AbstractThe full‐length provirus of human T‐cell leukemia virus type I (HTLV‐I) was isolated from MT‐2, a lymphoid cell line producing HTLV‐I. In transfected cells, structural proteins of HTLV‐I, the gag and env products, were formed and processed in the same manner as observed in MT‐2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open‐reading frame. The deduced amino acid sequences show that this open‐reading frame encodes a putative HTLV‐I protease.The protease gene (pro) of HTLV‐I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag‐ and protease‐coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans‐action. A change in the sequence Asp(64)‐Thr‐Gly, the catalytic core sequence among aspartyl proteases, to Gly‐Thr‐Gly was shown to abolish correct processing, suggesting that HTLV‐I protease may belong to the aspartyl protease group. The 76k gag‐pro precursor polyprotein was identified, implying that a cis‐acting function of HTLV‐I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans‐action of the released protease to form p19, p24, and p15.