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First published online 29 March 2005
doi: 10.1242/jcs.02299

Journal of Cell Science 118, 1733-1743 (2005)
Published by The Company of Biologists 2005
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Identification of the LEDGF/p75 HIV-1 integrase-interaction domain and NLS reveals NLS-independent chromatin tethering

Maria Vanegas1,2, Manuel Llano1, Sharon Delgado1, Daniah Thompson1, Mary Peretz1 and Eric Poeschla1,2,*

1 Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
2 Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA



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  		Fig. 1. PK-LEDGF/p75 fusion proteins. Transfections were in L cells. (A) Confocal microscopy. Lower panels show DAPI staining. (B) Immunoblotting. The primary antibody was an anti-Myc epitope mAb.

 


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  		Fig. 2. Basic residues 149-152 are required for nuclear localization. Transfections were in L cells. (A) Confocal microscopy. (B) Immunoblotting. Lane 1: Mock; lane 2: PK; lane 3: PK-LEDGF/146-156; lane 4: PK-LEDGF/RR146-147AA; lane 5: PK-LEDGF/RKRK149-152AAAA.

 


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  		Fig. 3 . LEDGF/p75 and LEDGF/p75.NLS–; subcellular location and interactions with chromatin, in different phases of the cell cycle. (A) Transient transfection into L cells. (B) Immunoblotting in L cells, using anti-LEDGF/p75 mAb. Endogenous p52 is also detected by the N-terminus antibody.

 


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  		Fig. 4. Co-expression of IN with LEDGF/p75 or LEDGF/p75.NLS– in L cells. (A) Panels a-d: HIV-1 IN was transfected alone. Panels e-h: cotransfection of HIV-1 IN with LEDGF/p75 (using pLEDGF/p75siMut). Panels i-l: cotransfection of HIV-1 IN with pLEDGF/p75.NLS–. (B) Immunoblotting of lysates from cells in (A).

 


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  		Fig. 5 . Stable expression of LEDGF/p75.NLS– in L cells; subcellular localization and interaction with IN. Stable cell lines were derived by puromycin selection. (A) Confocal immunofluorescence with anti-LEDGF/p75 mAb. DAPI staining (right) indicates chromatin (approximately 50% of the cells in this stable L cell line express the LEDGF/p75 mutant). (B) The same cells after transfection of HIV-1 IN. LEDGF/p75.NLS– and IN colocalize (overlay, right).

 


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  		Fig. 6 . C-terminal segments of LEDGF/p75 fused to NLS-GFP: expression and intracellular location. (A) Confocal microscopy of NLS-GFP-LEDGF fusions. (B) Immunoblotting with anti-GFP mAb. Lane 1: Mock; lane 2: NLS-GFP; lane 3: NLS-GFP/306-530; lane 4: NLS-GFP/330-417.

 


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  		Fig. 7. C-terminal segments of LEDGF/p75 fused to NLS-GFP: IN interaction. L cells were cotransfected with HIV-1 IN and either NLS-GFP, NLS-GFP/330-417, or NLS-GFP/306-530. shRNA target-site-mutated (siMut) versions of LEDGF/p75 were used in all expression plasmids. (A) Confocal microscopy. (B) Co-immunoprecipitation of NLS-GFP/330-417 and IN. Immunoprecipitation was carried out with a mAb to GFP, followed by immunoblotting for GFP (lower) and IN (upper).

 


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  		Fig. 8. LEDGF/p75 residues 340-417 are required for nuclear import of HIV IN. Transfections were done in L cells. (A) Confocal microscopy. (B) Immunoblotting.

 


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  		Fig. 9. Expression and subcellular localization of HRP-2 and HIV-1 IN. (A) Confocal microscopy of L cells cotransfected with pFlag-HRP-2 (top), HIV-1 IN (middle), or both (bottom). (B) Immunoblotting of L cells transfected with pFlag-HRP-2.

 


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  		Fig. 10 . Differential HRP-2 and LEDGF/p75 location in L cells. L cells were cotransfected with pFlag-HRP-2 without (a-h) or with (i-p) LEDGF/p75 (using pLEDGF/p75siMut).

 




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