Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation
Hung-Chih Yang, … , Joseph B. Margolick, Robert F. Siliciano
Hung-Chih Yang, … , Joseph B. Margolick, Robert F. Siliciano
Published October 1, 2009
Citation Information: J Clin Invest. 2009;119(11):3473-3486. https://doi.org/10.1172/JCI39199.
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Technical Advance AIDS/HIV

Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation

Abstract

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-κB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.

Authors

Hung-Chih Yang, Sifei Xing, Liang Shan, Karen O’Connell, Jason Dinoso, Anding Shen, Yan Zhou, Cynthia K. Shrum, Yefei Han, Jun O. Liu, Hao Zhang, Joseph B. Margolick, Robert F. Siliciano

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Figure 4

Establishment of in vitro HIV-1 latency in resting Bcl-2–transduced CD4+ T cells.

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Establishment of in vitro HIV-1 latency in resting Bcl-2–transduced CD4+...
(A) Genome structure of the reporter virus NL4-3-Δ6-drEGFP. It contains a truncated nef and premature stop codons in the ORFs of gag, vif, vpr, and vpu that alter the indicated amino acids shown in the standard single-letter code. A portion of env was replaced with destabilized EGFP, and the signal peptide of env was mutated to allow the destabilized EGFP to remain in the cytoplasm. The red letters indicate the mutated amino acids in the signal peptide. (B) Strategy for generating latently infected Bcl-2–transduced cells. (C) Detection of latently infected cells in the sorted GFP-negative population. The sorted GFP-negative cells were activated with anti-CD3 and anti-CD28 or PMA for 2 days and then analyzed by flow cytometry to quantify the number of GFP-positive cells. FL2-H, red fluorescence channel. (D) Latently infected cells contain integrated viral genomes. Latently infected Bcl-2–transduced cells were left untreated (upper panel) or were pretreated with either medium alone (middle panel) or 1 μM raltegravir (lower panel) for 1 day and then activated with anti-CD3 and anti-CD28 monoclonal antibodies for 2 days. Cells were analyzed using flow cytometry. (E) Cell-cycle status of latently infected cells was determined using Hoechst 33342/pyronin Y staining for DNA/RNA. The controls for the resting and activated cells are the same as in Figure 2B. The percentage of cells in each quadrant is indicated.