Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75.
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-alpha/beta-, 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 beta-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.
Maertens
Goedele
G
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Cherepanov
Peter
P
Debyser
Zeger
Z
Engelborghs
Yves
Y
Engelman
Alan
A
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001667:Binding, Competitive; D001693:Biological Transport, Active; D002467:Cell Nucleus / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000494:pharmacology; D019427:HIV Integrase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000502:physiology; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D028901:alpha Karyopherins / Q000502:physiology; D028961:beta Karyopherins / Q000502:physiology; D001616:beta-Galactosidase / Q000235:genetics; D020931:ran GTP-Binding Protein / Q000502:physiology
D000952:Antigens, Polyomavirus Transforming; D005456:Fluorescent Dyes; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; C120587:RAN protein, human; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C119380:lens epithelium-derived growth factor; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D019427:HIV Integrase; D001616:beta-Galactosidase; D020931:ran GTP-Binding Protein