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First published online July 30, 2004
doi: 10.1242/10.1242/jcs.01266

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Human Dlg protein binds to the envelope glycoproteins of human T-cell leukemia virus type 1 and regulates envelope mediated cell-cell fusion in T lymphocytes

¹ Département Biologie Cellulaire, CNRS UMR 8104 and INSERM U567, Institut Cochin, 22 rue Méchain, 75014 Paris, France
² Département Maladies Infectieuses, CNRS UMR 8104 and INSERM U567, Institut Cochin, 22 rue Méchain, 75014 Paris, France
³ Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif CEDEX, France
⁴ Department of Pharmacology and Cancer Center, University of Illinois College of Medicine, Chicago, IL 60607, USA
⁵ CNRS UPR 9051, Hôpital St.-Louis, 75475 Paris CEDEX 10, France

View larger version (18K): [in a new window]   Fig. 1. Schematic representation of hDlg and HTLV-1/HTLV-2 envelope transmembrane proteins. (A) Like other members of the MAGUK subfamily, hDlg is composed of three PDZ domains, one SH3, and one Guanylate kinase-like (GUK) domain. In addition, hDlg possesses a unique proline-rich N-terminal region. The portion of hDlg isolated during the yeast two-hybrid screen is indicated. (B) The transmembrane subunits of HTLV-1 and HTLV-2 envelope glycoproteins are composed of an extracellular domain (EC), a transmembrane domain (TM), and a cytoplasmic domain (CD). The entire primary sequences of the cytoplasmic domains are shown. Conserved consensus motifs involved in the interaction with PDZ domains are shown in grey and underlined.

View larger version (46K): [in a new window]   Fig. 2. Interaction between the cytoplasmic domains of several virus envelope glycoproteins and hDlg as determined by yeast two-hybrid assay. Yeast strain L40 was co-transformed with a plasmid encoding the fusion protein between the cytoplasmic domain of viral envelope and the LexA DNA-binding domain, and a plasmid encoding the fusion protein between hDlg (or its PDZ domains) and the Gal4 activation domain. Interaction between both fusion proteins leads to the activation of the lacZ reporter gene, which was visualized by the ß-galactosidase assay. Lamin protein was used as a negative control. (A) Interactions between the cytoplasmic domain of HTLV-1 envelope glycoproteins (CD) and the full-length hDlg protein (hDlg) or a segment of hDlg protein containing three PDZ domains (PDZ). (B) Interactions between mutated CDs and hDlg. (C) Interactions between envelope CDs of various members of the HTLV/BLV retrovirus genus and hDlg. (D) Interactions between hDlg and the CDs of Epstein-Barr virus (EBV) gp340 and rabies virus G glycoprotein.

View larger version (29K): [in a new window]   Fig. 3. Interaction between native HTLV-1 envelope proteins and hDlg. (A,B) Jurkat cells were lysed by several freeze/thaw cycles, and membrane and cytosol fractions were separated by centrifugation. The cytosol fraction was submitted to precipitation with either GST alone or GST fused to the CD of HTLV-1 envelope proteins (GST-CD). The precipitated proteins were detected either using a mAb specific for hDlg (A) or a mAb directed against the PDZ domains of MAGUKs (B). (C) Primary HTLV-1 infected CIB T-cells were metabolically labelled for 16 hours and lysed in a 0.5% NP40 buffer. Soluble material was subjected to precipitation with either GST alone or GST fused with the full-length hDlg (GST-hDlg). As a control, viral proteins were immunoprecipitated from cell lysate using sera obtained from HTLV-1 infected patients (IP anti-HTLV-1).

View larger version (35K): [in a new window]   Fig. 4. Concentration of HTLV-1 envelope glycoproteins and hDlg in restricted areas characteristic of cell contact sites in HTLV-1-infected primary T lymphocytes. Each row shows a single optical plane of HTLV-1 infected CIB cells, as recorded independently in the red and green channels by confocal microscopy. (A,B) HTLV-1 envelope glycoproteins were stained using sera obtained from infected patients, and Texas-red conjugated secondary antibodies before permeabilization (left panels). hDlg (A) and Gag (B) were stained on saponin-permeabilized cells using 2D11 and p19 monoclonal antibodies, respectively, followed by FITC-conjugated secondary antibodies (middle panels). On the right panels are presented overlay profiles of red and green channels (Overlay panels). Co-localization signal appears as yellow pixels. (B) HTLV-1 envelope glycoproteins were stained as described above (left panels). Staining of CD25, CD2 and CD4 were performed on non-permeabilized cells using either FITC-conjugated mAb, or mAb followed by FITC-conjugated secondary antibodies (middle panels). Staining of GM1 was done on non-permeabilized cells with biotin-conjugated cholera toxin and DTAF-conjugated streptavidin. Staining of Lck was performed on saponin-permeabilized cells using rabbit sera followed by FITC-conjugated secondary antibodies. Overlay profiles of red and green channels. Co-localization signal appears as yellow pixels (right panels, merge).

View larger version (68K): [in a new window]   Fig. 5. Mutant HTLV-1 unable to interact with hDlg shows reduced Env-triggered syncytia formation between T-lymphocytes. (A) Images of Jurkat T cells in culture 36 h after transfection with either wild-type (XMT-WT), envelope mutated (XMT-Env-S486L) or envelope deleted (XMT-delta Env) HTLV-1 provirus. The arrowheads point to gigantic syncitia detectable in cultures of cells expressing wild-type HTLV-1. (B) Thirty six hours after transfection, Jurkat T-cells were concentrated to 107 cells/ml and seeded onto glass slides for 30 minutes minutes to favour cell-to-cell contacts between transfected and nontransfected cells. Viral Env and Gag proteins are detected as described in the legend of Fig. 4A. The left rows show single optical planes as recorded in the red (Env) and green (Gag) channels by confocal microscopy; DAPI staining showing cells nucleus (DAPI) and a contrast phase recording showing the overall cell structure (contrast) are also presented.

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