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

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

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Title: High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay
Author: 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 R.; Demirci, Utkan; Henrich, Timothy J.

Note: Order does not necessarily reflect citation order of authors.

Citation: Yucha, R. W., K. S. Hobbs, E. Hanhauser, L. E. Hogan, W. Nieves, M. O. Ozen, F. Inci, et al. 2017. “High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.” EBioMedicine 20 (1): 217-229. doi:10.1016/j.ebiom.2017.05.006. http://dx.doi.org/10.1016/j.ebiom.2017.05.006.
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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.
Published Version: doi:10.1016/j.ebiom.2017.05.006
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478213/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:33490753
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