LEDGF/P75 DIRECTS HIV-1 TO INTEGRATE INTO HIGHLY SPLICED GENES DESPITE THEIR COMPARATIVELY LOW EXPRESSION

Abstract

Integration of HIV-1 into genes depends on multiple factors including transcription, splicing, LEDGF/p75, and CPSF6. But, the precise role of mRNA splicing in HIV-1 integration targeting is unclear. Transcriptional intron content can show high rate of splicing due to high transcription rate, whereas genes with higher number of introns can show high rate of splicing due to intron number, despite comparatively low transcription rate. To differentiate the roles of splicing and transcription in HIV-1 integration, we compared gene populations targeted by HIV-1 to random gene populations generated in silico (RIC, for random integration control). Analyzing gene groups with same numbers of introns, we determined that genes containing >10 introns were preferentially enriched for HIV-1 integration (ratio >1; R2 =0.9). We then parsed the human transcriptome into genes containing 0-10 introns (71% of all genes) and genes with minimally 11 introns (29% of all genes). Remarkably, we found that genes with >10 introns harbored 57% of all HIV-1 integration sites compare to 35% of sites in genes with <11 introns. This preference was independent of gene length: length-normalized genes with <11 introns were targeted 1.3 times greater than random whereas genes with >10 introns were targeted 2.1 times over the RIC (P < e-10). To assess the roles of virus-host interactions, we analyzed integration sites from LEDGF knockout (LKO) and CPSF6 knockout (CKO) cell lines. For genes with >10 introns, integration was similarly reduced by 20-21% in both cell types. Integration into genes with <11 introns was by contrast reduced by 2% and 7.6% in LKO and in CKO cells, respectively, indicating that LEDGF/p75 preferentially directs integration to genes with >10 introns. Consistent with this interpretation, analysis of a recently published LEDGF/p75 ChIP-Seq dataset revealed a strong correlation between number of genic LEDGF/p75 interaction sites and intron number (R2 =0.7; P < e-5), with 14 as the average number of introns for genes with minimally one LEDGF/p75 binding site. In LKO cells, integration into genes with LEDGF/p75 interaction sites was reduced by 14% (12% in genes with >10 introns), while integration into genes that lacked LEDGF/p75 interaction sites was reduced by 6% and 21% in LKO and CKO cells, respectively. Moreover, integration into genes with <11 introns that lacked LEDGF/p75 interaction sites was reduced in CKO but not in LKO cells. Our results reveal a previously unrecognized role for LEDGF/p75 in mediating HIV-1 integration into highly spliced genes that are expressed at comparatively low levels.