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.
Highlights
HIV-1 infects immune cells expressing CD4 and CCR5/CXCR4, which serve as HIV-1 receptor and co-receptor for entry, such as helper T-lymphocytes and macrophages
We designed a novel high through put screening workflow to investigate the early events of HIV-1 replication that are influenced by capsid protein (CA) (Fig 1A)
Jurkat CD4+ T-cells were co-infected with two VSV-G pseudotyped single cycle HIV-1 vectors, one containing wild type (WT) CA and expressing GFP (WT-GFP), and another containing the CA N74D mutation and expressing mCherry (N74D-CHE)
Summary
HIV-1 infects immune cells expressing CD4 and CCR5/CXCR4, which serve as HIV-1 receptor and co-receptor for entry, such as helper T-lymphocytes and macrophages. CA interacts with the mRNA processing factor CPSF6 and with the nuclear transport receptor Transportin 3 (TNPO3), which were shown to be important for HIV-1 pre- and post-nuclear entry steps [11,12,13,14,15]. Small amounts of HIV-1 CA have been detected inside the nuclei by biochemical fractionation [15], and the antibiotic Coumermycin-A1 was shown to impair HIV-1 integration by targeting CA [16]. These observations led to the proposal that CA is involved in post-nuclear entry steps leading to efficient integration [15]. CA binding to CPSF6 in the nucleus is critical for targeting HIV-1 integration near transcribed genes, whereas binding of host factor LEDGF/p75 to HIV-1 integrase seems more important for integration targeting within transcribed genes [23,24]
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