Olivier Rohr

Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing.

Following entry and reverse transcription, the HIV‐1 genome is integrated into the host genome. In contrast to productively infected cells, latently infected cells frequently harbor HIV‐1 genomes integrated in heterochromatic structures, allowing persistence of transcriptionally silent proviruses. Microglial cells are the main HIV‐1 target cells in the central nervous system and constitute an important reservoir for viral pathogenesis. In the present work, we show that, in microglial cells, the co‐repressor COUP‐TF interacting protein 2 (CTIP2) recruits a multienzymatic chromatin‐modifying complex and establishes a heterochromatic environment at the HIV‐1 promoter. We report that CTIP2 recruits histone deacetylase (HDAC)1 and HDAC2 to promote local histone H3 deacetylation at the HIV‐1 promoter region. In addition, DNA‐bound CTIP2 also associates with the histone methyltransferase SUV39H1, which increases local histone H3 lysine 9 methylation. This allows concomitant recruitment of HP1 proteins to the viral promoter and formation of local heterochromatin, leading to HIV‐1 silencing. Altogether, our findings uncover new therapeutic opportunities for purging latent HIV‐1 viruses from their cellular reservoirs.

Regulation of HIV‐1 gene transcription: from lymphocytes to microglial cells

Transcription is a crucial step for human immunodeficiency virus type 1 (HIV‐1) expression in all infected host cells, from T lymphocytes, thymocytes, monocytes, macrophages, and dendritic cells in the immune system up to microglial cells in the central nervous system. To maximize its replication, HIV‐1 adapts transcription of its integrated proviral genome by ideally exploiting the specific cellular environment and by forcing cellular stimulatory events and impairing transcriptional inhibition. Multiple cell type‐specific interplays between cellular and viral factors perform the challenge for the virus to leave latency and actively replicate in a great diversity of cells, despite the variability of its long terminal repeat region in different HIV strains. Knowledge about the molecular mechanisms underlying transcriptional regulatory events helps in the search for therapeutic agents that target the step of transcription in anti‐HIV strategies.

Molecular mechanisms of HIV-1 persistence in the monocyte-macrophage lineage

The introduction of the highly active antiretroviral therapy (HAART) has greatly improved survival. However, these treatments fail to definitively cure the patients and unveil the presence of quiescent HIV-1 reservoirs like cells from monocyte-macrophage lineage. A purge, or at least a significant reduction of these long lived HIV-1 reservoirs will be needed to raise the hope of the viral eradication. This review focuses on the molecular mechanisms responsible for viral persistence in cells of the monocyte-macrophage lineage. Controversy on latency and/or cryptic chronic replication will be specifically evoked. In addition, since HIV-1 infected monocyte-macrophage cells appear to be more resistant to apoptosis, this obstacle to the viral eradication will be discussed. Understanding the intimate mechanisms of HIV-1 persistence is a prerequisite to devise new and original therapies aiming to achieve viral eradication.

An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

Histone methyltransferase inhibitors induce HIV-1 recovery in resting CD4+ T cells from HIV-1-infected HAART-treated patients.

Objective:Reactivation of HIV-1 expression in persistent reservoirs together with an efficient HAART has been proposed as an adjuvant therapy aimed at reaching a functional cure for HIV. Previously, H3K9 methylation was shown to play a major role in chromatin-mediated repression of the HIV-1 promoter. Here, we evaluated the therapeutic potential of histone methyltransferase inhibitors (HMTIs) in reactivating HIV-1 from latency. Design:We evaluated the reactivation potential of two specific HMTIs (chaetocin and BIX-01294, two specific inhibitors of Suv39H1 and G9a, respectively) in ex-vivo cultures of resting CD4+ T cells isolated from HIV-1-infected HAART-treated individuals. Methods:We measured HIV-1 recovery in ex-vivo cultures treated with an HMTI alone or in combination with other HIV-1 inducers (in absence of IL-2 and of allogenic stimulation) of CD8+-depleted peripheral blood mononuclear cells (PBMCs) or of resting CD4+ T cells isolated from 67 HIV-infected, HAART-treated patients with undetectable viral load. Results:We demonstrated, for the first time, that chaetocin induced HIV-1 recovery in 50% of CD8+-depleted PBMCs cultures and in 86% of resting CD4+ T-cell cultures isolated from HIV-1-infected, HAART-treated patients, whereas BIX-01294 reactivated HIV-1 expression in 80% of resting CD4+ T-cell cultures isolated from similar patients. Moreover, we showed that combinatory treatments including one HMTI and either the histone deacetylase inhibitor suberoylanilide hydroxamic acid or the non-tumor-promoting NF-&kgr;B inducer prostratin had a higher reactivation potential than these compounds alone. Conclusion:Our results constitute a proof-of-concept for the therapeutic potential of HMTIs in strategies aiming at reducing the pool of latent reservoirs in HIV-infected, HAART-treated patient.

p21(WAF1) gene promoter is epigenetically silenced by CTIP2 and SUV39H1.

