High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.
EBioMedicine
; 20: 217-229, 2017 Jun.
Article
in En
| MEDLINE
| ID: mdl-28529033
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA, Viral
/
HIV Infections
/
Polymerase Chain Reaction
/
HIV-1
/
Virus Latency
/
High-Throughput Screening Assays
/
Single-Cell Analysis
Limits:
Adult
/
Humans
/
Middle aged
Language:
En
Journal:
EBioMedicine
Year:
2017
Document type:
Article
Affiliation country:
United States