scholarly journals Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency

2020 ◽  
Vol 6 (33) ◽  
pp. eaba6617
Author(s):  
Mateusz Stoszko ◽  
Abdullah M. S. Al-Hatmi ◽  
Anton Skriba ◽  
Michael Roling ◽  
Enrico Ne ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Rna Polymerase Ii ◽  
Ex Vivo ◽  
Elongation Factor ◽  
Bioactive Molecules ◽  
Reversal Agents ◽  
Small Nuclear Ribonucleoprotein ◽  
Latently Infected ◽  
Hiv 1

A leading pharmacological strategy toward HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

scholarly journals Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb and reverses HIV-1 latency

2019 ◽  
Author(s):  
Mateusz Stoszko ◽  
Abdullah M.S. Al-Hatmi ◽  
Anton Skriba ◽  
Michael Roling ◽  
Enrico Ne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Reversal Agents ◽  
Latently Infected ◽  
Infected Cells ◽  
Therapeutic Compounds ◽  
Hiv 1

AbstractA leading pharmacological strategy towards HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites (extrolites) as a source of bio-active molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the P-TEFb inhibitory 7SK snRNP complex to be significantly reduced upon GTX treatment of independent donor CD4+T cells. GTX disrupted 7SK snRNP, releasing active P-TEFb, which then phosphorylated RNA Pol II CTD, inducing HIV transcription. Our data highlight the power of combining a medium throughput bioassay, mycology and orthogonal mass spectrometry to identify novel potentially therapeutic compounds.

scholarly journals SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4+ T Cells

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Jenna M. Antonucci ◽  
Sun Hee Kim ◽  
Corine St. Gelais ◽  
Serena Bonifati ◽  
Tai-Wei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Leukemia Virus ◽  
Viral Latency ◽  
Viral Reservoir ◽  
Latently Infected ◽  
Ltr Promoter ◽  
Hiv 1

ABSTRACT Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in nondividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T cells, which are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 long terminal repeat (LTR) activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 LTR-driven gene expression at a transcriptional level. Tat coexpression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed the LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not that of murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D R207N [HD/RN]) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not the T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro. These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression potentially regulates viral latency in CD4+ T cells. IMPORTANCE A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in nondividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T cells.

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

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.

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

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.

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

2019 ◽  
Vol 93 (10) ◽  
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.

scholarly journals “A transient heritable memory regulates HIV reactivation from latency”

2020 ◽  
Author(s):  
Yiyang Lu ◽  
Abhyudai Singh ◽  
Roy D. Dar
Keyword(s):  
T Cells ◽  
Single Cells ◽  
Single Copy ◽  
Colony Growth ◽  
Skewed Distribution ◽  
Latent Reservoir ◽  
Reversal Agents ◽  
Fluctuation Test ◽  
Latently Infected ◽  
Hiv 1

AbstractReactivation of human immunodeficiency virus 1 (HIV-1) from latently infected T cells is a critical barrier to successfully eradicate HIV-1 from patients. Latency models in Jurkat T-cells reveal that individual cells reactivate in an all-or-none fashion when exposed to latency reversal agents (LRAs). Remarkably, this heterogeneity arises within a clonal cell population of genetically identical cells containing a single copy of the provirus integrated at the same genomic site. To characterize these single-cell responses, we leverage the classical Luria-Delbrück fluctuation test where single cells are isolated from a clonal population, and exposed to LRAs after a period of colony expansion. If cellular responses are purely random, then the fraction of reactivating cells should have minimal colony-to-colony fluctuations given the large number of cells present after weeks of colony growth. In contrast, data shows considerable colony-to-colony fluctuations with the fraction of reactivating cells following a skewed distribution. Systematic measurements of fluctuations over time in combination with mathematical modeling uncovers the existence of a heritable memory that regulates HIV-1 reactivation, where single cells are in a LRA-responsive state for a few weeks before switching back to an irresponsive state. These results have enormous implications for designing therapies to purge the latent reservoir and illustrate the utility of fluctuation-based assays to uncover hidden transient cellular states underlying phenotypic heterogeneity.

scholarly journals The Current Status of Latency Reversing Agents for HIV-1 Remission

2021 ◽  
Vol 8 (1) ◽  
pp. 491-514
Author(s):  
Anthony Rodari ◽  
Gilles Darcis ◽  
Carine M. Van Lint
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Viral Gene Expression ◽  
Current Status ◽  
Viral Gene ◽  
Latently Infected ◽  
Hiv 1 ◽  
Reversing Agents

