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.

Human homologue of the Drosophila Dlg tumor suppressor (hDlg) is a widely expressed scaffold protein implicated in the organization of multi-protein complexes at cell adhesion sites such as the neuronal synapse. hDlg contains three PDZ domains that mediate its binding to the consensus motifs present at the C-termini of various cell surface proteins, thus inducing their clustering and/or stabilization at the plasma membrane. Using a yeast two-hybrid screen, we identified hDlg as a cellular binding partner of a viral membrane integral protein, the envelope glycoprotein (Env) of human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 is a human retrovirus that infects CD4+ T lymphocytes and is preferentially transmitted via direct contacts between infected and target cells, through a structure referred to as the virological synapse. Here, we demonstrate that hDlg interacts with a classical PDZ domain-binding motif present at the C-terminus of the cytoplasmic domain of HTLV-1 Env and conserved in the related HTLV-2 virus. We further document that, in HTLV-1 infected primary T cells, hDlg and Env are concentrated in restricted areas of the plasma membrane, enriched in molecules involved in T-cell contacts. The presence of Gag proteins responsible for viral assembly and budding in these areas indicated that they constitute platforms for viral assembly and transmission. Finally, a mutant virus unable to bind hDlg exhibited a decreased ability to trigger Env mediated cell fusion between T lymphocytes. We thus propose that hDlg stabilizes HTLV-1 envelope glycoproteins at the virological synapse formed between infected and target cells, hence assisting the cell-to-cell transmission of the virus.

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 of 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.

The PPPY motif of human T-cell leukemia virus type 1 Gag protein is required early in the budding process.

Domains required late in the virus budding process (L domains) have been identified in the Gag proteins of a number of retroviruses. Here we show that the human T-cell leukemia virus type 1 candidate L domain motif PPPY is indeed required for virus production. Strikingly, however, mutation of this motif arrested virus particles at an earlier stage in the budding process than was seen for mutation of the L domain motifs thus far described for retroviruses. In view of the exchangeability of such domains, we propose that the retrovirus budding process may involve a continuum from bud formation to membrane fission.

Intracellular distribution of human T-cell leukemia virus type 1 Gag proteins is independent of interaction with intracellular membranes.

Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system.