EBioMedicine (Jun 2017)

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

  • Robert W. Yucha,
  • Kristen S. Hobbs,
  • Emily Hanhauser,
  • Louise E. Hogan,
  • Wildaliz Nieves,
  • Mehmet O. Ozen,
  • Fatih Inci,
  • Vanessa York,
  • Erica A. Gibson,
  • Cassandra Thanh,
  • Hadi Shafiee,
  • Rami El Assal,
  • Maja Kiselinova,
  • Yvonne P. Robles,
  • Helen Bae,
  • Kaitlyn S. Leadabrand,
  • ShuQi Wang,
  • Steven G. Deeks,
  • Daniel R. Kuritzkes,
  • Utkan Demirci,
  • Timothy J. Henrich

DOI
https://doi.org/10.1016/j.ebiom.2017.05.006
Journal volume & issue
Vol. 20, no. C
pp. 217 – 229

Abstract

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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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