scholarly journals Inhibition of Polo-like kinase 1 (PLK1) facilitates the elimination of HIV-1 viral reservoirs in CD4+ T cells ex vivo

2020 ◽  
Vol 6 (29) ◽  
pp. eaba1941
Author(s):  
Dawei Zhou ◽  
Tsuyoshi Hayashi ◽  
Maxime Jean ◽  
Weili Kong ◽  
Guillaume Fiches ◽  
...  
Keyword(s):  
T Cells ◽  
Nuclear Translocation ◽  
De Novo ◽  
Ex Vivo ◽  
Critical Role ◽  
Protein Stabilization ◽  
Viral Reservoirs ◽  
Promising Target ◽  
Latent Hiv ◽  
Hiv 1

Although combination antiretroviral therapy is effective in controlling HIV-1 infection, latent HIV-1 proviruses cannot be eliminated. HIV-1 reactivation induced by the mere use of latency-reversing agents is insufficient to render death of reservoir cells, indicating that certain intrinsic survival mechanisms exist. We report that Polo-like kinase 1 (PLK1) plays a critical role in survival of CD4+ T cells that undergo HIV-1 reactivation from latency or de novo infection. PLK1 is elevated in both scenarios, which requires HIV-1 Nef. HIV-1 enhances PLK1 SUMOylation, causing its nuclear translocation and protein stabilization. Inhibition or knockdown of PLK1 markedly facilitates death of HIV-1-infected CD4+ T cells. Furthermore, PLK1 inhibitors strikingly reduce the size of HIV-1 latent reservoirs in primary CD4+ T cells. Our findings demonstrate that HIV-1 infection hijacks PLK1 to prevent cell death induced by viral cytopathic effects, and that PLK1 is a promising target for chemical “killing” of HIV-1 reservoir cells.

scholarly journals Potential of the NKG2D/NKG2DL Axis in NK Cell-Mediated Clearance of the HIV-1 Reservoir

2019 ◽  
Vol 20 (18) ◽  
pp. 4490 ◽  
Author(s):  
Maria G. Desimio ◽  
Daniela A. Covino ◽  
Margherita Doria
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Virus Reactivation ◽  
Major Drawback ◽  
Virus Eradication ◽  
Latent Hiv ◽  
Hiv 1

Viral persistency in latently infected CD4+ T cells despite antiretroviral therapy (ART) represents a major drawback in the fight against HIV-1. Efforts to purge latent HIV-1 have been attempted using latency reversing agents (LRAs) that activate expression of the quiescent virus. However, initial trials have shown that immune responses of ART-treated patients are ineffective at clearing LRA-reactivated HIV-1 reservoirs, suggesting that an adjuvant immunotherapy is needed. Here we overview multiple lines of evidence indicating that natural killer (NK) cells have the potential to induce anti-HIV-1 responses relevant for virus eradication. In particular, we focus on the role of the NKG2D activating receptor that crucially enables NK cell-mediated killing of HIV-1-infected cells. We describe recent data indicating that LRAs can synergize with HIV-1 at upregulating ligands for NKG2D (NKG2DLs), hence sensitizing T cells that exit from viral latency for recognition and lysis by NK cells; in addition, we report in vivo and ex vivo data showing the potential benefits and drawbacks that LRAs may have on NKG2D expression and, more in general, on the cytotoxicity of NK cells. Finally, we discuss how the NKG2D/NKG2DLs axis can be exploited for the development of effective HIV-1 eradication strategies combining LRA-induced virus reactivation with recently optimized NK cell-based immunotherapies.

scholarly journals A Novel Single-Cell FISH-Flow Assay Identifies Effector Memory CD4+ T cells as a Major Niche for HIV-1 Transcription in HIV-Infected Patients

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Judith Grau-Expósito ◽  
Carla Serra-Peinado ◽  
Lucia Miguel ◽  
Jordi Navarro ◽  
Adrià Curran ◽  
...  
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Viral Reactivation ◽  
Viral Reservoir ◽  
Effector Memory ◽  
Specific Cell ◽  
Viral Reservoirs ◽  
Hiv Rna ◽  
Cell Subpopulations ◽  
Hiv 1

