Coaxing HIV-1 from resting CD4 T cells: histone deacetylase inhibition allows latent viral expression
Ylisastigui, Loydaa; Archin, Nancie Ma; Lehrman, Gingera; Bosch, Ronald Jc; Margolis, David Ma,b
From the aDepartment of Medicine, University of Texas Southwestern Medical Center and bNorth Texas Veterans Health Care Systems, Dallas, Texas and cHarvard School of Public Health, Boston, Massachusetts, USA.
Correspondence to Dr D. M. Margolis, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Y9-206a, Dallas, Texas 75390-9113, USA.
Received: 17 November 2003; revised: 27 January 2004; accepted: 16 February 2004.
Background: Histone deacetylase (HDAC), a host mediator of gene repression, inhibits HIV gene expression and virus production and may contribute to quiescence of HIV within resting CD4 T cells.
Objectives: To test the ability of valproic acid (VPA), an inhibitor of HDAC in clinical use, to induce expression of HIV from resting CD4 T cells.
Methods: Chromatin immunoprecipitation measured the capability of VPA to deacetylate the HIV promoter, a remodeling of chromatin linked to gene expression. The effect of VPA on resting CD4 T cell phenotype was measured by flow cytometric analysis, and its effect on de novo HIV infection of peripheral blood mononuclear cells was measured ex vivo. Outgrowth of HIV from resting CD4 T cells of aviremic, HIV-infected donors treated with highly active antiretroviral therapy was compared in limiting-dilution cultures after mitogen stimulation or exposure to VPA.
Results: VPA induced acetylation at the integrated HIV proviral promoter, but CD4 cells exposed to VPA did not become activated or more permissive for de novo HIV infection. VPA induced outgrowth of HIV from the resting CD4 cells of aviremic patients at concentrations achievable in vivo as frequently as did mitogen stimulation.
Conclusions: With advances in antiretroviral therapy, HIV infection might be cleared by intensive time-limited treatment coupled with practical strategies that disrupt latency without enhancing new infection. HDAC inhibitors are capable of inducing expression of quiescent provirus, without fully activating cells or enhancing de novo infection, and may be useful in future clinical protocols that seek to eradicate HIV infection.
Suppressive antiretroviral therapy is often clinically successful, but eradication of HIV infection is an ideal therapeutic goal. One of the major obstacles to eradication of HIV infection is the persistent, quiescent infection of resting CD4 memory T cells . Histone deacetylase (HDAC), a host mediator of gene inhibition, can inhibit HIV gene expression and virus production, and contributes to quiescence within resting cells harvested from HIV-positive patients [2–5].
Mechanisms that allow HIV to establish latency are unknown, but recent evidence suggests that modulation of histone architecture within the viral promoter participates in this process. The integrated HIV long terminal repeat (LTR) promoter requires remodeling to allow expression, and histone acetylation to respond to NF-kB activation. Its activation is augmented by HDAC inhibitors [6–12]. These observations imply a role for HDAC in the establishment or maintenance of quiescence. Local chromatin effects have long been thought to contribute to the durable suppression of HIV proviral expression, and latently infected cells recovered from a T lymphocyte cell line infected in vitro were recently found to contain HIV integrated in or close to alphoid repeat elements in heterochromatin [13,14].
HDAC1 is specifically recruited to the LTR at a domain adjacent to the transcriptional initiation site [2,3,15]. HDAC1 recruitment can deacetylate the adjacent nucleosome 1 (nuc 1) of the LTR and inhibits Tat activation, LTR expression, and viral production . Recently, we have shown that novel small molecules that specifically block HDAC recruitment to the LTR allow outgrowth of latent HIV from resting CD4 T cells obtained from HIV-infected patients .
While no available experimental system recapitulates all aspects of quiescent HIV infection within primary CD4 T cells, studies of quiescent HIV infection have been performed ex vivo in resting CD4 lymphocytes isolated from HIV-infected volunteers [16–21]. HIV is expressed from these cells if activated by mitogens or T cell signaling cytokines.
We hypothesized that inhibition of HDAC activity would allow outgrowth of HIV from latently infected resting CD4 T cells. Valproic acid (VPA) has recently been demonstrated to be a direct inhibitor of HDAC, and this capability has been linked to the clinical effects of VPA . VPA is in wide use and often given to HIV-infected patients to treat concomitant conditions. In early studies, HIV gene expression and virus production were increased in vitro in the presence of VPA [23,24]. Literature prior to the era of highly active antiretroviral therapy (HAART) advised against the use of VPA as it might increase plasma HIV RNA, although a small study recently observed no effect in the presence of HAART [25,26].
The present study examines the ability of the HDAC inhibitor VPA to induce acetylation at the integrated HIV LTR, the effect of VPA on de novo HIV infection, and the ability of VPA to induce expression of HIV from purified resting CD4 T cells obtained from HIV-positive donors.
