Genome-wide study of human methylation profile of J-Lat cells upon EGCG-treatment and UHRF1 knockdown
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE139320 | GEO | 2020/10/24
REPOSITORIES: GEO
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Project description:Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle towards HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression. We further validated our results using primary CD4+ T cells isolated from HIV+ individuals.
2018-03-13 | GSE111727 | GEO
Project description:A major obstacle for HIV eradication is the persistence of latent reservoirs, cells harboring replication competent yet transcriptionally silent proviruses. These reservoirs, composed primarily of CD4+ T cells, cannot be targeted by the host immune system or by combination anti-retroviral therapy (cART). Upon cessation of cART, a rapid viral rebound occurs. In order to develop effective HIV cure therapies, a better understanding of the factors and host programd governing latency establishment and/or maintenance must be obtained. To achieve this goal, we treated a CD4+T cell-based model of latency (J-Lat 10.6) with Latency Reversing Agents (LRAs) and performed bulk RNA-seq to predict host transcriptional programs activated, and potentially involved, in latent HIV reactivation. Our findings suggest that a novel small molecule (EPH-334) reactivates latent HIV by activating an oxidative stress transcriptional program linked to activation of MAPKs and downstream master regulators. Interestingly, the pan histone deacetylase inhibitor SAHA also induces oxidative stress programs potentially linking sensing of redox state aletartions with activation of cell signaling and transcriptioonal programs culminating on latent HIV reactivation.
2020-07-30 | GSE144552 | GEO
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:Suppressive HAART does not eradicate HIV-1 and viral DNA persists as a stably integrated form in the absence of viral particle production. As a consequence, latent reservoirs are refractory to antiretroviral drugs and invisible to immune surveillance. The largest latent reservoir consists of resting memory CD4+ T cells. These cells can resume viral infection when activated through antigen recognition, causing bursts of viremia (blips). Current therapies targeting latent HIV-1 have focused primarily on the M-bM-^@M-^\shock and killM-bM-^@M-^] approach, which employs M-bM-^@M-^\anti-latencyM-bM-^@M-^] drugs M-bM-^@M-^S most notably histone deacetylase (HDAC) inhibitors M-bM-^@M-^S to reactivate and flush latent provirus from its cellular reservoirs in the absence of global T cell activation. This approach is predicated on the notions that viral reactivation will lead to the demise of the infected cell, and that HAART will prevent spreading of the infection. On the contrary, recent evidence indicates that latently infected CD4+ T cells of HIV-1 patients on HAART survive in vitro viral reactivation with the HDAC inhibitor, SAHA, even when co-cultured with autologous CD8+ cytotoxic T lymphocytes (CTL). Moreover, it remains to be addressed the impact of anti-latency drugs on viral reservoirs undergoing low-level ongoing replication, inherently more resistant to the cytopathic effects of HIV-1 and residing in anatomical sites hard to reach for some antiretroviral drugs (e.g. macrophages). As a consequence, there is a need to develop alternative therapeutic approaches aimed at eliminating or decreasing the latent reservoir. Progress in that direction has been hindered by the lack of biomarkers uniquely or differentially expressed on latently infected compared to their uninfected counterparts. To gain insight into the cellular mechanisms that take place in the context of latency, and with the goal of identifying distinctive markers that distinguish latently infected CD4+ T cells, we have used an in vitro model developed in our laboratory to study the expression profile of latently infected CD4+ T cells by microarray analysis. We have used a culture system, previously established in our laboratory, to generate and isolate quiescent latently infected CD4+ T cells in vitro. In this in vitro HIV-1 latency model, CD4+ T cells are activated, infected with full length, replication competent HIV-1, and then returned to quiescence in the presence of IL-7, yielding a culture of quiescent latently infected and uninfected cells. We showed that HIV-1 p24gag expressed during viral replication persists in the cytoplasm of latently infected cells for several days before being degraded. Therefore, we exploited the presence of cytoplasmic p24gag to sort latently infected from uninfected cells by FACS from the same initial cell culture. Total RNA was isolated from sorted latently infected and uninfected cells generated from CD4+ T cells of four different donors. Paired RNA samples from infected and uninfected cells were labeled with Cy3 and Cy5 to allow dual-color competitive hybridization. Moreover, to control for the dye bias in our experiments, we implemented a dye swap protocol (reciprocal labeling) for paired RNA samples from 2 donors. Samples were analyzed by dual-color competitive hybridization on the Agilent whole human genome microarrays (41,000 unique probes). This is the first comparative genomic profiling of primary latently infected resting memory CD4+ T cells versus their uninfected counterparts sorted from the same culture. Microarray analyses performed in this study revealed profound differences between latently infected and uninfected cells. Of relevance are genes involved, not only in previously described pathways related with transcriptional and post-transcriptional regulation, but affecting proliferation, survival, cell cycle progression and cell metabolism. This could explain why latently infected cells have been resistant to reactivation with current anti-latency approaches. Thus, targeting of more downstream steps, such as the ones identified in this study, may be able to enhance viral flushing from refractory latent reservoirs. In addition, we identified a panel of surface makers differentially expressed in latently infected cells, which seem worth investigating for their potential use as biomarkers. Indeed, they might allow the enrichment of this latent reservoir for molecular in depth studies, for monitoring the size of the latent reservoir in the clinical setting, as well as for the development of new therapeutic strategies aimed at eradicating this reservoir.
