LEDGF/p75-dependent HIV-1 integration targeting into spliced genes: A role for the DNA damage response

Abstract

HIV-1 integration site selection is regulated at the level of transcription, splicing, histone modifications, super enhancer, nuclear architecture, and interactions of viral proteins with host factors. However, the mechanism of integration is not fully understood. Further, we lack a common understanding how these factors work together. HIV-1 integrase interacts with LEDGF/p75 and viral capsid interacts with another host factor CPSF6. Both interactions are important for HIV-1 integration in active genes, spliced genes and in gene-dense regions. LEDGF/p75 also interacts with several splicing factors (SFs) but we have not yet identified any SF with its possible role in HIV-1 integration. Therefore, we don’t understand how and why splicing plays a role in HIV-1 integration site selection. Here, we show that genes > 10 introns are highly targeted for integration. Further, we identified 15 different subsets of spliced genes that are less targeted in the absence of LEDGF/p75 or CPSF6 compared to WT cells. SFs that regulate these 15 spliced gene subsets are enriched for DNA damage response (DDR) proteins, suggesting a potential role of splicing factors in the repair of viral 5’ ends during integration. Moreover, we have determined that 160 out of 342 (46.8%) LEDGF/p75-interacting SFs1 are involved in DDR, suggesting enrichment of DDR proteins in LEDGF/p75-interacting SFs (P-value 4E-211, hypergeometric distribution, 367 DDR genes in population of 25,000 human genes). Additionally, we showed that alternative polyadenylation (APA) also plays a role in HIV-1 integration. Thus our results suggest that APA/splicing linked with DDR plays a role in HIV-1 integration. Recent study2 showed that genes with more than 1 integration in at least 2 out of 8 datasets are proximal to a super enhancer (SE). SEs contribute to 3D genome organization that is a driving force of integration in highly targeted genes. Our studies showed that all RefSeq genes of human hg19 were uniformly distanced from SEs. Genes > 10 introns, genes < 11 introns, intronless genes1 (least targeted) and highly targeted cancer genes1 were at the similar distance from SE, suggesting that highly targeted and less targeted genes are at similar distances from SEs. To test the role of SFs in integration targeting and 5’ end gap repair, we are knocking out/knocking down selective SFs to quantify3 associated defects.