CAGE-seq reveals that HIV-1 latent infection does not trigger unique cellular responses in a Jurkat T cell model.

The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a GFP reporter under the HIV-1 promoter and an mKO2 reporter under the internal EF1α promoter. This viral construct enables direct identification of HIV-1 both productively and latently infected cells. In this study we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using Cap Analysis of Gene Expression (CAGE). We deep sequenced CAGE tags in uninfected, latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus producing cells had differentially expressed TSSs related to T-cell activation and apoptosis when compared to uninfected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to uninfected cells. Among these, SPP1 and APOE were down-regulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have anti-viral properties. Components of the PI3K/mTOR pathway, MLST8, 4EBP and RPS6, were significant TSSs in productively infected cells, and S6K phosphorylation was increased compared to latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent. Importance: Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used CAGE to compare the transcriptional landscape of latently infected cells with that of non-infected or productively infected cells. We found that latently infected cells and non-infected cells show quite similar transcriptional profiles. Our data suggest that T-cells cannot recognize incoming viral components nor the integrated HIV-1 genome when infection remains latent. These findings should guide future research into widening our approaches to identify and target latent HIV-1 infected cells.

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Reactivation Kinetics of HIV-1 and Susceptibility of Reactivated Latently Infected CD4+T Cells to HIV-1-Specific CD8+T Cells

Journal of Virology ◽

10.1128/jvi.01454-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9631-9638 ◽

Cited By ~ 10

Author(s):

Victoria E. K. Walker-Sperling ◽

Valerie J. Cohen ◽

Patrick M. Tarwater ◽

Joel N. Blankson

Keyword(s):

T Cells ◽

T Cell ◽

Antiretroviral Therapy ◽

Time Frame ◽

Virus Release ◽

Latently Infected ◽

Shock And Kill ◽

Infected Cells ◽

Kinetics Of ◽

Hiv 1

ABSTRACTThe “shock and kill” model of human immunodeficiency virus type 1 (HIV-1) eradication involves the induction of transcription of HIV-1 genes in latently infected CD4+T cells, followed by the elimination of these infected CD4+T cells by CD8+T cells or other effector cells. CD8+T cells may also be needed to control the spread of new infection if residual infected cells are present at the time combination antiretroviral therapy (cART) is discontinued. In order to determine the time frame needed for CD8+T cells to effectively prevent the spread of HIV-1 infection, we examined the kinetics of HIV transcription and virus release in latently infected cells reactivatedex vivo. Isolated resting, primary CD4+T cells from HIV-positive (HIV+) subjects on suppressive regimens were found to upregulate cell-associated HIV-1 mRNA within 1 h of stimulation and produce extracellular virus as early as 6 h poststimulation. In spite of the rapid kinetics of virus production, we show that CD8+T cells from 2 out of 4 viremic controllers were capable of effectively eliminating reactivated autologous CD4+cells that upregulate cell-associated HIV-1 mRNA. The results have implications for devising strategies to prevent rebound viremia due to reactivation of rare latently infected cells that persist after potentially curative therapy.IMPORTANCEA prominent HIV-1 cure strategy termed “shock and kill” involves the induction of HIV-1 transcription in latently infected CD4+T cells with the goal of elimination of these cells by either the cytotoxic T lymphocyte response or other immune cell subsets. However, the cytotoxic T cell response may also be required after curative treatment if residual latently infected cells remain. The kinetics of HIV-1 reactivation indicate rapid upregulation of cell-associated HIV-1 mRNA and a 5-h window between transcription and virus release. Thus, HIV-specific CD8+T cell responses likely have a very short time frame to eliminate residual latently infected CD4+T cells that become reactivated after discontinuation of antiretroviral therapy following potentially curative treatment strategies.

