Digoxin reveals a functional connection between HIV-1 integration preference and Tcell activation

HIV-1 integrates more frequently into transcribed genes, however the biological significanceof HIV-1 integration targeting has remained elusive. Using a selective high-throughputchemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein.We confirmed that digoxin repressed viral gene expression by targeting the cellularNa+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mappingin infected cells demonstrated that digoxin inhibited expression of genes involved in Tcellactivation and cell metabolism. Analysis of >400,000 unique integration sites showedthat WT virus integrated more frequently than N74D mutant within or near genes susceptibleto repression by digoxin and involved in T-cell activation and cell metabolism. Two maingene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L byneutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thusthe selectivity of digoxin depends on a combination of integration targeting and repressionof specific gene networks. The drug unmasked a functional connection between HIV-1 integrationand T-cell activation. Our results suggest that HIV-1 evolved integration site selectionto couple its early gene expression with the status of target CD4+ T-cells, which mayaffect latency and viral reactivation