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

EBioMedicine
Robert W YuchaTimothy J Henrich

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 need...Continue Reading

Citations

Jan 31, 2018·Retrovirology·Alexander O Pasternak, Ben Berkhout
Jan 31, 2018·Retrovirology·Sofie RutsaertLinos Vandekerckhove
Apr 21, 2020·Critical Reviews in Biotechnology·Md Alamgir KabirWaseem Asghar
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Methods Mentioned

BETA
PCR
bioprinting
flow cytometry
density-gradient centrifugation
chips
chip
light microscopy
DropSeq

Software Mentioned

msRNA
Fluidigm
SPSS vs
[UNK]
usRNA
QX100
QuantaSoft
GraphPad Prism
ImageJ
LightCycler® 480

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