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Journal of Virology, May 2006, p. 4570-4579, Vol. 80, No. 9
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.9.4570-4579.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Hexamethylbisacetamide Remodels the Human Immunodeficiency Virus Type 1 (HIV-1) Promoter and Induces Tat-Independent HIV-1 Expression but Blunts Cell Activation

Vladimir Klichko,^1,2 Nancy Archin,³ Rupinderjeet Kaur,² Ginger Lehrman,² and David Margolis^3*

North Texas Veterans Health Care Systems, Dallas, Texas 75216,¹ University of Texas Southwestern Medical Center at Dallas, Texas 75390,² University of North Carolina at Chapel Hill, North Carolina 27599-7435³

Received 14 October 2005/ Accepted 9 February 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hexamethylbisacetamide (HMBA) induces human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected T-cell and monocytoid cell lines. We find that HMBA activation of viral expression is Tat independent but, like Tat, increases the efficiency of elongation of the HIV-1 promoter (long terminal repeat [LTR]) transcripts. Further, exposure to HMBA induces chromatin remodeling at nucleosome 1 (Nuc-1) near the start site of LTR transcription but does so without increasing histone acetylation or altering histone methylation near Nuc-1. Of note, despite enhanced proviral expression, HMBA suppressed HIV infection ex vivo in primary blood mononuclear cell (PBMC) cultures. Treatment with HMBA did not alter expression of the HIV coreceptors, CCR5 and CXCR4, in PBMCs but down-regulated CD4. Finally, HMBA interferes with cell proliferation and activation; it suppressed expression of Ki67 and CD25 and in PBMCs exposed to mitogen. As HMBA has been tested in oncology trials, its unusual properties make it a useful reagent for future studies of HIV promoter regulation and a novel prototype molecule for therapeutics that abort the latent proviral state of chronic HIV infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The introduction of highly active antiretroviral therapy (HAART) raised hopes for eradication of established human immunodeficiency virus (HIV) infection. This hope dimmed when latently infected CD4⁺ lymphocytes were found to persist in individuals despite years of viral suppression (11, 24, 75). Latent infection of memory T cells is established early after infection (12) and is unaffected by the antiviral immune response or antiviral therapies (15). The slow rate of disappearance of these cells during antiretroviral therapy led to estimates that eradication of infection would require decades of continuous therapy (23, 51, 63).

Activation of quiescent cells in the presence of HAART to drive latently infected cells out of the resting state has been explored as a therapeutic strategy. Unfortunately, intensive antiretroviral therapy in combination with the administration of interleukin-2 (IL-2) and/or anti-CD3 monoclonal antibody does not eradicate HIV infection (13, 18, 35, 49, 64, 71). Recently alternative strategies to disrupt latent HIV infection through the use of histone deacetylase (HDAC) inhibitors, IL-7, resveratrol, and prostratin have been proposed (7, 17, 34, 39, 40, 78).

Hexamethylbisacetamide (HMBA) was developed as an anticancer drug (54) as it can induce differentiation of leukemic and solid tumor cell lines (28, 42, 43, 58). Suboptimal antitumor activity at clinically tolerable doses has impeded the further development of HMBA (2).

HMBA is known to activate HIV expression in chronically infected cell lines (3, 62, 74) and in cell lines stably transfected with long terminal repeat (LTR)-reporter gene constructs (66, 81). While HMBA is structurally related to HDAC inhibitors, it does not inhibit HDACs or induce histone hyperacetylation (56). Indirect evidence suggests that HMBA and HDAC inhibitors induce cell differentiation by different pathways (57). Tumor cell lines resistant to the differentiation-inducing activity of HDAC inhibitors are not resistant to HMBA (56). The molecular mechanism underlying HDAC inhibitor induction of HIV expression is understood in some detail (17, 59, 70, 79). However, the mechanism by which HMBA induces HIV proviral expression has not been elucidated (80).