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Extensive proteomic and transcriptomic changes quench the TCR/CD3 activation signal of latently HIV-1 infected T cells

PLoS Pathogens ◽

10.1371/journal.ppat.1008748 ◽

2021 ◽

Vol 17 (1) ◽

pp. e1008748

Author(s):

Eric Carlin ◽

Braxton Greer ◽

Kelsey Lowman ◽

Alexandra Duverger ◽

Frederic Wagner ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Network Analysis ◽

Cell Lines ◽

Data Set ◽

Latently Infected ◽

Latent Hiv ◽

T Cell Lines ◽

Hiv 1

The biomolecular mechanisms controlling latent HIV-1 infection, despite their importance for the development of a cure for HIV-1 infection, are only partially understood. For example, ex vivo studies have recently shown that T cell activation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, but the molecular biology of this phenomenon is unclear. We demonstrate that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of T cell receptor (TCR)/CD3 activation-inert latently infected T cells. RNA-level analysis identified extensive transcriptomic differences between uninfected, TCR/CD3 activation-responsive and -inert T cells, but did not reveal a gene expression signature that could functionally explain TCR/CD3 signaling inertness. Network analysis suggested a largely stochastic nature of these gene expression changes (transcriptomic noise), raising the possibility that widespread gene dysregulation could provide a reactivation threshold by impairing overall signal transduction efficacy. Indeed, compounds that are known to induce genetic noise, such as HDAC inhibitors impeded the ability of TCR/CD3 activation to trigger HIV-1 reactivation. Unlike for transcriptomic data, pathway enrichment analysis based on phospho-proteomic data directly identified an altered TCR signaling motif. Network analysis of this data set identified drug targets that would promote TCR/CD3-mediated HIV-1 reactivation in the fraction of otherwise TCR/CD3-reactivation inert latently HIV-1 infected T cells, regardless of whether the latency models were based on T cell lines or primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell responsiveness prior to the use of any activating stimulus will likely have to be an element of future HIV-1 cure therapies.

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Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells

10.1101/526053 ◽

2019 ◽

Author(s):

Birgitta Lindqvist ◽

Sara Svensson Akusjarvi ◽

Anders Sonnerborg ◽

Marios Dimitriou ◽

J. Peter Svensson

Keyword(s):

T Cells ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Ex Vivo ◽

Immunodeficiency Virus ◽

Latently Infected ◽

Infected Cells ◽

Active Transcription ◽

Hiv 1

Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir. The reservoir that reinstates an active replication comprises only cells with intact provirus that can be reactivated. We confirmed that latently infected cells from patients exhibited active transcription throughout the provirus. To find transcriptional determinants, we characterized the establishment and maintenance of viral latency during proviral chromatin maturation in cultures of primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (i.e., H3K27ac) remained detectable, even after prolonged proviral silencing. After T-cell activation, the proviral activation occurred uniquely in cells with H3K27ac-marked proviruses. Our observations suggested that, after transient proviral activation, cells were actively returned to latency.

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Efficient inhibition of HIV using CRISPR/Cas13d nuclease system

10.1101/2021.07.21.453273 ◽

2021 ◽

Author(s):

Hoang Nguyen ◽

Hannah Wilson ◽

Sahana Jayakumar ◽

Viraj Kulkarni ◽

Smita Kulkarni

Keyword(s):

T Cells ◽

Alternative Treatment ◽

Treatment Modality ◽

Rna Viruses ◽

Guide Rnas ◽

Rna Targeting ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13 mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4+ T-cells and suppression of HIV-1 reactivated from latently infected cells using the CRISPR/Cas13d system. Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV.

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Humanized Mouse Model of HIV-1 Latency with Enrichment of Latent Virus in PD-1+and TIGIT+CD4 T Cells

Journal of Virology ◽

10.1128/jvi.02086-18 ◽

2019 ◽

Vol 93 (10) ◽

Cited By ~ 4

Author(s):

George N. Llewellyn ◽

Eduardo Seclén ◽

Stephen Wietgrefe ◽

Siyu Liu ◽

Morgan Chateau ◽

...

Keyword(s):

T Cells ◽

Mouse Model ◽

Ex Vivo ◽

Latent Reservoir ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

ABSTRACTCombination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used anex vivolatency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use theex vivolatency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishesin vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCEHIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body’s immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.