Mainly regulated at the transcriptional level, the cellular cyclin-dependent kinase inhibitor, CDKN1A/p21WAF1 (p21), is a major cell cycle regulator of the response to DNA damage, senescence and tumor suppression. Here, we report that COUP-TF-interacting protein 2 (CTIP2), recruited to the p21 gene promoter, silenced p21 gene transcription through interactions with histone deacetylases and methyltransferases. Importantly, treatment with the specific SUV39H1 inhibitor, chaetocin, repressed histone H3 lysine 9 trimethylation at the p21 gene promoter, stimulated p21 gene expression and induced cell cycle arrest. In addition, CTIP2 and SUV39H1 were recruited to the silenced p21 gene promoter to cooperatively inhibit p21 gene transcription. Induction of p21WAF1 gene upon human immunodeficiency virus 1 (HIV-1) infection benefits viral expression in macrophages. Here, we report that CTIP2 further abolishes Vpr-mediated stimulation of p21, thereby indirectly contributing to HIV-1 latency. Altogether, our results suggest that CTIP2 is a constitutive p21 gene suppressor that cooperates with SUV39H1 and histone methylation to silence the p21 gene transcription.

COUP-TF and Sp1 interact and cooperate in the transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat in human microglial cells.

We have recently reported that chicken ovalbumin upstream promoter transcription factor (COUP-TF) activates human immunodeficiency virus type 1 (HIV-1) gene transcription in glial and neuronal cells. Here, we have examined the role of COUP-TF in microglial cells, the major target cells for HIV-1 infection in brain. We show that COUP-TF activates gene expression from both the lymphotropic LAI and the macrophage-tropic JR-FL HIV-1 strains. Although COUP-TF binds to the −352/−320 nuclear receptor responsive element of the long terminal repeat, it functions as a transcriptional activator by acting on the −68/+29 minimal promoter. This region is a direct target of transcription factors Sp1 and Sp3. We report the discovery and features of a physical and functional interplay between COUP-TF and Sp1. Our cotransfection experiments provide evidence for a functional synergism between Sp1 and COUP-TF leading to enhanced transcriptional activity of the HIV-1 long terminal repeat through the Sp1 element. In contrast, Sp3 functions as a repressor of Sp1- or COUP-TF-induced activation. We further demonstrate that COUP-TF and Sp1 are capable of physically interacting, via the DNA-binding domain of COUP-TF, in vitro and in the cell. These findings reveal how the novel interplay of Sp1 and COUP-TF families of transcription factors regulate HIV-1 gene expression.

COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415–5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1α, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region.

HIV-1 regulation of latency in the monocyte-macrophage lineage and in CD4+ T lymphocytes

The introduction in 1996 of the HAART raised hopes for the eradication of HIV‐1. Unfortunately, the discovery of latent HIV‐1 reservoirs in CD4+ T cells and in the monocyte‐macrophage lineage proved the optimism to be premature. The long‐lived HIV‐1 reservoirs constitute a major obstacle to the eradication of HIV‐1. In this review, we focus on the establishment and maintenance of HIV‐1 latency in the two major targets for HIV‐1: the CD4+ T cells and the monocyte‐macrophage lineage. Understanding the cell‐type molecular mechanisms of establishment, maintenance, and reactivation of HIV‐1 latency in these reservoirs is crucial for efficient therapeutic intervention. A complete viral eradication, the holy graal for clinicians, might be achieved by strategic interventions targeting latently and productively infected cells. We suggest that new approaches, such as the combination of different kinds of proviral activators, may help to reduce dramatically the size of latent HIV‐1 reservoirs in patients on HAART.

Recruitment of Tat to Heterochromatin Protein HP1 via Interaction with CTIP2 Inhibits Human Immunodeficiency Virus Type 1 Replication in Microglial Cells

ABSTRACT The Tat protein of human immunodeficiency virus type 1 (HIV-1) plays a key role as inducer of viral gene expression. We report that Tat function can be potently inhibited in human microglial cells by the recently described nuclear receptor cofactor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2). Overexpression of CTIP2 leads to repression of HIV-1 replication, as a result of inhibition of Tat-mediated transactivation. In contrast, the related CTIP1 was unable to affect Tat function and viral replication. Using confocal microscopy to visualize Tat subcellular distribution in the presence of the CTIPs, we found that overexpression of CTIP2, and not of CTIP1, leads to disruption of Tat nuclear localization and recruitment of Tat within CTIP2-induced nuclear ball-like structures. In addition, our studies demonstrate that CTIP2 colocalizes and associates with the heterochromatin-associated protein HP1α. The CTIP2 protein harbors two Tat and HP1 interaction interfaces, the 145-434 and the 717-813 domains. CTIP2 and HP1α associate with Tat to form a three-protein complex in which the 145-434 CTIP2 domain interacts with the N-terminal region of Tat, while the 717-813 domain binds to HP1. The importance of this Tat binding interface and of Tat subnuclear relocation was confirmed by analysis of CTIP2 deletion mutants. Our findings suggest that inhibition of HIV-1 expression by CTIP2 correlates with recruitment of Tat within CTIP2-induced structures and relocalization within inactive regions of the chromatin via formation of the Tat-CTIP2-HP1α complex. These data highlight a new mechanism of Tat inactivation through subnuclear relocalization that may ultimately lead to inhibition of viral pathogenesis.