J89 cells (2 × 106 cells)  were washed with phosphate-buffered saline (PBS) and incubated overnight at 37°C under 5% carbon dioxide in media containing 0.5% fetal bovine serum (FBS; Invitrogen, Carlsbad, California, USA). Cells were then washed and incubated for 2 h in media without serum and containing 400 nmol/l trichostatin A (Sigma, St Louis, Missouri, USA) or 1–5 mmol/l VPA (Sigma), or they were were untreated. FBS was then added to a final concentration of 20% and cells were incubated only for an additional 2 h to avoid the induction of secondary gene effects. Cells were washed with PBS, cross-linked with 1% formaldehyde, washed again, snap-frozen in an ethanol/dry-ice bath and stored at −80°C. Chromatin immunoprecipitation assays were carried out as described  with modifications as follows. Briefly, cells were thawed on ice and incubated with 200 μl lysis buffer (Upstate, Waltham, Massachusetts, USA) containing 40 μl protease inhibitor cocktail (Sigma) for 10 min at 4°C; 300 μl dilution buffer (Upstate) was added to the cell lysates, which were then sonicated to fragment chromatin to 500 to 1000 base pairs. After centrifugation of sonicated cell lysates, 60 μg soluble chromatin was incubated overnight with 5 μl anti-acetylhistone H4 (Upstate) or rabbit pre-immune immunoglobulin G (Sigma). Immunoprecipitates were incubated with 50 μl salmon sperm DNA/protein A agarose beads (Upstate) for 1 h at 4°C. Agarose beads were recovered by centrifugation and washed using buffers from the chromatin immunoprecipitation assay kit (Upstate) following the manufacturers’ instructions. Immunoprecipitated DNA was eluted with 500 ml elution buffer (1% sodium dodceyl sulfate plus 0.1 mol/l sodium bicarbonate). DNA was reverse cross-linked by incubation in a solution containing 19 mg proteinase K [polymerase chain reaction (PCR) grade; Boehringer Mannheim, Germany] at 65°C for 90 min. DNA was extracted in phenol/chloroform/isoamyl alcohol, precipitated in ethanol, washed and resuspended in 50 ml water.
Quantitative duplex PCR assay was performed to analyze the amount of DNA precipitated by specified antibodies in proportion to input DNA. The sequence of primers for the HIV-1 LTR promoter was LTR-109F (5′-TACAAGGGACTTTCCGCTGG-3′) and LTR+82R (5′-AGCTTTATTGAGGCTTAAGC-3′). PCR was carried over 32–34 cycles using 5 μl precipitated DNA in a 25 μl PCR reaction. PCR products were resolved by 8% polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. PCR products were quantified by AlphaImager 2000 (Alpha Innotech Corp., San Leandro, California, USA).
Resting cells from HIV-infected patients were phenotyped immediately after isolation. Peripheral blood mononuclear cells (PBMC) were incubated with 20 U/ml interleukin-2 (IL-2; Chiron, Emeryville, California, USA), 2 μg/ml phytohemaglutinin (PHA; Boehringer Mannheim, Indianapolis, Indiana, USA) plus IL-2, 0.2 mmol/l VPA plus IL-2, or 1.0 mmol/l VPA plus IL-2 for 72 h in conditions identical to those used for outgrowth assays and then phenotyped. PBMC or resting CD4 T cells were washed and labeled with a cocktail of monoclonal antibodies for 30 min at 4°C: HLA-DR–fluoroscein isothiocyanate (FITC), CD25–phycoerythrin (PE), CD69–PE, CD38–PE, Ki-67–FITC, CXCR4–PE, CCR5–FITC, CD3–peridinin–chlorophyll a complex protein, and CD4–allophycocyanin (Beckton Dickinson, San Jose, California, USA). Stained cells were washed in PBS containing 2% FBS and subjected to cytofluorimetric analysis performed on a FACSCalibur (Becton Dickinson) equipped with Cell Quest (Macintosh, Sunnyvale, California, USA) software. For the analysis, total lymphocytes were first identified and gated by forward and side scatter. Then, cells were additionally gated for CD4 expression. A total of 10 000 gated events were collected for each sample.
Peripheral blood mononuclear cell infections
PBMC obtained from HIV-seronegative donor buffy coats were treated with 20 U/ml IL-2, 2 μg/ml PHA plus IL-2, 0.2 mmol/l VPA plus IL-2, or 1.0 mmol/l VPA plus IL-2 for 3 days. PBMC (4 × 106 cells) were then infected for 12 h with HIVLAI (10 ng p24) in the presence of 5 μg/ml polybrene. PBMC were washed and fed with media and IL-2; samples for p24 enzyme-linked immunosorbent assay (ELISA) were taken daily, and media replaced.
