Biology subjects

Browne, E. P.

Publications and source records attributed to Browne, E. P..

At least 19 recordsLinked to original sources

bioRxiv2026

SPP1+ Microglia Are Associated with Neuroimmune Rewiring and Glutamatergic Neuronal Injury in ART-Suppressed People Living with HIV

Abstract Antiretroviral therapy (ART) effectively suppresses systemic HIV replication but does not eradicate viral reservoirs in the brain, where their identity and contribution to neurological injury remain poorly defined. Using bulk, single-cell, and single-nucleus transcriptomics of postmortem human brain tissue, we identify an activated SPP1 microglial population that expands 5.8-fold during ART and serves as the primary central nervous system (CNS) reservoir, preferentially harboring HIV transcripts. These reservoir microglia adopt a distinct, immune-evasive reprogramming state marked by chronic type I interferon signaling and inflammasome activation, which we recapitulate in primary human microglia via prolonged interferon-{beta} exposure. We show that viral transcription in ART-suppressed brains is dominated by nef, which may sustain the viral reservoir by disrupting host HLA-A and HLA-E presentation machinery. This persistent, immune-evasive state is associated with TREM2-C1QC complement-mediated synaptic pruning and severe DNA damage response dysregulation, culminating in a profound loss of VGLUT1 and GAD67 glutamatergic neurons that persists despite viral suppression. Our findings establish SPP1 microglia as an active, pathogenic CNS reservoir, identifying the SPP1, MHC-I, and complement pathways as therapeutic targets to eliminate viral persistence and reverse HIV-associated neurocognitive dysfunction.

neuroscience↗

bioRxiv2026

RelB proximity proteomics and CRISPR screening define chromatin regulators of noncanonical NF-κB control of HIV latency and reactivation

Activation of the non-canonical NF-{kappa}B pathway via RelB/p52 signaling by SMAC mimetics such as AZD5582 is a promising strategy to induce HIV expression from latency, but the chromatin mechanisms linking RelB/p52 to proviral regulation remain poorly defined. Here, we combine RelB BioID proteomics, targeted HIV-CRISPR screening, and pharmacologic validation to identify ncNF-{kappa}B-associated regulators of HIV expression. RelB BioID revealed an extensive interaction network of chromatin and transcriptional regulators in basal and AZD5582-activated states. Basally associated RelB proteins included NSD2, SWI/SNF components, UHRF1, and DNMT1, while AZD5582-enriched proteins included p300, USP7, LSD1/KDM1A, NuRD components, SIN3A, and HBO1/KAT7. Functional screening using a custom guide RNA library targeting all BioID-identified factors identified regulators that promote HIV reactivation, including HBO1/KAT7, NSD2, and SIN3A, and regulators that restrict HIV expression, including p300, CHD4, and USP7. Because KAT7/HBO1 and p300 encode acetyltransferases with opposing screen phenotypes, we tested whether their catalytic activities contribute to HIV transcriptional regulation and found that, consistent with the CRISPR screen, KAT7/HBO1 inhibition reduced AZD5582-induced reactivation, whereas p300 inhibition enhanced it. Together, these data define a resource linking the RelB-associated chromatin landscape to HIV latency and reactivation.

cell biology↗

bioRxiv2025

Single cell analysis of HIV expression and integration sites reveals robust viral expression across diverse chromatin environments

Entry of HIV into latency is determined by a combination of factors, including fluctuations in the viral Tat protein, as well as the transcriptomic phenotype of the host cell. Determining the impact of the proviral integration site on viral expression has been challenging due to difficulty in measuring integration site and viral expression from the same cell. To investigate the influence of the HIV integration site on HIV expression, we analyzed a combined scRNAseq/scATACseq dataset from 117,610 HIV infected primary CD4 T cells. We used the scATACseq data to recover HIV integration site information from 1530 cells, and correlated this information with viral RNA reads in the scRNAseq data. We observed that, overall, HIV expression did not differ depending on the genomic features of viral integration, such as genic vs non genic, intron vs exon and forward versus reverse orientation. Furthermore, we found that there was no significant difference in HIV expression across 15 distinct chromatin compartments. Additionally, analysis of expression for host genes that were the target of proviral integration revealed strong upregulation of expression of the targeted host gene in [~]5% of the infected cells. This insertional activation occurred almost exclusively when HIV was integrated in the same orientation as the host cell gene and occurred as a result of integration within diverse positions across a gene. These findings suggest that HIV expression is relatively robust to the genomic context of the HIV integration site, and that HIV can strongly upregulate expression of integration site genes during infection.

