAGI cells, respectively. The effect of SIRT1 knockdown

Fig. 2. Effects of resveratrol on Tat-mediated inhibition of SIRT1 protein expression and SIRT1 activity in MAGI cells. MAGI cells were pretreated with 10, 25, and 50 μM resveratrol for 1 h, then transfected with Tat plasmid for 48 h. (A) The cells were prepared and the SIRT1 protein levels detected by western blotting analysis. β-actin was used as a loading control. The blots shown are representative of three independent experiments with similar results. 1, control group; 2, Tat group; 3, Tat + 10 μM resveratrol group; 4, Tat + 25 μM resveratrol group; 5, Tat + 50 μM resveratrol group. (B) Cell extracts were tested for SIRT1 activity. The data shown are the means of six independent experiments. **P b 0.01 (compared to control). #P b 0.05 (compared to Tat), ##P b 0.01 (compared to Tat).

H.-S. Zhang et al. / Life Sciences 85 (2009) 484–489

487

or overexpression of SIRT1 was confirmed by RT-PCR and western blot. As shown in Fig. 3, SIRT1 siRNA, but not control siRNA, effectively inhibited the mRNA or protein expression of SIRT1; overexpression with SIRT1 induced an increase in the mRNA or protein expression of SIRT1. As shown in Fig. 4B, overexpression of SIRT1 significantly reduced the β-Gal positive cell level to about 51.6% of the Tat alone level, whereas the β-Gal positive cell levels in siSIRT1-treated cells significantly increased to about 171.0% of the Tat treatment alone. Pretreatment with 50 μM resveratrol suppressed siRNA SIRT1induced increase in the β-Gal positive cell level in the presence of Tat, while it had no effect on control siRNA treatment. Treatment with SIRT1 H363Y plasmid or control siRNA had no effect on Tat-induced HIV-1 LTR transactivation. In the absence of Tat, no blue cells were detected after treatment with SIRT1 plasmid, siRNA SIRT1 or control siRNA alone (data not shown). Similar results were found when cells were treated with HIV-IIIB virus infection instead of treated with transfection of Tat plasmid (Fig. 5B). These results indicated that SIRT1 was involved in Tat-induced HIV-1 LTR transactivation. Discussion The molecular mechanism of the regulation of HIV-1 transcription provides a molecular basis for developing novel antiviral agents. There is a great need for developing therapeutic agents that can repress transcription of the HIV-1 to overcome the problem viral resistance, because such agents would prevent production of the genetic material for viral replication and the template for reverse transcriptase. Because Tat (transactivator protein) plays a central role in the transcription, inhibition of Tat-mediated HIV-1 transactivation can repress transcription and replication of HIV-1 (Baba 2006). In this study, we demonstrated that resveratrol inhibited HIV-1 Tat-mediated LTR transactivation through modulating NAD+-dependent SIRT1 activity. SIRT1 is a mammalian homologue of the yeast transcriptional repressor Sir2, an important factor governing longevity in yeast (Blander and Guarente 2004). Like Sir2, SIRT1 requires NAD+ as a cofactor, which links its activity to the metabolic state of the cell. SIRT1 is a possible candidate for redox modulation because its activity is regulated by NAD+ and it is, therefore, sensitive to the redox state and cellular metabolism (Prozorovski et al. 2008). Indeed, SIRT1 has been identified recently as a redox sensor in mouse embryonic fibroblasts and undifferentiated muscle cells, in which it controls proliferation, cell cycle arrest and differentiation (Fulco et al. 2003). NAD+ serves as a coenzyme in cellular redox reactions and is, thus, an essential component of metabolic pathways in all living cells. Numerous recent studies have demonstrated that NAD+ plays important roles in a variety of biological processes in mammals, such as cell survival and apoptosis, differentiation, and metabolism of carbohydrates and fat through the activity of SIRT1 (Li et al. 2006; Michan and Sinclair 2007; Yang and Sauve 2006; Yamamoto et al. 2007). In our study, we demonstrated that HIV-1 Tat caused intracellular NAD+ depletion and a decline in NAD+/NADH ratio, and it could be reversed by resveratrol. Our results indicated that intracellular NAD+ level played an important role in regulating Tat-induced HIV-1 transactivation.

Fig. 4. Effect of overexpression of SIRT1, siRNA SIRT1, resveratrol and nicotinamide on Tatinduced LTR transactivation in MAGI cells. (A) MAGI cells were pretreated with 10, 25, and 50 μM resveratrol (Res) or 1, 2, and 5 mM nicotinamide (NAM) for 1 h, then transfected with Tat plasmid for 48 h. (B) MAGI cells were transfected with Tat plasmid or cotansfected with 50 nM SIRT1 plasmid, 50 nM SIRT1H363Y plasmid, 50 nM control siRNA or 50 nM siRNA SIRT1 (siSIRT1) or 50 μM resveratrol for 48 h. The cells were fixed and stained with X-gal. Photographs of blue cells were taken 48 h post-infection using Olympus microscope. The data shown are means± S.D. of six independent experiments. **P b 0.01 (compared to Tat). ##P b 0.01 (compared to Tat+ siSIRT1).

