scholarly journals Phylogenetic approach to recover integration dates of latent HIV sequences within-host

2018 ◽  
Vol 115 (38) ◽  
pp. E8958-E8967 ◽  
Author(s):  
Bradley R. Jones ◽  
Natalie N. Kinloch ◽  
Joshua Horacsek ◽  
Bruce Ganase ◽  
Marianne Harris ◽  
...  
Keyword(s):  
Suppressive Therapy ◽  
Latent Reservoir ◽  
Rna Sequences ◽  
Hiv Rna ◽  
Latently Infected ◽  
Latent Hiv ◽  
Long Timescales ◽  
Age Range ◽  
Hiv Evolution

Given that HIV evolution and latent reservoir establishment occur continually within-host, and that latently infected cells can persist long-term, the HIV reservoir should comprise a genetically heterogeneous archive recapitulating within-host HIV evolution. However, this has yet to be conclusively demonstrated, in part due to the challenges of reconstructing within-host reservoir establishment dynamics over long timescales. We developed a phylogenetic framework to reconstruct the integration dates of individual latent HIV lineages. The framework first involves inference and rooting of a maximum-likelihood phylogeny relating plasma HIV RNA sequences serially sampled before the initiation of suppressive antiretroviral therapy, along with putative latent sequences sampled thereafter. A linear model relating root-to-tip distances of plasma HIV RNA sequences to their sampling dates is used to convert root-to-tip distances of putative latent lineages to their establishment (integration) dates. Reconstruction of the ages of putative latent sequences sampled from chronically HIV-infected individuals up to 10 y following initiation of suppressive therapy revealed a genetically heterogeneous reservoir that recapitulated HIV’s within-host evolutionary history. Reservoir sequences were interspersed throughout multiple within-host lineages, with the oldest dating to >20 y before sampling; historic genetic bottleneck events were also recorded therein. Notably, plasma HIV RNA sequences isolated from a viremia blip in an individual receiving otherwise suppressive therapy were highly genetically diverse and spanned a 20-y age range, suggestive of spontaneous in vivo HIV reactivation from a large latently infected cell pool. Our framework for reservoir dating provides a potentially powerful addition to the HIV persistence research toolkit.

scholarly journals A New Quinoline BRD4 Inhibitor Targets a Distinct Latent HIV-1 Reservoir for Reactivation from Other “Shock” Drugs

2018 ◽  
Vol 92 (10) ◽  
Author(s):  
Erik Abner ◽  
Mateusz Stoszko ◽  
Lei Zeng ◽  
Heng-Chang Chen ◽  
Andrea Izquierdo-Bouldstridge ◽  
...  
Keyword(s):  
Hdac Inhibitors ◽  
Jurkat Cells ◽  
Viral Reactivation ◽  
Viral Reservoir ◽  
Latently Infected ◽  
Core Histones ◽  
Latent Hiv ◽  
Expression Microarrays ◽  
Hiv 1

ABSTRACT Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol), that is able to reactivate viral transcription in several models of HIV latency, including J-Lat cells, through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or “shock” drugs, such as protein kinase C (PKC) agonists or histone deacetylase (HDAC) inhibitors. Here, we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation-sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a subset of latently integrated barcoded proviruses distinct from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of BET bromodomain inhibitors that, due to its minimalistic structure, holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV infection. IMPORTANCE The suggested “shock and kill” therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. However, to this day, clinical studies investigating the “shocking” element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a shock regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation in vivo . Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators and HDAC and bromodomain inhibitors all target different subsets of proviral integration. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory shock cocktail therapies for improved reservoir reactivation.

scholarly journals Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals

2020 ◽  
Vol 16 (11) ◽  
pp. e1009060
Author(s):  
Sara Moron-Lopez ◽  
Sushama Telwatte ◽  
Indra Sarabia ◽  
Emilie Battivelli ◽  
Mauricio Montano ◽  
...  
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Primary Cell ◽  
Cell Models ◽  
Transcriptional Initiation ◽  
Cellular Genes ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv

It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells.

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

2018 ◽  
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.

scholarly journals Relationship between Measures of HIV Reactivation and Decline of the Latent Reservoir under Latency-Reversing Agents

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Janka Petravic ◽  
Thomas A. Rasmussen ◽  
Sharon R. Lewin ◽  
Stephen J. Kent ◽  
Miles P. Davenport
Keyword(s):  
Clinical Trials ◽  
Life Span ◽  
Substantial Reduction ◽  
Latent Reservoir ◽  
Hiv Rna ◽  
Hiv Transcription ◽  
Latently Infected ◽  
Infected Cells ◽  
The Impact ◽  
Reversing Agents

ABSTRACT Antiretroviral-free HIV remission requires substantial reduction of the number of latently infected cells and enhanced immune control of viremia. Latency-reversing agents (LRAs) aim to eliminate latently infected cells by increasing the rate of reactivation of HIV transcription, which exposes these cells to killing by the immune system. As LRAs are explored in clinical trials, it becomes increasingly important to assess the effect of an increased HIV reactivation rate on the decline of latently infected cells and to estimate LRA efficacy in increasing virus reactivation. However, whether the extent of HIV reactivation is a good predictor of the rate of decline of the number of latently infected cells is dependent on a number of factors. Our modeling shows that the mechanisms of maintenance and clearance of the reservoir, the life span of cells with reactivated HIV, and other factors may significantly impact the relationship between measures of HIV reactivation and the decline in the number of latently infected cells. The usual measures of HIV reactivation are the increase in cell-associated HIV RNA (CA RNA) and/or plasma HIV RNA soon after administration. We analyze two recent studies where CA RNA was used to estimate the impact of two novel LRAs, panobinostat and romidepsin. Both drugs increased the CA RNA level 3- to 4-fold in clinical trials. However, cells with panobinostat-reactivated HIV appeared long-lived (half-life > 1 month), suggesting that the HIV reactivation rate increased by approximately 8%. With romidepsin, the life span of cells that reactivated HIV was short (2 days), suggesting that the HIV reactivation rate may have doubled under treatment. IMPORTANCE Long-lived latently infected cells that persist on antiretroviral treatment (ART) are thought to be the source of viral rebound soon after ART interruption. The elimination of latently infected cells is an important step in achieving antiretroviral-free HIV remission. Latency-reversing agents (LRAs) aim to activate HIV expression in latently infected cells, which could lead to their death. Here, we discuss the possible impact of the LRAs on the reduction of the number of latently infected cells, depending on the mechanisms of their loss and self-renewal and on the life span of the cells that have HIV transcription activated by the LRAs.

