Gene expression changes following latency reversal agents screening in a cellular model of HIV-1 latency
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE195855 | GEO | 2022/08/24
REPOSITORIES: GEO
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Project description:HIV-1 infected patients virally suppressed by antiviral treatment harbor a persistent reservoir of replication competent latent HIV-1 infected cells, which constitute the main roadblock to a cure. A main strategy for HIV cure aims to stimulate viral gene expression in latently infected cells so that they can be cleared. Crucial for the design of drugs referred to as “latency-reversing agents” (LRAs) is the identification of molecular targets for latency reversal. The regulatory factors physically associated with and repressing the latent HIV-1 promoter or 5’LTR would provide ideal putative molecular targets for latency reversal. However, due to technical limitations, the comprehensive and unbiased identification of host proteins associated with the latent or active integrated HIV LTR in infected cells not been possible. Here we use dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS), to purify the locus-associated dCas9 bait, guided downstream of the HIV-1 transcriptional start site (TSS) in latent and activated HIV-1 infected T cells to identify the 5’LTR bound latent and active regulatory complexes. Catchet-MS identified both previously described as well as novel host factors distinctly associated with the latent versus transcriptionally active HIV-1 5’LTR. Within the identified factors we find the transcription factor IKZF1 to be a novel repressor of the HIV-1 promoter required for maintenance of latency, and thus a molecular target for latency reversal. Finally, we identify the FDA approved drug, Iberdomide, which targets IKZF1 for degradation to be a novel LRA, which reversed latency in latent ex vivo HIV-1 infected primary CD4+ T cells and in cells isolated from HIV-1 infected, aviremic participants.
2022-05-31 | PXD032975 | Pride
Project description:The barrier to HIV-1 functional cure is caused by a small pool of latently infected resting CD4 T-cells that persist under antiretroviral therapy. Notably this latent reservoir of infected cells will produce replication-competent infectious virus once prolonged suppressive HAART is withdrawn. The reactivation of HIV-1 gene expression in T-cells harboring latent provirus in HIV-1 patients under HAART will likely result in depletion of this latent reservoir due to cytopathic effects and immune clearance. Many studies have investigated small molecules that reactivate HIV-1 gene expression but to date no latency reversal agent (LRA) has been identified to be specific, non-toxic, and effective in primary T-cells isolated from HIV-1 infected individuals undergoing long-term HAART. Stochastic fluctuations in HIV-1 tat gene expression have been attributed to be essential in the viral progression to latency. We hypothesized that exposing Tat to latently infected CD4 T-cells will result in potent latency reversal. Our results indicate the capacity of an engineered Tat to reactivate HIV-1 in latently infected cells from patients to a similar degree as the protein kinase C agonist PMA (Phorbol 12-Myristate 13-Acetate) while showing no T-cell activation nor any significant transcriptome perturbation in primary CD4 T-cells.
2023-09-05 | GSE235229 | GEO
Project description:The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo. We also detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells.
2024-09-17 | GSE276777 | GEO
Project description:A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has been challenging. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to probe the differential protein composition of the latent and activated HIV-1 5'LTR. Catchet-MS identified known and novel latent 5’LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs that, by targeting IKZF1 for degradation, reverse HIV-1 latency in CD4+T-cells isolated from virally suppressed people living with HIV-1.
2022-02-06 | GSE196059 | GEO
Project description:The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor are appealing towards inducing HIV reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 in both cell line models and in CD4 T cells from virally suppressed PLWH improves reactivation at the transcriptional level and is more specific than AZD5582 & I-BET151 treatment alone. INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observe more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151 indicating the combination may specifically overcome an elongation block to reactivation. We also observe viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 controls full-length HIV RNA and virus production ex vivo.
2024-09-17 | GSE277305 | GEO
Project description:The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor are appealing towards inducing HIV reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 in both cell line models and in CD4 T cells from virally suppressed PLWH improves reactivation at the transcriptional level and is more specific than AZD5582 & I-BET151 treatment alone. INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observe more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151 indicating the combination may specifically overcome an elongation block to reactivation. We also observe viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 controls full-length HIV RNA and virus production ex vivo.
2024-09-17 | GSE276747 | GEO
Project description:The latent viral reservoir represents one of the major barriers of curing HIV. Focus on the “kick and kill” approach, in which virus expression is reactivated then cells producing virus are selectively depleted, has led to the discovery of many latency reversing agents (LRAs) that can reactivate latently integrated virus and further our understanding of the mechanisms driving HIV latency and latency reversal. Thus far, individual compounds have yet to be robust enough to work as a therapy, highlighting the importance of identifying new compounds that target novel pathways and synergize with known LRAs. In this study, we identified a promising LRA, NSC95397, from a screen of ~4250 compounds in J-Lat cell lines. We validated that NSC95397 reactivates latent viral transcription and protein expression from cells with unique integration events. Co-treating cells with NSC95397 and known LRAs demonstrated that NSC95397 has the potential to synergize with different drugs under both standard normoxic and physiological hypoxic conditions. We showed that NSC95397 does not globally increase open chromatin, and bulk RNA sequencing revealed that NSC95397 does not greatly change cellular transcription. Instead, NSC95397 downregulates many pathways key to metabolism, cell growth, and DNA repair – highlighting the potential of these pathways in regulating HIV latency. Overall, we identified NSC95397 as a novel LRA that does not alter global transcription, that shows potential for synergy with known LRAs, and that may act through novel pathways not previously recognized for their ability to modulate HIV latency.
2024-08-31 | GSE241617 | GEO
Project description:T cells are the primary target of the virus HIV-1. Upon infection, the expression of the virus may come to a complete shutdown, a phenomenon known as latency. The molecular mechanisms responsible for the latency of HIV-1 are still poorly understood. To shed light on those mechanisms, we used the J-Lat A2 model for latency reversal, that consists of a Jurkat T cell clone containing a mini HIV construct that is transcriptionally silent. We treated J-Lat A2 and Jurkat cells with the latency-reversing drugs SAHA (suberoylanilide hydroxamic acid) and PMA (phorbol 12-myristate 13-acetate), and we performed single-cell RNA-seq to identify transcriptional signatures shared among the cells where HIV is reactivated.
2021-07-15 | GSE180133 | GEO
Project description:The goal of this study was to utilize CaptureSeq to be able to measure HIV-1 transcription after reversal of latency in primary cells from antiretroviral-treated HIV-1 infected individuals.
2021-06-01 | GSE152377 | GEO
Project description:Latency reversal and clearance strategies for HIV cure are beginning to employ IAP antagonists (IAPi) to induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with Bromodomain and Extra-Terminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single cell-RNAseq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bromodomain (BD)-selective BET, or selective BET isoform targeting in CD4 T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA but lesser induction of fully elongated and tat-rev RNA, especially compared to T cell activation positive controls. IAPi/BETi resulted in HIV protein induction in bulk cultures of CD4 T cells using an ultrasensitive p24 assay, but did not translate to enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. Overall, this study defines HIV transcriptional elongation and splicing as key barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their bromodomains in HIV latency, and provides rationale for testing of IAPi+BETi in animal models of HIV latency.
2022-02-10 | GSE196091 | GEO