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Natural antisense viral transcripts have been recognized in retroviruses, including human T-cell leukemia virus type 1 (HTLV-1), HIV-1, and feline immunodeficiency virus (FIV), and have been postulated to encode proteins important for the infection cycle and/or pathogenesis of the virus. The antisense strand of the HTLV-1 genome encodes HBZ, a novel nuclear basic region leucine zipper (b-ZIP) protein that in overexpression assays down-regulates Tax oncoprotein-induced viral transcription. Herein, we investigated the contribution of HBZ to HTLV-1–mediated immortalization of primary T lymphocytes in vitro and HTLV-1 infection in a rabbit animal model. HTLV-1 HBZ mutant viruses were generated and evaluated for viral gene expression, protein production, and immortalization capacity. Biologic properties of HBZ mutant viruses in vitro were indistinguishable from wild-type HTLV-1, providing the first direct …