Changes in the cellular NAD+ level would, thus, have a significant impact on mammal physiology, including humans, and NAD+ biosynthesis reactions should be tightly regulated; however, the mechanisms regulating the cellular content of NAD+ remain to be determined (Imai 2009). Nicotinamide phosphoribosyltransferase (Nampt), also known as pre-B cell colony-enhancing factor (PBEF), was originally identified as a cytokine that facilitates the clonal expansion and differentiation of pre-B cells (Garten et al. 2009). Increased amounts of Nampt in mammalian cells cause upregulation of NAD+. Nampt is a rate-limiting enzyme in the conversion of nicotinamide into NAD+, which is crucial for SIRT1 activation and regulation of transcription; Nampt could co-regulate the sirtuins and other NAD+-dependent processes by (i) lowering the NAM concentration and (ii) boosting NAD+ levels in the cytoplasm, nucleus and perhaps even in

Fig. 3. Effect of siRNA SIRT1 and overexpression of SIRT1 on SIRT1 mRNA and protein expression in MAGI cells. MAGI cells were transfected with 50 nM siRNA SIRT1 or control siRNA for 48 h. The cells were prepared and the SIRT1 mRNA (A) or protein (B) levels detected by RT-PCR or western blot analysis. GAPDH and β-actin were used as loading controls. The blots shown are representative of three independent experiments with similar results. 1 and 6, control group; 2 and 7, siRNA SIRT1 group; 3 and 8, control siRNA group; 4 and 9, SIRT1 group; 5 and 10 group, SIRT1H363Y group.

488

H.-S. Zhang et al. / Life Sciences 85 (2009) 484–489

HIV transcription with the metabolic state of the cell. NAD+ and NADH serve as cofactors in many metabolic and stress reactions involving oxidation and reduction. Changes in the cellular NAD+/NADH ratio may, therefore, regulate Tat activity through deacetylation by SIRT1. Tat inhibits SIRT1 activity and potentiates NF-κB transcriptional activity unveils a molecular mechanism by which hyperactivation of immune cells is promoted during HIV infection. It was suggested that the effects of resveratrol are mediated by the induction of SIRT1 and the consequent decrease in Tat-induced HIV-1 LTR transactivation. SIRT1 is inducibly transcribed in response to nutrient status, caloric restriction or fasting, suggesting a broad role in mammalian physiology as a mediator of adaptation to nutrient deprivation or the metabolic state of the cell. These functions imply that pharmacological modulation of SIRT1 activity is likely to have wide-reaching and not necessarily specific effects on human physiology. In HIV-1 infection conditions, both activation and inhibition of SIRT1 are worth considering as pharmacological approaches. Resveratrol has been shown to significantly increase SIRT1 activity through an allosteric interaction, resulting in the increase of SIRT1 affinity for both nicotinamide adenine dinucleotide and the acetylated substrate (Howitz et al. 2003). Resveratrol is an activator of sirtuins (Lagouge et al. 2006; Milne et al. 2007) and therefore it is possible that sirtuins are involved in the inhibition of Tat-induced HIV-1 LTR transactivation seen with resveratrol. Conclusions In summary, we have demonstrated SIRT1 plays an important role in Tat-induced HIV-1 LTR transactivation through modulating the redox status of the cell. Our study shows that resveratrol, a SIRT1 activator attenuates the transactive effects of Tat in MAGI cells. This observation suggests a novel therapeutic approach in anti-HIV-1 therapy. Fig. 5. Effect of overexpression of SIRT1, siRNA SIRT1, resveratrol and nicotinamide on HIV-IIIB-induced LTR transactivation in MAGI cells. (A) MAGI cells were pretreated with 10, 25, and 50 μM resveratrol (Res) or 1, 2, and 5 mM nicotinamide (NAM) for 1 h, then infected with HIV-IIIB virus for 48 h. (B) MAGI cells were transfected with 50 nM SIRT1 plasmid, 50 nM SIRT1H363Y plasmid, 50 nM control siRNA or 50 nM siRNA SIRT1 (siSIRT1) for 48 h, then infected with HIV-IIIB virus for 48 h. The cells were fixed and stained with X-gal. Photographs of blue cells were taken 48 h post-infection using Olympus microscope. The data shown are means ± S.D. of six independent experiments. **P b 0.01 (compared to HIV-IIIB). ##P b 0.01 (compared to HIV-IIIB + siSIRT1).

