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

Abstract

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection 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 cellular Na ⁺/K ⁺ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Author summary

HIV-1 integrates more frequently within transcribed host genes, however we do not understand the biological significance of this. We found that a drug called digoxin inhibits wild type HIV-1 more potently than an HIV-1 bearing a single point mutation in the capsid protein. Here we show that digoxin represses HIV-1 gene expression and in parallel inhibits CD4+ T-cell activation and metabolism. When we analysed the integration sites of wild type and mutant HIV-1, we discovered that wild type virus integrates within or near genes involved in CD4 ⁺ T-cell activation and metabolism more often than the mutant virus. Because these are the very same genes repressed by digoxin, the integration bias of wild type virus makes it more susceptible than mutant virus to silencing by the drug. Digoxin unmasked a functional link between HIV-1 integration and T-cell activation, which may affect HIV-1 latency and reactivation.