Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the LTR.

Macrophages are infected by HIV-1 in vivo and contribute to both viral spread and pathogenesis. Recent human and animal studies suggest that HIV-1-infected macrophages serve as a reservoir that contributes to HIV-1 persistence during anti-retroviral therapy. The ability of macrophages to serve as persistent viral reservoirs is likely influenced by the local tissue microenvironment, including interactions with pathogenic and commensal microbes. Here we show that the sexually transmitted pathogen Neisseria gonorrhoeae (GC) and the gut-associated microbe Escherichia coli (E. coli), which encode ligands for both Toll-like receptor 2 (TLR2) and TLR4, repressed HIV-1 replication in macrophages and thereby induced a state reminiscent of viral latency. This repression was mediated by signaling through TLR4 and the adaptor protein TRIF and was associated with increased production of type I interferons. Inhibiting TLR4 signaling, blocking type 1 interferon, or knocking-down TRIF reversed LPS- and GC-mediated repression of HIV-1. Finally, the repression of HIV-1 in macrophages was associated with the recruitment of interferon regulatory factor 8 (IRF8) to the interferon stimulated response element (ISRE) downstream of the 5' HIV-1 long terminal repeat (LTR). Our data indicate that IRF8 is responsible for repression of HIV-1 replication in macrophages in response to TRIF-dependent signaling during GC and E. coli co-infection. These findings highlight the potential role of macrophages as HIV-1 reservoirs as well as the role of the tissue microenvironment and co-infections as modulators of HIV-1 persistence.IMPORTANCE The major barrier toward the eradication of HIV-1 infection is the presence of a small reservoir of latently infected cells, which include CD4+ T cells and macrophages that escape immune-mediated clearance and the effects of anti-retroviral therapy. There remain crucial gaps in our understanding of the molecular mechanisms that lead to transcriptionally silent or latent HIV-1 infection of macrophages. The significance of our research is in identifying microenvironmental factors, such as commensal and pathogenic microbes, that can contribute to the establishment and maintenance of latent HIV-1 infection in macrophages. It is hoped that identifying key processes contributing to HIV-1 persistence in macrophages may ultimately lead to novel therapeutics to eliminate latent HIV-1 reservoirs in vivo.

Blimp-1, an intrinsic factor that represses HIV-1 proviral transcription in memory CD4+ T cells.

CD4(+) T cell subsets differentially support HIV-1 replication. For example, quiescent CD4(+) memory T cells are susceptible to HIV-1 infection but do not support robust HIV-1 transcription and have been implicated as the primary reservoir of latent HIV-1. T cell transcription factors that regulate maturation potentially limit HIV-1 transcription and mediate the establishment and maintenance of HIV-1 latency. We report that B lymphocyte-induced maturation protein-1 (Blimp-1), a critical regulator of B and T cell differentiation, is highly expressed in memory CD4(+) T cells compared with naive CD4(+) T cells and represses basal and Tat-mediated HIV-1 transcription. Blimp-1 binds an IFN-stimulated response element within HIV-1 provirus, and it is displaced following T cell activation. Reduction of Blimp-1 in infected primary T cells including CD4(+) memory T cells increases RNA polymerase II processivity, histone acetylation, and baseline HIV-1 transcription. Therefore, the transcriptional repressor, Blimp-1, is an intrinsic factor that predisposes CD4(+) memory T cells to latent HIV-1 infection.

Nuclear receptor signaling inhibits HIV-1 replication in macrophages through multiple trans-repression mechanisms.

Sexually transmitted pathogens activate HIV-1 replication and inflammatory gene expression in macrophages through engagement of Toll-like receptors (TLRs). Ligand-activated nuclear receptor (NR) transcription factors, including glucocorticoid receptor (GR), peroxisome proliferator-activated receptor gamma (PPARγ), and liver X receptor (LXR), are potent inhibitors of TLR-induced inflammatory gene expression. We therefore hypothesized that ligand-activated NRs repress both basal and pathogen-enhanced HIV-1 replication in macrophages by directly repressing HIV-1 transcription and by ameliorating the local proinflammatory response to pathogens. We show that the TLR2 ligand PAM3CSK4 activated virus transcription in macrophages and that NR signaling repressed both basal and TLR-induced HIV-1 transcription. NR ligand treatment repressed HIV-1 expression when added concurrently with TLR ligands and in the presence of cycloheximide, demonstrating that they act independently of new cellular gene expression. We found that treatment with NR ligands inhibited the association of AP-1 and NF-κB subunits, as well as the coactivator CBP, with the long terminal repeat (LTR). We show for the first time that the nuclear corepressor NCoR is bound to HIV-1 LTR in unstimulated macrophages and is released from the LTR after TLR engagement. Treatment with PPARγ and LXR ligands, but not GR ligands, prevented this TLR-induced clearance of NCoR from the LTR. Our data demonstrate that both classical and nonclassical trans-repression mechanisms account for NR-mediated HIV-1 repression. Finally, NR ligand treatment inhibited the potent proinflammatory response induced by PAM3CSK4 that would otherwise activate HIV-1 expression in infected cells. Our findings provide a rationale for studying ligand-activated NRs as modulators of basal and inflammation-induced HIV-1 replication.