scholarly journals A Critical Review of the Evidence Concerning the HIV Latency Reversing Effect of Disulfiram, the Possible Explanations for Its Inability to Reduce the Size of the Latent Reservoir In Vivo, and the Caveats Associated with Its Use in Practice

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Harry D. J. Knights
Keyword(s):  
Immune Deficiency Syndrome ◽  
Deficiency Syndrome ◽  
Latent Reservoir ◽  
Major Barrier ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Hiv 1

Combination antiretroviral therapy (cART) effectively suppresses the replication of human immunodeficiency virus type 1 (HIV-1), improves immune function, and decreases the morbidity of acquired immune deficiency syndrome (AIDS). However, it is unable to eradicate the virus because it does not eliminate latently infected cells. The latent reservoir poses the major barrier to an HIV-1 cure. The “shock and kill” strategy aims to reactivate the virus and destroy latently infected cells. Many latency reversing agents (LRAs) reactivate HIV in vitro, but the absence of damaging side-effects and efficacy in vivo make disulfiram particularly promising. However, in clinical trials to date, disulfiram treatment has not resulted in a reduction in the size of the latent reservoir. In this article I will therefore discuss the evidence for the latency reversing effect of disulfiram, the possible explanations for its inability to reduce the size of the latent reservoir in vivo, and the caveats associated with its use in practice. These considerations will help to inform judgements about the prospect of an HIV cure from disulfiram based treatments.

scholarly journals In vivo analysis of the effect of panobinostat on cell-associated HIV RNA and DNA levels and latent HIV infection

Retrovirology ◽  
2016 ◽  
Vol 13 (1) ◽  
Author(s):  
Perry Tsai ◽  
Guoxin Wu ◽  
Caroline E. Baker ◽  
William O. Thayer ◽  
Rae Ann Spagnuolo ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Hiv Rna ◽  
In Vivo Analysis ◽  
Latent Hiv ◽  
Rna And Dna

scholarly journals Experimental Systems for Measuring HIV Latency and Reactivation

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1279
Author(s):  
Koh Fujinaga ◽  
Daniele C. Cary
Keyword(s):  
The Body ◽  
Viral Reservoir ◽  
Latent Reservoir ◽  
Hiv Latency ◽  
Viral Loads ◽  
Experimental Systems ◽  
Latently Infected ◽  
Latent Hiv ◽  
Underlying Mechanisms

The final obstacle to achieving a cure to HIV/AIDS is the presence of latent HIV reservoirs scattered throughout the body. Although antiretroviral therapy maintains plasma viral loads below the levels of detection, upon cessation of therapy, the latent reservoir immediately produces infectious progeny viruses. This results in elevated plasma viremia, which leads to clinical progression to AIDS. Thus, if a HIV cure is ever to become a reality, it will be necessary to target and eliminate the latent reservoir. To this end, tremendous effort has been dedicated to locate the viral reservoir, understand the mechanisms contributing to latency, find optimal methods to reactivate HIV, and specifically kill latently infected cells. Although we have not yet identified a therapeutic approach to completely eliminate HIV from patients, these efforts have provided many technological breakthroughs in understanding the underlying mechanisms that regulate HIV latency and reactivation in vitro. In this review, we summarize and compare experimental systems which are frequently used to study HIV latency. While none of these models are a perfect proxy for the complex systems at work in HIV+ patients, each aim to replicate HIV latency in vitro.

scholarly journals Proliferation of latently infected CD4+ T cells carrying replication-competent HIV-1: Potential role in latent reservoir dynamics

2017 ◽  
Vol 214 (4) ◽  
pp. 959-972 ◽  
Author(s):  
Nina N. Hosmane ◽  
Kyungyoon J. Kwon ◽  
Katherine M. Bruner ◽  
Adam A. Capoferri ◽  
Subul Beg ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Productive Infection ◽  
Latent Reservoir ◽  
Full Genome Sequencing ◽  
Latently Infected ◽  
Infected Cells ◽  
Hiv 1

A latent reservoir for HIV-1 in resting CD4+ T lymphocytes precludes cure. Mechanisms underlying reservoir stability are unclear. Recent studies suggest an unexpected degree of infected cell proliferation in vivo. T cell activation drives proliferation but also reverses latency, resulting in productive infection that generally leads to cell death. In this study, we show that latently infected cells can proliferate in response to mitogens without producing virus, generating progeny cells that can release infectious virus. Thus, assays relying on one round of activation underestimate reservoir size. Sequencing of independent clonal isolates of replication-competent virus revealed that 57% had env sequences identical to other isolates from the same patient. Identity was confirmed by full-genome sequencing and was not attributable to limited viral diversity. Phylogenetic and statistical analysis suggested that identical sequences arose from in vivo proliferation of infected cells, rather than infection of multiple cells by a dominant viral species. The possibility that much of the reservoir arises by cell proliferation presents challenges to cure.

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