In contrast to inducers such as tumor necrosis factor alpha and phorbol-12-myristate-13-acetate, HMBA-mediated induction of HIV-1 expression does not require NF-B binding sites at the HIV-1 LTR, whereas Sp1 binding sites are required for HMBA response (3, 74). HMBA mediates a number of changes in cellular metabolism (6, 26, 41, 44, 45) and induces expression of globin genes and changes in expression of the proto-oncogenes c-myb, c-myc, c-fos, and p53 (9, 52). Treatment with HMBA blocks cell cycle progression in G₁. It also induces a rapid decrease in the cdk4 protein level, followed by suppression of cdk4-associated pRB kinase activity and an increase in cyclin D3 (32).

We sought to expand our understanding of the mechanisms through which HMBA induces expression of quiescent HIV provirus. We find that HMBA increases both initiation and elongation of the HIV-1 LTR in the absence of Tat. Surprisingly, HMBA increased DNA accessibility and induced nucleosome remodeling without histone acetylation. However, unlike mitogen activation, HMBA did not increase cell susceptibility to HIV infection or the expression of cell surface markers of activation. In fact, HMBA down-regulated the surface expression of the HIV receptor, CD4. As a likely result of this effect, HMBA did not enhance de novo cell infection, and it suppressed HIV propagation in ex vivo primary blood mononuclear cell (PBMC) cultures.

In summary, our findings suggest that HMBA is a novel prototype for therapeutics designed to interrupt latent HIV infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell culture and viral stocks. PBMCs were separated from whole blood of healthy donors by density centrifugation with Ficoll Histopaque (Amersham, Piscataway, NJ). PBMCs were cultured in medium consisting of Iscove's modified Dulbecco's medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cellgro, Herndon, VA), 2 mM glutamine, penicillin/streptomycin (Invitrogen), and 20 U/ml recombinant IL-2 (Sigma, St. Louis, MO).

THP89GFP and J89GFP cell lines were obtained from D. Levy (36). The HeLa-CD4-LTR cell line (16, 76), HLM1 cell line (60, 61), U1 cell line (25), and HIV-1 molecular clone pNL4-3 (1) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program repository. Cells were grown and maintained as previously described (1, 16, 25, 36, 60, 61, 76). Virus stock was prepared by transient transfection of 293T cells, and p24 production was measured by enzyme-linked immunosorbent assay (Coulter, Miami, FL).

Flow cytometry analysis. Conjugated antibodies used for fluorescence-activated cell sorting (FACS) included the following: anti-CD3-peridinin chlorophyll protein, anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-allophycocyanin, anti-CCR5-FITC, anti-HL-DR-FITC, anti-CD69-phycoerythrin (PE), anti-CD38-PE, anti-CD25-PE, anti-Ki67-FITC (all from BD Pharmingen, San Diego, CA), and anti-p24-FITC (Coulter, Miami, FL). A total of 1 x 10⁶ PBMCs were washed twice with ice-cold 2% FBS-phosphate-buffered saline (PBS) and stained in 0.1 ml of PBS-FBS with a cocktail of antibodies for surface markers or isotype control in the dark for 30 min at 4°C. Stained cells were fixed and permeabilized (Fix and Perm Kit; Caltag Laboratories, Burlingame, CA) and restained for intracellular markers at room temperature for 20 min. FACS was performed on a FACSCalibur, and data were analyzed with Cellquest software (Becton Dickinson, San Jose, CA). For the analysis, total lymphocytes were first identified and gated by forward and side scatter. A total of 10,000 gated events were collected for each sample.

PBMC infections. PBMCs obtained from HIV-seronegative donor buffy coats were stimulated with 2 µg/ml of phytohemagglutinin (PHA) (Remel, Lanexa, KS) in Iscove's modified Dulbecco's medium supplemented with 10% FBS and 20 U/ml of IL-2 for 3 days. Stimulated cells were washed, and 1 x 10⁶ PBMCs per condition were infected with viral stock (50 ng of p24) in 0.2 ml for 1 h. PBMCs were washed and cultured at a density of 1 x 10⁶ cells/ml. After 3 days cells were washed twice with 2% FBS-PBS, fixed, permeabilized, and stained with antibody against p24 HIV antigen, and cells positive for intracellular p24 were quantitated by flow cytometry.