PPARgamma and LXR signaling inhibit dendritic cell-mediated HIV-1 capture and trans-infection.

Dendritic cells (DCs) contribute to human immunodeficiency virus type 1 (HIV-1) transmission and dissemination by capturing and transporting infectious virus from the mucosa to draining lymph nodes, and transferring these virus particles to CD4+ T cells with high efficiency. Toll-like receptor (TLR)-induced maturation of DCs enhances their ability to mediate trans-infection of T cells and their ability to migrate from the site of infection. Because TLR-induced maturation can be inhibited by nuclear receptor (NR) signaling, we hypothesized that ligand-activated NRs could repress DC-mediated HIV-1 transmission and dissemination. Here, we show that ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor (LXR) prevented proinflammatory cytokine production by DCs and inhibited DC migration in response to the chemokine CCL21 by preventing the TLR-induced upregulation of CCR7. Importantly, PPARgamma and LXR signaling inhibited both immature and mature DC-mediated trans-infection by preventing the capture of HIV-1 by DCs independent of the viral envelope glycoprotein. PPARgamma and LXR signaling induced cholesterol efflux from DCs and led to a decrease in DC-associated cholesterol, which has previously been shown to be required for DC capture of HIV-1. Finally, both cholesterol repletion and the targeted knockdown of the cholesterol transport protein ATP-binding cassette A1 (ABCA1) restored the ability of NR ligand treated cells to capture HIV-1 and transfer it to T cells. Our results suggest that PPARgamma and LXR signaling up-regulate ABCA1-mediated cholesterol efflux from DCs and that this accounts for the decreased ability of DCs to capture HIV-1. The ability of NR ligands to repress DC mediated trans-infection, inflammation, and DC migration underscores their potential therapeutic value in inhibiting HIV-1 mucosal transmission.

RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement.

Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603-607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837-847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-alpha, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.

FcγRIIB regulation of BCR/TLR-dependent autoreactive B-cell responses.

Crosslinking of Fc γ receptor II B (FcγRIIB) and the BCR by immune complexes (IC) can downregulate antigen-specific B-cell responses. Accordingly, FcγRIIB deficiencies have been associated with B-cell hyperactivity in patients with systemic lupus erythematosus and mouse models of lupus. However, we have previously shown that murine IgG2a-autoreactive AM14 B cells respond robustly to chromatin-associated IC through a mechanism dependent on both the BCR and the endosomal TLR9, despite FcγRIIB coexpression. To further evaluate the potential contribution of FcγRIIB to the regulation of autoreactive B cells, we have now compared the IC-triggered responses of FcγRIIB-deficient and FcγRIIB-sufficient AM14 B cells. We find that FcγRIIB-deficient cells respond significantly better than FcγRIIB-sufficient cells when stimulated with DNA IC that incorporate low-affinity TLR9 ligand (CG-poor dsDNA fragments). AM14 B cells also respond to RNA-associated IC through BCR/TLR7 coengagement, but such BCR/TLR7-dependent responses are normally highly dependent on IFN-α costimulation. However, we now show that AM14 FcγRIIB(-/-) B cells are very effectively activated by RNA IC without supplemental IFN-α priming. These results demonstrate that FcγRIIB can effectively modulate both BCR/TLR9 and BCR/TLR7 endosomal-dependent activation of autoreactive B cells.

Requirement for DNA CpG content in TLR9-dependent dendritic cell activation induced by DNA-containing immune complexes.

