Dynamics of HIV Latency and Reactivation in a Primary CD4+ T Cell Model
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE95297 | GEO | 2017/02/24
SECONDARY ACCESSION(S): PRJNA376596
REPOSITORIES: GEO
Similar Datasets
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: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
Project description:Despite the success of antiretroviral therapy, HIV cannot be cured because of a reservoir of latently infected cells that evades therapy. To understand the mechanisms of HIV latency, we employed an integrated single-cell RNA-seq/ATAC-seq approach to simultaneously profile the transcriptomic and epigenomic characteristics of ~125,000 latently infected primary CD4 cells after reactivation using three different latency-reversing agents.
2023-12-28 | GSE242997 | 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: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:Herein expression trends of host miRNA were measured in HIV-1 latently infected and persistent replication cells, as well as the control cells. HIV-1 latency infection was established by infecting CEM-SS lymphocytes with HIV-1 Bru strain. After selection and long-term culture, the chronically infected cell showed the characteristics of latency definition: 1. The provirus was intergrated in to the host genome.2. No viral expression could be detected during culture.3 .Cell stimulators, such as TNFa,PMA, etc, reactivate the viral expression. As expected, miRNA trend was different in HIV-1 latency when compared to the control or HIV-1 replication. A subset of miRNAs is enriched in HIV-1 latency model. The observation reinforces the concept of active HIV-1 interplay with host small RNAs that modulate HIV-1 infection mode. In this study, the in vitro steady cell culture was used to explore the miRNA transcription signatures in HIV-1 infection. miRNA profiles were performed and compared among normal control,HIV-1 latency and HIV-1 replication .
2012-12-01 | E-GEOD-38388 | biostudies-arrayexpress
Project description:HIV infection is not curable due to viral latency. Compelling reports studying proteins such as CD2, PD-1, LAG-3 and TIGIT suggest that there is a distinct profile of surface proteins that can be used for targeting latently infected cells. We have recently reported that glycoproteins were differentially secreted from HIV latently infected ACH-2 cells compared to the parental A3.01 cells. This observation suggests that glyco-phenotype might be different in these two cell lines. To determine the difference, the ACH-2 and A3.01 cell lines were subjected to a glycoproteomic analysis. A total number of 940 unique N-linked glycosite-containing peptides from 515 glycoproteins were identified. Among the glycoproteins, 365 and 104 were annotated as cell surface and membrane-associated proteins, respectively. Label free quantitative LC-MS/MS analysis revealed a change of 236 glycosite-containing peptides from 172 glycoproteins between the two cell lines without reactivation. Bioinformatic analysis suggests that cell adhesion, immune response, glycoprotein metabolic process, cell motion and cell activation were associated with the changed proteins. After reactivation of latency by phorbol myristate acetate (PMA), changes in glycosite-containing peptides were observed in both cell lines. Changes in 49 glycosite-containing peptides from 45 glycoproteins might be due to viral replication. The changed proteins suggest that cell migration, response to wounding and immune response were impaired in reactivated latently infected cells. Our study provides important glycoproteomic data in respect to HIV latently infected cells. Glycoproteomics merits future application using primary cells to discover reveal mechanisms in HIV pathogenesis.
2016-05-23 | PXD003561 | Pride
Project description:A leading pharmacological strategy towards HIV cure requires “shock” or activation of expression of the HIV genome in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. As a source of bio-active molecules we used fungal secondary metabolites (extrolites) in a screen for novel LRAs. We used orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, and identified gliotoxin (GTX) to potently reverse latency. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA to be most significantly downregulated in independent donors upon GTX treatment of CD4+ T cells. GTX disrupted the 7SK snRNP complex causing release of P-TEFb, which induced phosphorylation of RNA Pol II CTD, and increased HIV transcription. Our data highlight the power of combining low throughput bioassays, mycology and orthogonal mass spectrometry as an approach to screen for and characterize novel LRAs.
2020-08-31 | GSE135184 | 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:Herein expression trends of host miRNA were measured in HIV-1 latently infected and persistent replication cells, as well as the control cells. HIV-1 latency infection was established by infecting CEM-SS lymphocytes with HIV-1 Bru strain. After selection and long-term culture, the chronically infected cell showed the characteristics of latency definition: 1. The provirus was intergrated in to the host genome.2. No viral expression could be detected during culture.3 .Cell stimulators, such as TNFa,PMA, etc, reactivate the viral expression. As expected, miRNA trend was different in HIV-1 latency when compared to the control or HIV-1 replication. A subset of miRNAs is enriched in HIV-1 latency model. The observation reinforces the concept of active HIV-1 interplay with host small RNAs that modulate HIV-1 infection mode.
2012-12-01 | GSE38388 | GEO