Abstract HIV-1 transcriptional inhibitors have immense potential in functional cure approaches and could transform the way we treat HIV infections. Unlike current antiretroviral therapy (ART), transcriptional inhibitors offer the prospect of reducing residual viremia derived from reservoir of long-lived cells containing integrated proviruses, likely reducing ongoing the chronic immune activation, inflammation and HIV-associated co-morbidities still experienced by ART-adherent individuals living with HIV. Furthermore, we believe transcriptional inhibitors are amenable to block-and-lock functional cure approaches, aimed at the durable suppression of HIV in the absence of daily therapy, through permanent epigenetic silencing of integrated proviruses. This hypothesis was founded on the activity of the potent Tat inhibitor didehydro-Cortistatin A (dCA). In in vitro and in vivo models of HIV latency, dCA inhibition of HIV transcription over time prompts the viral promoter into deep transcriptional inhibition, limiting viral reactivation upon treatment interruption or with latency reactivating agents (LRAs). Despite their great potential, there are still no HIV transcriptional inhibitors in the clinic, and challenges with the cost of large-scale production of dCA are slowing its progression towards clinical studies. Here we propose to investigate the repurposing the FDA approved aldosterone antagonist Spironolactone (SP) for HIV transcriptional inhibition. An off-target activity of SP is the degradation of the XPB subunit of the general transcription factor TFIIH, a key player in RNAPII initiation at the transcriptional start site (TSS) of genes. We demonstrated in vitro that SP treatment or shRNA knockdown of XPB selectively inhibits HIV transcription and blocks viral reactivation from latency without global transcriptomic defects. This study highlighted the host factor XPB as a novel drug target and SP as a potential block-and-lock agent. Here we propose to explore the potential of SP, alone or in combination with dCA, as a block-and-lock agent in the humanized bone-marrow, thymus liver (BLT) mouse model of HIV infection by: 1) Determine the relationship between SP treatment length with residual viremia levels in tissues and correlates of chronic immune activation/inflammation in HIV infected ART- suppressed BLT mice.; 2) Assess the ability of SP to maintain deep latency as a single drug in the absence of ART and study viral resistance evolution; 3) Impact of dCA and SP in combination as front-line therapy on the size of the established viral reservoir and time to viral rebound. We predict that longer treatment lengths of HIV infected BLT mice with SP will correlate with improved reduction of low-grade HIV persistent transcription from the viral reservoir and likely chronic immune activation. Importantly, we seek to demonstrate that once deep transcriptional suppression is established, SP alone blocks viral rebound. In addition, when used as front-l...