microbiology↗

bioRxiv2025

Single cell multiomic analysis of the impact of Delta-9-tetrahydrocannabinol on HIV infected CD4 T cells

Cannabis use is prevalent among individuals living with HIV in the United States, but the impact of cannabis exposure on the reservoir of latently infected cells that persists during antiretroviral therapy (ART) remains unclear. To address this gap, we analyzed the effect of {Delta}-9-tetrahydrocannabinol (THC) on primary CD4 T cells that were latently infected with HIV. We found that THC had no detectable effect on baseline or latency reversing agent (LRA) stimulated HIV expression, or on expression of an activation marker (CD38). However, using an integrated multiomic single-cell analysis of genome-wide chromatin accessibility and gene expression, we observed altered expression of several hundred genes in HIV infected CD4 T cells after THC exposure, including transcriptional downregulation of genes involved in protein translation and antiviral pathways, indicating that THC suppresses innate immune activation in infected cells. Additionally, chromatin accessibility analysis demonstrated upregulated chromatin binding activity for the transcriptional regulator CTCF, and reduced activity for members of the ETS transcription factor family in infected cells after THC exposure. These findings provide insights into the mechanisms by which cannabis use could influence the persistence of HIV within cellular reservoirs and the molecular phenotype of latently infected cells. Further elucidation of the underlying mechanisms involved in THC-mediated changes to HIV infected cells, will lead to an improved understanding of the impact of cannabis use on the HIV reservoir. ImportanceCannabis use is common among individuals living with HIV, but the long-term effects of cannabis use on the HIV reservoir are not yet studied completely. We employed advanced single-cell technologies to reveal how cannabis components, specifically THC, influence HIV-infected immune cells and their pattern of gene expression. We found that, while THC doesnt reactivate virus in latently infected cells, it alters the molecular characteristics of these infected immune cells. These findings are important because they underscore how cannabis could regulate persistent infection in people living with HIV. Understanding these cellular changes in response to THC could be helpful for successful treatment for people living with HIV.

molecular biology↗

bioRxiv2025

A screen of chromatin-targeting compounds identifies TAF1 as a novel regulator of HIV latency

Antiretroviral therapy (ART) suppresses HIV replication but fails to eliminate the virus due to the persistence of a transcriptionally silent reservoir, which remains the primary barrier to a cure. HIV latency is maintained through chromatin-mediated repression, making epigenetic regulators attractive therapeutic targets. To identify new modulators of latency, we screened a focused library of 84 chromatin-targeting small molecules. This screen identified BAY-299, a bromodomain inhibitor selective for TAF1 and BRD1, as a latency-modulating compound. BAY-299 reactivated HIV expression and enhanced the efficacy of established latency-reversing agents (LRAs), including vorinostat, prostratin, and iBET-151, in cell line models. CRISPR/Cas9-mediated knockout experiments demonstrated that TAF1, but not BRD1, is essential for maintaining HIV latency and that TAF1 depletion selectively increases HIV transcription with minimal effects on host gene expression. Dual knockout of TAF1 and Tat revealed that the reactivation effect is partially Tat dependent. CUT&RUN analysis further showed that TAF1 depletion increases RNA Polymerase II occupancy across the HIV gene body, suggesting enhanced transcriptional elongation. These findings identify TAF1 as a novel regulator of HIV latency and demonstrate the utility of targeted chemical screening to uncover therapeutic vulnerabilities within the latent reservoir. ImportanceHIV remains incurable due to the persistence of a transcriptionally silent reservoir in infected cells that is not eliminated by antiretroviral therapy. This transcriptionally silent state, known as latency, is controlled by host cell factors that regulate access to the viral genome. In this study, we identified the host protein TAF1 as a key regulator that maintains HIV in a latent state. Using both genetic and chemical approaches, we demonstrated that reducing TAF1 levels selectively increases HIV gene expression without broadly disrupting host gene transcription. These findings highlight a previously unrecognized mechanism of HIV latency control and identify TAF1 as a potential therapeutic target. Understanding how host chromatin regulators contribute to latency is essential for developing strategies that aim to eliminate the persistent HIV reservoir.

microbiology↗

bioRxiv2025

HIV-1 accessory protein Vpr possesses a cryptic p300-dependent transcription-promoting activity that is blocked by histone deacetylases in CD4+ T cells.

