(Elsevier, 2017) 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; El Assal, Rami; Kiselinova, Maja; Robles, Yvonne P.; Bae, Helen; Leadabrand, Kaitlyn S.; Wang, ShuQi; Deeks, Steven G.; Kuritzkes, Daniel; Demirci, Utkan; Henrich, Timothy J.
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.