ABSTRACT Cells that actively transcribe HIV-1 have been defined as the “active viral reservoir” in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow) assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs) to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4+ T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4+ T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4+ T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation. IMPORTANCE Persons infected with HIV-1 contain several cellular viral reservoirs that preclude the complete eradication of the viral infection. Using a novel methodology, we identified effector memory CD4+ T cells, immune cells preferentially located in inflamed tissues with potent activity against pathogens, as the main cells encompassing the transcriptionally active HIV-1 reservoir in patients on antiretroviral therapy. Importantly, the identification of such cells provides us with an important target for new therapies designed to target the hidden virus and thus to eliminate the virus from the human body. In addition, because of its ability to identify cells forming part of the viral reservoir, the assay used in this study represents an important new tool in the field of HIV pathogenesis and viral persistence. IMPORTANCE Persons infected with HIV-1 contain several cellular viral reservoirs that preclude the complete eradication of the viral infection. Using a novel methodology, we identified effector memory CD4+ T cells, immune cells preferentially located in inflamed tissues with potent activity against pathogens, as the main cells encompassing the transcriptionally active HIV-1 reservoir in patients on antiretroviral therapy. Importantly, the identification of such cells provides us with an important target for new therapies designed to target the hidden virus and thus to eliminate the virus from the human body. In addition, because of its ability to identify cells forming part of the viral reservoir, the assay used in this study represents an important new tool in the field of HIV pathogenesis and viral persistence.

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 Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4+ T Cells

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Deanna A. Kulpa ◽  
Aarthi Talla ◽  
Jessica H. Brehm ◽  
Susan Pereira Ribeiro ◽  
Sally Yuan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Cell Surface Markers ◽  
Cell Cycle Entry ◽  
Latent Hiv ◽  
Hiv 1

ABSTRACT During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal. IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells.

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 The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation

2019 ◽  
Vol 11 (513) ◽  
pp. eaaw5589 ◽  
Author(s):  
Melissa-Rose Abrahams ◽  
Sarah B. Joseph ◽  
Nigel Garrett ◽  
Lynn Tyers ◽  
Matthew Moeser ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Infection ◽  
Latent Reservoir ◽  
Viral Reservoirs ◽  
Art Initiation ◽  
Therapy Initiation ◽  
Hiv Positive Women ◽  
Latent Hiv ◽  
Viral Sequences ◽  
Hiv 1

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4+ T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.

scholarly journals Identification of celastrol as a novel HIV-1 latency reversal agent by an image-based screen

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0244771
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

Although 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 Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

2020 ◽  
Author(s):  
Lin Chen ◽  
Ariane Zutz ◽  
Julia Phillippou-Massier ◽  
Tim Liebner ◽  
Oliver T. Keppler ◽  
...  
Keyword(s):  
T Cells ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
De Novo ◽  
Treatment Strategies ◽  
Proteome Analysis ◽  
Global Changes ◽  
Hiv Replication ◽  
Latent Hiv ◽  
Hiv 1

AbstractWhile current combined antiretroviral therapy (cART) allows control of HIV replication in patients and effectively suppresses plasma viral loads, it is unable to target latent reservoirs, which are responsible for virus rebound after discontinuation of therapy. Several histone deacetylase inhibitors (HDACIs) have been shown to target reservoirs and to reactivate latent HIV. While this effect is highly desired, it carries the risk that HIV-1 may be reactivated in tissue compartments were cART concentrations are insufficient and thus leading to de novo infections in this sites. To address this concern, we evaluated the effect of different HDACIs for their ability to reverse HIV latency and to modulate de novo infections. Two of the inhibitors, sodium butyrate and bufexamac, significantly inhibited de novo HIV-1 infection in activated CD4+ T-cells. Transcriptome and proteome analysis indicated global changes of protein abundancies, exhibited reduced proliferation of CD4+ T-cells, and revealed butyrate-based proteasomal degradation of EP300, an important factor for HIV-1 replication. Our results disclose new potential treatment strategies and minimizes the concern of potential reservoir reseeding by HDACIs.

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