Antiretroviral therapy (ART) has dramatically improved the clinical prognosis for people with HIV and prevents HIV transmission. However, ART does not cure HIV infection because of a persistent, latent reservoir in long-lived cells such as central memory CD4+ T (TCM) cells. Eliminating or preventing reservoir formation will require a better understanding of HIV-1 latency establishment. We and others have recently shown that host cell factors such as histone deacetylases (HDACs) are critical cellular factors that allow HIV-1 entry into latency. Whether HDACs interact with specific viral factors to regulate latency establishment, however, is unknown. To examine the role of individual HIV-1 accessory proteins, we constructed a panel of HIV-1 reporter strains, each expressing a single HIV-1 accessory protein, and examined them in a primary CD4+ T-cell latency model. Interestingly, we found that the HDAC inhibitor (HDACi) vorinostat potently enhances the effect of the HIV-1 protein Vpr in promoting HIV expression in infected cells, suggesting that Vpr possesses a cryptic transcription-promoting activity that is restricted by HDACs. This activity was dependent on a p300-binding domain of Vpr and inhibited by a selective p300 histone acetyltransferase inhibitor. Interestingly, Vpr expression also resulted in a significant increase in the proportion of infected cells with a central memory (TCM) phenotype. Furthermore, we observed that TCM cells were more resistant to Vpr-induced apoptosis/cell death than other CD4+ T-cell subtypes, indicating that Vpr expression during reservoir formation selects for latent proviruses in TCM cells. Overall, these findings suggest that Vpr plays an important role in shaping the latent reservoir and that HIV-1 latency results, in part, from an HDAC-mediated restriction on Vprs transcription-promoting activity. Understanding how viral factors shape the latent reservoir and how host and viral factors interact during HIV-1 latency establishment in CD4+ T cells will aid in the development of new latency-targeting therapies. Author SummaryAlthough antiretroviral therapy is effective at treating HIV, a cure remains elusive. The primary obstacle to HIV cure is the presence of a long-lived reservoir of latently infected cells in which the virus persists despite therapy. Recent work has shown that a sizable fraction of this latent reservoir forms near the time that therapy is initiated, suggesting it may be possible to prevent some of the reservoir from forming. However, latency prevention will require a better understanding of how HIV enters latency, including how viral gene expression is silenced. We therefore sought to examine the role of the interaction between viral proteins and host factors in turning off viral gene expression and found that, whereas the HIV protein Vpr turns on viral gene expression, host histone deacetylases block this activity. Second, we observed that Vpr expression in infected cells leads to an increase in the relative proportion of central memory CD4+ T cells, a cell type that harbors latent virus. Our findings on the role of the viral protein Vpr in the silencing of viral gene expression and the persistence of certain memory cell types during infection will be important for developing new approaches to targeting latently infected cells.

microbiology↗

bioRxiv2025

NF-κB dependent gene expression and plasma IL-1β, TNFα and GCSF drive transcriptomic diversity and CD4:CD8 ratio in people with HIV on ART.

Despite antiretroviral therapy (ART), people with HIV (PWH) on ART experience higher rates of morbidity and mortality vs. age-matched HIV negative controls, which may be driven by chronic inflammation due to persistent virus. We performed bulk RNA sequencing (RNA-seq) on peripheral CD4+ T cells, as well as quantified plasma immune marker levels from 154 PWH on ART to identify host immune signatures associated with immune recovery (CD4:CD8) and HIV persistence (cell-associated HIV DNA and RNA). Using a novel dimension reduction tool - Pairwise Controlled Manifold Approximation (PaCMAP), we defined three distinct participant transcriptomic clusters. We found that these three clusters were largely defined by differential expression of genes regulated by the transcription factor NF-{kappa}B. While clustering was not associated with HIV reservoir size, we observed an association with CD4:CD8 ratio, a marker of immune recovery and prognostic factor for mortality in PWH on ART. Furthermore, distinct patterns of plasma IL-1{beta}, TNF- and GCSF were also strongly associated with the clusters, suggesting that these immune markers play a key role in CD4+ T cell transcriptomic diversity and immune recovery in PWH on ART. These findings reveal novel subgroups of PWH on ART with distinct immunological characteristics, and define a transcriptional signature associated with clinically significant immune parameters for PWH. A deeper understanding of these subgroups could advance clinical strategies to treat HIV-associated immune dysfunction.