Although TLR9 was originally thought to specifically recognize microbial DNA, it is now evident that mammalian DNA can be an effective TLR9 ligand. However, the DNA sequence required for TLR9 activation is controversial, as studies have shown conflicting results depending on the nature of the DNA backbone, the route of DNA uptake, and the cell type being studied. In systemic lupus erythematosus, a major route whereby DNA gains access to intracellular TLR9, and thereby activates dendritic cells (DCs), is through uptake as a DNA-containing immune complex. In this report, we used defined dsDNA fragments with a natural (phosphodiester) backbone and show that unmethylated CpG dinucleotides within dsDNA are required for murine DC TLR9 activation induced by a DNA-containing immune complex. The strongest activation is seen with dsDNA fragments containing optimal CpG motifs (purine-purine-CpG-pyrimidine-pyrimidine) that are common in microbial DNA but rare in mammalian DNA. Importantly, however, activation can also be induced by CpG-rich DNA fragments that lack these optimal CpG motifs and that we show are plentiful in CpG islands within mammalian DNA. No activation is induced by DNA fragments lacking CpG dinucleotides, although this CpG-free DNA can induce DC activation if internalized by liposomal transfection instead of as an immune complex. Overall, the data suggest that the release of CpG-rich DNA from mammalian DNA may contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and psoriasis in which activation of TLR9 in DCs by self DNA has been implicated in disease pathogenesis.

Monoallelic IRF5 deficiency in B cells prevents murine lupus.

Gain-of-function polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. We now show that monoallelic IRF5 deficiency in B cells markedly reduced disease in a murine lupus model. In contrast, similar reduction of IRF5 expression in macrophages, monocytes, and neutrophils did not reduce disease severity. B cell receptor and TLR7 signaling synergized to promote IRF5 phosphorylation and increase IRF5 protein expression, with these processes being independently regulated. This synergy increased B cell-intrinsic IL-6 and TNF-α production, both key requirements for germinal center (GC) responses, with IL-6 and TNF-α production in vitro and in vivo being substantially lower with loss of 1 allele of IRF5. Mechanistically, TLR7-dependent IRF5 nuclear translocation was reduced in B cells from IRF5-heterozygous mice. In addition, we show in multiple lupus models that IRF5 expression was dynamically regulated in vivo with increased expression in GC B cells compared with non-GC B cells and with further sequential increases during progression to plasmablasts and long-lived plasma cells. Overall, a critical threshold level of IRF5 in B cells was required to promote disease in murine lupus.

Autoreactive B cells discriminate CpG-rich and CpG-poor DNA and this response is modulated by IFN-alpha.

Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs) through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a-reactive B cells. In contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR crosslinking alone is insufficient to activate low-affinity autoreactive B cells. Importantly, priming B cells with IFN-alpha lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Taken together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-alpha can contribute to the pathogenesis of systemic lupus erythematosus.

Toll-like receptor-dependent immune complex activation of B cells and dendritic cells.

High titers of autoantibodies reactive with DNA/RNA molecular complexes are characteristic of autoimmune disorders such as systemic lupus erythematosus (SLE). In vitro and in vivo studies have implicated Toll-like receptor 9 (TLR9) and Toll-like receptor 7 (TLR7) in the activation of the corresponding autoantibody producing B cells. Importantly, TLR9/TLR7-deficiency results in the inability of autoreactive B cells to proliferate in response to DNA/RNA-associated autoantigens in vitro, and in marked changes in the autoantibody repertoire of autoimmune-prone mice. Uptake of DNA/RNA-associated autoantigen immune complexes (ICs) also leads to activation of dendritic cells (DCs) through TLR9 and TLR7.The initial studies from our lab involved ICs formed by a mixture of autoantibodies and cell debris released from dying cells in culture. To better understand the nature of the mammalian ligands that can effectively activate TLR7 and TLR9, we have developed a methodology for preparing ICs containing defined DNA fragments that recapitulate the immunostimulatory activity of the previous "black box" ICs. These reagents reveal an important role for nucleic acid sequence, even when the ligand is mammalian DNA.

DNA and RNA autoantigens as autoadjuvants.

AM14 B cells are a prototype for those low affinity autoreactive B cells that routinely mature as naïve cells in peripheral lymphoid tissues. These cells express a transgene-encoded receptor specific for IgG2a and can be effectively activated by immune complexes that incorporate either mammalian DNA or mammalian RNA that has been released from dead or dying cells. Activation depends on the ability of the B-cell receptor to deliver antigen to an internal vesicular compartment containing either Toll-like receptor-9 (TLR9) or TLR7. Since TLR9 and TLR7 are thought to recognize microbial DNA and RNA preferentially, it is important to determine under what conditions mammalian DNA and RNA become effective TLR ligands, and whether the determining factor is delivery or structure. This issue has been addressed by using IgG2a mAbs to deliver immune complexes preloaded with defined fragments of DNA or RNA, or by using modified ODNs/ORNs. The data demonstrate that only certain nucleic acid sequences or structures can induce autoreactive B-cell proliferation, even when delivery to the appropriate TLR compartment is facilitated by uptake through the B-cell receptor (BCR).

