Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors

SummaryTranscriptional 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.

Download Full-text

Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors

PLoS Pathogens ◽

10.1371/journal.ppat.1009346 ◽

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.

Download Full-text

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

10.1101/303198 ◽

2018 ◽

Cited By ~ 1

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.

Download Full-text

Harmine enhances the activity of the HIV-1 latency-reversing agents ingenol A and SAHA

10.1101/2020.01.31.927368 ◽

2020 ◽

Author(s):

Jared P. Taylor ◽

Lucas H. Armitage ◽

Daniel L. Aldridge ◽

Melanie N. Cash ◽

Mark A. Wallet

Keyword(s):

Transcription Factors ◽

Cell Model ◽

Hdac Inhibitors ◽

Negative Regulator ◽

Viral Reactivation ◽

Latent Reservoir ◽

Transcriptional Elongation ◽

Latently Infected ◽

Infected Cells ◽

Hiv 1

AbstractInfection of HIV-1 remains incurable because long-lived, latently-infected cells persist during prolonged antiretroviral therapy. Attempts to pharmacologically reactivate and purge the latent reservoir with latency reactivating agents (LRAs) such as protein kinase C (PKC) agonists (e.g. ingenol A) or histone deacetylase (HDAC) inhibitors (e.g. SAHA) have shown promising but incomplete efficacy. Using the J-Lat T cell model of HIV latency, we found that the plant-derived compound harmine enhanced the efficacy of existing PKC agonist LRAs in reactivating latently-infected cells. Treatment with harmine increased not only the number of reactivated cells but also increased HIV transcription and protein expression on a per-cell basis. Importantly, we observed an additive effect when harmine was used in combination with ingenol A and the HDAC inhibitor SAHA. An investigation into the mechanism revealed that harmine, when used with LRAs, increased the availability of transcription factors needed for viral reactivation such as NFκB, MAPK p38, and ERK1/2. We also found that harmine treatment resulted in reduced expression of HEXIM1, a negative regulator of transcriptional elongation. Despite harmine’s reported inhibitory effects on DYRK1A and consequent enhancement of NFAT signaling, the HIV reactivating effects of harmine occurred independent of DYRK1A and NFAT. Harmine increases the efficacy of LRAs by increasing the availability of HIV-1 transcription factors and decreasing expression of HEXIM1. Combination therapies with harmine and LRAs could benefit patients by achieving deeper reactivation of the latent pool of HIV provirus.

Download Full-text

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

Frontiers in Immunology ◽

10.3389/fimmu.2021.697604 ◽

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.

Download Full-text

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

Frontiers in Immunology ◽

10.3389/fimmu.2021.720697 ◽

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.

Download Full-text

Rapid Expression of Human Immunodeficiency Virus following Activation of Latently Infected Cells

Journal of Virology ◽

10.1128/jvi.80.3.1599-1603.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1599-1603 ◽

Cited By ~ 22

Author(s):

Philip A. Arlen ◽

David G. Brooks ◽

Lian Y. Gao ◽

Dimitrios Vatakis ◽

Helen J. Brown ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protein Production ◽

Cell Activation ◽

Viral Gene Expression ◽

Viral Rna ◽

Viral Gene ◽

Quiescent Cells ◽

Immunodeficiency Virus ◽

Latently Infected ◽

Infected Cells

ABSTRACT The host cell activation state impacts the nature of human immunodeficiency virus infection. Activated cells facilitate productive infections; quiescent cells enable the virus to enter a latent state, the major obstacle to viral clearance. We wanted to understand how these differences affected viral gene expression. In quiescent cells activated prior to infection, viral RNA was seen 12 h postinfection; when cells were stimulated postinfection, viral RNA was not seen until 36 h postinfection. Up-regulation of viral RNA in latently infected cells occurred within 2 h poststimulation. This hierarchy also held true for viral protein production. These results may explain the rapid reemergence of viremia following termination of therapy.

Download Full-text

Viral gene expression in murine sarcoma virus(murine leukemia virus)-infected cells.

Journal of Virology ◽

10.1128/jvi.27.3.768-775.1978 ◽

1978 ◽

Vol 27 (3) ◽

pp. 768-775 ◽

Cited By ~ 8

Author(s):

D Dina ◽

E E Penhoet

Keyword(s):

Gene Expression ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Viral Gene Expression ◽

Viral Gene ◽

Murine Sarcoma Virus ◽

Sarcoma Virus ◽

Infected Cells ◽

Murine Sarcoma ◽

Murine Leukemia

Download Full-text

Patterns of accumulation of miRNAs encoded by herpes simplex virus during productive infection, latency, and on reactivation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1422657112 ◽

2014 ◽

Vol 112 (1) ◽

pp. E49-E55 ◽

Cited By ~ 34

Author(s):

Te Du ◽

Zhiyuan Han ◽

Guoying Zhou ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cultured Cells ◽

Viral Gene Expression ◽

The Body ◽

Productive Infection ◽

Viral Gene ◽

Infected Cells ◽

Simplex Virus ◽

Portal Of Entry

The key events in herpes simplex virus (HSV) infections are (i) replication at a portal of entry into the body modeled by infection of cultured cells; (ii) establishment of a latent state characterized by a sole latency-associated transcript and microRNAs (miRNAs) modeled in murine peripheral ganglia 30 d after inoculation; and (iii) reactivation from the latent state modeled by excision and incubation of ganglia in medium containing anti-NGF antibody for a timespan of a single viral replicative cycle. In this report, we examine the pattern of synthesis and accumulation of 18 HSV-1 miRNAs in the three models. We report the following: (i) H2-3P, H3-3P, H4-3P, H5-3P, H6-3P, and H7-5P accumulated in ganglia harboring latent virus. All but H4-3P were readily detected in productively infected cells, and most likely they originate from three transcriptional units. (ii) H8-5P, H15, H17, H18, H26, and H27 accumulated during reactivation. Of this group, only H26 and H27 could be detected in productively infected cells. (iii) Of the 18 we have examined, only 10 miRNAs were found to accumulate above background levels in productively infected cells. The disparity in the accumulation of miRNAs in cell culture and during reactivation may reflect differences in the patterns of regulation of viral gene expression during productive infection and during reactivation from the latent state.

Download Full-text

The gammaherpesvirus 68 viral cyclin facilitates reactivation by promoting latent gene expression

10.1101/761700 ◽

2019 ◽

Author(s):

Brian F Niemeyer ◽

Joy E Gibson ◽

Jennifer N Berger ◽

Lauren M Oko ◽

Eva Medina ◽

...

Keyword(s):

Gene Expression ◽

Critical Role ◽

Productive Infection ◽

Viral Gene ◽

Cellular Distribution ◽

Viral Cyclin ◽

Latently Infected ◽

Infected Cells ◽

Virally Encoded ◽

The Impact

AbstractGammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into more active lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on latent gene expression, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::βla), to enumerate both the cellular distribution and frequency of latent gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::βla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::βla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential latent gene expression, and that a primary function of the viral cyclin is to promote latent gene expression within infected cells in vivo.AUTHOR SUMMARYGammaherpesviruses are ubiquitous viruses with oncogenic potential that establish latency for the life of the host. These viruses can emerge from latency through reactivation, a process that is controlled by the immune system. Control of viral latency and reactivation is thought to be critical to prevent γHV-associated disease. This study focuses on a virally-encoded cyclin that is required for reactivation from latency. By characterizing how the viral cyclin influences latent infection in pure cell populations, we find that the viral cyclin has a vital role in promoting viral gene expression during latency. This work provides new insight into the function of a virally encoded cyclin in promoting reactivation from latency.

Download Full-text