Limiting dilution cultures of latently infected CD4 T cells from HIV-positive donors
To obtain a sufficient number of infected resting CD4 T cells, leukopheresis was performed for five stably treated, aviremic HIV-positive volunteers (plasma HIV-1 RNA < 50 copies /ml for more than 6 months; CD4 cell counts > 400 × 106 cells/l). Informed consent was obtained from all patients. Resting CD4 T cells were negatively selected from leukopheresis samples using magnetic bead elution. HIV outgrowth assays were performed as previously described  with the following modifications. Cells were maintained without IL-2 for 3–4 days in culture in the presence of 10 μmol/l L-731,988, an investigational integrase inhibitor , and a reverse transcriptase inhibitor to which the donor had not been exposed. L-731,988 at 10 μmol/l is reported to inhibit completely integration of HIV . Efavirenz (gift of Bristol-Meyers Squibb) or abacavir (gift of Glaxo SmithKline) were used at 15 nmol/l and 4 μmol/l, respectively, concentrations 10 times above the median inhibitory concentration for wild-type HIV.
After this period of incubation, in limiting dilution format, 5.0–0.1 × 106 resting CD4 cells were (a) activated with 2 μg/ml PHA-L, 6 × 106 allogeneic irradiated PBMC from an HIV-seronegative donor, and 100 U/ml IL-2; (b) treated with 0.2–1.0 mmol/l VPA and 100 U/ml IL-2; or (c) cultured in 100 U/ml IL-2 alone. After 72 h, the cultures were expanded with the addition of 106 CD8-depleted seronegative donor PBMC. Cultures were fed with media and 20 U/ml IL-2, and supernatants harvested every 3–4 days. Additional CD8-depleted PBMC were added every 7 days; virus was detected by p24 antigen capture ELISA. Cultures were carried for up to 28 days.
Valproic acid induced acetylation at the integrated HIV long terminal repeat
The nucleosome nuc 1 is positioned about the HIV LTR between +1 and +155 with respect to the transcription start site of HIV-1 LTR . Studies have suggested that disruption of this nucleosome accompanies activation of transcription of the integrated LTR [6–10]. The exposure of cell lines containing a single HIV integrant to the potent HDAC inhibitor trichostatin A results in the displacement of HDAC1 from nuc 1 about the HIV LTR initiation region, in association with increased acetylation of histone H4 and increased LTR expression .
To confirm that VPA could induce chromatin remodeling via HDAC inhibition, it would be desirable to perform similar assays on resting CD4 T cells from HIV-infected individuals. However, this is not technically feasible because of the rarity of replication-competent integrated HIV and the frequency of non-integrated HIV DNA in cells obtained from HIV-positive patients. To document changes induced by VPA in histone H4 acetylation at nuc 1 in vivo, chromatin immunoprecipitation assays were performed in J89 T cell lines containing a single integrated HIV genome . These T cell lines are a model of latency in which expression of integrated provirus can be induced by activation, and in which quiescence is reestablished when activating stimulus is withdrawn.
Using an antibody directed against acetylated histone H4, DNA fragments associated with acetylated histone H4 were precipitated from cell extracts. The precipitated HIV LTR DNA was quantified by PCR with primers spanning the nuc 1 region of LTR. Amplification of serial dilutions of DNA demonstrated that a twofold increase in density units of PCR product represented at least a fourfold increase in target DNA . As chromatin immunoprecipitation is a qualitative assay of factor occupancy at a specific DNA sequence, any twofold increase in PCR product observed in these assay conditions represents a significant qualitative increase in occupancy. Cells were assayed after only a 4 h exposure to VPA, as longer periods of time might allow the observation of the chromatin remodeling events that occur during cell cycle progression or in response to the expression of other host genes. As shown in Fig. 1, exposure of cells to 1 mmol/l VPA for 2 h results in a significant increase in acetylated histone H4 at nuc 1.
Valproic acid induced outgrowth of HIV from the resting CD4 T cells of aviremic HIV-positive donors
A major latent reservoir of HIV infection resides in resting memory CD4 T cells, which harbor integrated, functional, but quiescent proviral HIV genomes. This reservoir can be detected, quantified, and studied by negative selection of resting CD4 T cells on the basis of surface markers, incubation of limiting dilutions of cells with an activator of T cell proliferation or signaling, and amplifying the resultant output virus by the addition of CD4 lymphoblasts from uninfected donors [16–21]. The selection of HIV-infected donors with prolonged suppression of viremia minimizes the potential for the outgrowth of actively replicating HIV in cultures of resting cells.
Lymphocytes were obtained by leukopheresis from five volunteers with CD4 cell counts ranging from 558 to 898 × 106 cell/l. Four of these subjects had been aviremic (< 50 copies/ml HIV-1 RNA) for 24–28 months; one was aviremic for significantly longer than 11 months but documentation of the last date of detectable HIV RNA was unavailable. More than 110 × 106 resting cells were studied in each of the four subjects; 52 × 106 resting cells were available for study in the fifth subject. In total, > 490 × 106 resting CD4 T cells were tested in 176 limiting-dilution cultures.
