T cell stimulation alters the HIV-1 reservoir by differential activation of proviral chromatin
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE183275 | GEO | 2021/10/04
REPOSITORIES: GEO
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Project description:Human immunodeficiency virus (HIV) infection is a chronic condition, where viral DNA integrates into the genome. The fate of the provirus determines the infection course. Latently infected cells form a persistent, heterogeneous reservoir. The reservoir that reinstates an active infection comprises cells with intact provirus that can be reactivated. We confirmed that latent cells from patients exhibited active transcription throughout the provirus. To find transcriptional determinants, we characterized the establishment and maintenance of latency during proviral chromatin maturation in primary CD4+ T-cells for four months after HIV infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible and lost activation potential. In a subset of infected cells, active marks (i.e., H3K27ac) remained detectable, even after prolonged proviral silencing. After T-cell activation, the proviral activation occurred uniquely in cells with H3K27ac-marked proviruses. Our observations suggested that, after transient proviral activation, cells were actively returned to latency.
2019-12-16 | GSE121055 | GEO
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reservoir of infected cells will produce replication-competent infectious virus once prolonged suppressive HAART is withdrawn.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: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: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: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:Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected persons (“elite controllers”, ECs), despite the presence of a replication-competent viral reservoir. Such an ability to spontaneously maintain undetectable plasma viremia is a major objective of functional cure efforts, yet the characteristics of proviral reservoirs in ECs remain to be determined. Using single-genome, near full-length next-generation sequencing and chromosomal integration site analysis, we here show that proviral reservoirs of ECs frequently consist of oligoclonal to near monoclonal clusters of identical intact proviral sequences. In contrast to persons treated with long-term antiretroviral therapy, intact proviral species from ECs displayed highly distinct chromosomal integration sites in the human genome and were preferentially located in centromeric satellite DNA or in KRAB-ZNF genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, integration sites of intact proviruses from ECs showed increased distance to host transcriptional start sites and accessible chromatin and were enriched for repressive chromatin marks. These data suggest that a distinct proviral reservoir configuration represents a structural correlate of natural viral control, and that quality rather than quantity of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, failure to detect intact proviral sequences despite analyzing > 1.5 billion peripheral blood mononuclear cells in one EC raises the possibility that a sterilizing cure of HIV-1 infection, previously only observed following allogeneic hematopoietic stem cell transplantation, may be feasible in rare instances.
2020-06-15 | GSE144329 | GEO
Project description:Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected persons (“elite controllers”, ECs), despite the presence of a replication-competent viral reservoir. Such an ability to spontaneously maintain undetectable plasma viremia is a major objective of functional cure efforts, yet the characteristics of proviral reservoirs in ECs remain to be determined. Using single-genome, near full-length next-generation sequencing and chromosomal integration site analysis, we here show that proviral reservoirs of ECs frequently consist of oligoclonal to near monoclonal clusters of identical intact proviral sequences. In contrast to persons treated with long-term antiretroviral therapy, intact proviral species from ECs displayed highly distinct chromosomal integration sites in the human genome and were preferentially located in centromeric satellite DNA or in KRAB-ZNF genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, integration sites of intact proviruses from ECs showed increased distance to host transcriptional start sites and accessible chromatin and were enriched for repressive chromatin marks. These data suggest that a distinct proviral reservoir configuration represents a structural correlate of natural viral control, and that quality rather than quantity of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, failure to detect intact proviral sequences despite analyzing > 1.5 billion peripheral blood mononuclear cells in one EC raises the possibility that a sterilizing cure of HIV-1 infection, previously only observed following allogeneic hematopoietic stem cell transplantation, may be feasible in rare instances.
