Nedd4.1-mediated ubiquitination and subsequent recruitment of Tsg101 ensure HTLV-1 Gag trafficking towards the multivesicular body pathway prior to virus budding

One of the most exciting recent developments in the field of retroviruses is the finding that their Gag proteins hijack cellular proteins from the mutivesicular body (MVB) pathway during the budding process. The Gag proteins of oncoretroviruses possess a PPxY motif that recruits a ubiquitin ligase from the Nedd4 family, whereas those of the human immunodeficiency virus interact through a PTAP motif with Tsg101, a protein of the ESCRT-1 complex. It is currently assumed that Nedd4 and Tsg101 represent equivalent entry gates towards the same cellular process leading to budding, and that both partners are recruited to the plasma membrane where viral budding occurs. However, we report here that the budding of the human oncoretrovirus HTLV-1, the Gag proteins of which possess tandem PPPY/PTAP motifs, requires both Nedd4 and Tsg101. We show that Nedd4.1, but not Nedd4.2, is recruited by the PPPY motif or Gag and subsequently catalyzes Gag ubiquitination. We also demonstrate that Gag interacts first with Nedd4.1 at the plasma membrane and then with Tsg101 in late endosomes/MVBs. Consistently, we found that HTLV-1 particles mutated in the PPPY motif remain underneath the plasma membrane, blocked at an early step of the budding process, whereas PTAP-mutated viruses accumulate in intracellular vesicles, blocked at a later step. Our findings indicate that Nedd4.1 and Tsg101 act successively in the assembly process of HTLV-1 to ensure proper Gag trafficking through the endocytic pathway up to late endosomes where the late steps of retroviral release occur.