microbiology↗

bioRxiv2024

Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal

The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo, and we detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells. Finally, we found that INTS12 more generally limits the efficacy of a variety of LRAs with different mechanisms of action.

microbiology↗

bioRxiv2024

A targeted CRISPR screen identifies ETS1 as a regulator of HIV latency.

Human Immunodeficiency virus (HIV) infection is regulated by a wide array of host cell factors that combine to influence viral transcription and latency. To understand the complex relationship between the host cell and HIV latency, we performed a lentiviral CRISPR screen that targeted a set of host cell genes whose expression or activity correlates with HIV expression. We further investigated one of the identified factors - the transcription factor ETS1 and found that it is required for maintenance of HIV latency in a primary CD4 T cell model. Interestingly, ETS1 played divergent roles in actively infected and latently infected CD4 T cells, with knockout of ETS1 leading to reduced HIV expression in actively infected cells, but increased HIV expression in latently infected cells, indicating that ETS1 can play both a positive and negative role in HIV expression. CRISPR/Cas9 knockout of ETS1 in CD4 T cells from ART-suppressed people with HIV (PWH) confirmed that ETS1 maintains transcriptional repression of the clinical HIV reservoir. Transcriptomic profiling of ETS1-depleted cells from PWH identified a set of host cell pathways involved in viral transcription that are controlled by ETS1 in resting CD4 T cells. In particular, we observed that ETS1 knockout increased expression of the long non-coding RNA MALAT1 that has been previously identified as a positive regulator of HIV expression. Furthermore, the impact of ETS1 depletion on HIV expression in latently infected cells was partially dependent on MALAT1. Overall, these data demonstrate that ETS1 is an important regulator of HIV latency and influences expression of several cellular genes, including MALAT1, that could have a direct or indirect impact on HIV expression. Author SummaryHIV latency is a major obstacle for the eradication of HIV. However, molecular mechanisms that restrict proviral expression during therapy are not well understood. Identification of host cell factors that silence HIV would create opportunities for targeting these factors to reverse latency and eliminate infected cells. Our study aimed to explore mechanisms of latency in infected cells by employing a lentiviral CRISPR screen and CRISPR/Cas9 knockout in primary CD4 T cells. These experiments revealed that ETS1 is essential for maintaining HIV latency in primary CD4 T cells and we further confirmed ETS1s role in maintaining HIV latency through CRISPR/Cas9 knockout in CD4 T cells from antiretroviral therapy (ART)-suppressed individuals with HIV. Transcriptomic profiling of ETS1-depleted cells from these individuals identified several host cell pathways involved in viral transcription regulated by ETS1, including the long non-coding RNA MALAT1. Overall, our study demonstrates that ETS1 is a critical regulator of HIV latency, affecting the expression of several cellular genes that directly or indirectly influence HIV expression.

microbiology↗

bioRxiv2023

The histone methyltransferase SETD2 regulates HIV expression and latency through a post-transcriptional mechanism.

