Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease.

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.     

Expression of human T-cell leukemia virus type I protease in Escherichia coli.

Human T-cell leukemia virus type I (HTLV-I) genome is believed to encode its own protease, although the protease has not yet been detected. To identify the HTLV-I protease, an in-frame gag (3' portion)-prt region was expressed in Escherichia coli. The 14-kDa product was detected using antisera against a synthetic peptide mimicking the fragment of HTLV-I protease, although the molecular weight of the primary translational product was 27,000. A cell extract had a proteolytic activity to cleave a synthetic peptide substrate containing the cleavage site of gag p19/p24 at the correct site in vitro. Replacement of the putative active site Asp-64 with Gly abolished both in vivo processing activity and in vitro proteolytic activity. These results suggest that the 14-kDa product is the mature enzymatically active HTLV-I protease generated through posttranslational autoprocessing in E. coli.