Because of the stringent conditions and the small numbers of latently infected cells, replication-competent HIV was only recovered in 39 of 176 cultures. However, exposure of resting CD4 cells to safe and clinically achievable concentrations of VPA resulted in outgrowth of HIV at a frequency comparable to that following full cell activation with PHA (Table 1). HIV was recovered in 21 cultures activated with PHA and 18 cultures exposed to VPA. Using a maximum likelihood method , the infectious units per 106 resting CD4 cells was calculated for each patient sample using the data from PHA stimulation or VPA exposure. Differences in this estimated value within each subject between PHA and VPA were not statistically significant.
Outgrowth was not consistently detected in the cultures containing the greatest numbers of patient resting cells, perhaps because of suboptimal viability of cells in high-input cultures. No virus was detected in negative control cultures with an equivalent range of input cells maintained in IL-2 for up to 30 days.
Valproic acid did not induce de novo infection or expression of activation markers
As the goal of induction of latent HIV expression would be to allow eradication of chronic viral infection, reagents used to induce viral outgrowth should not simultaneously increase the likelihood of de novo infection. Global inhibition of HDAC activity is reported to alter significantly the expression of only 2% of host genes .
Cell surface phenotype analysis and de novo infection in the presence of VPA was examined to explore the global effect of VPA on PBMC in relation to HIV-1 infection.
PBMC were pretreated with IL-2, PHA plus IL-2, or VPA plus IL-2 for 3 days. Pretreated PBMC were then infected with a CXCR4-tropic clone (HIVLAI) and p24 assayed over 2 weeks. Whereas VPA induced HIV expression from latently infected CD4 cells, infection of seronegative donor cells in the presence of 0.2–1 mmol/l VPA and IL-2 did not result in a change in production of HIV compared with cells cultured with IL-2 alone (Fig. 2). To eliminate the possibility that VPA exposure increased HIV entry and/or reverse transcription, despite the fact that it did not upregulate de novo productive infection, HIV DNA PCR was performed. Equivalent amounts of HIV DNA were detected in cells infected in the presence of VPA/IL-2 or IL-2, more than fourfold less than in PHA-activated cells (not shown). Findings using a CCR5-tropic viral clone (HIVBaL) were similar (not shown).
Expression of activation markers on the surface of lymphocytes was unaltered by VPA. Exposure of PBMC to up to 1 mmol/l VPA and 20 U/ml IL-2 did not significantly upregulate cell surface expression of CD38, CD69, CD25, or HLA-DR on CD3+CD4+ or CD3+CD8+ cells compared with cells cultured in 20U/ml IL-2 alone (Fig. 3).
Even in the face of a highly potent antiviral immune response, a proportion of replication competent HIV escapes viral clearance and establishes quiescence within resting CD4 cells. Disease can be prevented by chronic suppressive antiviral therapy, but this is fraught with toxicities and costs, and it is threatened by the potential of HIV to develop drug resistance. Clearance of HIV infection should be a therapeutic goal, although it may be a difficult one. While advances in antiviral and immunotherapy may allow the termination of low-level viral replication in a variety of cellular compartments and reservoirs, HIV will remain a chronic viral infection unless there are therapeutic answers to persistent, latent infection of resting CD4 T cells. Selective induction of latent HIV would allow antiretroviral drugs and the antiviral immune response to access this residual HIV infection.
Several studies have shown that intensive antiretroviral therapy in combination with IL-2 or global T cell activators fails to eradicate HIV infection [31–36]. Modeling studies suggest that global T cell activation may induce viral replication and increase the number of susceptible uninfected target cells beyond the threshold that can be contained by antiretroviral therapy . An alternative approach could use reagents that selectively activate quiescent proviral genomes but have limited effects on the host cell; this might allow outgrowth of latent HIV and avoid the pitfalls of global T cell activation.
Prostratin, a unique phorbol ester, and the human cytokine interleukin-7 have been reported to reactivate latent HIV [38–40]. These reagents, however, are not yet available for use in humans. As HDAC contributes to quiescence within the resting cells of HIV-positive patients [2–5], it was interesting to see if VPA, an HDAC inhibitor that is often given to HIV-infected patients for other conditions, could induce expression of latent HIV from their cells ex vivo.
