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.
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