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. 2004 Sep;78(17):9524-37.
doi: 10.1128/JVI.78.17.9524-9537.2004.

LEDGF/p75 determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes

Manuel Llano  1 , Maria Vanegas, Oliver Fregoso, Dyana Saenz, Susan Chung, Mary Peretz, Eric M Poeschla
Affiliations

LEDGF/p75 determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes

Manuel Llano et al. J Virol. 2004 Sep.

Abstract

Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integrase-to-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.

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Figures

FIG. 1.
FIG. 1.
Intron A is necessary and sufficient for Rev-independent HIV-1 and FIV integrase expression. (A) Expression plasmids; (B) intron A dependence. 293T cells transfected with the indicated plasmids were analyzed by Western blotting (WB) with an anti-Myc MAb. +, present; −, absent.
FIG. 2.
FIG. 2.
Karyophilic properties of retroviral integrase proteins in stable cell lines. (A) Confocal immunofluorescence microscopy of puromycin-selected 293T cells stably transfected with pHIN.ip, pFIN.ip, and pMIN.ip. The primary antibody used in the left three panels was an anti-Myc MAb. In the rightmost panel, an anti-MoMLV IN antibody was used. The bottom panels are overlays with nuclear DAPI staining. Photographic exposure times were equal. (B) Immunoblotting of the same cell lines. HIV-1, FIV, and MoMLV integrase are 34, 32, and 46 kDa, respectively.
FIG. 3.
FIG. 3.
Lentiviral integrases lack transferable NLSs. (A) Cellular distribution of PK fusion proteins. 293T cells were transfected with pPK, pPK-M9, pPKHIN, or pPKFIN and analyzed by confocal immunofluorescence microscopy with anti-Myc antibody. (B) Immunoblotting of cells shown in panel A with an anti-Myc MAb.
FIG. 3.
FIG. 3.
Lentiviral integrases lack transferable NLSs. (A) Cellular distribution of PK fusion proteins. 293T cells were transfected with pPK, pPK-M9, pPKHIN, or pPKFIN and analyzed by confocal immunofluorescence microscopy with anti-Myc antibody. (B) Immunoblotting of cells shown in panel A with an anti-Myc MAb.
FIG. 4.
FIG. 4.
Interaction of lentiviral integrases with LEDGF/p75. (A) FIV integrase colocalizes with endogenous LEDGF/p75. Cells stably expressing FIV integrase were double stained with an anti-Myc polyclonal antibody and an anti-LEDGF MAb and analyzed by confocal immunofluorescence microscopy. The arrow indicates a mitotic figure. (B) FIV and HIV-1 integrases coimmunoprecipitate with endogenous LEDGF/p75. Samples were immunoprecipitated (IP) with a feline anti-FIV antiserum or rabbit anti-HIV integrase antiserum and then probed with an anti-LEDGF MAb.
FIG. 5.
FIG. 5.
Generation of LEDGF/p75-deficient cell lines. (A) Immunoblotting. LEDGF/p75 protein expression was evaluated by Western blotting with anti-LEDGF MAb in siScram cells and si1340 cells (left) and si1340/1428 cells (right). (B) mRNA levels. LEDGF/p75 mRNA was quantitated by RT-PCR in the same cell lines. (C) LEDGF/p75 protein expression in Jurkat cell lines.
FIG. 6.
FIG. 6.
LEDGF is required for nuclear localization of lentiviral integrase proteins. (A) Stable HIV-1 and FIV integrase expression in si1340 cells and siScram cells; (B and C) subcellular distributions of lentiviral integrase proteins in the same cells were analyzed by confocal immunofluorescence microscopy with anti-Myc antibody.
FIG. 6.
FIG. 6.
LEDGF is required for nuclear localization of lentiviral integrase proteins. (A) Stable HIV-1 and FIV integrase expression in si1340 cells and siScram cells; (B and C) subcellular distributions of lentiviral integrase proteins in the same cells were analyzed by confocal immunofluorescence microscopy with anti-Myc antibody.
FIG. 7.
FIG. 7.
LEDGF/p75 is a component of functional PICs. HIV-1 vector PICs were isolated and used in an in vitro DNA integration assay before (left) and after (right) immunoprecipitation with an MAb to LEDGF/p75 or a control (anti-Myc) MAb. Integration products are detected as a larger band that is a fraction of the nonintegrated PICs. The reaction is shown to require both a functional integrase (IN mutant tested is D64V) and the presence of the target DNA. Single asterisk, HIV-1 PICs prominently immunoprecipitated with anti-LEDGF/p75 MAb; double asterisk, integrated HIV-1 vector genomes detected in reactions with anti-LEDGF/p75 MAb-immunoprecipitated samples. The same results were obtained with FIV PICs (data not shown).
FIG. 8.
FIG. 8.
Lentiviral infection of LEDGF/p75-deficient cells. (A) Two-LTR circle formation in growth-arrested control and LEDGF/p75-deficient cells infected with HIV eGFP or an FIV lacZ vector (CT26). (B) Infection of LEDGF/p75-deficient si1340/1428 cells (squares) and siScram (diamonds) with pNL43.Luc.R−E− or pNL43.Luc.R+E−. Luminescence units are shown on the y axes. (C) Back complementation of si1340JK clones with LEDGF/p75. (D) HIV-1 NL4-3 replication in si1340JK clones with or without back complementation with LEDGF/p75. Jurkat BC cells are wild-type Jurkat E6 cells which were stably transfected with the same LEDGF/p75 expression construct.
FIG. 8.
FIG. 8.
Lentiviral infection of LEDGF/p75-deficient cells. (A) Two-LTR circle formation in growth-arrested control and LEDGF/p75-deficient cells infected with HIV eGFP or an FIV lacZ vector (CT26). (B) Infection of LEDGF/p75-deficient si1340/1428 cells (squares) and siScram (diamonds) with pNL43.Luc.R−E− or pNL43.Luc.R+E−. Luminescence units are shown on the y axes. (C) Back complementation of si1340JK clones with LEDGF/p75. (D) HIV-1 NL4-3 replication in si1340JK clones with or without back complementation with LEDGF/p75. Jurkat BC cells are wild-type Jurkat E6 cells which were stably transfected with the same LEDGF/p75 expression construct.
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