A high-throughput, microplate reader-based method to monitor in vitro HIV latency reversal in the absence of flow cytometry

Abstract

J-Lat cells are derivatives of the Jurkat CD4⁺ T cell line that contain a non-infectious, inducible HIV provirus with a GFP tag. While these cells have substantially advanced our understanding of HIV latency, their use by many laboratories in low and middle-income countries is restricted by limited access to flow cytometry. To overcome this barrier, we describe a modified J-Lat assay using a standard microplate reader that detects HIV-GFP expression following treatment with latency-reversing agents (LRAs). We show that HIV reactivation by control LRAs like prostratin and romidepsin is readily detected with dose dependence and with significant correlation and sensitivity to standard flow cytometry. For example, 10 μM prostratin induced a 20.1 ± 3.3-fold increase in GFP fluorescence in the microplate reader assay, which corresponded to 64.2 ± 5.0% GFP-positive cells detected by flow cytometery. Similarly, 0.3 μM prostratin induced a 1.7 ± 1.2-fold increase compared to 8.7 ± 5.7% GFP-positive cells detected. Using this method, we screen 79 epigenetic modifiers and identify CUDC-101, molibresib, and quisinostat as novel LRAs. This microplate reader-based method offers accessibility to researchers in resource-limited regions to work with J-Lat cells and more actively participate in global HIV cure research efforts.