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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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