The Interaction of LEDGF/p75 with Integrase Is Lentivirus-specific and Promotes DNA Binding

Katrien Busschots,Jo Vercammen,Stéphane Emiliani,Richard Benarous,Yves Engelborghs,Frauke Christ,Zeger Debyser

doi:10.1074/jbc.m411681200

Abstract

We have previously shown that the p75 isoform of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF) interacts tightly with human immunodeficiency virus (HIV)-1 integrase (IN) and is essential for nuclear targeting of this protein in human cells (Cherepanov, P., Maertens, G., Proost, P., Devreese, B., Van Beeumen, J., Engelborghs, Y., De Clercq, E., and Debyser, Z. (2003) J. Biol. Chem. 278, 372-381; Maertens, G., Cherepanov, P., Pluymers, W., Busschots, K., De Clercq, E., Debyser, Z., and Engelborghs, Y. (2003) J. Biol. Chem. 278, 33528-33539). Here the interaction between recombinant LEDGF/p75 and HIV-1 IN was examined in a pull-down binding test. LEDGF/p75 was shown to increase the solubility of HIV-1 IN. Next, fluorescent correlation spectroscopy was used to measure the interaction of LEDGF/p75 or the complex of HIV-1 IN and LEDGF/p75 with a specific double-stranded DNA oligonucleotide. Whereas LEDGF/p75 displayed only a moderate affinity for DNA, it strongly promoted the binding of HIV-1 IN to DNA. This effect was specific for the p75 isoform of LEDGF and was not seen with p52. In the pull-down assay LEDGF/p75 interacted with HIV-1, HIV-2, and feline immunodeficiency virus IN, but not with the IN of human T-cell lymphotropic virus type 2, Moloney murine leukemia virus, or Rous sarcoma virus. These results strongly suggest that the interaction of LEDGF/p75 with IN is specific to lentiviridae. LEDGF/p75 stimulated the binding of HIV-1 and HIV-2 IN, but not Moloney murine leukemia virus or Rous sarcoma virus IN, to an aspecific DNA. These results provide supporting evidence for our hypothesis that LEDGF/p75 plays a role in the tethering of lentiviral IN to the chromosomal DNA.

Highlights

We have previously shown that the p75 isoform of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF) interacts tightly with human immunodeficiency virus (HIV)-1 integrase (IN) and is essential for nuclear targeting of this protein in human cells (Cherepanov, P., Maertens, G., Proost, P., Devreese, B., Van Beeumen, J., Engelborghs, Y., De Clercq, E., and Debyser, Z. (2003) J
We have previously demonstrated that LEDGF/p75 but not p52 interacts with HIV-1 IN in this test [16]
First we examined the effect of a N- versus C-terminal His6 tag on the interaction between LEDGF/p75 and HIV-1 IN as well as the effect of the NaCl concentration in the buffer

Summary

EXPERIMENTAL PROCEDURES

Plasmids for Bacterial Expression—The constructs pCPnat and pKBnat were used for the bacterial expression of non-tagged LEDGF/ p75 and p52 proteins as described previously [16]. 1 ␮g of recombinant His6-tagged IN was incubated with 1–3 ␮g of LEDGF/p75 in 200 ␮l of binding buffer supplemented with 1 mg/ml bovine serum albumin. Bound proteins were eluted in 40 ␮l of binding buffer supplemented with 200 mM imidazole and 1% SDS and analyzed by 11% SDS-PAGE followed by staining with Coomassie Blue R-250 [16]. The effect of LEDGF/p75 on the DNA binding properties of IN was tested in the reaction buffer normally used for enzymatic assays This buffer contained the following final concentrations: 20 mM Tris-HCl (pH 7.6), 50 mM NaCl, 10 mM MgCl2, 5 mM dithiothreitol, 10% dimethyl sulfoxide, and 5% polyethylene glycol 8000 and will be referred to as the reaction buffer. The data were subsequently analyzed as described previously, using the quantile plot analysis method [12]

RESULTS

DISCUSSION

Fold increase in binding