Toll-Like Receptor-Dependent Immune Complex Activation of B Cells and Dendritic Cells.

High titers of autoantibodies reactive with DNA/RNA molecular complexes are characteristic of autoimmune disorders such as systemic lupus erythematosus (SLE). In vitro and in vivo studies have implicated the endosomal Toll-like receptor 9 (TLR9) and Toll-like receptor 7 (TLR7) in the activation of the corresponding autoantibody producing B cells. Importantly, TLR9/TLR7-deficiency results in the inability of autoreactive B cells to proliferate in response to DNA/RNA-associated autoantigens in vitro, and in marked changes in the autoantibody repertoire of autoimmune-prone mice. Uptake of DNA/RNA-associated autoantigen immune complexes (ICs) also leads to activation of dendritic cells (DCs) through TLR9 and TLR7. The initial studies from our lab involved ICs formed by a mixture of autoantibodies and cell debris released from dying cells in culture. To better understand the nature of the mammalian ligands that can effectively activate TLR7 and TLR9, we have developed a methodology for preparing ICs containing defined DNA fragments that recapitulate the immunostimulatory activity of the previous "black box" ICs. As the endosomal TLR7 and TLR9 function optimally from intracellular acidic compartments, we developed a facile methodology to monitor the trafficking of defined DNA ICs by flow cytometry and confocal microscopy. These reagents reveal an important role for nucleic acid sequence, even when the ligand is mammalian DNA and will help illuminate the role of IC trafficking in the response.

Porphyromonas gingivalis-mediated signaling through TLR4 mediates persistent HIV infection of primary macrophages.

Periodontal infections contribute to HIV-associated co-morbidities in the oral cavity and provide a model to interrogate the dysregulation of macrophage function, inflammatory disease progression, and HIV replication during co-infections. We investigated the effect of Porphyromonas gingivalis on the establishment of HIV infection in monocyte-derived macrophages. HIV replication in macrophages was significantly repressed in the presence of P. gingivalis. This diminished viral replication was due partly to a decrease in the expression of integrated HIV provirus. HIV repression depended upon signaling through TLR4 as knock-down of TLR4 with siRNA rescued HIV expression. Importantly, HIV expression was reactivated upon removal of P. gingivalis. Our observations suggest that exposure of macrophages to Gram-negative bacteria influence the establishment and maintenance of HIV persistence in macrophages through a TLR4-dependent mechanism.

Comparison of CpG s-ODNs, chromatin immune complexes, and dsDNA fragment immune complexes in the TLR9-dependent activation of rheumatoid factor B cells.

Synthetic single-stranded oligodeoxynucleotides (15-30 bp) containing CpG motifs and phosphorothioate backbones (CpG s-ODN), immune complexes consisting of anti-nucleosome mAbs and mammalian chromatin (chromatin IC), and immune complexes consisting of anti-hapten mAbs and haptenated-double stranded DNA fragments ( approximately 600 bp) can all effectively stimulate transgenic B cells expressing a rheumatoid factor receptor by a TLR9-dependent process. However, differential sensitivity to both s-ODN and small molecule inhibitors suggests that stimulatory CpG sODN and chromatin IC may either access TLR9 via different routes or depend on discrete activation parameters. These data have important implications regarding the therapeutic application of TLR9 inhibitors to the treatment of systemic autoimmune diseases.

Interleukin 1 beta and interleukin 6 potentiate retinoic acid-mediated repression of human immunodeficiency virus type 1 replication in macrophages.

All-trans-retinoic acid (RA) has been shown either to activate or repress human immunodeficiency virus type 1 (HIV-1) replication in primary monocyte-derived-macrophages (MDMs). We systematically investigated the contribution that cell donor and virus differences make to this variability. We found that the effect of RA was cell donor dependent. In addition, the ability of RA to repress HIV-1 replication varied between different virus stocks. In no case did RA affect either virus entry or integration but instead affected the accumulation of viral mRNAs in infected cells. Despite the complex variability in RA responsiveness in untreated cells, we found that RA consistently repressed virus replication when the MDMs were treated with concentrations of interleukin 1 beta (IL-1 beta) and IL-6 that are expected at local sites of infection, where HIV-1-infected macrophages reside in vivo.

The stimulation of Toll-like receptors by nuclear antigens: a link between apoptosis and autoimmunity.

