CPSF6 LICENSES HIV-1 INTEGRATION INTO POL II-PAUSED GENES REGULATED by P-TEFb and U2 snRNP

Abstract

Pre-mRNA splicing regulates promoter-proximal Pol II pausing and alternative polyadenylation (APA). Splicing inhibitors increase pausing and use of proximal polyadenylation sites (PAS) for comparatively long (>4 introns) transcripts by impairing P-TEFb recruitment, which is a core component of the super elongation complex (SEC). Similar to splicing, the CFIm complex composed of CPSF6 and CPSF5 promotes the use of distal PAS. CPSF6 binds capsid (CA) to target integration into highly spliced genes and gene-dense (GD) regions. We previously reported that HIV-1 preferentially integrates into genes harboring >10 introns and Pol II-paused genes. Based on the connection between intron number, pausing, and APA, we hypothesized that CPSF6 targets HIV-1 integration into paused genes regulated by U2 snRNP and P-TEFb. U2 snRNP-regulated APA genes, which represent 5% of the transcribed genome, contained 24% of integration sites (3x compared to RIC or random integration control; p<1E-5). In contrast, nonregulated genes were targeted similarly to all genes (p< 0.2). Moreover, we found that paused genes regulated by PAF-1, which is important for APA, were preferentially targeted (3.5x RIC; p<1E-5), whereas the reciprocal gene set was preferentially avoided (p<1E-5). To test the role of splicing, we mapped integration sites in Jurkat T cells in the presence of the U2 snRNP inhibitor Pladenolide B (Plad B) or SEC inhibitor KL-2. Plad B significantly reduced genic integration in PAF-1 paused genes but not in unpaused genes. We defined chromosomes with reduced genic integrations as Plad B sensitive chromosomes (PBSC) and the remaining chromosomes as Plad B insensitive chromosomes (PBIC). KL-2 reduced genic integration significantly for PBSC but not for PBIC, suggesting that splicing targets HIV-1 into genes regulated by P-TEFb. To test the roles of integration targeting cofactors, we mapped sites for CPSF6- defective CA mutant viruses or WT HIV-1 in LEDGF/p75 knockout (LKO) cells. Despite predictably similar levels of splicing, PBSC (avg. intron content 9.6 and GD 11.8/Mb) were targeted 2.6x to RIC, whereas PBIC (avg. intron 9.5 and 6.6 genes/Mb) were but 1.2x. 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, CPSF6-CA interactions preferentially target HIV-1 integration into paused genes regulated by the SEC and U2 snRNP. Acknowledgment: HUCFAR 5P30AI060354-18.