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Can HIV Be Eradicated?
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"Histone Deacetylation Dampens HIV Expression in Activated CD4 Cells"
NEW YORK (Reuters Health) Oct 13 - Histone deacetylase (HDAC) -1, a chromatin-remodeling enzyme, appears to be important in mechanisms that lead to persistent quiescent reservoirs of HIV-infected resting CD4+ T cells, researchers report in the October 15th issue of The Journal of Infectious Diseases.
The findings, senior investigator Dr. David M. Margolis told Reuters Health, "suggest that natural mechanisms which restrict human gene expression also act to regulate HIV gene expression. This opens the door to specific therapeutic strategies which may enable us to root out virus that lies dormant in non-activated T cells, one of the most daunting obstacles to the eradication of HIV infection."
Dr. Margolis of the University of Texas Southwestern Medical Branch at Dallas and colleagues used four different polyamides that specifically block HDAC-1 associated processes to treat cultures of resting CD4+ T cells from HIV-infected donors.
Following this procedure, replication-competent HIV was recovered from cultures from six of eight patients whose viremia had been suppressed by therapy.
No HIV was recovered after exposure to control mismatched polyamides, but activation of T cells led to viral recovery in seven of these eight samples.
In further work, Dr. Margolis added that he and his colleagues investigated whether histone deacetylase inhibitors might be useful in strategies that disrupt latency without enhancing new infection. "We are now testing this idea in patients."
J Infect Dis Oct 15, 2004;190:1429-1437.
Polyamides Reveal a Role for Repression in Latency within Resting T Cells of HIV-Infected Donors
Loyda Ylisastigui,1 Jason J. Coull,1,a Victor C. Rucker,5,a Christian Melander,5,a Ronald J. Bosch,3 Scott J. Brodie,4,a Lawrence Corey,4 Donald L. Sodora,1 Peter B. Dervan,5 and David M. Margolis1,2
1University of Texas Southwestern Medical Center at Dallas, Department of Medicine, Division of Infectious Diseases, and 2North Texas Veterans Health Care Systems, Dallas; 3Harvard School of Public Health, Boston, Massachusetts; 4University of Washington, Department of Laboratory Medicine, Seattle; 5California Institute of Technology, Division of Chemistry and Chemical Engineering, Pasadena
"...Attempts have been made to purge the resting cell reservoir of HIV infection through the use of simultaneous immune activation and intensive ART...
...in our study... polyamides did not facilitate the outgrowth of latent HIV.
... it has recently been shown that IL-7 can induce the expression of latent HIV without inducing cellular proliferation...
... In the future, intensive but time-limited ART, if coupled with therapies designed to induce the expression of latent HIV, might allow the clearance of HIV infection...
Background. The persistence of human immunodeficiency virus (HIV) type 1 within resting CD4+ T cells poses a daunting therapeutic challenge. Histone deacetylase (HDAC)--1, a chromatin-remodeling enzyme that can mediate gene silencing, is recruited to the HIV-1 long terminal repeat by the host transcription factor LSF. Pyrrole-imidazole polyamides, small molecules that target specific DNA sequences, can access the nucleus of cells and specifically block transcription-factor binding.
Methods. We used polyamides to directly test the role of chromatin remodeling in HIV quiescence in primary resting CD4+ T cells obtained from HIV-infected patients.
Results. After exposure to any of 4 different polyamides that specifically block HDAC-1 recruitment by LSF to the HIV promoter, replication-competent HIV was recovered from cultures of resting CD4+ T cells in 6 of 8 HIV-infected patients whose viremia had been suppressed by therapy. In comparison, HIV was not recovered after exposure to control, mismatched polyamides but was recovered from 7 of 8 of these patients' samples after the activation of T cells.
Conclusions. We identify histone deacetylation as a mechanism that can dampen viral expression in infected, activated CD4+ T cells and establish a persistent, quiescent reservoir of HIV infection.
AUTHOR DISCUSSION
The present studies, which were performed directly in unmodified cells from HIV-infected patients, has shown that host factors within resting CD4+ T cells can restrict the expression of HIV in a biologically meaningful way. Pyrrole-imidazole polyamides are useful tools to modulate the DNA-binding activity of transcription factors, which allows mechanistic studies of the biological role of a targeted gene in living, unmanipulated cells. RCS-binding polyamides can inhibit LSF binding to the LTR in vitro and in vivo and increase the basal level of expression of the LTR. Using these molecules, we have shown that a mechanism of transcriptional repression is operational within primary lymphocytes; RCS-binding polyamides specifically allowed the expression of latent HIV within primary resting CD4+ cells obtained from HIV-infected donors. HDAC recruitment to the LTR must therefore play a role in the maintenance of viral quiescence within resting CD4+ T cells.