2013-03-15 | E-GEOD-40550 | biostudies-arrayexpress
Project description:Suppressive HAART does not eradicate HIV-1 and viral DNA persists as a stably integrated form in the absence of viral particle production. As a consequence, latent reservoirs are refractory to antiretroviral drugs and invisible to immune surveillance. The largest latent reservoir consists of resting memory CD4+ T cells. These cells can resume viral infection when activated through antigen recognition, causing bursts of viremia (blips). Current therapies targeting latent HIV-1 have focused primarily on the “shock and kill” approach, which employs “anti-latency” drugs – most notably histone deacetylase (HDAC) inhibitors – to reactivate and flush latent provirus from its cellular reservoirs in the absence of global T cell activation. This approach is predicated on the notions that viral reactivation will lead to the demise of the infected cell, and that HAART will prevent spreading of the infection. On the contrary, recent evidence indicates that latently infected CD4+ T cells of HIV-1 patients on HAART survive in vitro viral reactivation with the HDAC inhibitor, SAHA, even when co-cultured with autologous CD8+ cytotoxic T lymphocytes (CTL). Moreover, it remains to be addressed the impact of anti-latency drugs on viral reservoirs undergoing low-level ongoing replication, inherently more resistant to the cytopathic effects of HIV-1 and residing in anatomical sites hard to reach for some antiretroviral drugs (e.g. macrophages). As a consequence, there is a need to develop alternative therapeutic approaches aimed at eliminating or decreasing the latent reservoir. Progress in that direction has been hindered by the lack of biomarkers uniquely or differentially expressed on latently infected compared to their uninfected counterparts. To gain insight into the cellular mechanisms that take place in the context of latency, and with the goal of identifying distinctive markers that distinguish latently infected CD4+ T cells, we have used an in vitro model developed in our laboratory to study the expression profile of latently infected CD4+ T cells by microarray analysis.
2013-03-15 | GSE40550 | GEO
Project description:HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic and metabolomic analysis, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, the antioxidant NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a “shock and kill effect” decreasing proviral DNA in cells from people-living-with-HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.
2021-06-16 | GSE163979 | GEO
Project description:HIV-1 latency results from a combination of tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. In an effort to elucidate the molecular players that govern viral latency, we previously performed a dCas9 chromatin immunoprecipitation coupled with mass spectrometry (Catchet-MS) and identified proteins bound differentially to the latent LTR that putatively promote HIV-1 latency. Here we characterize the Catchet-MS identified PCI domain-containing 2 (PCID2) protein to play a dual role in promoting HIV-1 latency by enforcing both HIV-1 transcription repression as well as post-transcriptional blocks. PCID2 bound the latent HIV-1 LTR and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and reversal of latency. Immunoprecipitation coupled to Mass Spectrometry identified the PCID2 interacting proteins to include members of the spliceosome and other splicing regulators, including negative regulators of viral RNA alternative splicing. PCID2 depletion resulted in over-splicing of intron-containing HIV-1 RNA and mis-regulated expression of vRNA splice variants. Finally, consistent with its role in NXF1-mediated nascent RNA nucleocytoplasmic export as part of the TREX2 complex, PCID2 modulates export of completely spliced vRNA. In summary, we demonstrate that PCID2 is a previously unidentified factor involved in HIV-1 latency regulation which plays a dual role in blocking HIV-1 gene expression by acting on transcription initiation as well as viral RNA processing.
2024-06-16 | PXD043334 | Pride
Project description:Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The â??shock and killâ?? strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64).  We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a â??hotspotâ?? for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple different in vitro latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT). While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs. In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus. In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a â??functional cureâ?? of HIV/AIDS. Three experimental conditions (sgRNA control, TNF treated and sgRNA against the LTR of HIV-1) were analyzed in triplicate using two sequencing lanes
2016-08-20 | E-GEOD-72259 | biostudies-arrayexpress
Project description:HIV-1 latency is a critical hurdle to a cure for HIV-1 infection, but our understanding of the molecular biology of latency control is still incomplete. We provide experimental evidence that HIV-1 infection of primary T cells and T cell lines generates a substantial amount of TCR/CD3 activation-inert latently infected T cells. Proteomic and genome-wide RNA-level analysis comparing CD3-responsive and CD3-inert latently HIV-1 infected T cells, followed by software-based data integration suggested two phenomena to govern CD3-inertness: (i) the presence of extensive transcriptomic noise that affected the efficacy of CD3 signaling and (ii) defined changes to specific signaling pathways. Validation experiments demonstrated that compounds known to increase transcriptomic noise further diminished the ability of TCR/CD3 stimulation to trigger HIV-1 reactivation. Conversely, targeting specific central nodes in the generated PINs such as STAT3 improved the ability of TCR/CD3 activation to trigger HIV-1 reactivation in T cell lines and primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. We discuss the implications of these findings for the idea of using single drug therapies to trigger HIV-1 reactivation in the latent T cell reservoir in patients.
2020-06-19 | GSE152788 | GEO
Project description:Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy 1. Current research efforts to cure HIV-1 infection include “shock and kill” strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells 2. However, the modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy 3,4. Aminobisphosphonates that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies 5. Here, we show the use of aminobisphosphonates as a novel class of LRA: we found in ex vivo assays using primary cells from ART-suppressed people living with HIV-1 that aminobisphosphonates induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin. RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further of alendronate-mediated latency reversal and activation of immune effector cells.Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of aminobisphosphonates to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination.
2023-09-13 | GSE224546 | GEO