The role of the X region of the genome of the human T-cell leukemia virus type I (HTLV-I) in the hmmortalization of lymphocytes has been difficult to distinguish from its role in viral replication as this region encodes at least two genes, tax and rex, required for replication and the expression of viral proteins. To determine whether the X region does encode immortalizing functions, a fragment of the HTLV-I provirus capable of expressing known X-region proteins was inserted into the genome of a transformation-defective, replication-competent Herpesvirus saimiri. Infection of fresh mitogen-activated human cord blood and thymocytes yielded immortal T-cell lines that had the same phenotype (CD4+, CD5+, HLA class HII, interleukin 2 receptor a-chain +) as lymphocytes transformed by cocultivation with HTLV-I. These experiments demonstrate that the X region encodes the functions of HTLV-I that immortalize a distinct subpopulation of human T cells. The experiments also demonstrate the utility of the H.saimiri vector for the transduction of heterologous genes into human T cells.

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.

Human T cell leukemia virus type I (HTLV-I) is a persistent virus that causes adult T cell leukemia and tropical spastic paraparesis/HTLV-I–associated myelopathy. Studies on rabbits have shown that viral proteins encoded by the open reading frames pX-I and pX-II are required for the establishment of the persistent infection. To examine the in vivo production of these proteins in humans, we have investigated whether cytotoxic T lymphocytes isolated from HTLV-I–infected individuals recognized pX-I and pX-II peptides. CD8+ T lymphocytes to pX-I and pX-II peptides were detected in HTLV-I–infected individuals, whatever their clinical status, and even in the absence of any antigenic restimulation. These findings indicate that the HTLV-I pX-I and pX-II proteins are chronically synthesized in vivo, and are targets of the natural immune response to the virus.