HIV outgrowth in cultures exposed to LTR-binding polyamides is not the result of nonspecific cell activation or the up-regulation of cellular genes—4 distinct polyamides targeted to 3 significantly different sequences within the RCS (figure 2) specifically activated LTR expression and allowed virus production from resting CD4+ cells, but 3 control, mismatched polyamides containing a single altered residue did not. It is important to recognize the sequence specificity of this effect, given that 3 of 4 binding polyamides targeted completely different sequences within the general region in which LSF binds, whereas the control nonbinding polyamides did not bind within the RCS, yet both groups of molecules bound at numerous sites within the genome. However, any single binding polyamide can induce viral expression.
Furthermore, polyamides do not nonspecifically improve the infection of allogeneic feeder cells added during the outgrowth phase of experiments or enhance the production of virus or proliferation of cells grown in IL-2, because the exposure of activated PBMCs to binding polyamides during HIV infection does not measurably alter viral output (data not shown). Additionally, the exposure of cells to polyamides has no effect on resting CD4+ cell morphology or surface-marker expression. We cannot exclude the possibility that IL-2 has a contributory effect to the ability of RCS-binding polyamides to induce viral outgrowth in resting cells from patients with HIV. However, despite the provision of activated target cells and IL-2, control polyamides did not facilitate the outgrowth of latent HIV.
Dudouet et al. recently measured the effect of 2 polyamides that target similar sequences that occur in the genome every 〜2000 bp. Global gene-expression analysis of 〜18,000 genes found that these polyamides affected 21 genes: 11 genes were inhibited by 1 of the polyamides, and 10 genes were inhibited by both polyamides; however, no genes were activated. The largest change observed was a 2.7-fold down-regulation. Although subtle effects of polyamides on the host cell cannot be ruled out, HIV outgrowth was allowed by 4 RCS-binding polyamides recognizing 3 unique sequences but not by 3 control polyamides of similar composition but slightly altered sequence specificity, and this is not likely to have been due to the modulation of host gene expression.
Our results clearly illustrate a role for transcriptional repression in the regulation of HIV expression. It is not yet clear whether inhibition of the HDAC function in vivo will be sufficient to induce the expression of latent HIV. HDAC inhibition augments HIV LTR expression in cell line models of latency [9]. Recently, in assays similar to those described here, our laboratory found that replication-competent HIV can be recovered from cells from patients after ex vivo exposure to an HDAC inhibitor.
Attempts have been made to purge the resting cell reservoir of HIV infection through the use of simultaneous immune activation and intensive ART. Subsequent modeling studies have suggested that, under such conditions, viral replication might be activated beyond the threshold that can be contained by ART. It may be difficult to use therapies to purge the resting cell reservoir that induce nonspecific T cell activation without unintended deleterious consequences. In a thymic explant model, it has recently been shown that IL-7 can induce the expression of latent HIV without inducing cellular proliferation.
Current ART is not yet potent enough to block all active HIV replication, but, with improvements in ART, this goal might be achieved. In the future, intensive but time-limited ART, if coupled with therapies designed to induce the expression of latent HIV, might allow the clearance of HIV infection.
INTRODUCTION
Multiple mechanisms contribute to persistence of HIV infection, despite host immunity and potent antiretroviral therapy (ART). A rare population of infected resting memory CD4+ cells T is one important contributor to the persistence of HIV infection. Years of continuous ART may be required to eliminate this reservoir of virus, given that the half-life of HIV within resting CD4+ T cells may be 44 months. Improvements in therapy are unlikely to alter the stability of this reservoir. The mechanisms that allow the provirus to establish latency in resting CD4+ T cells are unknown but may result if an infected activated cell is able to return to the resting state.
HIV gene expression may be ineffective without sufficient cellular or viral factors to allow the efficient initiation, elongation, or transport of HIV mRNA, a trans mechanism of latency. The chromatin environment into which an HIV provirus integrates may contribute to quiescence in cis. Acetylated histones allow activation by NF-κB. Histone deacetylase (HDAC) inhibitors augment the expression of HIV, which implies a role for HDAC in the establishment or maintenance of proviral quiescence. Jordan et al. found that integration sites of cell lines with quiescent HIV were frequently found in or close to alphoid repeat elements in heterochromatin. In contrast, integration in or near heterochromatin is disfavored in productive infection. Another recent analysis showed that 69% of integration sites occur in actively transcribed genes. Latency might also result after integration by the subsequent formation of heterochromatin about the provirus.
The host factors LSF and YY1 recruit HDAC-1 to a long-terminal repeat (LTR) site, the repressor complex sequence. HDAC1 recruitment can deacetylate the adjacent nucleosome 1 of the LTR and inhibit Tat activation, LTR expression, and viral production. If HDAC recruitment contributes to HIV proviral quiescence, a blockade of repression might allow the outgrowth of HIV from latently infected resting CD4+ T cells.
No available experimental system recapitulates all aspects of quiescent HIV infection within primary resting CD4+ T cells. Latency in cell-line models may be due to unique characteristics selected in culture. Recently, HIV latency has been modeled in SCID-hu mice with transplanted thymic grafts. Findings in this model system have suggested that specific signals may activate viral outgrowth without inducing T cell proliferation.
