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

Biology Open ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. bio052969
Author(s):  
Jared P. Taylor ◽  
Lucas H. Armitage ◽  
Daniel L. Aldridge ◽  
Melanie N. Cash ◽  
Mark A. Wallet
Keyword(s):  
Cell Model ◽  
Hdac Inhibitors ◽  
Negative Regulator ◽  
Latent Reservoir ◽  
Transcriptional Elongation ◽  
Hiv Transcription ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTInfection with human immunodeficiency virus 1 (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 a synergistic 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 activity of NFκB, MAPK p38, and ERK1/2. Harmine treatment also resulted in reduced expression of HEXIM1, a negative regulator of transcriptional elongation. Thus, harmine enhanced the effects of LRAs by increasing the availability of transcription factors needed for HIV reactivation and promoting transcriptional elongation. Combination therapies with harmine and LRAs could benefit patients by achieving deeper reactivation of the latent pool of HIV provirus.

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

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.

scholarly journals Induction of Autophagy to Achieve a Human Immunodeficiency Virus Type 1 Cure

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1798
Author(s):  
Grant R. Campbell ◽  
Stephen A. Spector
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Functional Cure ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Hiv 1

Effective antiretroviral therapy has led to significant human immunodeficiency virus type 1 (HIV-1) suppression and improvement in immune function. However, the persistence of integrated proviral DNA in latently infected reservoir cells, which drive viral rebound post-interruption of antiretroviral therapy, remains the major roadblock to a cure. Therefore, the targeted elimination or permanent silencing of this latently infected reservoir is a major focus of HIV-1 research. The most studied approach in the development of a cure is the activation of HIV-1 expression to expose latently infected cells for immune clearance while inducing HIV-1 cytotoxicity—the “kick and kill” approach. However, the complex and highly heterogeneous nature of the latent reservoir, combined with the failure of clinical trials to reduce the reservoir size casts doubt on the feasibility of this approach. This concern that total elimination of HIV-1 from the body may not be possible has led to increased emphasis on a “functional cure” where the virus remains but is unable to reactivate which presents the challenge of permanently silencing transcription of HIV-1 for prolonged drug-free remission—a “block and lock” approach. In this review, we discuss the interaction of HIV-1 and autophagy, and the exploitation of autophagy to kill selectively HIV-1 latently infected cells as part of a cure strategy. The cure strategy proposed has the advantage of significantly decreasing the size of the HIV-1 reservoir that can contribute to a functional cure and when optimised has the potential to eradicate completely HIV-1.

scholarly journals Similar frequency and inducibility of intact HIV-1 proviruses in blood and lymph nodes

2020 ◽  
Author(s):  
Alyssa R Martin ◽  
Alexandra M Bender ◽  
Jada Hackman ◽  
Kyungyoon J Kwon ◽  
Briana A Lynch ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Nodes ◽  
Cd4 T Cells ◽  
Viral Rna ◽  
Latent Reservoir ◽  
Similar Frequency ◽  
Latently Infected ◽  
Infected Cells ◽  
Secondary Lymphoid Organs ◽  
Hiv 1

Abstract Background The HIV-1 latent reservoir (LR) in resting CD4 + T cells is a barrier to cure. LR measurements are commonly performed on blood samples and therefore may miss latently infected cells residing in tissues, including lymph nodes. Methods We determined the frequency of intact HIV-1 proviruses and proviral inducibility in matched peripheral blood (PB) and lymph node (LN) samples from ten HIV-1-infected patients on ART using the intact proviral DNA assay and a novel quantitative viral induction assay. Prominent viral sequences from induced viral RNA were characterized using a next-generation sequencing assay. Results The frequencies of CD4 + T cells with intact proviruses were not significantly different in PB vs LN (61vs104/10 6CD4 + cells), and were substantially lower than frequencies of CD4 + T cells with defective proviruses. The frequencies of CD4 + T cells induced to produce high levels of viral RNA were not significantly different in PB vs LN (4.3/10 6 vs 7.9/10 6), but were 14-fold lower than the frequencies of cells with intact proviruses. Sequencing of HIV-1 RNA from induced proviruses revealed comparable sequences in paired PB and LN samples. Conclusions These results further support the use of PB as an appropriate proxy for the HIV-1 LR in secondary lymphoid organs

scholarly journals eCD4-Ig Variants That More Potently Neutralize HIV-1

2018 ◽  
Vol 92 (12) ◽  
Author(s):  
Ina Fetzer ◽  
Matthew R. Gardner ◽  
Meredith E. Davis-Gardner ◽  
Neha R. Prasad ◽  
Barnett Alfant ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Entry Inhibitor ◽  
Immune Effector ◽  
Effector Cells ◽  
Antibody Recognition ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Prophylaxis Therapy ◽  
Hiv 1

