scholarly journals Entry of Polarized Effector Cells into Quiescence Forces HIV Latency

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Curtis Dobrowolski ◽  
Saba Valadkhan ◽  
Amy C. Graham ◽  
Meenakshi Shukla ◽  
Angela Ciuffi ◽  
...  
Keyword(s):  
T Cells ◽  
Molecular Mechanisms ◽  
Primary Cell ◽  
T Cell Subsets ◽  
Hiv Latency ◽  
Cell Models ◽  
Homogeneous Population ◽  
Wide Range ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT The latent HIV reservoir is generated following HIV infection of activated effector CD4 T cells, which then transition to a memory phenotype. Here, we describe an ex vivo method, called QUECEL (quiescent effector cell latency), that mimics this process efficiently and allows production of large numbers of latently infected CD4+ T cells. Naïve CD4+ T cells were polarized into the four major T cell subsets (Th1, Th2, Th17, and Treg) and subsequently infected with a single-round reporter virus which expressed GFP/CD8a. The infected cells were purified and coerced into quiescence using a defined cocktail of cytokines, including tumor growth factor beta, interleukin-10 (IL-10), and IL-8, producing a homogeneous population of latently infected cells. Flow cytometry and transcriptome sequencing (RNA-Seq) demonstrated that the cells maintained the correct polarization phenotypes and had withdrawn from the cell cycle. Key pathways and gene sets enriched during transition from quiescence to reactivation include E2F targets, G2M checkpoint, estrogen response late gene expression, and c-myc targets. Reactivation of HIV by latency-reversing agents (LRAs) closely mimics RNA induction profiles seen in cells from well-suppressed HIV patient samples using the envelope detection of in vitro transcription sequencing (EDITS) assay. Since homogeneous populations of latently infected cells can be recovered, the QUECEL model has an excellent signal-to-noise ratio and has been extremely consistent and reproducible in numerous experiments performed during the last 4 years. The ease, efficiency, and accuracy of the mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency. IMPORTANCE Current primary cell models for HIV latency correlate poorly with the reactivation behavior of patient cells. We have developed a new model, called QUECEL, which generates a large and homogenous population of latently infected CD4+ memory cells. By purifying HIV-infected cells and inducing cell quiescence with a defined cocktail of cytokines, we have eliminated the largest problems with previous primary cell models of HIV latency: variable infection levels, ill-defined polarization states, and inefficient shutdown of cellular transcription. Latency reversal in the QUECEL model by a wide range of agents correlates strongly with RNA induction in patient samples. This scalable and highly reproducible model of HIV latency will permit detailed analysis of cellular mechanisms controlling HIV latency and reactivation.

scholarly journals Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals

2020 ◽  
Vol 16 (11) ◽  
pp. e1009060
Author(s):  
Sara Moron-Lopez ◽  
Sushama Telwatte ◽  
Indra Sarabia ◽  
Emilie Battivelli ◽  
Mauricio Montano ◽  
...  
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Primary Cell ◽  
Cell Models ◽  
Transcriptional Initiation ◽  
Cellular Genes ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv

It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells.

scholarly journals Single cell analysis of quiescent HIV infection reveals host transcriptional profiles that regulate proviral latency

2018 ◽  
Author(s):  
Todd Bradley ◽  
Guido Ferrari ◽  
Barton F Haynes ◽  
David M Margolis ◽  
Edward P Browne
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Latent Reservoir ◽  
Hiv Latency ◽  
Wide Range ◽  
Cell Assays ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv

SummaryThe latent HIV reservoir is diverse, but most studies of HIV latency have used bulk cell assays. Here we characterized cell line and primary cell models of HIV latency with single cell qPCR (sc-qPCR) for viral RNA (vRNA), and single cell RNAseq (scRNAseq). sc-qPCR revealed distinct populations of cells transcribing vRNA across a wide range of levels. Strikingly, scRNAseq of latently infected primary cells revealed a relationship between vRNA levels and the transcriptomic profiles within the population. Cells with the greatest level of HIV silencing expressed a specific set of host genes including markers of central memory T cells. By contrast, latently infected cells with higher levels of HIV transcription expressed markers of activated and effector T cells. These data reveal that heterogeneous behaviors of HIV proviruses within the latent reservoir are influenced by the host cell transcriptional program. Therapeutic modulation of these programs may reverse or enforce HIV latency.

scholarly journals Shared Mechanisms Govern HIV Transcriptional Suppression in Circulating CD103+ and Gut CD4+ T Cells

