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

Abstract

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 pl9, 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 vmimic 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.

Among all human T-cell leukemia virus type 1 proteins, tax, polymerase, and envelope proteins are predicted as preferential targets for the HLA-A2-restricted cytotoxic T-cell response

“…In order to be effective, an HTLV-1 vaccine should therefore have the capacity to induce both the production of antibodies, including neutralizing antibodies, and a cellular CTL response. HTLV-1 encodes the regulatory proteins Tax, Rex, Rof, and Tof and the structural proteins Gag, protease, polymerase, and envelope (5,29,34,44). The global immune response is generally considered to be higher in TSP/HAM patients than in asymptomatic carriers (10,23,24), although this has been the matter of recent discussion for the CTL response (7).…”

Characterization of ribosomal frameshifting for expression of pol gene products of human T-cell leukemia virus type I

“…1), we digested pHT5 to completion with XmaIII and Bsu36I. The 6.8-kb XmaIII-Bsu36I fragment harboring the vector sequence was isolated from plasmid p7.5gagfsl9AspII, described in a previous report (23).…”

“…In addition to the mutational studies of Jacks et al (13), sequencing of the natural viral transframe protein has determined the first frameshift site (gag-pro) of MMTV (9). Nam et al (23) reported previously that the Gag-Pro fusion protein of HTLV-I is also translated via ribosomal frameshifting in a vaccinia virus expression vector system. Site-specific mutagenesis experiments showed that the six consecutive adenine residues found in the gag-pro overlap are involved in the slippage of tRNA to the -1 pro frame to generate the Gag-Pro fusion protein that is the precursor of the PR (23).…”

Generation and characterization of monoclonal antibodies against multiple epitopes on the C‐terminal half of envelope gp46 of human T‐cell leukemia virus type‐I (HTLV‐I)

“…Recombinant vaccinia viruses (RVV) containing HTLV-I entire envelope gene (WR-proenvl), gag gene (WR-gag) and pX gene (WR-27") have been reported previously (Shida et al, 1987;Nam et al, 1988). For the expression of HTLV-I antigen by RVV, HeLa cells were infected with RVV for 1 hr at a multiplicity of infection (m.0.i.)…”