ChIP assay. Anti-acetyl-H3, anti-acetyl-H4, and anti-dimethyl-lysine 4-H3 were purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies recognizing amino acids 124 to 135 of H3 were purchased from Abcam (Cambridge, MA). Immunoprecipitation of chromatin was performed using a chromatin immunoprecipitation (ChIP) assay kit (Upstate Biotechnology) according to the manufacturer's protocol. Immunoprecipitated DNA was quantitated by PCR using two sets of primers: LTR-109F (5'-TACAAGGGACTTTCCGCTGG-3') and LTR+82R (5'-AGCTTTATTGAGGCTTAAGC-3') for the HIV-1 promoter and CCR5F (5'-TACCTGCTCAACCTGGCCAT-3') and CCR5R (5'-TTCCAAAGTCCCACTGGGC-3') for the CCR5 receptor. A total of 30 PCR cycles were carried out with 2 to 10 µl of precipitated DNA in a 25-µl reaction mixture containing 12.5 µl of master mix (QIAGEN, Valencia, CA) and a 400 nM concentration of each primer. PCR products were resolved by 8% polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. Images of gels were quantified using AlphaImager 2000 with Alpha Ease FC software (Alpha Innotech Corp., San Leandro, CA). Serial twofold dilutions of input DNA were also subjected to PCR to ensure linear amplification in each experiment. All values represent the average of at least three independent experiments. Differences were compared using a two-tailed Student's t test assuming independence of variance.

Analysis of LTR nucleosome 1 (Nuc-1) chromatin structure. Restriction enzyme digestion of purified nuclei with AlfII and HinfI was performed as previously described (70, 73). Thirty micrograms of purified DNA per condition was digested to completion with PstI, and the fragments were separated by electrophoresis in 1.5% agarose. Digoxigenin-labeled probe A (see Fig. 1) (72) was used for Southern blot analysis. The probe was generated using the primer set EV1 and EV2 (72) and a PCR DIG Probe Synthesis Kit (Roche Applied Science, Indianapolis, IN). Alkaline phosphatase-conjugated antibody and chemiluminescent CDP-Star substrate (Roche Applied Science) were used for detection. Blots were exposed to X-ray film, and images were quantified using AlphaImager 2000 with Alpha Ease FC. All values represent the average of least three independent experiments. Differences were compared using a two-tailed Student's t test assuming independent variance.

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FIG. 1. HMBA activates the HIV LTR expression of HeLa-CD4-LTR-CAT cells. Cells transfected with control vector pCMV (lanes 1 to 3) or with plasmid pCMV-Tat, encoding HIV Tat protein (lane 4). After transfection cells were cultured in medium alone (lanes 1 and 4), treated with 400 nM TSA (lane 2), or treated with 5 mM HMBA (lane 3). The level of LTR-driven CAT expression, normalized to the background level of CAT expressed in the absence of Tat, is indicated. Cm, chloramphenicol; bCm, butyrlated chloramphenicol. Results are representative of three independent transfections.

Real-time PCR assay. Total RNA was isolated from cells using Triazol (Invitrogen, Carlsbad, CA) and treated with DNase I amplification grade (Invitrogen). cDNA was synthesized in the presence of 0.75 µM concentrations of the gene-specific primers (37) HIV-SHORT-RT (5'-GGGTTCCCTAGTT-3') and HIV-Long-RT (5'-CGCCACTGCTAG-3') and 5 µg/ml concentrations of random hexamers (Invitrogen) using Moloney murine leukemia virus reverse transcriptase according to the manufacturer's protocol (Promega, Madison, WI). Real-time PCR for detection of total and elongated HIV-1 transcripts was performed using a modified version of a previously described set of primers (30, 37): HIV-START-M, 5'-GGTCTCTCTGGTTAGACCAGATCTGAGCC-3'; HIV-SHORT-M, 5'-GGGTTCCCTAGTTAGCCAGAGAGCTCC-3'; and HIV-LONG-M, 5'-CGCCACTGCTAGAGATTTTCCACACTGAC-3'. As an input control, a primer set that detects glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) mRNA was used (GAPDH-F, 5'-TCACCACCATGGAGAAGGCTGGG-3'; GAPDH-RA, 5'-GGCATCAGCAGAGGGGGCAGA-3'). PCR was performed by heating the reaction mixture to 95°C for 10 min, followed by 45 cycles at 95°C for 15 seconds and 60°C for 1 min. Amplification and detection were performed using an ABI Prism 7700 Sequence Detector system and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Triplicate 50-µl reaction mixtures contained 200 pg of cDNA and a 400 nM concentration of each PCR primer. Changes in transcript concentrations were estimated using the comparative cycle threshold method.

