Author:

Abstract:

HIV proviral integration favors transcriptionally active regions. The transcriptional co-activator LEDGF/p75 is the dominant cellular co-factor for HIV-1 integrase (IN) tethering the PIC to chromatin; however, the exact orchestration of this process remains unknown. LEDGF/p75 coordinates the nuclear accumulation and chromosomal localization of HIV IN via an N-terminal domain containing an ensemble of chromatin binding elements. HIV IN interacts with the C-terminal end of LEDGF/p75, specifically the IN binding domain (IBD, aa 347-429). Here we show that lentiviral integration can be targeted away from genes using an artificial tethering factor. We generated a strong knock-down cell line using miRNA-based hairpins directed against LEDGF/p75. HeLaP4 cells were stably transduced with MLV-based vectors and monoclonal cell lines were selected. No LEDGF/p75 protein could be detected by Western, Q-PCR revealed >97% knock-down and immunocytochemistry demonstrated LEDGF/p75 depletion from the nucleus. LEDGF/p75 depleted cells were backcomplemented in parallel using chimeric constructs of which the N-terminal chromatin binding elements of LEDGF/p75 (aa 1-324) were replaced with alternative chromatin binding domains or proteins, such as human histone H1, histone 2B (H2B), hepatoma-derived growth factor (HDGF), HDGF-related protein 2 (HRP2) or CBX1 (HP-1, heterochromatin binding protein-1). The chimeric proteins supported viral replication and transduction of lentiviral vectors. CBX1 binds histone H3 di- or tri-methylated on K9, which are associated with pericentric heterochromatin and intergenic regions. The CBX1 chimeric construct directed integration outside of genes, near bound CBX1. Despite integration in regions rich in epigenetic marks associated with gene silencing, lentiviral vector expression remained efficient. Thus engineered LEDGF/p75 chimeras provide technology for controlling integration site selection by lentiviral vectors.