mRNA splicing targets HIV integration into PAF-1-regulated genes.

Abstract

Pre-mRNA co-transcriptional splicing is coupled with promoter-proximal Pol II pausing and alternative polyadenylation (APA)1,2. Splicing inhibitors increase pausing and the use of upstream or proximal polyadenylation sites (PASs) by impairing the recruitment of positive transcription elongation factor b (P-TEFb)1,2, which is a core component of the super elongation complex (SEC). The cleavage factor Im (CFIm) complex consisting of cleavage and polyadenylation specificity factor (CPSF) 6 and CPSF5 regulates APA by promoting the use of distal PASs. CPSF6 binds capsid (CA) to license HIV-1 intranuclear trafficking and integration targeting into highly spliced or intron rich genes. Based on the interconnections between splicing, pausing, and APA, we hypothesized that APA and Pol II elongation might play a role in HIV-1 integration targeting. Indeed, in Jurat T cells3, APA-regulated genes dependent on U2 snRNP (4.6% of human genes) for the selection of distal PASs harbored 24% of HIV-1 integration sites (3x compared to RIC or random integration control; p<1E-5). In contrast, genes independent of U2 snRNP for APA regulation were targeted similarly to all human genes (p< 0.2). Apart from splicing and CFIm complex, Pol II associated factor-1 (PAF-1) regulates pausing4 and the selection of distal PASs5. Additionally, PAF-1 regulates the expression of integrated proviral DNA. We observed that paused genes regulated by PAF-1 (14% of human genes) were preferentially targeted (3.5x RIC, containing 40% HIV-1 sites; p<1E-5), whereas the reciprocal gene set was preferentially avoided (p<1E-5). To test the role of splicing, we infected Jurkat T cells in the presence of the U2 snRNP inhibitor Pladienolide B (Plad B) or the SEC inhibitor KL-2 and determined sites of HIV-1 integration. Whereas Plad B significantly reduced integration into PAF-1 paused genes, it failed to significantly effect integration into the reciprocal set of human genes. We called chromosomes with reduced genic integrations (p<1E-04) as Plad B sensitive chromosomes (PBSC) and the remaining chromosomes with increased genic integrations as Plad B insensitive chromosomes (PBIC; p<0.02)). Although both PBSC and PBIC genes had the same average number of introns, PBSC were comparatively gene enriched (12 genes/Mb) whereas PBIC were gene-poor (7 genes/Mb; the average genes/Mb in human genome is 9). KL-2 reduced genic integration significantly for PBSC but not for PBIC, suggesting that both splicing and SEC inhibitors reduced HIV-1 integration into the same sets of genes. To test the roles of integration targeting cofactors, we mapped sites for CPSF6-defective CA mutant viruses or wild type (WT) HIV-1 in LEDGF/p75 knockout (LKO) cells. PBSC supported significantly less genic integration for CA mutants and for WT virus in LKO cells (p<1E-7). However, while PBIC were significantly less targeted by WT virus in LKO cells, these genes were significantly more targeted by CA mutants (p<1E-7 for both comparisons). Thus, the CPSF6-CA interaction is critical for preferential HIV-1 integration targeting of paused genes and APA genes regulated through P-TEFb/SEC and splicing. Currently, we are planning to differentiate the role of CPSF6-dependent APA from CPSF6-dependent trafficking of CA in the targeting of paused and APA-regulated genes.