HIV can enter a state of transcriptional latency in CD4 T cells, allowing the virus to evade the host immune system and persist during antiretroviral therapy. Thus, understanding the mechanisms that drive HIV latency, and developing strategies to reactivate viral expression in latently infected cells, are key goals for achieving a cure for HIV. The full spectrum of mechanisms behind the regulation of HIV expression and latency are unclear but include covalent modifications to cellular histones that are associated with the integrated provirus. Here we investigate the role of the SETD2 histone methyltransferase, which deposits H3K36 trimethylation (H3K36me3) cotranscriptionally at genes, in HIV infection. We show that prevention of H3K36me3 through addition of a potent and selective inhibitor of SETD2 (EPZ-719) in human T cells leads to reduced post-integration viral gene expression and accelerated the emergence of a latently infected pool within a population of infected cells. CRISPR/Cas9-mediated knockout of SETD2 in HIV infected primary CD4 T cells confirmed the role of SETD2 in HIV expression. Intriguingly, EPZ-719 exposure also enhanced responsiveness of latently infected cells to latency reversal with the HDAC inhibitor vorinostat. Transcriptomic profiling of EPZ-719 exposed HIV-infected cells identified numerous cellular pathways impacted by EPZ-719. Finally, we show SETD2 inhibition does not affect HIV viral transcription, but instead leads to a shift in the pattern of viral RNA splicing - a result that would be predicted to reduce HIV expression. These results identify SETD2 and H3K36me3 as novel regulators of HIV expression and latency through a post-transcriptional mechanism.

microbiology↗

bioRxiv2023

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy.

Understanding the interplay between the HIV reservoir and the host immune system may yield insights into HIV persistence during antiretroviral therapy (ART) and inform strategies for a cure. Here, we applied machine learning approaches to cross-sectional high-parameter HIV reservoir and immunology data in order to characterize host-reservoir associations and generate new hypotheses about HIV reservoir biology. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and measurement of genetically intact and total HIV proviral DNA frequencies were performed on peripheral blood samples from 115 people with HIV (PWH) on long-term ART. Analysis demonstrated that both intact and total proviral DNA frequencies were positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A Leave-One-Covariate-Out (LOCO) inference approach identified specific HIV reservoir and clinical-demographic parameters, such as age and biological sex, that were particularly important in predicting immunophenotypes. Overall, immune parameters were more strongly associated with total HIV proviral frequencies than intact proviral frequencies. Uniquely, however, expression of the IL-7 receptor alpha chain (CD127) on CD4 T cells was more strongly correlated with the intact reservoir. Unsupervised dimension reduction analysis identified two main clusters of PWH with distinct immune and reservoir characteristics. Using reservoir correlates identified in these initial analyses, decision tree methods were employed to visualize relationships among multiple immune and clinical-demographic parameters and the HIV reservoir. Finally, using random splits of our data as training-test sets, machine learning algorithms predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA. The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology.

microbiology↗

bioRxiv2023

A novel high throughput microwell outgrowth assay for HIV infected cells

Although antiretroviral therapy (ART) is highly effective at suppressing HIV replication, a viral reservoir persists that can reseed infection if ART is interrupted. Curing HIV will require elimination or functional containment of this reservoir, but the size of the HIV reservoir is highly variable between individuals. To evaluate the overall size of the HIV reservoir, several assays have been developed, including PCR based assays for viral DNA, the Intact Proviral DNA Assay (IPDA), and the Quantitative Viral Outgrowth Assay (QVOA). QVOA is the gold standard assay for measuring inducible replication competent proviruses, but this assay is technically challenging and time consuming. To provide a more rapid and less laborious tool for quantifying cells infected with replication competent HIV, we developed the Microwell Outgrowth Assay (MOA), in which HIV infected CD4 T cells are cocultured with an HIV-detecting reporter cell line in a polydimethylsiloxane (PDMS)/polystyrene array of nanoliter sized wells (rafts). Transmission of HIV from infected cells to the reporter cell line induces fluorescent reporter protein expression that is detected by automated scanning across the array. We show that this assay can detect HIV infected cells with a high degree of sensitivity and precision. Using this approach, we were able to detect HIV infected cells from ART-naive people with HIV (PWH) and from PWH on ART. Furthermore, we demonstrate that infected cells can be recovered from individual rafts and used to analyze the diversity of viral sequences. This assay may be a useful tool for quantifying and characterizing infected cells from PWH. Author summaryMeasuring the size of the HIV reservoir in people with HIV (PWH) will be important for determining the impact of HIV cure strategies. However, measuring this reservoir is challenging. We report a new method for quantifying HIV infected cells that involves culturing cells from PWH in an array of microwells with a cell line that detects HIV infection. We show that this approach can detect rare HIV infected cells and derive detailed virus sequence information for each infected cell.

microbiology↗

bioRxiv2023

Automated microarray for single-cell sorting and collection of lymphocytes following HIV reactivation

A promising strategy to cure HIV infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of various LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5 %, 11.2 %, and 12.1 % percentage of cells respectively, reactivated similar to that observed in other experimental systems. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found. These results advance our conceptual understanding of HIV reactivation dynamics at the single-cell level toward a cure for HIV.

bioengineering↗

bioRxiv2022

Impact of cannabis use on immune cell populations and the viral reservoir in people with HIV on suppressive antiretroviral therapy.

