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J. Biol. Chem., Vol. 279, Issue 32, 33421-33429, August 6, 2004

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Identification and Characterization of a Functional Nuclear Localization Signal in the HIV-1 Integrase Interactor LEDGF/p75^*

Goedele Maertens¶||, Peter Cherepanov**, Zeger Debyser¶, Yves Engelborghs, and Alan Engelman**

From the Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Heverlee B-3001, Belgium, the ^**Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, and ^¶Rega Institute for Medical Research and KULAK, Katholieke Universiteit Leuven, Leuven B-3000, Belgium

Human lens epithelium-derived growth factor (LEDGF)/p75 protein forms a specific nuclear complex with human immunodeficiency virus type 1 (HIV-1) integrase and is essential for nuclear localization and chromosomal association of the viral protein. We now studied nuclear import of LEDGF/p75 in live and semipermeabilized cells. We show that nuclear import of LEDGF/p75 is GTP-, Ran-, importin-/-, and energy-dependent and that the protein competes with the canonical SV40 large T antigen nuclear localization signal (NLS) for nuclear import receptors. We identified the NLS of LEDGF/p75 through deletion analysis and site-directed mutagenesis. The LEDGF/p75 NLS, ¹⁴⁸GRKRKAEKQ¹⁵⁶, belongs to the canonical SV40-like family. Fusion of this short peptide to the amino terminus of Escherichia coli -galactosidase rendered the fusion protein nuclear, confirming that the LEDGF/p75 NLS is transferable. Moreover, a single amino acid change in the NLS was sufficient to exclude the mutant LEDGF/p75 protein from the nucleus and abolish nuclear import of HIV-1 integrase.

Received for publication, April 28, 2004

^* This work was funded by National Institutes of Health Grant AI52014 (to A. E.). Work at the KULeuven was financially supported by the SBO program from the IWT Flanders (Belgium). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| An Aspirant of the Fund for Scientific Research (Fonds voor Wetenschappelijk Onderzoek) Flanders.

To whom correspondence should be addressed. Tel.: 617-632-4361; Fax: 617-632-3113; E-mail: Alan_Engelman{at}dfci.harvard.edu.

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