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Background:  We have previously shown that the p75, but not the p52, isoform of the transcriptional activator LEDGF (lens epithelium-derived growth factor) interacts tightly with HIV-1 integrase and is essential for nuclear targeting of this protein in human cells. Recently, doubts were raised regarding the role of LEDGF/p75 during HIV-1 replication.

Methods:  We have now selected polyclonal HeLaCD4 cell lines with a stable, partial knock-down of p75 after transduction with a lentiviral vector encoding a short hairpin against LEDGF/p75 mRNA. As controls HeLaCD4 cell lines were selected after transduction with lentiviral vectors encoding a double mismatch or an irrelevant (eGFP) shRNA. As a stringent control, we successfully back-complemented the HeLaCD4-p75 knock-down cell line with a plasmid encoding for LEDGF/p75 but resistant to the specific shRNA. LEDGF/p75 expression levels in the different cell lines were determined by Western blotting. Fluorescent correlation spectroscopy was used to measure the increased DNA binding of LEDGF/p75 or the protein complex of various integrases and LEDGF/p75 with a specific or aspecific double-stranded DNA oligonucleotide.

Results:  Stable knock-down of LEDGF/p75 resulted in a 2-fold reduction in HIV-1 replication that was completely rescued by back-complementation ruling out aspecific RNAi effects. In a direct binding assay, LEDGF/p75, but not p52, dramatically enhanced the direct binding of lentiviral but not retroviral integrases to DNA.

Conclusions:  LEDGF/p75 is the first cellular co-factor of HIV integrase that has been unambiguously shown to be important for HIV replication. LEDGF/p75 seems to play a role in the nuclear trapping of integrase and its tethering to the chromosomal DNA. This mechanism is apparently specific for lentiviridae.

Keywords: integration; co-factors; LEDGF