mitochondria (Skokowa et al. 2009; van Gool et al. 2009). Nampt which converts nicotinamide (NAM) into NAD+ could be the target of resveratrol rather than SIRT1. In our study, pretreatment with NAM potentiated Tat-induced HIV-1 transactivation in MAGI cells. It seems that the increasing ratio of NAD+/NAM is more important than NAD+/NADH in the activation of SIRT1. In addition to its enzymatic activity on histone substrates in vitro, SIRT1 predominantly targets nonhistone proteins for deacetylation, such as p53, nuclear factor κB (NF-κB), forkhead transcription factor (FOXO). HIV-1 Tat protein is a substrate for the deacetylase activity of SIRT1 (Pagans et al. 2005). In vitro, HIV-1 Tat is acetylated by the acetyltransferase activities of p300, the human homologue of the yeast general control of amino acid synthesis 5 (GCN5) protein and its close relative, PCAF (Stevens et al. 2006). The acetylation site of p300 has been mapped to lysine 50, a highly conserved lysine within the arginine-rich motif of Tat (Pagans et al. 2005). This Tat modification has also been detected in chromatin immunoprecipitation (ChIP) assays, demonstrating that Tat undergoes acetylation at lysine 50 during transactivation of the HIV promoter in vivo. Lysine 50 in Tat is deacetylated by the class III deacetylase SIRT1. Sirtuins, unlike other deacetylases, require NAD+ as a cofactor, inducing the hydrolysis of the cofactor to acetyl-ADP ribose and nicotinamide. The demonstration that Tat transactivating activity is controlled by SIRT1 connects

Acknowledgments This study was supported in part by the National Natural Sciences Foundation of China (No. 30800580, to Zhang H-S); Beijing Nova Program (No. 2007B014, to Zhang H-S), Beijing Natural Science Foundation (No. 5093025, to Zhang H-S), Beijing Talents Foundation (No. 20071D0501500213, to Zhang H-S), BJUT Science Foundation for Youths (No. 97015999200702, to Zhang H-S), BJUT Scientific Research Starting Foundation for Doctor (No. 52015999200703, to Zhang H-S). We would like to thank Dr. Kouzarides and Ping-Kun Zhou for their kind gifts of the plasmids. We would also like to thank Mrs. York for her help for language editing. References Athar M, Back JH, Tang X, Kim KH, Kopelovich L, Bickers DR, Kim AL. Resveratrol: A review of preclinical studies for human cancer prevention. Toxicology and Applied Pharmacology 224, 274–283, 2007. Baba M. Recent status of HIV-1 gene expression inhibitors. Antiviral Research 71, 301–306, 2006. Blander G, Guarente L. The Sir2 family of protein deacetylases. Annual Review of Biochemistry 73, 417–435, 2004. Fulco M, Schiltz RL, Iezzi S, King MT, Zhao P, Kashiwaya Y, Hoffman E, Veech RL, Sartorelli V. Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state. Molecular Cell 12, 51–62, 2003. Fulda S, Debatin KM. Resveratrol modulation of signal transduction in apoptosis and cell survival: A minireview. Cancer Detection and Prevention 30, 217–223, 2006. Garten A, Petzold S, Körner A, Imai S, Kiess W. Nampt: Linking NAD biology, metabolism and cancer. Trends Endocrinology & Metabolism 20, 130–138, 2009. Harrich D, McMillan N, Munoz L, Apolloni A, Meredith L. Will diverse Tat interactions lead to novel antiretroviral drug targets? Current Drug Targets 7, 1595–1606, 2006. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425, 191–196, 2003. Imai S. The NAD World: A new systemic regulatory network for metabolism and agingSirt1, systemic NAD biosynthesis, and their importance. Cell Biochemistry and Biophysics 53, 65–74, 2009.