Combinatory antiretroviral therapy (cART) reduces human immunodeficiency virus type 1 (HIV-1) replication but is not curative because cART interruption almost invariably leads to a rapid rebound of viremia due to the persistence of stable HIV-1-infected cellular reservoirs. These reservoirs are mainly composed of CD4+ T cells harboring replication-competent latent proviruses. A broadly explored approach to reduce the HIV-1 reservoir size, the shock and kill strategy, consists of reactivating HIV-1 gene expression from the latently infected cellular reservoirs (the shock), followed by killing of the virus-producing infected cells (the kill). Based on improved understanding of the multiple molecular mechanisms controlling HIV-1 latency, distinct classes of latency reversing agents (LRAs) have been studied for their efficiency to reactivate viral gene expression in in vitro and ex vivo cell models. Here, we provide an up-to-date review of these different mechanistic classes of LRAs and discuss optimizations of the shock strategy by combining several LRAs simultaneously or sequentially.

scholarly journals Identification of Celastrol as a novel HIV-1 Latency Reversal Agent by an Image-Based Screen

2020 ◽  
Author(s):  
Hongbing Liu ◽  
Pei-Wen Hu ◽  
Julien Dubrulle ◽  
Fabio Stossi ◽  
Bryan C. Nikolai ◽  
...  
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Research Effort ◽  
Viral Reservoir ◽  
Reversal Agent ◽  
Reversal Agents ◽  
Healthy Donors ◽  
Latent Hiv ◽  
Hiv 1

AbstractAlthough current antiretroviral therapies (ART) are successful in controlling HIV-1 infection, a stable viral reservoir reactivates when ART is discontinued. Consequently, there is a major research effort to develop approaches to disrupt the latent viral reservoir and enhance the immune system’s ability to clear HIV-1. A number of small molecules, termed latency reversal agents (LRAs), have been identified which can reactivate latent HIV-1 in cell lines and patients’ cells ex vivo. However, clinical trials have suggested that combinations of LRAs will be required to efficiently reactivate HIV-1 in vivo, especially LRAs that act synergistically by functioning through distinct pathways. To identify novel LRAs, we used an image-based assay to screen a natural compound library for the ability to induce a low level of aggregation of resting primary CD4+ T cells from healthy donors. We identified celastrol as a novel LRA. Celastrol functions synergistically with other classes of LRA to reactivate latent HIV-1 in a Jurkat cell line, suggesting a novel mechanism in its LRA activity. Additionally, celastrol does not appear to activate resting CD4+ T cells at levels at which it can reactivate latent HIV-1. Celastrol appears to represent a novel class of LRAs and it therefore can serve as a lead compound for LRA development.

scholarly journals Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth

2021 ◽  
Vol 118 (48) ◽  
pp. e2105927118
Author(s):  
Tomas Raul Wiche Salinas ◽  
Yuwei Zhang ◽  
Daniele Sarnello ◽  
Alexander Zhyvoloup ◽  
Laurence Raymond Marchand ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Th17 Cell ◽  
Th17 Cells ◽  
Nuclear Hormone Receptor ◽  
Long Terminal Repeats ◽  
T Helper 17 ◽  
Hiv Replication ◽  
Latently Infected ◽  
Hiv 1

Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2− cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.

scholarly journals Infection with a newly-designed dual fluorescent reporter HIV-1 effectively identifies latently infected CD4+ T cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jinfeng Cai ◽  
Hongbo Gao ◽  
Jiacong Zhao ◽  
Shujing Hu ◽  
Xinyu Liang ◽  
...  
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Stable Cell Line ◽  
Fluorescent Reporter ◽  
Major Barrier ◽  
Latently Infected ◽  
Integration Sites ◽  
Compound Screening ◽  
Infected Cells ◽  
Hiv 1

The major barrier to curing HIV-1 infection is a small pool of latently infected cells that harbor replication-competent viruses, which are widely considered the origin of viral rebound when ART is interrupted. The difficulty of distinguishing latently infected cells from the vast majority of uninfected cells has represented a significant bottleneck precluding comprehensive understandings of HIV-1 latency. Here we reported and validated a newly-designed dual fluorescent reporter virus, DFV-B, infection with which in primary CD4+ T cells can directly label latently infected cells and generate a latency model that was highly physiological relevant. Applying DFV-B infection in Jurkat T cells, we generated a stable cell line model of HIV-1 latency with diverse viral integration sites. High-throughput compound screening with this model identified ACY-1215 as a potent latency reversing agent, which could be verified in other cell models and in primary CD4+ T cells from ART-suppressed individuals ex vivo. In summary, we have generated a meaningful and feasible model to directly study latently infected cells, which could open up new avenues to explore the critical events of HIV-1 latency and become a valuable tool for the research of AIDS functional cure.

Sign in / Sign up

Export Citation Format

Share Document