ABSTRACTThe human immunodeficiency virus type 1 (HIV-1) entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralized all HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates that it has been tested against, suggesting that it may be useful in clinical settings, where antibody escape is a concern. Here, we characterize three new eCD4-Ig variants, each with a different architecture and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as the original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented the promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications.IMPORTANCEHIV-1 bNAbs have properties different from those of antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral life cycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the utility of antibodies as a treatment for HIV-1 infection or as part of an effort to eradicate latently infected cells. eCD4-Ig is an antibody-like entry inhibitor that closely mimics HIV-1's obligate receptors. eCD4-Ig appears to be qualitatively different from antibodies, since it neutralizes all HIV-1, HIV-2, and SIV isolates. Here, we characterize three new structurally distinct eCD4-Ig variants and show that each excels in a key property useful to prevent, treat, or cure an HIV-1 infection. For example, one variant neutralized HIV-1 most efficiently, while others best enlisted natural killer cells to eliminate infected cells. These observations will help generate eCD4-Ig variants optimized for different clinical applications.

scholarly journals Rational Design of Drugs That Induce Human Immunodeficiency Virus Replication

2003 ◽  
Vol 77 (19) ◽  
pp. 10227-10236 ◽  
Author(s):  
Dean H. Hamer ◽  
Sven Bocklandt ◽  
Louise McHugh ◽  
Tae-Wook Chun ◽  
Peter M. Blumberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cell Activation ◽  
Rational Design ◽  
Latent Reservoir ◽  
Viral Gene ◽  
Human Immunodeficiency Virus Replication ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Hiv 1

ABSTRACT Drugs that induce human immunodeficiency virus type 1 (HIV-1) replication could be used in combination with highly active antiretroviral therapy (HAART) to reduce the size of the latent reservoir that is in part responsible for viral persistence. Protein kinase C (PKC) is a logical target for such drugs because it activates HIV-1 transcription through multiple mechanisms. Here we show that HIV-1 gene expression can be induced by potent synthetic analogues of the lipid second messenger diacylglycerol (DAG) synthesized on a five-member ring platform that reduces the entropy of binding relative to that of the more flexible DAG template. By varying the alkyl side chains of these synthetic DAG lactones, it was possible to maximize their potency and ability to render latently infected T cells sensitive to killing by an anti-HIV-1 immunotoxin while minimizing the side effects of CD4 and CXCR4 downregulation and tumor necrosis factor alpha upregulation. The two lead compounds, LMC03 and LMC07, regulated a series of PKC-sensitive genes involved in T-cell activation and induced viral gene expression in peripheral blood mononuclear cells from HIV-1-infected individuals. These studies demonstrate the potential for the rational design of agents that, in conjunction with HAART and HIV-specific toxins, can be used to decrease or eliminate the pool of latently infected reservoirs by forcing viral expression.

scholarly journals Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1

2000 ◽  
Vol 74 (17) ◽  
pp. 7824-7833 ◽  
Author(s):  
Theodore Pierson ◽  
Trevor L. Hoffman ◽  
Joel Blankson ◽  
Diana Finzi ◽  
Karen Chadwick ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Chemokine Receptor ◽  
Latent Reservoir ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Hiv 1

ABSTRACT Latently infected resting CD4+ T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1 env genes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4+ T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4+ T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established.

scholarly journals The dynamics of the HIV-1 latent reservoir – considering the heterogeneous subpopulations

2019 ◽  
Author(s):  
Ruian Ke ◽  
Kai Deng
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Latent Reservoir ◽  
Major Barrier ◽  
Clinical Observations ◽  
Wide Range ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Ctl Escape ◽  
Hiv 1

AbstractA major barrier to finding a cure for human immunodeficiency virus type-I (HIV-1) infection is the existence and persistence of the HIV-1 latent reservoir. Although the size of the reservoir is shown to be extremely stable under effective antiretroviral therapy, multiple lines of evidence suggest that the reservoir is composed of dynamic and heterogeneous subpopulations. Quantifying the dynamics of these subpopulations and the processes that maintain the latent reservoir is crucial to the development of effective strategies to eliminate this reservoir. Here, we constructed a mathematical model to consider four latently infected subpopulations, according to their ability to proliferate and the type of virus they are infected. Our model explains a wide range of clinical observations, including variable estimates of the reservoir half-life and dynamical turnover of cytotoxic T lymphocyte (CTL) escape viruses in the reservoir. It suggests that very early treatment leads to a reservoir that is small in size and is composed of less stable latently infected cells (compared to the reservoir in chronically infected individuals). The shorter half-lives estimated from individuals treated during acute infection is likely driven by cells that are less prone to proliferate; in contrast, the remarkably consistent estimate of the long half-lives in individuals who are treated during chronic infection are driven by fast proliferating cells that are likely to be infected by CTL escape mutants. Our model shed light on the dynamics of the reservoir in the absence and presence of antiretroviral therapy. More broadly, it can be used to estimate the turnover rates of subpopulations of the reservoir as well as to design and evaluate the impact of various therapeutic interventions to purge the HIV-1 reservoir.Author summaryHuman immunodeficiency virus (HIV) infects tens of millions of people globally and causes approximately a million death each year. Current treatment for HIV infection suppresses viral load but does not eradicates the virus. A major barrier to cure HIV infection is the existence and persistence of populations of cells that are latently infected by HIV, i.e. the HIV latent reservoir. Understanding and quantifying the kinetics of the reservoir is therefore critical for developing and evaluating effective therapies to purge the reservoir. Recent studies suggested that this reservoir is heterogenous in their population dynamics; yet most previous mathematical models consider this reservoir as a homogenous population. Here we developed a model explicitly tracking the heterogenous subpopulations of the reservoir. We show that this model explains a wide range of clinical observations, and then demonstrate its utility to make quantitative predictions about varies interventions that aim to restrict or reduce the size of the reservoir.

