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In this work we demonstrate the effect of DEAD-box polypeptide 3, X-Linked (DDX3) inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). RNA sequencing analysis revealed that while overall gene expression was minimally dysregulated, DDX3 inhibition in independent donor CD4+ T cells led to significant downregulation of BIRC5 and HSPB1A, genes critical to cell survival during HIV-1 infection. We used single-cell FISH-Flow technology to characterise latency reversal and the contribution of viral RNA to inducing cell death pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3, upregulation of IFNβ and selective induction of apoptosis in viral RNA-expressing CD4+ T cells from PLWHIV but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV in an in vitro culture model over five days resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir as determined by quantitation of CA HIV-1 RNA, by TILDA, as well as by FISH-Flow technology. Our data support the translation of DDX3 inhibitor class compounds into HIV-1 curative strategies and provide proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards elimination of the inducible reservoir. Overall design: Primary CD4+ T cells from two healthy donors were mock treated with DMSO, with 2uM DDX3 inhibitor RK-33 or 1uM DDX3 inhibitor FH-1321. Cells were lysed and RNA was collected 4 hours and 18 hours post treatment and subjected to RNA-seq. The goal was to check what genes were differentially expressed upon DDX3 inhibition.ENAfalseEffect of DDX3 inhibitors on CD4+ T cellsEffect of DDX3 inhibitors on CD4+ T cells2025-09-242021-02-27PRJNA704943GSE167553960633931637