Studies of HIV latency have been performed in resting lymphocytes isolated from HIV-infected volunteers, but the mechanisms that regulate latency have not been fully described. Pyrrole-imidazole polyamides, synthetic molecules that contain N-methylpyrrole and N-methylimidazole amino acids that are capable of binding to specific DNA sequences with affinities comparable to those of natural DNA-binding transcriptional regulatory proteins, can modulate gene expression. We demonstrated that LTR-binding polyamides targeted to the repressor complex sequence block late SV40 transcription factor (LSF) binding, decrease the occupancy of LSF, increase histone H4 acetylation at the integrated LTR, and increase LTR expression in model systems of quiescent HIV infection (D.M.M., unpublished data). Of note, these effects depend on the specific binding sequence recognized by LTR-binding polyamides, because none of these effects are seen with control polyamides that differ from LTR-binding polyamides by a single residue and do not bind the LTR within the repressor complex sequence.
To test the relevance of transcriptional repression in the regulation of latent HIV, we tested the effect of polyamides on the expression of HIV in resting CD4+ cells obtained from HIV-infected donors. Polyamides targeted to the repressor complex sequence specifically allowed HIV outgrowth from these cells. Mechanisms that recruit HDAC to the integrated HIV promoter may thus contribute to the regulation of expression of HIV. In agreement with the results of Brooks et al, our findings suggest that the expression of quiescent provirus may be induced without global T cell activation.
EXCERPTS FROM RESULTS
HIV was recovered from the cells from 22 donors when cells were treated with 1 or 2 RCS-binding polyamides as frequently as when they were treated with PHA. HIV was recovered more frequently when cultures were exposed to PHA or polyamides 1, 2, 3, or 4 than when they were unstimulated or treated with the mismatch polyamides M1, M2, or M3/4 (P < .001).
Outgrowth of latent HIV from the resting CD4+ T cells of persistently aviremic HIV-infected donors treated with highly active ART after exposure to RCS-binding polyamides. To avoid these confounding factors, we then studied outgrowth of HIV from resting CD4+ cells obtained from aviremic HIV-infected donors who were receiving optimal ART. In 5 subjects without detectable viremia (<50 copies HIV-1 RNA/mL), replication-competent HIV was never detected in control cultures. HIV was detected in 1 of 5 samples from patient after activation with PHA and in that patient's sample after exposure to RCS-binding polyamides. These findings suggest that binding polyamides could allow the outgrowth of latent HIV.
However, given the infrequency of latently infected cells, we harvested larger numbers of cells by leukopheresis of samples from HIV-infected donors in whom plasma levels of HIV-1 RNA had been durably suppressed (>6 months) to <50 copies/mL; 10--20 × 109 lymphocytes were obtained from each patient, and 200--500 × 106 resting CD4+ cells were isolated. Recently infected cells are rare in such donors. To further reduce the likelihood of the outgrowth of rare circulating or preintegrated virus, cells were maintained for 2--3 days in the presence of both an HIV integrase inhibitor and an HIV reverse-transcriptase inhibitor with which the donor had not been treated. This additional procedure prolongs the ex vivo manipulation of cells without growth factors or stimulation and is likely to have decreased the recovery of HIV in our assays. It thus increases the confidence that the HIV detected represents replication-competent, preintegrated HIV. Allogeneic CD8+-depleted PBMCs obtained from an HIV-seronegative donor were then added, to allow the amplification and detection of rare replication-competent provirus in the resting cells of these aviremic patients.
The results of limiting-dilution outgrowth assays performed in 8 subjects (table 2) confirmed that only specific RCS-binding polyamides allow viral outgrowth from the resting cell reservoir, whereas control polyamides that contain a single altered residue do not. Replication-competent HIV was recovered from resting CD4+ cells in 6 of 8 samples from patients after exposure to polyamides that bind the RCS and in 7 of 8 patient samples exposed to mitogen. There was no observable difference in the frequency of viral outgrowth between binding polyamides; the largest number of experiments was performed by use of polyamide 2. In every sample from which HIV outgrowth was induced by exposure to binding polyamides, virus was also recovered after T cell activation. Outgrowth curves from 4 representative donors from whom virus was recovered from equivalent cell inputs are shown. The outgrowth of virus was generally observed during the second or third week of culture. Outgrowth in the presence of IL-2 alone or control polyamides with IL-2 was never observed. Control cultures remained without viral outgrowth for up to 2 weeks after the detection of virus in polyamide-treated or -activated cultures or for up to 28 days in culture.
Under these conditions, HIV outgrowth was rare. From each subject, 40--80 × 106 resting CD4+ cells were assayed in up to 100 individual limiting-dilution cultures. Replication-competent virus was recovered in, at most, 10 cultures/patient.
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