High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay
eScholarship, University of California, 2017
Online
academicJournal
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.
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High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay
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Autor/in / Beteiligte Person: | Yucha, Robert W ; Hobbs, Kristen S ; Hanhauser, Emily ; Hogan, Louise E ; Nieves, Wildaliz ; Ozen, Mehmet O ; Inci, Fatih ; York, Vanessa ; Gibson, Erica A ; Thanh, Cassandra ; Shafiee, Hadi ; Assal, Rami El ; Kiselinova, Maja ; Robles, Yvonne P ; Bae, Helen ; Leadabrand, Kaitlyn S ; Wang, Shu ; Qi ; Deeks, Steven G ; Kuritzkes, Daniel R ; Demirci, Utkan ; Henrich, Timothy J |
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Veröffentlichung: | eScholarship, University of California, 2017 |
Medientyp: | academicJournal |
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