H.-S. Zhang et al. / Life Sciences 85 (2009) 484–489 Karn J. Tackling Tat. Journal of Molecular Biology 293, 235–254, 1999. Kimpton J, Emerman M. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. Journal of Virology 66, 2232–2239, 1992. Kwon HS, Brent MM, Getachew R, Jayakumar P, Chen LF, Schnolzer M, McBurney MW, Marmorstein R, Greene WC, Ott M. Human immunodeficiency virus type 1 Tat protein inhibits the SIRT1 deacetylase and induces T cell hyperactivation. Cell Host & Microbe 3, 158–167, 2008. Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F, Messadeq N, Milne J, Lambert P, Elliott P, Geny B, Laakso M, Puigserver P, Auwerx J. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell 127, 1109–1122, 2006. Langley E, Pearson M, Faretta M, Bauer UM, Frye RA, Minucci S, Pelicci PG, Kouzarides T. Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. EMBO Journal 21, 2383–2396, 2002. Li F, Chong ZZ, Maiese K. Cell life versus cell longevity: The mysteries surrounding the NAD+ precursor nicotinamide. Current Medicinal Chemistry 13, 883–895, 2006. Michan S, Sinclair D. Sirtuins in mammals: Insights into their biological function. Biochemical Journal 404, 1–13, 2007. Milne JC, Lambert PD, Schenk S, Carney DP, Smith JJ, Gagne DJ, Jin L, Boss O, Perni RB, Vu CB, Bemis JE, Xie R, Disch JS, Ng PY, Nunes JJ, Lynch AV, Yang H, Galonek H, Israelian K, Choy W, Iffland A, Lavu S, Medvedik O, Sinclair DA, Olefsky JM, Jirousek MR, Elliott PJ, Westphal CH. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 450, 712–716, 2007. Ott M, Dorr A, Hetzer-Egger C, Kaehlcke K, Schnolzer M, Henklein P, Cole P, Zhou MM, Verdin E. Tat acetylation: A regulatory switch between early and late phases in HIV transcription elongation. Novartis Foundation Symposium 259, 182–193, 2004. Pagans S, Pedal A, North BJ, Kaehlcke K, Marshall BL, Dorr A, Hetzer-Egger C, Henklein P, Frye R, McBurney MW, Hruby H, Jung M, Verdin E, Ott M. SIRT1 regulates HIV transcription via Tat deacetylation. PLoS Biology 3, e41, 2005. Prozorovski T, Schulze-Topphoff U, Glumm R, Baumgart J, Schrotter F, Ninnemann O, Siegert E, Bendix I, Brüstle O, Nitsch R, Zipp F, Aktas O. Sirt1 contributes critically to the redox-dependent fate of neural progenitors. Nature Cell Biology 10, 385–394, 2008. Robinson R. A simple feedback resistor switch keeps latent HIV from awakening. PLoS Biology 5, e25, 2007.

489

Richter SN, Palu G. Inhibitors of HIV-1 Tat-mediated transactivation. Current Medicinal Chemistry 13, 1305–1315, 2006. Skokowa J, Lan D, Thakur BK, Wang F, Gupta K, Cario G, Brechlin AM, Schambach A, Hinrichsen L, Meyer G, Gaestel M, Stanulla M, Tong Q, Welte K. NAMPT is essential for the G-CSF-induced myeloid differentiation via a NAD(+)-sirtuin-1-dependent pathway. Nature Medicine 15, 151–158, 2009. Stevens M, De Clercq E, Balzarini J. The regulation of HIV-1 transcription: Molecular targets for chemotherapeutic intervention. Medical Research Reviews 26, 595–625, 2006. Sun Y, Huang YC, Xu QZ, Wang HP, Bai B, Sui JL, Zhou PK. HIV-1 Tat depresses DNA-PK (CS) expression and DNA repair, and sensitizes cells to ionizing radiation. International Journal of Radiation Oncology Biology Physics 65, 842–850, 2006. van Gool F, Gallí M, Gueydan C, Kruys V, Prevot PP, Bedalov A, Mostoslavsky R, Alt FW, De Smedt T, Leo O. Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner. Nature Medicine 15, 206–210, 2009. van Lint C, Emiliani S, Ott M, Verdin E. Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation. EMBO Journal 15, 1112–1120, 1996. Weinberger LS, Shenk T. An HIV feedback resistor: Auto-regulatory circuit deactivator and noise buffer. PLoS Biology 5, e9, 2007. Yang T, Sauve A. NAD metabolism and sirtuins: Metabolic regulation of protein deacetylation in stress and toxicity. Aaps Journal 8, E632–E643, 2006. Yamamoto H, Schoonjans K, Auwerx J. Sirtuin functions in health and disease. Molecular Endocrinology 21, 1745–1755, 2007. Zhang HS, Wang SQ. Salvianolic acid B inhibits tumor necrosis factor-α (TNF-α)induced MMP-2 upregulation in human aortic smooth muscel cells via suppression of NAD(P)H oxidase-derived reactive oxygen species. Journal of Molecular and Cellular Cardiology 41, 138–148, 2006a. Zhang HS, Wang SQ. Notoginsenoside R1 inhibits TNF-α-induced fibronectin production in smooth muscle cells via ROS/ERK pathway. Free Radicals Biology and Medicine 40, 1664–1674, 2006b. Zhang HS, Wang SQ. Nrf2 is involved in the effect of tanshinone IIA on intracellular redox status in human aortic smooth muscle cells. Biochemical Pharmacology 73, 1358–1366, 2007.