2020 ◽  
Vol 95 (2) ◽  
pp. e01331-20
Author(s):  
Steven A. Yukl ◽  
Shahzada Khan ◽  
Tsui-Hua Chen ◽  
Martin Trapecar ◽  
Frank Wu ◽  
...  
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Cd4 T Cells ◽  
Molecular Mechanisms ◽  
Hiv Latency ◽  
Expression Levels ◽  
Hiv Rna ◽  
Infected Cells ◽  
Latent Hiv ◽  
Main Barrier

ABSTRACTLatent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor β (TGF-β), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-β signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103+ and CD103− CD4 T cells from the blood and rectum of HIV-negative (HIV−) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV+) individuals. Like gut CD4+ T cells, circulating CD103+ cells harbored more HIV DNA than did CD103− cells but transcribed less HIV RNA per provirus. Circulating CD103+ cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103− cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103+ CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency.IMPORTANCE The ability of HIV to establish a reversibly silent, “latent” infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103+ CD4+ T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103+ T cells share a cellular transcriptome that more closely resembles that of CD4+ T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4+ or circulating CD103+ T cells from circulating CD103− T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV.

scholarly journals Establishment of HIV Latency in Primary CD4+ Cells Is due to Epigenetic Transcriptional Silencing and P-TEFb Restriction

2010 ◽  
Vol 84 (13) ◽  
pp. 6425-6437 ◽  
Author(s):  
Mudit Tyagi ◽  
Richard John Pearson ◽  
Jonathan Karn
Keyword(s):  
T Cells ◽  
Histone Deacetylases ◽  
Fluorescent Protein ◽  
Transcriptional Silencing ◽  
Hiv Latency ◽  
Primary Cells ◽  
Protein Marker ◽  
Necrosis Factor Alpha ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT The development of suitable experimental systems for studying HIV latency in primary cells that permit detailed biochemical analyses and the screening of drugs is a critical step in the effort to develop viral eradication strategies. Primary CD4+ T cells were isolated from peripheral blood and amplified by antibodies to the T-cell receptor (TCR). The cells were then infected by lentiviral vectors carrying fluorescent reporters and either the wild-type Tat gene or the attenuated H13L Tat gene. After sorting for the positive cells and reamplification, the infected cells were allowed to spontaneously enter latency by long-term cultivation on the H80 feeder cell line in the absence of TCR stimulation. By 6 weeks almost all of the cells lost fluorescent protein marker expression; however, more than 95% of these latently infected cells could be reactivated after stimulation of the TCR by α-CD3/CD28 antibodies. Chromatin immunoprecipitation assays showed that, analogously to Jurkat T cells, latent proviruses in primary CD4+ T cells are enriched in heterochromatic markers, including high levels of CBF-1, histone deacetylases, and methylated histones. Upon TCR activation, there was recruitment of NF-κB to the promoter and conversion of heterochromatin structures present on the latent provirus to active euchromatin structures containing acetylated histones. Surprisingly, latently infected primary cells cannot be induced by tumor necrosis factor alpha because of a restriction in P-TEFb levels, which can be overcome by activation of the TCR. Thus, a combination of restrictive chromatin structures at the HIV long terminal repeat and limiting P-TEFb levels contribute to transcriptional silencing leading to latency in primary CD4+ T cells.

scholarly journals Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors

2021 ◽  
Vol 17 (2) ◽  
pp. e1009346
Author(s):  
Stuart R. Jefferys ◽  
Samuel D. Burgos ◽  
Jackson J. Peterson ◽  
Sara R. Selitsky ◽  
Anne-Marie W. Turner ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factors ◽  
Hiv Infection ◽  
Cd4 T Cells ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Hiv Latency ◽  
Latently Infected ◽  
Infected Cells

Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency.

scholarly journals The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

2018 ◽  
Vol 92 (13) ◽  
pp. e02225-17 ◽  
Author(s):  
Simin D. Rezaei ◽  
Hao K. Lu ◽  
J. Judy Chang ◽  
Ajantha Rhodes ◽  
Sharon R. Lewin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Egfp Expression ◽  
Hiv Latency ◽  
Latently Infected ◽  
Infected Cells ◽  
Virus Expression

