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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01095

Journal of Cell Science 117, 2357-2367 (2004)
Published by The Company of Biologists 2004
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Nedd4.1-mediated ubiquitination and subsequent recruitment of Tsg101 ensure HTLV-1 Gag trafficking towards the multivesicular body pathway prior to virus budding

Vincent Blot1, Fabien Perugi1, Bernard Gay2, Marie-Christine Prévost3, Laurence Briant2, Frédéric Tangy4, Hugues Abriel5, Olivier Staub5, Marie-Christine Dokhélar1 and Claudine Pique6,*

1 Département de Biologie Cellulaire, CNRS UMR 8104 and INSERM U567, Institut Cochin, 75014 Paris, France
2 CNRS UMR 5121, Institut de Biologie, 34960 Montpellier, France
3 Plate-forme de microscopie électronique, CNRS URA 1930, Institut Pasteur, 75015 Paris, France
4 Unité des Virus Lents, CNRS URA 1930, Institut Pasteur, 75015 Paris, France
5 Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland
6 CNRS UPR 9051, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, 75010 Paris, France



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  		Fig. 1. Nedd4.1 is required for HTLV-1 release and catalyses Gag ubiquitination. (A) Anti-MAp19 ELISA was used to quantify virus particles released from 293T cells after co-transfection of different plasmid ratios of wild-type provirus and dominant negative mutants of either Nedd4.1 (Nedd4.1-C867S) or Nedd4.2 (Nedd4.2-C801S) proteins, or of the Flag epitope-tagged WW domains of Nedd4.1 (WW). For comparison, virus production of the PPPY-mutated provirus (Y121D) is indicated. For each condition, Gag intracellular production and extracellular virion-associated CAp24 as detected by immunoblot are also shown. The ELISA results are means and standard errors of at least three independent experiments and immunoblots correspond to one representative experiment. (B) Detection of ubiquitinated Gag products when the wild-type (XMT WT) or the PPPY-mutated (XMT Y121D) provirus is expressed alone or in combination with wild-type (Nedd4.1 WT) or dominant negative mutant (Nedd4.1-C867S) of Nedd4.1, or with the dominant negative mutant of Nedd4.2 (Nedd4.2-C801S). The position of Gag p55 is indicated, as are those of mono and tri-ubiquitinated Gag products (approximately 63 kDa and 79 kDa, respectively, asterisks).

 


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  		Fig. 2. The PTAP motif is also required for HTLV-1-Gag release. (A) Schematic representation of the HTLV-1 Gag protein. The sequence of the L domain at the C-terminus of the MA domain is indicated, and the PPPY and PTAP motifs are shown in bold characters. Positions of the introduced Y121D, P124L and P127L mutations are indicated. (B) Immunoprecipitation of HTLV-1 proteins from 293T cells transfected with either the wild-type (WT), PTAP-mutated (P127L) or PPPY-mutated (Y121D) provirus. Virus proteins were immunoprecipitated from either cell lysates (Cells), or from sucrose-cushion purified virions (Virions). Immature (Gagp55) and mature (CAp24, MAp19 and NCp15) Gag products are indicated. (C) Anti-MAp19 ELISA was used to quantify virus particles released from 293T cells transfected with either the wild-type (WT), PTAP-mutated (P127L) or PPPY-mutated (Y121D) provirus. The results are the means and standard errors of three independent experiments.

 


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  		Fig. 3. Tsg101 is required for HTLV-1 release. (A) Anti-Tsg101 immunoblot analysis of 293T cells transfected with either control (ctrl) or Tsg101-specific siRNAs. (B) Anti-MAp19 ELISA was used to quantify wild-type virus release from 293T cells after depletion of Tsg101 by siRNAs. For comparison, the amount of virus particles produced by the PTAP-mutated provirus (P127L) is indicated. For each condition, Gag intracellular production and extracellular virion-associated CAp24 as detected by immunoblots are also shown. The ELISA results are the means and standard errors of at least three independent experiments, and immunoblots correspond to one representative experiment.

 


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  		Fig. 4. Wild-type HTLV-1-Gag proteins localize mainly in the endocytic pathway in 293T cells at steady state. (A) Gag co-staining with the whole endocytic pathway revealed by 1-hour uptake of FITC-conjugated dextran. (B) Gag co-staining with early/recycling endosomes revealed by 1-hour uptake of cyanin 3-conjugated transferrin. (C) Gag co-staining with the late endosomes/MVBs marker CD63. Each row shows a median section of cells recorded by confocal microscopy using a 63x objective and a 4x zoom. Overlays of red and green pictures are shown as well as bright light pictures. Zoomed images of colocalization areas are also presented (boxed).

 


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  		Fig. 5. PPPY/Nedd4.1 and PTAP/Tsg101 interactions take place in different compartments. (A) Gag/CD63 co-staining in 293T cells expressing the Y121D-mutated provirus. (B) Gag/Nedd4.1 co-staining in 293T cells transfected with the wild-type provirus and Nedd4.1-C867S-expressing construct. (C) Gag/CD63 co-staining in 293T cells expressing the P127L-mutated provirus. (D) Gag/CD63 co-staining in 293T cells transfected with the wild-type provirus and Tsg101 siRNAs. Each row shows a median section of cells recorded by confocal microscopy using a 63x objective and a 2x (A) or 4x (B,C,D) zoom. Overlays of red and green pictures are shown as well as bright light pictures. Zoomed images of colocalization areas are also presented (boxed).

 


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  		Fig. 6. Gag/Nedd4.1 interaction at the plasma membrane precedes Gag/Tsg101 interaction in late endosomes. (A) Gag/Nedd4.1 co-staining in 293T cells co-transfected with wild-type XMT provirus, Tsg101 siRNAs and Nedd4.1-C867S construct. (B) Gag/Nedd4.1 co-staining in 293T cells co-transfected with XMT P127L provirus and Nedd4.1-C867S construct. (C) Gag/CD63 co-staining in 293T cells co-transfected with XMT Y121D provirus and Tsg101 siRNAs. Each row shows a median section of cells recorded by confocal microscopy using a 63x objective and a 4x (A,C) or 2x (B) zoom. Overlays of red and green pictures are shown as well as bright light pictures.

 


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  		Fig. 7. Wild-type, Y121D and P127L virus particles display distinct cellular localizations and morphologies. (A) Electron micrographs of 293T cells transfected with the wild-type XMT provirus. Arrows point to electron dense thickenings underneath the plasma membrane. Scale bars represent 100 nm. (B) Electron micrographs of 293T cells transfected with the Y121D-mutated provirus. Arrows point to electron dense thickenings underneath the plasma membrane. Scale bars represent 50 nm. (C) Electron micrographs of 293T cells transfected with the P127L-mutated provirus. Arrows point to particles still tethered to cellular membrane. Bars, 100 nm.

 

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© The Company of Biologists Ltd 2004