CAT assay. HeLa-CD4-LTR-chloramphenicol acetyltransferase (CAT) cells were transfected with plasmids pCMV-Tat (where CMV is cytomegalovirus), encoding the HIV Tat protein, or control vector pCMV using Lipofectamine (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. Each transfection used 5 µg of total DNA. Drugs were added 4 h after transfection, and all cultures were then incubated for 24 h with 10% FBS in a 5% CO₂ incubator. Cells were then washed and treated with trypsin, and cell lysates were made as previously described (29). Extracts were normalized for protein content, and thin-layer chromatography CAT assays were performed (Promega, Madison, WI). Silica plates were exposed to X-ray film, and densitometric quantification of the CAT activities was performed using the AlphaImager 2000 with Alpha Ease FC software.

Limiting dilution cultures of latently infected CD4 T cells from HIV-positive donors. Lymphocytes were obtained by continuous-flow leukopheresis from stable, HIV-infected volunteers on antiretroviral therapy with plasma HIV-1 RNA levels of <50 copies/ml for more than 12 months and CD4 cell counts of >300 cells/µl. Human Subjects Committee-approved informed consent was obtained from all patients. Isolation of CD4 T cells and recovery and quantification of replication-competent HIV were performed as described in detail previously (14, 39, 78). After isolation of resting CD4⁺ cells and treatment as described, cells were activated or exposed to HMBA for 72 h. Cells were then fed with fresh medium and target feeder cells and cultured as detailed previously (78).

Top Abstract Introduction Materials and Methods Results Discussion References

 
HMBA activation of HIV LTR is Tat independent. In latently infected resting T cells, HIV provirus transcription is absent or persists at very low levels. Ideally, drugs that interrupt HIV latency should be able to stimulate basal expression by Tat-independent mechanisms. Early studies differed as to the ability of HMBA to induce HIV expression in the absence of Tat (62, 72). To resolve these uncertainties, we studied the effect of HMBA in cell lines stably transfected with an LTR-reporter gene construct and three cell lines carrying HIV provirus with functioning and defective Tat activation pathways.

HeLa-CD4-LTR-CAT cells, which carry one copy of an LTR-reporter gene construct, were used to compare the effect of HMBA to the HDAC inhibitor, trichostatin A (TSA), and viral transactivator Tat. We found that both HMBA and TSA induced LTR expression, but levels of induction in cells exposed to HMBA or TSA were much lower than the level following Tat activation (Fig. 1).

HMBA enhanced LTR expression in both the J89GFP Jurkat T-cell line and monocytoid THP89GFP cell line, which carry a single copy of provirus with functioning Tat (Fig. 2A and B). Further, HMBA strongly enhanced Tat-deficient provirus expression and virion production in the HLM1 cell line (Fig. 2C). Thus, HMBA is able to increase the basal level of HIV-1 expression through a Tat-independent mechanism.

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FIG. 2. HMBA induces LTR expression in latently infected cell lines. Enhanced green fluorescent protein expression of THP89GFP (A) and J89GFP (B) cells that possess functional Tat and p24 antigen production of HLM1 cells that lack Tat (C) treated with 1 and 5 mM HMBA. Viability of cells shown in panels A, B, and C is given in panels D, E, and F, respectively. Viability of PBMCs treated with HMBA is shown in panel G. MFI, mean fluorescence intensity. Times after HMBA exposure are indicated as follows: open bars, 24 h; gray bars, 48 h; black bars, 72 h. Results are representative of three independent experiments.

Exposure to 1 to 5 mM HMBA induces HIV expression in latently infected cell lines, with most studies finding maximal induction at 5 mM (3, 62, 74). We reevaluated the effect of 0.2 to 25 mM HMBA in PBMCs and cell lines chosen for our studies and confirmed that 5 mM HMBA induces maximal expression without an appreciable effect on cell viability (Fig. 2D to G).

