A highly stringent high-throughput screening assay for identifying transcription-modulating agents of HIV-1 latency

HIV-1 latency remains a central obstacle to curing infection, and current latency-modulating agents (LMAs) suffer from poor specificity and inconsistent efficacy. To enable discovery of small molecules (SMs) that directly target the viral master transcriptional regulatory circuit (MTRC), we developed a highly stringent, dual-reporter high-throughput screening (HTS) assay based on a natural HIV-1 subtype C long terminal repeat (LTR) variant, LRhR-HC, which exhibits markedly reduced transcriptional noise and a high activation threshold. We engineered Jurkat cells to stably harbour two independent reporter cassettes driven by the tough-to-activate LRhR-HC-LTR and the canonical LR-HHC-LTR, enabling simultaneous detection of latency-promoting and latency-reversing activities through both fluorescent and secreted enzymatic reporters. This dual-reporter line responded robustly and predictably to conventional latency-reversal agents (LRAs) and latency-promoting agents (LPAs), validating assay responsiveness. Z'-factor measurements demonstrated excellent assay performance, with values ranging from approximately 0.7 across different formats, confirming a strong dynamic range and reproducibility. Screening of the 1,520-compound Prestwick chemical library (PCL), which includes FDA-approved drugs, identified 27 candidate LPAs, including known agents such as Spironolactone and Aminacrine, thereby validating the assay specificity while revealing several novel inhibitory molecules. A mechanistically focused panel of 10 additional compounds yielded two putative LPAs and one LRA, with secondary analyses confirming their latency-modulating activities and cytotoxicity profiles. Collectively, this HIV-1C-derived dual-reporter platform provides a stringent and flexible HTS system for identifying LMAs with potential applications in both block-and-lock and shock-and-kill cure strategies.