Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag)

Abstract

Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55(Gag) structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55(Gag) and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55(Gag). The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.

Figure 1

Identification of the region in Pr55^Gag required for Tsg101 binding by using the two-hybrid assay. Reporter gene activation was quantified by determination of β-galactosidase units. (A) The interaction of Gag and p1-p6 with Tsg101. β-Galactosidase activity of Gag and p1-p6 was equivalent in two independent trials. (B) The interaction of p1-p6 with Tsg101 (taken as 100%) ranged from ≈10 to 30 β-galactosidase units in 20 independent trials. Negative interactions were equivalent to that obtained when p1-p6 was cotransformed with vector lacking Tsg101 (<0.3 units). The figure shows averaged values obtained for mutants in six independent trials as a percentage of the wild-type interaction ±1%.

Figure 2

Binding of Pr55^Gag and Tsg101 in vitro. (A) Autoradiography to detect immune-captured radiolabeled Tsg101. Lane 1, Tsg101 synthesized in RRL. Lanes 2–9, determination of binding of radioactive Tsg101 to unlabeled Pr55^Gag bound to anti-T7 (lane 3), anti-CA (lanes 7 and 8), or anti-p6 (lane 9) IgG immobilized on protein A beads. Antibodies are as defined in Materials and Methods. The amount of RRL used in lanes 2–9 was 5-fold greater than the amount used in lane 1. (B) Confirmation of the presence of Pr55^Gag on the beads by Western analysis. A monoclonal antibody against an antigenic site in the CA domain was used to visualize the Gag proteins immunoprecipitated with the antibodies used in A. Molecular mass markers (kDa) are on the left.

Figure 3

Coimmunoprecipitation of Pr55^Gag and Tsg101 from cytoplasmic extracts. (A) Total cytoplasmic extract. Extracts were prepared from cells transfected with rev (lane 1), rev, gag, and pol (lane 2), or rev, gagΔp6, and pol (lane 3). (B) Immunoprecipitation with anti-Tsg101 monoclonal antibody by using extracts of cells transfected with rev (lane 1); rev, wild-type gag and pol (lane 2); or rev, gag^Δp6, and pol (lane 3) as detected by the anti-NC polyclonal antibody. The blot was reprobed with an anti-Tsg101 polyclonal antibody to confirm the presence of Tsg101 in the immunoprecipitates (lanes 4–6). (C) Immunoprecipitation with anti-CA polyclonal antibody by using extracts from cells transfected with rev (lane 1) or rev, gag, and pol (lane 2) as detected by anti-Tsg101 monoclonal antibody. The blot was reprobed with anti-CA monoclonal antibody to confirm the presence of Gag in the immunoprecipitate (lanes 3 and 4). Molecular mass markers (kDa) are on the left.

Figure 4

Identification of the region in Tsg101 required for Pr55^Gag binding. Truncation (A) and substitution (B and C) mutants of Tsg101 were tested in the two-hybrid assay for interaction with the p1-p6 fusion protein. The figure shows averaged values as a percentage of the wild-type interaction with ±2–6% error. Notations are as in legend to Fig. 1.