HMBA increases the rate of initiation of HIV-1 transcription. In the absence of Tat, HIV transcription pauses at a point downstream of the transcription initiation site, in the transactivation response element (TAR). Tat increases the efficiency of elongation (22) and stimulates transcription complex assembly (50). We analyzed the temporal profile of expression of paused and elongated transcripts in HLM1 cells treated with HMBA compared to the HDAC inhibitor TSA (Fig. 3). An increase in HIV RNA expression was detected after only 2.5 h of treatment (P < 0.05), and maximal induction was reached at 5.5 h of treatment. TSA and HMBA increased the initiation rate 2.2- and 3.6-fold, respectively, and elongated transcripts increased 3.9- and 7.1-fold, respectively (Fig. 3). These results show that TSA and HMBA enhance elongation beyond TAR, augmenting HIV expression in the absence of Tat.

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FIG. 3. Temporal profile of initiation and elongation rates of proviral transcription in HLM1 cells treated with 5 mM HMBA (A) and 400 nM TSA (B). Changes in concentrations of total and elongated transcripts were measured by real-time reverse transcription-PCR. Graphs indicate changes in concentrations (conc) of all LTR transcripts (both short, nonprocessive and full-length; open bars) and full-length transcripts (gray bars). Results are representative of three independent experiments.

HMBA induces HIV-1 LTR chromatin remodeling. The nucleosome positioned near the start site of HIV LTR transcription (Nuc-1) presents an elongation barrier to polymerase, and its remodeling plays a role in the reactivation of the HIV promoter from latency (69). Histones within Nuc-1 are remodeled by acetylation upon stimulation in response to Tat, mitogens, and HDAC inhibitors (19, 70, 73).

We examined changes in histone modification at Nuc-1 after 2.5 h of HMBA treatment of J89GFP and THP89GFP cells by using a ChIP assay with antibodies specific for acetylated H3, acetylated H4, and dimethylated lysine 4 of H3. Performing the ChIP assay after a short period of HMBA exposure makes it unlikely that secondary, nonspecific effects will be observed. Precipitated DNA was analyzed by PCR with primers spanning the Nuc-1 region and the CCR5 receptor gene. Amplification of serial dilutions of DNA demonstrated that a twofold increase in density of the PCR product band represents more than a threefold increase in target DNA (Fig. 4A). It is important to point out that the semiquantitative nature of these assays allows us to qualitatively measure significant changes in occupancy but not to compare the relative quantity of different histone modifications. As expected, TSA induced acetylation of H3 and H4 and dimethylation of H3 lysine 4. Surprisingly, we found that H3 and H4 acetylation and H3 lysine 4 methylation were not altered in J89GFP lymphocytic cells treated with HMBA (Fig. 4B and C). Similar results were seen in THP89GFP monocytoid cells. Although HMBA appeared to reproducibly decrease the acetylation of H3 in a statistically significant way, it is difficult to demonstrate that such a decrease is quantitatively significant.

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FIG. 4. Activation of HIV expression with HMBA is not accompanied by histone modification at the 5' LTR Nuc-1. (A) Semiquantitation of ChIP assay. Twofold increases in DNA input result in less than a twofold increase in PCR product. We therefore consider any twofold change in PCR band density to represent a significant qualitative change in occupancy. Chromatin isolation for ChIP analysis was performed after 2.5 h of drug exposure. (B) Histone modifications within Nuc-1 as detected by ChIP assay in THP89GFP and J89GFP cells treated with 400 nM TSA or 5 mM HMBA. Antibodies used in ChIP assays against acetylated H3, acetylated H4, dimethylated lysine 4 of H3, and total H3 are shown at left. (C) Relative levels of histone modification and total level of H3 averaged from three independent ChIP experiments. Statistically significant changes are indicated by an asterisk (P < 0.05). Open, light gray, dark gray, and black bars are relative levels of H3 acetylation, H4 acetylation, lysine 4 of H3 dimethylation, and total H3, respectively. Untr, untreated.

Taken at face value, our results would suggest that, contrary to the histone code hypothesis (31, 65, 67), histone acetylation at the Nuc-1 of the HIV-1 LTR is largely unchanged after HMBA exposure, despite the induction of transcription. However, another possible interpretation of these findings is that HMBA induces a rapid loss of contact of histones with DNA at Nuc-1. This phenomenon has recently been observed in other systems (38, 53).

