The central region of human T-cell leukemia virus type 1 Tax protein contains distinct domains involved in subunit dimerization

Abstract

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) can form homodimers. Tax dimerization contributes to optimal Tax activity involved in transactivation of the HTLV-1 promoter. The mechanisms used to form specific Tax dimers are poorly understood because the domains that mediate such interactions have not been clearly characterized. Here we have used different approaches (the two-hybrid assay in yeast, the glutathione S-transferase pull-down assay, and the Spot method) to study Tax-Tax interactions. Our results indicate that the integrity of the sequence of Tax, except for the last 16 amino acids (residues 338 to 353), is critical, suggesting that Tax dimerization is dictated more by secondary structure than by primary structure. We were, however, able to delimit a central region involved in Tax self-association that encompasses the residues 127 to 228. This region can be divided into three subdomains of dimerization: DD1 (residues 127 to 146), DD2 (residues 181 to 194), and DD3 (residues 213 to 228). Moreover, the Tax mutants M22 (T130A and L131S) and M29 (K189A and R190S), with amino acid substitutions located in DD1 and DD2, respectively, were found to be impaired in Tax self-association.

FIG. 1.

Study of Tax self-association by yeast two-hybrid assay. (A) Schematic representation of Tax proteins fused to the GAL4 activation domain: the cysteine-rich zinc-binding domain is represented by a black box. The Y190 yeast cells were transformed with either WT Tax or truncations of Tax fused to the activation domain of GAL4 and the plasmid pAS-Tax (WT Tax fused to the GAL4-DNA-binding domain). The β-galactosidase assay with CPRG as substrate was carried out on three independent colonies per transformation. Symbols: +, β-galactosidase activity corresponding to the level of interaction obtained with WT Tax; −, no stimulation of β-galactosidase activity. (B) Stability of the different mutant Tax proteins fused to the GAL4 activation domain in S. cerevisiae. Yeast extracts were analyzed by SDS-PAGE and Western blotting with GAL4 AD monoclonal antibodies. Molecular size markers (in kilodaltons) are shown on the left.

FIG. 1.

Study of Tax self-association by yeast two-hybrid assay. (A) Schematic representation of Tax proteins fused to the GAL4 activation domain: the cysteine-rich zinc-binding domain is represented by a black box. The Y190 yeast cells were transformed with either WT Tax or truncations of Tax fused to the activation domain of GAL4 and the plasmid pAS-Tax (WT Tax fused to the GAL4-DNA-binding domain). The β-galactosidase assay with CPRG as substrate was carried out on three independent colonies per transformation. Symbols: +, β-galactosidase activity corresponding to the level of interaction obtained with WT Tax; −, no stimulation of β-galactosidase activity. (B) Stability of the different mutant Tax proteins fused to the GAL4 activation domain in S. cerevisiae. Yeast extracts were analyzed by SDS-PAGE and Western blotting with GAL4 AD monoclonal antibodies. Molecular size markers (in kilodaltons) are shown on the left.

FIG. 2.

Study of Tax-Tax interactions by GST-pull down assay. (A) In vitro-translated WT Tax in the presence of [³⁵S]methionine and [³⁵S]cysteine (lanes 1, 8, and 15) was incubated with equal amounts of purified GST (lanes 2, 9, and 16), GST-WT (lanes 3, 10, and 17), or GST-truncated Tax (lanes 4, 5, 6, 7, 11, 12, 13, 14, 18, 19, 20, and 21) immobilized on glutathione-Sepharose beads. The upper panels show Western blot analyses performed to detect the amounts of protein for the different mutant Tax proteins fused to the GST by using peroxidase-conjugated anti-GST. Molecular size markers are shown on the left in kilodaltons. In the lower panels, the bound ³⁵S-labeled Tax was revealed, following incubation, by autoradiography after SDS-PAGE and then quantified by scanning with Scion Image. The results are reported in calibrated units (CU) under each lane. (B) Coomassie blue-stained SDS-PAGE gels show the purity rate of the GST-fused proteins.

FIG. 3.

Results of the study of Tax self-association obtained by GST pull-down assay. Schematic representation of Tax proteins fused to GST. The cysteine-rich zinc-binding domain is represented by a black box. The ³⁵S-radiolabeled Tax was incubated with equal amounts of GST, GST-WT, or truncated Tax. Each mutant was analyzed three times, with similar results. Symbols: +, signal intensity corresponding the interaction between ³⁵S-labeled Tax and GST-WT Tax; −, interaction between ³⁵S-labeled Tax and GST.

FIG. 4.

Identification by the Spot method of Tax peptides recognized by GST-Tax. The set of membrane-bound peptides was incubated with GST-c-Jun (A) or GST-Tax (B). The binding was revealed by peroxidase-conjugated anti-GST.

FIG. 5.

Amino acid sequence of Tax and mapping of the peptides recognized by Tax by the Spot method. The peptides recognized by GST-Tax are underlined. The dotted lines correspond to weak interactions between the peptides and GST-Tax.

FIG. 6.

Analysis of interactions of mutant Tax proteins in yeast. (A) The Y190 yeast cells were transformed with pAS-Tax and mutant Tax proteins fused to the GAL4 activation domain (pGAD-M22, -M29, -M37, or -M47). The β-galactosidase assay with CPRG as substrate was carried out on three independent colonies per transformation. The β-galactosidase activity was calculated in Miller units. (B) Stability of the different mutant Tax proteins fused to the GAL4 activation domain in S. cerevisiae. Yeast extracts were analyzed by SDS-PAGE and Western blotting with GAL4 AD monoclonal antibodies.

FIG. 7.

Analysis of interactions of mutant Tax proteins in vitro. Radiolabeled WT Tax (lanes 1, 7, 13, and 19), M22 (lane 4), M29 (lane 10), M37 (lane 16), or M47 (lane 22) was incubated with GST (lanes 2, 5, 8, 11, 14, 17, 20, and 23) or GST-WT Tax (lanes 3, 6, 9, 12, 15, 18, 21, and 24). The bound ³⁵S-labeled Tax was analyzed as described in the legend of Fig. 2 and quantified by scanning. The results are reported in calibrated units (CU) under each lane.