VPA was surprisingly effective at inducing outgrowth of HIV from resting cells harvested from the peripheral blood, no less effective than activation with the mitogen PHA. Our findings differ from those of Brooks et al. , who found the more potent HDAC inhibitor trichostatin ineffective in inducing outgrowth of latent HIV. One possible explanation for this discrepancy is that more potent HDAC inhibition induces cellular toxicity or alters host gene expression in a way that prevents viral outgrowth ex vivo. We have found that latent HIV was only rarely recovered when cells were treated with 5 mmol/l VPA (data not shown). In addition, thymocytes are implanted and infected in the mouse model studied by Brooks et al., resulting in latently infected naive CD4 T cells. Latent HIV may occur less frequently within naive cells than in peripheral memory CD4 cells , and expression of provirus may be regulated differently in naive cells than in the memory cell populations we have studied.
It should be noted that CD4 lymphocytes were maintained ex vivo in these studies with IL-2. Although virus was not recovered in our culture conditions when cells were maintained with IL-2 alone, IL-2 in higher concentrations can itself induce viral replication . A cooperative effect between VPA and IL-2 in allowing viral outgrowth cannot be ruled out.
We found that VPA induced outgrowth of HIV from the resting CD4 cells of aviremic patients exposed ex vivo to VPA concentrations achievable in vivo. When exposed to VPA, CD4 cells did not become activated or more permissive for viral replication. VPA is, therefore, a drug that may be used clinically to perturb the quiescent reservoir of HIV infection within resting CD4 T cells.
Current therapy is not yet potent enough to block all active HIV replication [43–47]. However, the clinical experience with VPA may make it possible to test the ability to deplete the latent reservoir in test-of-principal clinical experiments. If future advances in immunotherapeutics and antiretroviral chemotherapy can terminate viral replication, interventions that derepress the LTR and result in selective expression of the quiescent HIV may deplete the reservoir of persistent HIV infection and make it possible to eliminate HIV infection.
We thank H. Wise for primary study coordination, with assistance from M. B. Kvanli and D. Turner. We thank J. P. Kassirer, J. V. Garcia, R. Gaynor, and G. D. Miralles for advice and careful review.
Sponsorship: This and related work was supported by an amfAR award and N.I.H. grants (RO1-AI 45297 and UO1-AI46376) to D.M.M.
1. Pierson T, McArthur J, Siliciano RF. Reservoirs for HIV-1: mechanisms for viral persistence in the presence of antiviral immune responses and antiretroviral therapy. Annu Rev Immunol
. 2000, 18
2. Coull JJ, Romerio F, Sun JM, Volker JL, Galvin KM, Davie JR, et al
. The human factors YY1 and LSF repress the human immunodeficiency virus type 1 long terminal repeat via recruitment of histone deacetylase 1. J Virol
3. Coull JJ, He G, Melander C, Rucker V, Dervan PB, Margolis DM. Targeted derepression of the human immunodeficiency type 1 virus long terminal repeat by pyrrole-imidazole polyamides. J Virol
4. He G, Margolis DM. Counter-regulation of chromatin acetylation and histone deacetylase occupancy at the integrated promoter of human immunodeficiency virus type 1 by the HIV-1 activator Tat and the HIV-1 repressor YY1. Mol Cell Biol
5. Ylisastigui L, Coull JJ, Rucker V, Brodie SJ, Bosch RJ, Sodora D, et al. Polyamides reveal a role for repression in viral latency within HIV-infected donors’ resting CD4+ T cells. J Infect Dis
2004, in press.
6. Verdin E, Paras Jr P, van Lint C. Chromatin disruption in the promoter of human immunodeficiency virus type 1 during transcriptional activation. EMBO J
7. Pazin MJ, Sheridan PL, Cannon K, Cao Z, Keck JG, Kadonaga JT, et al
. NF-κB-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro. Genes Dev
8. 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 J
. 1996, 15
9. Sheridan PL, Mayall TP, Verdin E, Jones KA. Histone acetyltrans-ferases regulate HIV-1 enhancer activity in vitro. Genes Dev
10. El Kharroubi A, Piras G, Zensen R, Martin MA. Transcriptional activation of the integrated chromatin associated human immunodeficiency virus type 1 promoter. Mol Cell Biol
11. Benkirane M, Chun RF, Xiao H, Ogryzko VV, Howard BH, Nakatani Y, et al. Activation of integrated provirus requires histone acetyltransferase. p300 and P/CAF are coactivators for HIV-1 Tat. J Biol Chem
12. Quivy V, Adam E, Collette Y, Demonte D, Chariot A, Vanhulle C, et al
. Synergistic activation of human immunodeficiency virus type 1 promoter activity by NF-κB and inhibitors of deacetylases: potential perspectives for the development of therapeutic strategies. J Virol
13. Jordan A, Bisgrove D, Verdin E. HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J
14. Winslow BJ, Pomerantz RJ, Bagasra O, Trono D. HIV-1 latency due to the site of proviral integration. Virology
15. Romerio F, Gabriel MN, Margolis DM. Repression of HIV-1 through the novel cooperation of the human factors YY1 and LSF. J Virol
16. Chun T-W, Finzi D, Margolick J, Chadwick K, Schwartz D, Siliciano RF. In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat Med
17. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, et al
. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science
18. Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CI, Spina CA, et al
. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science
19. Chun T-W, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, et al
. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci USA
20. Chun T-W, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, et al
. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature
21. Siliciano JD, Kajdas J, Finzi D, Quinn TC, Chadwick K, Margolick JB, et al. Long-term studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T. Nat Med
22. Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem
23. Moog C, Kuntz-Simon G, Caussin-Schwemling C, Obert G. Sodium valproate, an anticonvulsant drug, stimulates human immunodeficiency virus type 1 replication independently of glutathione levels. J Gen Virol
24. Witvrouw M, Schmidt J-C, van Romoortel B, Daelmans D, Este JA, Vandamme A-M, et al
. Cell type-dependent effect of sodium valproate on human immunodeficiency virus type 1 replication in vitro. AIDS Res Hum Retroviruses
25. Maggi JD, Halman MH. The effect of divalproex sodium on viral load: a retrospective review of HIV-positive patients with manic syndromes. Can J Psychiatry
26. Jennings HR Romanelli F. The use of valproic acid in HIV-positive patients. Ann Pharmacother
27. Kutsch O, Benveniste EN, Shaw GM, Levy DN. Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency. J Virol
28. Hazuda DJ, Felock P, Witmer M, Wolfe A, Stillmock K, Grobler JA, Espeseth A, et al
. Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science
29. Myers LE, McQuay LJ, Hollinger FB. Dilution assay statistics. J Clin Microbiol
30. van Lint C, Emiliani S, Verdin E. The expression of a small fraction of cellular genes is changed in response to histone hyperacetylation. Gene Expr
31. Chun TW, Engel D, Mizell SB, Hallahan CW, Fischette M, Park S, et al
. Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy. Nat Med
32. Prins JM, Jurriaans S, van Praag RM, Blaak H, van Rij R, Schellekens PT, et al
. Immuno-activation with anti-CD3 and recombinant human IL-2 In HIV-1- infected patients on potent antiretroviral therapy. AIDS
33. van Praag RM Prins JM, Roos MT, Schellekens PT, Ten Berge IJ, Yong SL, et al
. OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion. J Clin Immunol
34. Kulkosky J, Nunnari G, Otero M, Calarota S, Dornadula G, Zhang H, et al
. Intensification and stimulation therapy for human immunodeficiency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy. J Infect Dis
35. Stellbrink HJ, van Lunzen J, Westby M, O'Sullivan E, Schneider C, Adam A, et al
. Effects of interleukin-2 plus highly active antiretroviral therapy on HIV-1 replication and proviral DNA (COSMIC trial). AIDS
36. Dybul M, Hidalgo B, Chun TW, Belson M, Migueles SA, Justement JS, et al
. Pilot study of the effects of intermittent interleukin-2 on human immunodeficiency virus (HIV)-specific immune responses in patients treated during recently acquired HIV infection. J Infect Dis
37. Fraser C, Fergeson, NM, Ghani AC, Prins JM, Lange JM, Goudsmit J, et al
. Reduction of the HIV-1 infected T cell reservoir by immune activation treatment is dose-dependent and restricted by the potency of antiretroviral drugs. AIDS
38. Kulkosky J, Culnan DM, Roman J, Dornadula G, Schnell M, Boyd MR, et al. Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART. Blood
39. Brooks DG, Hamer DH, Arlen PA, Gao L, Bristol G, Kitchen CM, et al. Molecular characterization, reactivation, and depletion of latent HIV. Immunity
40. Brooks DG, Arlen PA, Gao L, Kitchen CM, Zack JA. Identification of T cell-signaling pathways that stimulate latent HIV in primary cells. Proc Natl Acad Sci USA
41. Shen A, Zink MC, Mankowski JL, Chadwick K, Margolick JB, Carruth LM, et al
. Resting CD4+ T lymphocytes but not thymocytes provide a latent viral reservoir in a simian immunodeficiency virus-Macaca nemestrina model of human immunodeficiency virus type 1-infected patients on highly active antiretroviral therapy. J Virol
42. Kinter AL, Poli G, Fox L, Hardy E, Fauci AS. HIV replication in IL-2-stimulated peripheral blood mononuclear cells is driven in an autocrine/paracrine manner by endogenous cytokines. J Immunol
43. Dornadula G, Zhang H, van Uitert B, Stern J, Livornese L Jr, Ingerman MJ, et al
. Residual HIV-1 RNA in blood plasma of patients taking suppressive highly active antiretroviral therapy. JAMA
44. Gunthard HF, Frost SD, Leigh-Brown AJ, Ignacio CC, Kee K, Perelson AS, et al
. Evolution of envelope sequences of human immunodeficiency virus type 1 in cellular reservoirs in the setting of potent antiviral therapy. J Virol
45. Natarajan V, Bosche M, Metcalf JA, Ward DJ, Lane HC, Kovacs JA. HIV-1 replication in patients with undetectable plasma virus receiving HAART. Highly active antiretroviral therapy. Lancet
46. Zhang L, Ramratnam B, Tenner-Racz K, He Y, Vesanen M, Lewin S, et al
. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med
47. Ramratnam B, Mittler JE, Zhang L, Boden D, Hurley A, Fang F, et al
. The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged antiretroviral therapy. Nat Med
This article has been cited 67 time(s).