To test whether histone occupancy was lost at Nuc-1 following exposure to HMBA, we measured occupancy of total H3 at Nuc-1 using an antibody that recognizes an unmodified epitope at the carboxy terminus of histone H3. We did not detect changes in H3 occupancy at Nuc-1 in either cell line (J89GFP or THP89GFP) after HMBA and TSA treatment compared to untreated control cells (Fig. 4). Thus, the lack of upregulation of histone acetylation after HMBA exposure and LTR expression is not the result of the complete loss of histones from this region of chromatin.

An alternative explanation is that HMBA induces novel histone modifications which result in increased DNA accessibility. Methylation of lysine 4 in H3 is associated with activation of transcription (27, 33), and we found that activation of the HIV-1 promoter with TSA was accompanied by dimethylation of lysine 4 of H3. However, no changes were detected at the level of dimethylation in HMBA-treated cells (Fig. 4B and C).

Chromatin remodeling at the HIV-1 promoter can result in increased accessibility of the DNA to nucleases (70-73). In order to directly examine chromatin accessibility, we determined the susceptibility of the HIV-1 LTR to restriction endonucleases following exposure to HMBA. The chronically infected U1 cell line, carrying two copies of proviruses, each with defective Tat transactivating genes (20, 21), were used to assess the effect of HMBA.

After treatment of U1 cells with HMBA or TSA for 5 h, nuclei were isolated and digested with AlfII or HinfI, which have cleavage sites in the Nuc-1 region (Fig. 5A). DNA was purified from the nuclei and digested to completion with PstI, which has a recognition site downstream of the LTR sequence. The digested products were analyzed by Southern blotting (Fig. 5B). TSA was used in these experiments as a positive control, as Nuc-1 disruption has been demonstrated following HDAC inhibitor treatment (70). We found that the region corresponding to Nuc-1 became accessible to digestion by restriction enzymes following treatment with HMBA. The increase in accessibility of the underlying DNA in Nuc-1 is similar to that observed after TSA treatment. This effect was specific for the Nuc-1 region because, in marked contrast, restriction by HinfI in the nucleosome-free region was not enhanced by either treatment. Strikingly, unlike TSA, HMBA did not induce acetylation of chromatin, typically associated with nucleosome mobilization, and uniformly associated with activation of the HIV LTR (Fig. 5D).

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FIG. 5. HMBA induces HIV LTR Nuc-1 remodeling. (A) Diagram shows the position of nucleosomes and the location of restriction enzyme sites and the probe used for Southern blotting (73). Letters identify the restriction fragments shown in panel B. (B) U1 cells were treated for 5 h with 400 nM TSA or 5 mM HMBA; nuclear DNA was partially digested with AlfII or HinfI, digested to completion with PstI, and analyzed by Southern blotting. M, molecular weight marker III. (C) Densitometric quantification of the accessibility of DNA at the Nuc-1 region after TSA (open bars) or HMBA (gray bars) treatment, relative to untreated cells. Results are representative of three independent experiments. (D) Histone modifications within Nuc-1 as detected by ChIP assay in U1 cells treated with 400 nM TSA or 5 mM HMBA for 4 to 5 h. Antibodies used in ChIP against acetylated H3 and acetylated H4 are indicated at left. Results are representative of three independent experiments. IP, immunoprecipitation.

HMBA suppresses CD4, Ki67, and CD25 expression in activated PBMCs but does not affect expression of HIV coreceptors or other surface markers of activation. Reagents used to reactivate latent HIV should not simultaneously increase the susceptibility of cells to new rounds of infection. Therefore, we investigated the effect of HMBA on the expression of CD4 and HIV coreceptors in human PBMC ex vivo cultures. Four culture conditions were used in this experiment. PBMCs were stimulated with 2 µg/ml PHA for 3 days in the presence or absence of HMBA. Non-PHA-stimulated PBMCs were also cultured for 3 days with or without 5 mM HMBA. FACS analysis showed that HMBA down-regulated surface CD4 expression, particularly after PHA stimulation. HMBA had little effect on CCR5 and CXCR4 expression (Fig. 6).

