Parallel analysis of transcription, integration, and sequence of single HIV-1 proviruses
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE168337 | GEO | 2022/01/12
REPOSITORIES: GEO
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Project description:CD4 T cells with latent HIV-1 infection persist despite treatment with currently available antiretroviral agents and represent the main barrier to a cure of HIV-1 infection. Pharmacological disruption of viral latency may increase the immunological vulnerability of HIV-1-infected cells, but the clinical efficacy of latency-reversing agents for reducing HIV-1 persistence remains to be proven. Here, we show in a randomized, controlled human clinical trial that the histone deacetylase inhibitor panobinostat, when administered in combination with pegylated interferon-a2a, induces a structural transformation of the HIV-1 reservoir cell pool, characterized by a disproportionate overrepresentation of HIV-1 proviruses integrated in ZNF genes and in chromatin regions with reduced H3K27ac marks, the molecular target site for panobinostat. In contrast, proviruses near H3K27ac marks appeared to be actively selected against, likely due to increased susceptibility to panobinostat. These data suggest that latency-reversing treatment with panobinostat can accelerate immune selection of HIV-1 proviruses.
2024-02-29 | GSE234623 | GEO
Project description:CD4 T cells with latent HIV-1 infection persist despite treatment with currently available antiretroviral agents and represent the main barrier to a cure of HIV-1 infection. Pharmacological disruption of viral latency may increase the immunological vulnerability of HIV-1-infected cells, but the clinical efficacy of latency-reversing agents for reducing HIV-1 persistence remains to be proven. Here, we show in a randomized, controlled human clinical trial that the histone deacetylase inhibitor panobinostat, when administered in combination with pegylated interferon-a2a, induces a structural transformation of the HIV-1 reservoir cell pool, characterized by a disproportionate overrepresentation of HIV-1 proviruses integrated in ZNF genes and in chromatin regions with reduced H3K27ac marks, the molecular target site for panobinostat. In contrast, proviruses near H3K27ac marks appeared to be actively selected against, likely due to increased susceptibility to panobinostat. These data suggest that latency-reversing treatment with panobinostat can accelerate immune selection of HIV-1 proviruses.
2024-02-29 | GSE234624 | GEO
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 reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure the reservoir of activatable proviruses should be removed, whereas permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. Latently HIV-1 infected cells were transduced with a zinc finger protein specifically binding to the HIV-1 promoter, producing a fluorescent signal as the viral transactivator Tat is recruited to the HIV-1 promoter. In these cells we assessed the proviral chromatin composition simultaneously with the proviral activation. By linking the Tat promoter recruitment to viral activation, we dissected the mechanisms of HIV-1 reactivation and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasts to the continuous production of viral proteins. As expected, promoter H3K4me3 led to massive expression of the provirus following T cell stimulation. However, the activation induced cell cycle arrest and death resulted in an expanded surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this model can be used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determine the effect of CBP/P300-inhibitor GNE049. We found that only active enhancers, associated with H3K4me1 and H3K27ac, efficiently recruit Tat, as GNE049 that specifically inhibits enhancer H3K27ac also depletes promoter Tat. Despite the absence of Tat, HIV-1 latency reversal still occurred. Single cell assessment of the chromatin composition of the latent HIV-1 proviruses revealed how T cell stimulation modulates the proviral activity and the subsequent fate of the infected cell.
2021-10-04 | GSE183275 | GEO
Project description:HIV integrates semi-randomly into the genome of immune cells and thus proviruses that persist in patients under long-term, highly active suppressive therapy can be detected in various positions (inside and outside) and orientations (same, convergent and divergent) respective to genes, promoters, and enhancers. Thus, this integration landscape heterogeneity can influence HIV transcription activity thereby dictating proviral fate (active vs latent). However, the effect of the integration site to proviral transcription activity has so far remained elusive. Here we integrate open-source, large-scale datasets including epigenetics, transcriptome, and 3D genome architecture to interrogate the chromatin states, transcription activity landscape, nuclear sub-compartments, and topological associated domains around HIV integration sites in CD4+ T cell-based models to decipher ‘codes’ in the human genome shaping proviral transcription. As part of this integrated approach, here we collect nucleosome occupancy profiles in Jurkat CD4+ T cells.
2021-05-05 | GSE144753 | GEO
Project description:Antiretroviral therapy controls but does not cure HIV-1 infection due to a reservoir of rare CD4 + T cells harboring latent proviruses. Little is known about the transcriptional program of latent cells. Here we report a novel strategy to enrich clones of latent cells carrying intact, replication-competent HIV-1 proviruses from blood based on their expression of unique T cell receptors. Latent cell enrichment enabled single cell transcriptomic analysis of 1,050 CD4 + T cells belonging to expanded clones harboring intact HIV-1 proviruses from 6 different individuals. The analysis revealed that most of these cells are T effector memory cells that are enriched for expression of HLA-DR, HLA-DP, CD74, CCL5, Granzymes A and K, cystatin F, LYAR and DUSP2. We conclude that expanded clones of latent cells carrying intact HIV-1 proviruses persist preferentially in a distinct CD4 + T cell population opening new possibilities for eradication.
2022-06-03 | GSE204756 | 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:Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a major impediment for HIV cure research. To address this, we developed single-cell viral ASAPseq to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from antiretroviral treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by novel and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered novel epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.
2022-10-21 | GSE199727 | GEO
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:The restructuring of chromatin architecture following lentiviral integration is not well elucidated. We jointly interrogate (HIV-distal & -local) chromatin organization (via Hi-C & ATAC-seq) and the RNA landscape around defined sites of proviral integration using HIV-inducible cellular models. We report chromatin interaction networks and nuclear ultrastructure around integrated HIV-1 are predominantly preserved, suggesting HIV integration does not induce large scale remodeling of cellular chromatin. Instead, we find that induction of proviral transcription leads to stark local changes in nucleosome organization with chromatin accessibility increasing at the intergenic junction between the HIV-1 3’ LTR and flanking cellular genome. This result suggests subtle changes in chromatin structure may be mediating proviral activation. Using long-read Nanopore RNA-seq, we interrogate the local host & HIV transcriptomes, observing a small fraction of HIV-1 transcripts are chimeric read-through products, where transcription initiates at the HIV-1 5’ LTR promoter and continues extensively into the flanking cellular genome. Despite provirus-driven read-through, HIV-1 appears to have only a modest effect on the local transcriptional environment. The changes in chromatin accessibility and read-through at activated proviruses closely resembles lytic Herpes simplex virus type 1 (HSV-1) induced cellular chromatin reprogramming. We propose chromatin “opening” at the 3’ LTR HIV-host junction is important for sustained proviral activity, and overall, HIV proviruses do not significantly alter local host transcription and chromatin structure. Our studies provide the first in-depth integrative investigation of 3D chromatin organization, nucleosome density, and HIV-host transcriptomes at HIV-host genic boundaries.
2021-10-26 | GSE184345 | GEO