scholarly journals Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique

2016 ◽  
Vol 90 (20) ◽  
pp. 9018-9028 ◽  
Author(s):  
G. Martrus ◽  
A. Niehrs ◽  
R. Cornelis ◽  
A. Rechtien ◽  
W. García-Beltran ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Gag Protein ◽  
Novel Approach ◽  
Latently Infected ◽  
Infected Cells ◽  
First Time ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACTHIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection ofgagpolmRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4+T cells, four cell populations were detected according to their expression levels of viral mRNA and protein.gagpolmRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4+T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle.IMPORTANCEEarly after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency-reversing agents. Here we describe a novel approach allowing for the first time quantification of the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation orde novoinfection on the single-cell level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently infected andde novo-infected cells dependent on the presence of viral RNA or protein.

scholarly journals Quantification of Unintegrated HIV-1 DNA at the Single Cell Level In Vivo

PLoS ONE ◽  
2012 ◽  
Vol 7 (5) ◽  
pp. e36246 ◽  
Author(s):  
Rodolphe Suspène ◽  
Andreas Meyerhans
Keyword(s):  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Hiv 1

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

2015 ◽  
Vol 89 (18) ◽  
pp. 9631-9638 ◽  
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.

scholarly journals FISHing Out the Hidden Enemy: Advances in Detecting and Measuring Latent HIV-Infected Cells

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Vinayaka R. Prasad ◽  
Ganjam V. Kalpana
Keyword(s):  
Immune Surveillance ◽  
Gag Protein ◽  
Link Type ◽  
Latently Infected ◽  
Hiv Eradication ◽  
Infected Cells ◽  
Latent Hiv ◽  
Cell Host Microbe ◽  
Unique Cell ◽  
Hiv 1

ABSTRACT The indomitable aspect of HIV-1 infection is not that HIV-1 proviral DNA is integrated into host DNA but that it can also turn itself off, remaining invisible to drug or immune surveillance. Thus, the goals of eradication include ways to precisely excise HIV-1 DNA or wake up the silent HIV-1 provirus and eliminate the infected cells thus identified. Methods to identify and fish out the latently infected cells or to delineate their characteristics are being rapidly developed. In 2016, Baxter et al. (A. E. Baxter, J. Niessl, R. Fromentin, J. Richard, F. Porichis, R. Charlebois, M. Massanella, N. Brassard, N. Alsahafi, G. G. Delgado, J. P. Routy, B. D. Walker, A. Finzi, N. Chomont, and D. E. Kaufmann, Cell Host Microbe 20:368–380, 2016, https://doi.org/10.1016/j.chom.2016.07.015 ) and Martrus et al. (G. Martrus, A. Niehrs, R. Cornelis, A. Rechtien, W. García-Beltran, M. Lütgehetmann, C. Hoffmann, and M. Altfeld, J Virol 90:9018–9028, 2016, https://doi.org/10.1128/JVI.01448-16 ) reported using the fluorescence in situ hybridization-flow cytometry technique to identify and quantify cells expressing HIV-1 RNA and Gag protein, as well as bearing unique cell surface markers. In a recent article in mBio, Grau-Expósito et al. (J. Grau-Expósito, C. Serra-Peinado, L. Miguel, J. Navarro, A. Curran, J. Burgos, I. Ocaña, E. Ribera, A. Torrella, B. Planas, R. Badía, J. Castellví, V. Falcó, M. Crespo, and M. J. Buzon, mBio 8:e00876-17, 2017, https://doi.org/10.1128/mBio.00876-17 !) reported a similar method that they claim to be more sensitive. With these methods, researchers are one step closer to measuring latent reservoirs and eliminating critical barriers to HIV eradication.

scholarly journals Prediction of single-cell gene expression for transcription factor analysis

GigaScience ◽  
2020 ◽  
Vol 9 (11) ◽  
Author(s):  
Fatemeh Behjati Ardakani ◽  
Kathrin Kattler ◽  
Tobias Heinen ◽  
Florian Schmidt ◽  
David Feuerborn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Single Cell ◽  
Cell Types ◽  
Single Cell Level ◽  
Cell Level ◽  
Novel Approach ◽  
Cell Gene Expression ◽  
Specific Regulation ◽  
Cell Gene

Abstract Background Single-cell RNA sequencing is a powerful technology to discover new cell types and study biological processes in complex biological samples. A current challenge is to predict transcription factor (TF) regulation from single-cell RNA data. Results Here, we propose a novel approach for predicting gene expression at the single-cell level using cis-regulatory motifs, as well as epigenetic features. We designed a tree-guided multi-task learning framework that considers each cell as a task. Through this framework we were able to explain the single-cell gene expression values using either TF binding affinities or TF ChIP-seq data measured at specific genomic regions. TFs identified using these models could be validated by the literature. Conclusion Our proposed method allows us to identify distinct TFs that show cell type–specific regulation. This approach is not limited to TFs but can use any type of data that can potentially be used in explaining gene expression at the single-cell level to study factors that drive differentiation or show abnormal regulation in disease. The implementation of our workflow can be accessed under an MIT license via https://github.com/SchulzLab/Triangulate.

scholarly journals Low-Level Ionizing Radiation Induces Selective Killing of HIV-1-Infected Cells with Reversal of Cytokine Induction Using mTOR Inhibitors

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 885
Author(s):  
Daniel O. Pinto ◽  
Catherine DeMarino ◽  
Thy T. Vo ◽  
Maria Cowen ◽  
Yuriy Kim ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Mtor Inhibitors ◽  
Viral Reservoirs ◽  
Viral Loads ◽  
Viral Spread ◽  
Novel Approach ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Hiv 1

HIV-1 infects 39.5 million people worldwide, and cART is effective in preventing viral spread by reducing HIV-1 plasma viral loads to undetectable levels. However, viral reservoirs persist by mechanisms, including the inhibition of autophagy by HIV-1 proteins (i.e., Nef and Tat). HIV-1 reservoirs can be targeted by the “shock and kill” strategy, which utilizes latency-reversing agents (LRAs) to activate latent proviruses and immunotarget the virus-producing cells. Yet, limitations include reduced LRA permeability across anatomical barriers and immune hyper-activation. Ionizing radiation (IR) induces effective viral activation across anatomical barriers. Like other LRAs, IR may cause inflammation and modulate the secretion of extracellular vesicles (EVs). We and others have shown that cells may secrete cytokines and viral proteins in EVs and, therefore, LRAs may contribute to inflammatory EVs. In the present study, we mitigated the effects of IR-induced inflammatory EVs (i.e., TNF-α), through the use of mTOR inhibitors (mTORi; Rapamycin and INK128). Further, mTORi were found to enhance the selective killing of HIV-1-infected myeloid and T-cell reservoirs at the exclusion of uninfected cells, potentially via inhibition of viral transcription/translation and induction of autophagy. Collectively, the proposed regimen using cART, IR, and mTORi presents a novel approach allowing for the targeting of viral reservoirs, prevention of immune hyper-activation, and selectively killing latently infected HIV-1 cells.

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