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FIG. 6. HMBA does not increase cell activation or expression of HIV receptors and coreceptors. PBMCs were stimulated with 2 µg/ml PHA in the presence or absence of 5 mM HMBA for 3 days. Unstimulated PBMCs were cultured for this time in medium with or without HMBA. Flow cytometry analysis was performed at 3 days after stimulation to evaluate the levels of expression of viral receptor, coreceptors, and activation markers. Dual-parameter plots demonstrate the effect of HMBA on the expression of CD25 and Ki67 in panel A and CD4 and CXCR4 in panel B. Numbers displayed in each quadrant of these plots are percentages of positive cells. In panel C results of flow cytometry analysis are presented as histogram plots. The results shown are representative of three different experiments with PBMCs from three donors.

As cell cycle and activation state also influence the efficiency of productive viral infection, we investigated the influence of HMBA on markers of activation and proliferation. HMBA completely suppressed PHA-mediated upregulation of the intracellular proliferation marker Ki67 and abolished the PHA-induced expression of the activation marker CD25, the alpha chain of the IL-2 receptor. In contrast, expression of CD38, CD69, and HLA-DR were not significantly altered by HMBA (Fig. 6). These findings suggest that HMBA may induce HIV proviral expression but interfere with the propagation of viral infection.

HMBA suppresses de novo infection in cultured PBMCs. To examine the possibility that HMBA inhibits HIV infection, we studied infection in PBMC cultures. PBMCs were stimulated with PHA for 3 days and infected with a virus generated from a CXCR4-tropic HIV molecular clone (NL4-3) in medium with or without HMBA. After 1 h of infection, the cells were washed and cultured in medium with or without HMBA. On day 3 after infection, intracellular p24 expression was measured by flow cytometry (Table 1). The presence of HMBA during infection did not affect virus production, but viral expression was substantially reduced when infected cells were cultured in the presence of HMBA. This finding suggests that HMBA did not directly inactivate virus, but its effects on CD4 expression and/or cell proliferation substantially inhibited virus propagation over time in culture.

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TABLE 1. HMBA restricts HIV propagation in PBMC cultures

We then tested the ability of HMBA to induce expression of quiescent provirus in resting CD4 T cells obtained from aviremic, HAART-treated, HIV-infected donors, as described previously (39, 40). While virus was recovered from the cells of three donors when maximally activated by PHA and irradiated allogeneic cells, exposure to 0.5 to 5 mM HMBA rarely resulted in the recovery of HIV from these patients' resting CD4⁺ T cells. This result might be explained by weak induction of proviral outgrowth at lower HMBA concentrations and, at higher HMBA concentrations, by the inhibition of viral spread required to detect rare replication-competent HIV in this ex vivo outgrowth system.

Top Abstract Introduction Materials and Methods Results Discussion References

 
HMBA induces HIV expression in chronically infected T lymphocytic (ACH-2) and monocytoid (U1) cell lines with defects in the Tat activation axis (20, 21, 74). HMBA also activates expression of the LTR in the absence of Tat in cells stably transfected with LTR-reporter gene constructs (66, 81).

We find that HMBA enhances Tat-independent transcription in cell lines carrying an LTR-reporter gene construct and in a latently HIV-infected HIV-1 cell line, HLM1 (Fig. 2). As expected, the level of activation with HMBA in HeLa-CD4-LTR-CAT reporter cell lines is lower than that induced by Tat (Fig. 1). Further, real-time reverse transcription-PCR analysis examining the mechanism of activation of LTR expression found that both HMBA and TSA enhance initiation and elongation beyond TAR in the absence of Tat (Fig. 3). Tat increases elongation by recruiting the positive transcriptional elongation factor b (p-TEFb) with subsequent phosphorylation of the C-terminal domain of RNA polymerase II. However, although HMBA appears to exert a similar transcriptional phenotype, HMBA has been shown to inhibit transcription mediated by p-TEFb by inducing the expression of the p-TEFb cellular inhibitor HEXIM1 (77). Further study is needed to elucidate the unique mechanism by which HMBA induces HIV LTR expression.

Dynamic changes in chromatin are thought to induce expression, in part by making DNA more accessible to transcription factors. Two general classes of multiprotein complexes remodel chromatin by modifying the core histone tails (31, 65, 67) or using ATP hydrolysis to alter chromatin structure (4, 48).