Expert Opinion on Biological TherapyAntibody-based candidate therapeutics against HIV-1: implications for virus eradication and vaccine designExpert Opinion on Biological Therapy
Plos OneAntiretroviral Intensification and Valproic Acid Lack Sustained Effect on Residual HIV-1 Viremia or Resting CD4+Cell InfectionPlos One
Plos OneResting Regulatory CD4 T Cells: A Site of HIV Persistence in Patients on Long-Term Effective Antiretroviral TherapyPlos One
Journal of VirologyHigh-frequency epigenetic repression and silencing of retroviruses can be antagonized by histone deacetylase inhibitors and transcriptional activators, but uniform reactivation in cell clones is restricted by additional mechanismsJournal of Virology
Journal of Infectious DiseasesEliminating persistent HIV infection: Getting to the end of the rainbowJournal of Infectious Diseases
Embo JournalCBF-1 promotes transcriptional silencing during the establishment of HIV-1 latencyEmbo Journal
Future MicrobiologyCCAAT/enhancer-binding proteins and the pathogenesis of retrovirus infectionFuture Microbiology
Journal of Virology
Mitogen-activated protein kinases regulate LSF occupancy at the human immunodeficiency virus type 1 promoter
Journal of Virology, 79():
Journal of Infectious DiseasesHexamethylbisacetamide and disruption of human immunodeficiency virus type 1 latency in CD4(+) T cellsJournal of Infectious Diseases
Journal of VirologyA Limited Group of Class I Histone Deacetylases Acts To Repress Human Immunodeficiency Virus Type 1 ExpressionJournal of Virology
RetrovirologyMolecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategiesRetrovirology
Journal of VirologyEstablishment of HIV Latency in Primary CD4(+) Cells Is due to Epigenetic Transcriptional Silencing and P-TEFb RestrictionJournal of Virology
Seizure-European Journal of EpilepsyFrequency of seizures and epilepsy in neurological HIV-infected patientsSeizure-European Journal of Epilepsy
Future VirologyEstablishment and maintenance of HIV latency: model systems and opportunities for interventionFuture Virology
RetrovirologyPRMT6 diminishes HIV-1 Rev binding to and export of viral RNARetrovirology
Drug Discovery TodayEmerging host cell targets for hepatitis C therapyDrug Discovery Today
BloodInduction of HIV-1 latency and reactivation in primary memory CD4(+) T cellsBlood
Nucleic Acids ResearchCOUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cellsNucleic Acids Research
RetrovirologyLatency: the hidden HIV-1 challengeRetrovirology
Journal of NeurovirologyValproic acid does not affect markers of human immunodeficiency virus disease progressionJournal of Neurovirology
Journal of VirologyIdentification of cellular proteins that maintain retroviral epigenetic silencing: Evidence for an antiviral responseJournal of Virology
Journal of Infectious Diseases
Polyamides reveal a role for repression in latency within resting T cells of HIV-infected donors
Journal of Infectious Diseases, 190(8):
Future VirologyHIV latency: present knowledge and future directionsFuture Virology
Journal of Theoretical BiologyModeling HIV persistence, the latent reservoir, and viral blipsJournal of Theoretical Biology
Current Hiv Research
HAART-persistent HIV-1 latent reservoirs: Their origin, mechanisms of stability and potential strategies for eradication
Current Hiv Research, 4(2):
Current Pharmaceutical Design
Chromatin modifications (acetylation/deacetylation/methylation) as new targets for HIV therapy
Current Pharmaceutical Design, 12():
Genome BiologyAttacking pathogens through their hostsGenome Biology
Annual Review of MedicineHide-and-seek: The challenge of viral persistence in HIV-1 infectionAnnual Review of Medicine
HIV/AIDS epidemiology, pathogenesis, prevention, and treatment
Hiv Clinical Trials
HIV Pharmacology: Barriers to the eradication of HIV from the CNS
Hiv Clinical Trials, 7(3):
Amino AcidsIsoform-specific determinants in the HP1 binding to histone 3: insights from molecular simulationsAmino Acids
Epilepsy seizures linked to HIV infection in Africa
Journal of VirologyEpigenetic Silencing of Human Immunodeficiency Virus (HIV) Transcription by Formation of Restrictive Chromatin Structures at the Viral Long Terminal Repeat Drives the Progressive Entry of HIV into LatencyJournal of Virology