ABSTRACTHIV infection requires lifelong antiretroviral therapy because of the persistence of latently infected CD4+T cells. The induction of virus expression from latently infected cells occurs following T cell receptor (TCR) activation, but not all latently infected cells respond to TCR stimulation. We compared two models of latently infected cells using an enhanced green fluorescent protein (EGFP) reporter virus to infect CCL19-treated resting CD4+(rCD4+) T cells (preactivation latency) or activated CD4+T cells that returned to a resting state (postactivation latency). We isolated latently infected cells by sorting for EGFP-negative (EGFP−) cells after infection. These cells were cultured with antivirals and stimulated with anti-CD3/anti-CD28, mitogens, and latency-reversing agents (LRAs) and cocultured with monocytes and anti-CD3. Spontaneous EGFP expression was more frequent in postactivation than in preactivation latency. Stimulation of latently infected cells with monocytes/anti-CD3 resulted in an increase in EGFP expression compared to that for unstimulated controls using the preactivation latency model but led to a reduction in EGFP expression in the postactivation latency model. The reduced EGFP expression was not associated with reductions in the levels of viral DNA or T cell proliferation but depended on direct contact between monocytes and T cells. Monocytes added to the postactivation latency model during the establishment of latency reduced spontaneous virus expression, suggesting that monocyte-T cell interactions at an early time point postinfection can maintain HIV latency. This direct comparison of pre- and postactivation latency suggests that effective strategies needed to reverse latency will depend on how latency is established.IMPORTANCEOne strategy being evaluated to eliminate latently infected cells that persist in HIV-infected individuals on antiretroviral therapy (ART) is to activate HIV expression or production with the goal of inducing virus-mediated cytolysis or immune-mediated clearance of infected cells. The gold standard for the activation of latent virus is T cell receptor stimulation with anti-CD3/anti-CD28. However, this stimulus activates only a small proportion of latently infected cells. We show clear differences in the responses of latently infected cells to activating stimuli based on how latent infection is established, an observation that may potentially explain the persistence of noninduced intact proviruses in HIV-infected individuals on ART.

scholarly journals Plasma Extracellular Vesicles Enhance HIV-1 Infection of Activated CD4+ T Cells and Promote the Activation of Latently Infected J-Lat10.6 Cells via miR-139-5p Transfer

2021 ◽  
Vol 12 ◽  
Author(s):  
Isobel Okoye ◽  
Lai Xu ◽  
Olaide Oyegbami ◽  
Shima Shahbaz ◽  
Desmond Pink ◽  
...  
Keyword(s):  
T Cells ◽  
Extracellular Vesicles ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Hiv Latency ◽  
Healthy Controls ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

HIV latency is a challenge to the success of antiretroviral therapy (ART). Hence patients may benefit from interventions that efficiently reactivate the latent virus to be eliminated by ARTs. Here we show that plasma extracellular vesicles (pEVs) can enhance HIV infection of activated CD4+ T cells and reactivate the virus in latently infected J-Lat 10.6 cells. Evaluation of the extravesicular miRNA cargo by a PCR array revealed that pEVs from HIV patients express miR-139-5p. Furthermore, we found that increased levels of miR-139-5p in J-Lat 10.6 cells incubated with pEVs corresponded with reduced expression of the transcription factor, FOXO1. pEV treatment also corresponded with increased miR-139-5p expression in stimulated PD1+ Jurkat cells, but with concomitant upregulation of FOXO1, Fos, Jun, PD-1 and PD-L1. However, J-Lat 10.6 cells incubated with miR-139-5p inhibitor-transfected pEVs from HIV ART-naïve and on-ART patients expressed reduced levels of miR-139-5p than cells treated with pEVs from healthy controls (HC). Collectively, our results indicate that pEV miR-139-5p belongs to a network of miRNAs that can promote cell activation, including latent HIV-infected cells by regulating the expression of FOXO1 and the PD1/PD-L1 promoters, Fos and Jun.

scholarly journals The Effect of JAK1/2 Inhibitors on HIV Reservoir Using Primary Lymphoid Cell Model of HIV Latency

2021 ◽  
Vol 12 ◽  
Author(s):  
Lesley R. de Armas ◽  
Christina Gavegnano ◽  
Suresh Pallikkuth ◽  
Stefano Rinaldi ◽  
Li Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Cell Model ◽  
Productive Infection ◽  
Latent Reservoir ◽  
Anatomical Site ◽  
Hiv Latency ◽  
Hiv Reservoir ◽  
Latently Infected ◽  
Infected Cells

HIV eradication is hindered by the existence of latent HIV reservoirs in CD4+ T cells. Therapeutic strategies targeting latent cells are required to achieve a functional cure, however the study of latently infected cells from HIV infected persons is extremely challenging due to the lack of biomarkers that uniquely characterize them. In this study, the dual reporter virus HIVGKO was used to investigate latency establishment and maintenance in lymphoid-derived CD4+ T cells. Single cell technologies to evaluate protein expression, host gene expression, and HIV transcript expression were integrated to identify and analyze latently infected cells. FDA-approved, JAK1/2 inhibitors were tested in this system as a potential therapeutic strategy to target the latent reservoir. Latent and productively infected tonsillar CD4+ T cells displayed similar activation profiles as measured by expression of CD69, CD25, and HLADR, however latent cells showed higher CXCR5 expression 3 days post-infection. Single cell analysis revealed a small set of genes, including HIST1-related genes and the inflammatory cytokine, IL32, that were upregulated in latent compared to uninfected and productively infected cells suggesting a role for these molecular pathways in persistent HIV infection. In vitro treatment of HIV-infected CD4+ T cells with physiological concentrations of JAK1/2 inhibitors, ruxolitinib and baricitinib, used in clinical settings to target inflammation, reduced latent and productive infection events when added 24 hr after infection and blocked HIV reactivation from latent cells. Our methods using an established model of HIV latency and lymphoid-derived cells shed light on the biology of latency in a crucial anatomical site for HIV persistence and provides key insights about repurposing baricitinib or ruxolitinib to target the HIV reservoir.

