Differential Interaction of HIV-1 Integrase and JPO2 with the C Terminus of LEDGF/p75 Koen Bartholomeeusen 1 , Jan De Rijck 1 , Katrien Busschots 1 Linda Desender 1 , Rik Gijsbers 1 , Stéphane Emiliani 2 Richard Benarous 2 , Zeger Debyser 1 ⁎ and Frauke Christ 1 1 Molecular Medicine, KULeuven and IRC KULAK Leuven, Belgium 2 Institut Cochin, U567 INSERM, UPR8104 CNRS Departement of Infectious Diseases, 27 Rue du Faubourg St. Jacques, 75014 Paris, France The transcriptional co-activator lens epithelium-derived growth factor (LEDGF) has been shown to protect cells against environmental stress. The protein has been implicated in auto-immunity and cancer, and is present in cells as the p52 or p75 splice variant. Recently, LEDGF/p75, but not p52, was identified as the prominent interaction partner of human immunode- ficiency virus type 1 (HIV-1) integrase. This interaction of HIV-1 integrase with the C-terminal integrase-binding domain of LEDGF/p75 is crucial for HIV-1 replication. To gain insight into the cell biology of LEDGF/p75, we were interested in identifying cellular binding partners of its C-terminal domain. By yeast-two-hybrid screening with a CEMC7 cDNA-library, we were able to identify JPO2 as a binding partner of the C-terminal part of LEDGF/p75. The specific interaction between JPO2 and LEDGF/p75 was verified by pull-down, AlphaScreen, and co-immunoprecipitation. Compe- tition assays using recombinant proteins show a mutually exclusive binding of either JPO2 or HIV-1 integrase to LEDGF/p75. However, differing mechanisms of binding were suggested by continuing interaction of JPO2 with some LEDGF/p75 mutants (I365A, D366A, F406A) that are totally defective for interaction with HIV-1 integrase. This finding is of significance for the development of specific inhibitors targeting only the interaction between LEDGF/p75 and HIV-1 integrase, without disturbing interaction with other cellular factors. Over-expression of JPO2 resulted in a modest but reproducible inhibition of HIV-1 replication, consistent with competition between integrase and JPO2 for binding to LEDGF/p75. Furthermore, JPO2 over-expression activated transcription from the HIV-1 LTR. © 2007 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: JPO2; LEDGF/p75; HIV-1; integrase; D366A Introduction Cells respond to environmental stress by modulat- ing their gene expression patterns. The products of the lens epithelium-derived growth factor (LEDGF) gene play a role in this survival response. Via alter- native splicing, the LEDGF gene yields two co- activators of transcription diverging in their potency: the stronger co-activator p52 (exons 1–9 plus part of intron 9) and the weaker p75 (exons 1–15). 1,2 Both are karyophilic proteins, but differ in their nuclear localization pattern. 3 LEDGF/p75 was first described as the transcrip- tion cofactor PC4 interacting protein. 1,4 It rescues cells under stress by up-regulating the expression of a variety of proteins via its association with stress responsive (STRE) and heat shock elements (HSE). 5–7 Its transcriptional co-activator function has been implicated in human prostate cancer, 8 as well as in autoimmunity during chronic inflamma- Abbreviations used: LEDGF, lens epithelium-derived growth factor; STRE, stress responsive; HSE, heat shock element; HIV-1, human immunodeficiency virus type 1; HDGF, hepatoma-derived growth factor; IN, integrase; IBD, integrase-binding domain; MAO, monoamine oxidase; SID, specific interaction domain; MBP, maltose-binding protein; DMEM, Dulbecco's modified Eagle's medium. E-mail address of the corresponding author: Zeger.Debyser@med.kuleuven.be doi:10.1016/j.jmb.2007.06.090 J. Mol. Biol. (2007) 372, 407–421 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.