LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes

Abstract

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin

“…3F). Consistent with a recent report (42), LKO changed the shape of the entire curve, with integration now highly favored within the initial 30% and disfavored to below the MRC for latter halves of genes (Fig. 3F).…”

“…6, WT cell). LKO was reported recently to decrease integration into genes enriched for intron content to near random (42). Interestingly, we found that CPSF6 more prominently affected integration into intron-dense genes than did LEDGF/ p75 (Fig.…”

“…LEDGF/p75 is implicated in mRNA splicing (42,43), and CFI m components CPSF6 and CPSF7 interact with several spliceosome components (44)(45)(46). To investigate the relationship between splicing and integration targeting, frequencies of genetropic integration were correlated to intron density.…”

“…3A), we normalized our datasets to integrations within genes. Integration density kb −1 biases data with fewer gene-tropic integrations toward random because it fails to account for the decreased preference for genes upon LKO (42). Thus, although LEDGF/p75 influences integration targeting to genes, CPSF6 is the primary factor targeting HIV-1 to intron-rich genes.…”

Structural basis for spumavirus GAG tethering to chromatin

“…It is not surprising, therefore, that retroviruses evolved genus-specific and contrasting traits with respect to the types of chromosomal loci they preferentially target. HIV-1, a highly pathogenic lentivirus, channels integration toward genedense chromosomal domains, integrating predominantly within active transcription units (4,5). Recent studies uncovered a hierarchical mechanism that depends on HIV-1 capsid, the major viral structural protein (a product of the viral gag gene), and integrase, which interact with cellular proteins CPSF6 and LEDGF/p75, respectively (6)(7)(8)(9)(10)(11).…”