In our experiments, using a nuclease accessibility assay to assess the state of chromatin, we found that HMBA induced LTR Nuc-1 remodeling to the same extent as does the HDAC inhibitor TSA (Fig. 5). Strikingly, in contrast to HDAC inhibitors or tumor necrosis factor alpha (19, 70, 73), HMBA does not induce histone hyperacetylation or alter dimethylation of H3 lysine 4 at Nuc-1 (Fig. 4). Chromatin remodeling could cause the dissociation of histones from a promoter region, resulting in an apparent absence of or decrease in the histone modification signal (e.g., acetylation) measured by a ChIP assay. In Saccharomyces cerevisiae, nucleosome occupancy at proximal gene regulatory regions is inversely proportional to the transcriptional initiation rate of the promoter (38, 53). Similarly, at the mammalian IL-2 and granulocyte-macrophage colony stimulation factor promoters, apparent loss of histone acetylation and phosphorylation was found to be caused by decreased nucleosome occupancy (10). We found that HMBA did not alter histone H3 occupancy, and so our results cannot be explained by a general loss of histones at the 5' LTR.

Another explanation for the finding that HMBA increased DNA accessibility without typical increases of acetylation is that other modifications of histone structure render the proximal LTR susceptible to nuclease restriction. Recently, the potential of SWI/SNF complex to cause an ATP-dependent increase in histone exchange was demonstrated in yeast (8). Perhaps HMBA stimulates similar ATP-dependent remodeling. Although HMBA-mediated remodeling of the LTR Nuc-1 might be a downstream effect of HMBA action, its rapid onset of action makes this less likely.

The cellular effects of HMBA differ from other reagents that induce the expression of latent HIV. HDAC inhibitors do not alter the expression of activation markers on the surface lymphocytes (78), while prostratin enhances the expression of CD25 and CD69 (5). HMBA, however, down-regulates the activation marker CD25 and the proliferation marker Ki67 in PBMCs. Similar to prostratin, HMBA decreased expression of viral receptor CD4 but, unlike prostratin, only slightly affected the expression of viral coreceptors CXCR4 and CCR5. HDAC inhibitors do not enhance de novo HIV infection (78). Prostratin restricts viral entry and reverse transcription but facilitates integration of the HIV genome in nondividing CD4 T cells (5). Here, we found that HMBA significantly inhibits HIV-1 infection in PBMCs. Our data suggest that HMBA may interfere with completion of the other steps of the viral cycle. Remarkably, induction of the IL-2 receptor CD25 in PBMCs stimulated with PHA was abolished in the presence of HMBA (Fig. 6). This is reminiscent of the effect of antibodies against CD25 that block IL-2 signaling (46, 68).

As HMBA induces cell differentiation, it seems plausible that affected cellular regulatory factors might also induce HIV promoter activity. In a teratocarcinoma cell line, Zeichner and colleagues found evidence that factors binding to sites located at the HIV core promoter and enhancer played a role in the HIV LTR response to HMBA (47, 81).

In summary, our results indicate that HMBA is able to activate HIV LTR expression yet suppresses de novo infection. Other structurally related compounds such as polymethylene bisacetamide (55) and diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate demonstrate effects similar to the effect of HMBA on cell metabolism (56). Therefore, although HMBA itself might be too toxic for immediate clinical use (2), it may represent a prototype pharmacophore for reagents to approach the difficult goal of depleting the HIV-1 latent reservoir.

 
This study was supported by a VA Merit Award and National Institutes of Health grant RO1 AI-45297 to D.M.

We are grateful to F. Scott and J. Kramer for technical assistance, to HIV-seropositive volunteers for sample donation, and to H. Wise and J. Wagner for clinical coordination.

 
* Corresponding author. Mailing address: University of North Carolina at Chapel Hill, 3302 Michael Hooker Research Ctr., CB#7435, Chapel Hill, NC 27599-7435. Phone: (919) 966-6388. Fax: (919) 966-0584. E-mail: dmargo{at}med.unc.edu.

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Journal of Virology, May 2006, p. 4570-4579, Vol. 80, No. 9
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