HIV-1 Integration Favors Genes Associated With Promoter-Proximal Pol II Pausing

Abstract

HIV-1 integration targeting is mediated by interactions of viral integrase and capsid with LEDGF/p75 and CPSF6, respectively. HIV-1 also prefers to integrate into highly spliced genes, but the role of pre-mRNA splicing in HIV-1 integration is unclear. Splicing inhibitors reduce the association of exon junction complex (EJC) with genes containing introns but not with intronless genes. The EJC causes promoter-proximal Pol II pausing, which is released by super elongation complex (SEC). To assess the role of Pol II pausing in integration, genes were stratified as paused versus unpaused, which revealed 50%/40% respective HIV-1 integration targeting preferences in HEK293T cells; random paused and unpaused values were 15%/34%. In LEDGF/p75 knockout (LKO), CPSF6 knockout (CKO), and double knockout (DKO) cells, unpaused gene targeting dropped marginally from 40% to 37%, 38%, and 35%, respectively. However, in paused genes, 50% targeting reduced to 31%, 24%, and 20% in respective LKO, CKO and DKO cells. Thus, HIV-1 prefers integration into Pol II-paused genes. To test the role of splicing, we mapped integration sites in the presence of splicing inhibitor Pladienolide B (PladB) in WT and LKO Jurkat T cells. Genic integration in WT cells was reduced in a dose-dependent manner, with maximum reduction of 2.7% (P <2E-31) at 9 nM. In LKO cells, genic integration was reduced by 3.6% (P <4E-13), but only at 9 nM inhibitor, suggesting both LEDGF/p75-dependent and independent roles for pre- mRNA splicing in integration targeting. Similar to HEK293T cells, integration in paused and unpaused genes was 55% and 38% in WT Jurkat cells, while in LKO cells, these respective values were 25% and 35%. PladB significantly reduced integration into paused genes in both WT and LKO, but only at 9 nM in LKO cells. By contrast, whereas PladB significantly increased integration into unpaused genes (1.2% at 9 nM; P <2E-16) in WT cells, it had no effect in LKO cells. To rule out the role of pausing itself, we assessed the SEC inhibitor KL-2 in HEK293T cells, which revealed significant reductions in the amount of integration in genes, paused genes, and speckle-associated domains. Yet, integration into unpaused genes was unaffected. Thus, our results reveal an interplay between pre-mRNA splicing with Pol II pausing and transcriptional elongation in HIV-1 integration targeting. Acknowledgment: Harvard University Center for AIDS Research (HU CFAR NIH/NIAID fund 5P30AI060354-17).