Antimicrobial Agents and ChemotherapyHuman immunodeficiency virus type 1 latency model for high-throughput screeningAntimicrobial Agents and Chemotherapy
Embo JournalNF-kappa B p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiationEmbo Journal
Journal of VirologyHexamethylbisacetamide remodels the human immunodeficiency virus type 1 (HIV-1) promoter and induces tat-independent HIV-1 expression but blunts cell activationJournal of Virology
Current Pharmaceutical Design
The pharmaceutical potential of histone deacetylase inhibitors
Current Pharmaceutical Design, 13():
Plos OneSynergistic Activation of HIV-1 Expression by Deacetylase Inhibitors and Prostratin: Implications for Treatment of Latent InfectionPlos One
Expert Reviews in Molecular MedicinePharmaceutical approaches to eradication of persistent HIV infectionExpert Reviews in Molecular Medicine
Antiviral ResearchStpC-based gene therapy targeting latent reservoirs of HIV-1Antiviral Research
Biochemical PharmacologyHDAC inhibitors: Clinical update and mechanism-based potentialBiochemical Pharmacology
Prolonged valproic acid treatment does not reduce the size of latent HIV reservoir
Valproic acid without intensified antiviral therapy has limited impact on persistent HIV infection of resting CD4+ T cells
Neurologic ClinicsTreatment of HTLV-I-associated myelopathy/tropical spastic paraparesis: Toward rational targeted therapyNeurologic Clinics
Biochimica Et Biophysica Acta-Gene Regulatory MechanismsChromatin dynamics associated with HIV-1 Tat-activated transcriptionBiochimica Et Biophysica Acta-Gene Regulatory Mechanisms
Molecular PharmacologyEffects of valproic acid derivatives on inositol trisphosphate depletion, teratogenicity, glycogen synthase kinase-3 beta inhibition, and viral replication: A screening approach for new bipolar disorder drugs derived from the valproic acid core structureMolecular Pharmacology
Depletion of latent HIV-1 infection in vivo: a proof-of-concept study
Journal of Infectious DiseasesStability of the latent reservoir for HIV-1 in patients receiving valproic acidJournal of Infectious Diseases
Clinical and Experimental Rheumatology
HIV/AIDS: epidemic update, new treatment strategies and impact on autoimmunity
Clinical and Experimental Rheumatology, 26(1):
Valproic acid: a potential role in treating latent HIV infection
Nature Reviews MicrobiologyChromatin control of herpes simplex virus lytic and latent infectionNature Reviews Microbiology
AIDS Research and Human RetrovirusesExpression of Latent HIV Induced by the Potent HDAC Inhibitor Suberoylanilide Hydroxamic AcidAIDS Research and Human Retroviruses
AIDS Research and Human RetrovirusesShort Communication: Activation of Latent HIV Type 1 Gene Expression by Suberoylanilide Hydroxamic Acid (SAHA), an HDAC Inhibitor Approved for Use to Treat Cutaneous T Cell LymphomaAIDS Research and Human Retroviruses
Antiviral ResearchHIV reservoirs, latency, and reactivation: Prospects for eradicationAntiviral Research
BioessaysFrom reactivation of latent HIV-1 to elimination of the latent reservoir: The presence of multiple barriers to viral eradicationBioessays
Drug Discovery TodayTargeting HIV latency: pharmacologic strategies toward eradicationDrug Discovery Today
Journal of General VirologyRecent developments in human immunodeficiency virus-1 latency researchJournal of General Virology
Current Hiv Research
Histone Deacetylase Inhibitor MC1293 Induces Latent HIV-1 Reactivation by Histone Modification In Vitro Latency Cell Lines
Current Hiv Research, 11(1):
Viruses-BaselLost in Transcription: Molecular Mechanisms that Control HIV LatencyViruses-Basel
Plos PathogensPhosphorylation of CDK9 at Ser175 Enhances HIV Transcription and Is a Marker of Activated P-TEFb in CD4(+) T LymphocytesPlos Pathogens
Journal of VirologyTargeting I kappa B Proteins for HIV Latency Activation: the Role of Individual I kappa B and NF-kappa B ProteinsJournal of Virology
Drugs of the FutureThe Potential Role of Hdac Inhibitors in De-Silencing Latent Hiv VirusDrugs of the Future
Journal of VirologyAn AP-1 Binding Site in the Enhancer/Core Element of the HIV-1 Promoter Controls the Ability of HIV-1 To Establish Latent InfectionJournal of Virology
RetrovirologyFeline immunodeficiency virus latencyRetrovirology
latency; histone deacetylase; resting CD4 T cells
© 2004 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.