As immunologists have long understood, effective responses to foreign antigens require adjuvants. It is now apparent that the initiation of autoimmune disease is comparably facilitated by adjuvant activity. In the case of antinuclear antibodies, it seems that DNA itself can serve as an endogenous adjuvant. Similar to many of the microbial adjuvants, mammalian DNA mediates its effect through a Toll-like receptor--in this case, TLR9.

Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

Toll-like receptors, endogenous ligands, and systemic autoimmune disease.

The critical role of Toll-like receptors (TLRs) as mediators of pathogen recognition by the innate immune system is now firmly established. Such recognition results in the initiation of an inflammatory immune response and subsequent instruction of the adaptive immune system, both of which are designed to rid the host of the invading pathogen. More controversial is the potential role of TLRs in the recognition of endogenous ligands and what effect this might have on the consequent development of autoimmune or other chronic sterile inflammatory disorders. An increasing number of studies implicate TLRs as being involved in the immune response to self-molecules that have in some way been altered from their native state or accumulate in non-physiologic sites or amounts, although questions have been raised about possible contaminants in certain of these studies. In this review, we discuss the evidence for endogenous ligand-TLR interactions with particular emphasis on mammalian chromatin, systemic lupus erythematosus, and atherosclerosis. Overall, the data support the general concept of a role for TLRs in the recognition of endogenous ligands. However, the precise details of the interactions and the extent to which they may contribute to the pathogenesis of human disease remain to be clarified.

Retinoid-dependent restriction of human immunodeficiency virus type 1 replication in monocytes/macrophages.

Vitamin A deficiency has been correlated with increased severity of human immunodeficiency virus type 1 (HIV-1)-associated disease. Moreover, vitamin A supplementation can reduce AIDS-associated morbidity and mortality. Our group and others have shown that retinoids, the bioactive metabolites of vitamin A, repress HIV-1 replication in monocytic cell lines and primary macrophages by blocking long-terminal-repeat (LTR)-directed transcription. Based on these studies, we hypothesize that retinoids are natural repressors of HIV-1 in vivo. We show here that all-trans-retinoic acid (RA)-mediated repression of HIV-1 activation requires pretreatment for at least 12 h and is blocked by the protein synthesis inhibitors cycloheximide and puromycin. Studies of the kinetics of RA-mediated repression in U1 cells and primary monocyte-derived macrophages (MDMs) reveal that the repressive effects of RA on HIV-1 expression are long-lasting but reversible. We demonstrate that HIV-1 expression is activated when U1 cells or MDMs are cultured in retinoid-free synthetic medium and show that physiological concentrations of RA repress this activation. In addition, the synthetic pan-retinoic acid receptor antagonist BMS-204 493 activates HIV-1 replication in U1 cells in a dose-dependent manner, suggesting that RA-induced transactivation of cellular gene expression is required for HIV-1 repression. Together, these data support the hypothesis that retinoids present in tissue culture media in vitro and serum in vivo maintain HIV-1 in a transcriptionally repressed state in monocytes/macrophages.

Retinoic acid inhibition of chromatin remodeling at the human immunodeficiency virus type 1 promoter. Uncoupling of histone acetylation and chromatin remodeling.

All-trans retinoic acid (RA) represses HIV-1 transcription and replication in cultured monocytic cells and in primary monocyte-derived macrophages. Here we examine the role of histone acetylation and chromatin remodeling in RA-mediated repression. RA pretreatment of latently infected U1 promonocytes inhibits HIV-1 expression in response to the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). TSA is thought to activate HIV-1 transcription by inducing histone hyperacetylation within a regulatory nucleosome, nuc-1, positioned immediately downstream from the transcription start site. Acetylation of nuc-1 is thought to be a critical step in activation that precedes nuc-1 remodeling and, subsequently, transcriptional initiation. Here we demonstrate that TSA treatment induces H3 and H4 hyperacetylation and nuc-1 remodeling. Although RA pretreatment inhibits nuc-1 remodeling and HIV-1 transcription, it has no effect on histone acetylation. This suggests that acetylation and remodeling are not obligatorily coupled. We also show that growth of U1 cells in retinoid-deficient medium induces nuc-1 remodeling and HIV-1 expression but does not induce histone hyperacetylation. These findings suggest that remodeling, not histone hyperacetylation, is the limiting step in transcriptional activation in these cells. Together, these data suggest that RA signaling maintains the chromatin structure of the HIV-1 promoter in a transcriptionally non-permissive state that may contribute to the establishment of latency in monocyte/macrophages.