HIV infection remains incurable due to the persistence of a viral reservoir during antiretroviral therapy. Cannabis (CB) use is prevalent amongst people with HIV (PWH), but the impact of CB on the latent HIV reservoir has not been investigated. Peripheral CD4 and CD8 T cells from a cohort of CB-using PWH and a matched cohort of non-users on antiretroviral therapy were evaluated for expression of maturation/activation markers, HIV-specific T cell responses, and the frequency of intact proviral DNA. CB use was associated with increased abundance of naive T cells, reduced effector T cells, and reduced expression of activation markers. CB users also exhibited reduced levels of exhausted and senescent T cells compared to non-using controls. HIV-specific CD8 T cell responses were unaffected by CB use. While the abundance of intact proviruses was not significantly affected by CB use across the whole cohort, we observed that, for participants with high frequency of NKG2A or CD16 expression in NK cells, CB use was associated with a smaller intact HIV reservoir. This analysis is consistent with the hypothesis that CB use reduces activation, exhaustion and senescence in the T cells of PWH and may influence the size of the HIV reservoir.

microbiology↗

bioRxiv2022

A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency

Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR, which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent - AZD5582, an activator of the non-canonical NF{kappa}B pathway - to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR, and only in the combination of ING3 knockout with the activation of non-canonical NF{kappa}B via AZD5582 is there dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.

microbiology↗

bioRxiv2022

Integrated single-cell multiomic analysis of HIV latency reversal reveals novel regulators of viral reactivation.

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 ~4000 latently infected cells after reactivation using three different latency-reversing agents (LRAs). Differentially expressed genes and differentially accessible motifs were used to examine transcriptional pathways and transcription factor (TF) activities across the cell population. We identify cellular transcripts and TFs whose expression/activity was correlated with viral reactivation and demonstrate that a machine learning model trained on these data was 68% accurate at predicting viral reactivation. Finally, we validate the role of a new candidate HIV-regulating factor, GATA3, in the viral response to prostratin stimulation. These data demonstrate the power of integrated multimodal single-cell analysis to uncover novel relationships between host cell factors and HIV latency.

microbiology↗

bioRxiv2022

A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV infected cells

Approximately 70% of the HIV-1 latent reservoir originates from infections of CD4 T cells that occur in the months near the time of ART initiation, raising the possibility that interventions during this period might prevent reservoir seeding and reduce reservoir size. We identify class 1 histone deacetylase inhibitors (HDACi) as potent agents of latency prevention. Inhibiting HDACs in productively infected cells caused extended maintenance of HIV expression and this activity was associated with persistently elevated H3K9 acetylation and reduced H3K9 methylation at the viral LTR promoter region. HDAC inhibition in HIV-infected CD4 T cells during effector-to-memory transition led to striking changes in the memory phenotype of infected cells. Proviral silencing is accomplished through distinct activities of HDAC1/2 and HDAC3. Thus HDACs regulate a critical gateway process for HIV latency establishment and are required for the development of CD4 T-cell memory subsets that preferentially harbor long-lived, latent provirus.

molecular biology↗

bioRxiv2020

Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors.

Transcriptional silencing of HIV generates a reservoir of latently infected cells, but the mechanisms that lead to this outcome are not well understood. We characterized a primary cell model of HIV latency, and observed that latency is a stable, heritable viral state that is rapidly reestablished after stimulation. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit reduced proviral accessibility, elevated activity of Forkead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs. Latency reversing agents caused distinct patterns of chromatin reopening across the provirus. Furthermore, depletion of a chromatin domain insulator, 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.

microbiology↗