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EBioMedicine. 2017 Jun;20:217-229. doi: 10.1016/j.ebiom.2017.05.006. Epub 2017 May 4.

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

Author information

1
Division of Infectious Diseases, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, United States.
2
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States.
3
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States.
4
Division of Renal Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, United States; Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, United States.
5
HIV Translational Research Unit, Department of Internal Medicine, Ghent University and Ghent University Hospital, Ghent, Belgium.
6
Harvard University, Faculty of Arts & Sciences, Cambridge, MA 02138, United States.
7
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affliliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China; Institute for Translational Medicine, Zhejiang University, China.
8
Positive Health Program, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States.
9
Division of Infectious Diseases, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, United States; Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, United States.
10
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States. Electronic address: utkan@stanford.edu.
11
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States. Electronic address: timothy.henrich@ucsf.edu.

Abstract

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous-increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

KEYWORDS:

Digital PCR; HIV reactivation; HIV reservoirs; Histone deacetylase inhibitors; Human Immunodeficiency Virus (HIV); Single cell quantification

PMID:
28529033
PMCID:
PMC5478213
DOI:
10.1016/j.ebiom.2017.05.006
[Indexed for MEDLINE]
Free PMC Article

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