scholarly journals Epigenetic Silencing of Human Immunodeficiency Virus (HIV) Transcription by Formation of Restrictive Chromatin Structures at the Viral Long Terminal Repeat Drives the Progressive Entry of HIV into Latency

2008 ◽  
Vol 82 (24) ◽  
pp. 12291-12303 ◽  
Author(s):  
Richard Pearson ◽  
Young Kyeung Kim ◽  
Joseph Hokello ◽  
Kara Lassen ◽  
Julia Friedman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Lentiviral Vectors ◽  
Hiv Transcription ◽  
Wide Range ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT The molecular mechanisms utilized by human immunodeficiency virus (HIV) to enter latency are poorly understood. Following the infection of Jurkat T cells with lentiviral vectors that express Tat in cis, gene expression is progressively silenced. Silencing is greatly enhanced when the lentiviral vectors carry an attenuated Tat gene with the H13L mutation. Individual clones of lentivirus-infected cells showed a wide range of shutdown rates, with the majority showing a 50% silencing frequency between 30 to 80 days. The silenced clones characteristically contained a small fraction (0 to 15%) of activated cells that continued to express d2EGFP. When d2EGFP+ and d2EGFP− cell populations were isolated from the shutdown clones, they quickly reverted to the original distribution of inactive and active cells, suggesting that the d2EGFP+ cells arise from stochastic fluctuations in gene expression. The detailed analysis of transcription initiation and elongation using chromatin immunoprecipitation (ChIP) assays confirms that Tat levels are restricted in the latently infected cells but gradually rise during proviral reactivation. ChIP assays using clones of latently infected cells demonstrate that the latent proviruses carry high levels of deacetylated histones and trimethylated histones. In contrast, the cellular genes IκBα and GAPDH had high levels of acetylated histones and no trimethylated histones. The levels of trimethylated histone H3 and HP1-α associated with HIV proviruses fell rapidly after tumor necrosis factor alpha activation. The progressive shutdown of HIV transcription following infection suggests that epigenetic mechanisms targeting chromatin structures selectively restrict HIV transcription initiation. This decreases Tat production below the levels that are required to sustain HIV gene expression.

scholarly journals Similar frequency and inducibility of intact HIV-1 proviruses in blood and lymph nodes

2020 ◽  
Author(s):  
Alyssa R Martin ◽  
Alexandra M Bender ◽  
Jada Hackman ◽  
Kyungyoon J Kwon ◽  
Briana A Lynch ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Nodes ◽  
Cd4 T Cells ◽  
Viral Rna ◽  
Latent Reservoir ◽  
Similar Frequency ◽  
Latently Infected ◽  
Infected Cells ◽  
Secondary Lymphoid Organs ◽  
Hiv 1

Abstract Background The HIV-1 latent reservoir (LR) in resting CD4 + T cells is a barrier to cure. LR measurements are commonly performed on blood samples and therefore may miss latently infected cells residing in tissues, including lymph nodes. Methods We determined the frequency of intact HIV-1 proviruses and proviral inducibility in matched peripheral blood (PB) and lymph node (LN) samples from ten HIV-1-infected patients on ART using the intact proviral DNA assay and a novel quantitative viral induction assay. Prominent viral sequences from induced viral RNA were characterized using a next-generation sequencing assay. Results The frequencies of CD4 + T cells with intact proviruses were not significantly different in PB vs LN (61vs104/10 6CD4 + cells), and were substantially lower than frequencies of CD4 + T cells with defective proviruses. The frequencies of CD4 + T cells induced to produce high levels of viral RNA were not significantly different in PB vs LN (4.3/10 6 vs 7.9/10 6), but were 14-fold lower than the frequencies of cells with intact proviruses. Sequencing of HIV-1 RNA from induced proviruses revealed comparable sequences in paired PB and LN samples. Conclusions These results further support the use of PB as an appropriate proxy for the HIV-1 LR in secondary lymphoid organs

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