2020-06-15 | GSE144332 | GEO
Project description:To comprehensively compare the HIV-1 integration sites between viral producing cells (GFP+ cells) and non-producing cells (GFP- cells), linker-mediated PCR is performed by using the samples obtained from HIV1-GFP-infected humanized mice. Additionally, to reactivate latently infected cells, viral non-producing cells (GFP- cells) were stimulated with PMA and ionomycin ex vivo. Then, the viral integration sites in potential latently infected cells (GFP-_GFP+ cells) and the cells with dead/inactivated proviruses (GFP-_GFP- cells) were analyzed by linker-mediated PCR.
2020-07-16 | GSE137962 | GEO
Project description:HIV-1 infection of resting memory CD4+ T cells forms a barrier to curing HIV-1. Identification of immune biomarkers that correlate with HIV-1 reservoir size could aid with assessing efficacy of HIV-1 eradication strategies, especially in pediatric infections where blood sampling is limited. In adults, the immune exhaustion marker PD-1 on central memory CD4+ T cells (Tcm) correlates with HIV-1 reservoir size. Immune correlates of HIV-1 reservoir size are less defined in perinatal infection. Using multi-parameter flow cytometry, we examined immune activation (CD69, CD25, HLA-DR), and exhaustion (PD-1, TIGIT, LAG-3 and TIM-3) markers on CD4+ T cell subsets (naïve (Tn), central memory (Tcm), and a combination (Ttem) of transitional (Ttm) and effector memory (Tem) in 10 children living with HIV-1 (median age 15.9 years; median duration of virologic suppression 7.0 years), in whom HIV-1 reservoir size was determined with both the Intact Proviral HIV-1 DNA assay (IPDA) and the Tat/Rev limiting dilution assay (TILDA). Total HIV-1 DNA in CD4+ T cells was also measured. Correlations between immune activation, and exhaustion markers on T cell subsets with the various markers of proviral reservoir size, and baseline CD4+ T cell transcriptomes were examined. The median total HIV-1 DNA concentration was 211.9 copies per million CD4+ T cells, with a median intact proviral load in individuals with HIV-1 subtype B of 8.0 copies per million CD4+ T cells. Levels of HLA-DR and TIGIT on Ttem were strongly correlated with total HIV-1 DNA (r=0.758, p=0.015) and (r=0.721, p=0.023), respectively, but not with intact proviral load or inducible reservoir size. HIV-1 DNA load was also positively correlated with transcriptional clusters associated with HLA-DR. In contrast, PD-1 expression on Tcm was inversely correlated with both total HIV-1 DNA (r=-0.67, p=0.039) and HLA-DR in Ttem (r=-0.89, p=0.060). Gene expression profiles for HLA-DR and PD-1 were also inversely correlated. In conclusion, with virologically suppressed perinatal HIV-1 infection, HLA-DR and TIGIT on Ttem CD4+ T cells correlate with total HIV-1 DNA, and may serve as immune biomarkers for transcriptionally active proviruses, including defectives persisting on ART.
2023-03-20 | GSE209828 | GEO
Project description:HIV infection remains incurable due to the long-term persistence of latently infected cells, capable of refueling viremia upon antiretroviral therapy interruption. Shock-and-kill strategies aim to purge this latent reservoir by inducing the expression of viral genes, making infected cells visible for cleanup by the immune system. Here, we present a Tat mRNA formulation that, when combined with panobinostat, enables latency reversal at levels >3-fold higher than those reached with the most potent mitogens. We demonstrate that this mRNA formulation does not alter the transcriptome or phenotype of CD4 T cells, enabling the ex vivo assessment of infected cells upon latency reversal in a near-native state. Taking advantage of this characteristic, we show transcriptomic and proteomic differences between infected cells upon latency reversal and non-infected cells, including the upregulation of the cytotoxic protein granzyme A and a novel long non-coding RNA. Overall, we demonstrate that a Tat mRNA formulation enables potent reactivation of latent HIV, presenting a valuable research tool for studying the inducible HIV reservoir, as well as paving the way to a more effective shock-and-kill functional cure.
2023-11-09 | GSE247402 | GEO