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Antigen responsive CD4+ T cell clones contribute to the HIV-1 latent reservoir

10.1101/2020.01.10.902155 ◽

2020 ◽

Cited By ~ 1

Author(s):

Pilar Mendoza ◽

Julia R. Jackson ◽

Thiago Oliveira ◽

Christian Gaebler ◽

Victor Ramos ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Half Life ◽

Latent Reservoir ◽

Small Subset ◽

T Cell Clones ◽

Cell Clones ◽

Latently Infected ◽

Infected Cells ◽

Hiv 1

AbstractAntiretroviral therapy suppresses but does not cure HIV-1 infection due to the existence of a long-lived reservoir of latently infected cells. The reservoir has an estimated half-life of 44 months and is largely composed of clones of infected CD4+ T cells. The long half-life appears to result in part from expansion and contraction of infected CD4+ T cell clones. However, the mechanisms that govern this process are poorly understood. To determine whether the clones might result from, and be maintained by exposure to antigen, we measured responses of reservoir cells to a small subset of antigens from viruses that produce chronic or recurrent infections. Despite the limited panel of test antigens, clones of antigen responsive CD4+ T cells containing defective or intact latent proviruses were found in 7 out of 8 individuals studied. Thus, chronic or repeated exposure to antigen may contribute to the longevity of the HIV-1 reservoir by stimulating the clonal expansion of latently infected CD4+ T cells.

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CD32+and PD-1+Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals

Journal of Virology ◽

10.1128/jvi.00901-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 18

Author(s):

Alessandra Noto ◽

Francesco A. Procopio ◽

Riddhima Banga ◽

Madeleine Suffiotti ◽

Jean-Marc Corpataux ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Hiv Infection ◽

Cell Population ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

Cell Populations ◽

Latently Infected ◽

Infected Cells ◽

Hiv 1

ABSTRACTA recent study conducted in blood has proposed CD32 as the marker identifying the “elusive” HIV reservoir. We have investigated the distribution of CD32+CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1+CD4 T cells. The frequency of CD32+CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32+cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32+CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32+and PD-1+CD4 T cells compared to CD32−and PD-1−cells in both viremic and treated individuals, but there was no difference between CD32+and PD-1+cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32+versus PD-1+cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32+PD-1+CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32−PD-1−(averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32+PD-1−(2.2-fold in treated individuals and 4.3-fold in viremics), and CD32−PD-1+(2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32+PD-1−and CD32−PD-1+CD4 T cells. Interestingly, the proportion of CD32+and PD-1+CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.IMPORTANCEThe existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

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Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System

Viruses ◽

10.3390/v13091850 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1850

Author(s):

Hoang Nguyen ◽

Hannah Wilson ◽

Sahana Jayakumar ◽

Viraj Kulkarni ◽

Smita Kulkarni

Keyword(s):

T Cells ◽

Alternative Treatment ◽

Treatment Modality ◽

Rna Viruses ◽

Guide Rnas ◽

Rna Targeting ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13-mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct, non-overlapping sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4+ T-cells and suppression of HIV-1 reactivated from latently infected cells using the CRISPR/Cas13d system. Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV.

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Inhibition of the H3K27 demethylase UTX enhances the epigenetic silencing of HIV proviruses and induces HIV-1 DNA hypermethylation but fails to permanently block HIV reactivation

10.1101/2021.08.02.454353 ◽

2021 ◽

Author(s):

Kien Nguyen ◽

Jonathan Karn ◽

Won Kyung ◽

Curtis Dobrowolski ◽

Meenakshi Shukla

Keyword(s):

Dna Methylation ◽

T Cells ◽

Cell Receptor ◽

Epigenetic Silencing ◽

Dna Hypermethylation ◽

Dual Inhibitor ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

One strategy for a functional cure of HIV-1 is block and lock, which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. To suppress latent HIV-1 in a stable fashion, we depleted the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and were associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and induced DNA methylation at specific sites in the 5' LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. Thus, epigenetic silencing by itself appears to be insufficient to permanently silence HIV-1 proviral transcription.

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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.

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