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 Quantifying the turnover of transcriptional subclasses of HIV-1-infected cells

2013 ◽  
Author(s):  
Christian L Althaus ◽  
Beda Joos ◽  
Alan S Perelson ◽  
Huldrych F Günthard
Keyword(s):  
Peripheral Blood ◽  
Chronic Infection ◽  
Burst Size ◽  
Acute Infection ◽  
The Body ◽  
Latent Reservoir ◽  
Published Data ◽  
Latently Infected ◽  
Infected Cells ◽  
Hiv 1

Background: HIV-1-infected cells in peripheral blood can be grouped into different transcriptional subclasses. Quantifying the turnover of these cellular subclasses can provide important insights into the viral life cycle and the generation and maintenance of latently infected cells. Results: We used previously published data from five patients chronically infected with HIV-1 that initiated combination antiretroviral therapy (cART). Patient-matched PCR for unspliced and multiply spliced viral RNAs combined with limiting dilution analysis provided measurements of transcriptional profiles at the single cell level. Furthermore, measurement of intracellular transcripts and extracellular virion-enclosed HIV-1 RNA allowed us to distinguish productive from non-productive cells. We developed a mathematical model describing the dynamics of plasma virus and the transcriptional subclasses of HIV-1-infected cells. Fitting the model to the data allowed us to better understand the phenotype of different transcriptional subclasses and their contribution to the overall turnover of HIV-1 before and during cART. The average number of virus-producing cells in peripheral blood is small during chronic infection (25.7 cells per ml). We find that 14.0%, 0.3% and 21.2% of infected cells become defectively, latently and persistently infected cells, respectively. Assuming that the infection is homogenous throughout the body, we estimate an average in vivo viral burst size of 2.1 x 10^4 virions per cell. Conclusions: Our study provides novel quantitative insights into the turnover and development of different subclasses of HIV-1-infected cells. The model predicts that the pool of latently infected cells becomes rapidly established during the first months of acute infection and continues to increase slowly during the first years of chronic infection. Having a detailed understanding of this process will be useful for the evaluation of viral eradication strategies that aim to deplete the latent reservoir of HIV-1.

scholarly journals Loss of the Brm-Type SWI/SNF Chromatin Remodeling Complex Is a Strong Barrier to the Tat-Independent Transcriptional Elongation of Human Immunodeficiency Virus Type 1 Transcripts

2009 ◽  
Vol 83 (22) ◽  
pp. 11569-11580 ◽  
Author(s):  
Taketoshi Mizutani ◽  
Aya Ishizaka ◽  
Mariko Tomizawa ◽  
Takuya Okazaki ◽  
Nobutake Yamamichi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Competent Cell ◽  
Transcriptional Elongation ◽  
Gfp Expression ◽  
Proviral Integration ◽  
Hiv Transcription ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT To elucidate the epigenetic regulation of Tat-independent human immunodeficiency virus (HIV) transcription following proviral integration, we constructed an HIV type 1 (HIV-1)-based replication-defective viral vector that expresses a reporter green fluorescent protein (GFP) product from its intact long terminal repeat (LTR). We transduced this construct into human tumor cell lines that were either deficient in or competent for the Brm-type SWI/SNF complex. One day after transduction, single cells that expressed GFP were sorted, and the GFP expression profiles originating from each of these clones were analyzed. Unlike clones of the SWI/SNF-competent cell line, which exhibited clear unimodal expression patterns in all cases, many clones originating from Brm-deficient cell lines either showed a broad-range distribution of GFP expression or were fully silenced. The resorting of GFP-negative populations of these isolated clones showed that GFP silencing is either reversible or irreversible depending upon the proviral integration sites. We further observed that even in these silenced clones, proviral gene transcription initiates to accumulate short transcripts of around 60 bases in length, but no elongation occurs. We found that this termination is caused by tightly closed nucleosome-1 (nuc-1) at the 5′ LTR. Also, nuc-1 is remodeled by exogenous Brm in some integrants. From these results, we propose that Brm is required for the occasional transcriptional elongation of the HIV-1 provirus in the absence of Tat. Since the Brm-type SWI/SNF complex is expressed at marginal levels in resting CD4+ T cells and is drastically induced upon CD4+ T-cell activation, we speculate that it plays crucial roles in the early Tat-independent phase of HIV transcription in affected patients.

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