FIG. 4.

Intracellular interaction of Gag with SIV Env proteins having an ER retention signal. HeLa T4 cells coexpressing the Gag protein with wt Env, Env164RS, Env37RS, or Env18RS were labeled with [³⁵S]methionine/cysteine for 3 h and permeabilized with saponin for 2 min. The cells were incubated with anti-Gag antibody (α p28) for 60 min, lysed in CHAPS buffer, and processed as described in Materials and Methods. The protein complexes were resolved by SDS-PAGE. (A) Lane 1 shows coimmunoprecipitation of wt Env (ENV) and Gag (GAG) with Gag antibody; lanes 2 to 4 show the coimmunoprecipitation of Gag with Env164RS (164RS), Env37RS (37RS), or Env18RS (18RS) with Gag antibodies. For a control, lysates from cells separately expressing wt Env and Gag proteins were mixed and one portion was immunoprecipitated with Gag antibodies (lane 5) and the other portion was immunoprecipitated with SIV antiserum (lane 6). Lane 7 is a control showing the proteins from vaccinia virus-infected but untransfected cells immunoprecipitated with Gag antiserum. Lanes 8 to 11 show Env-Gag complexes detected by a rabbit antibody to the Gag protein. (B) Cells expressing EnvΔ164aa (Δ164aa) alone (lanes 1 and 3) or coexpressed with Gag (lanes 2 to 4) and cells expressing a fusion protein (E-TC25) having the extracellular domain of HIV-1 gp160 and the anchor and 25 amino acids in the cytoplasmic domains of CD4 alone (lanes 5 and 7) or in combination with Gag (lanes 6 and 8). Rhesus SIV antiserum was used in lanes 1 and 2, and mouse p28 antibody was used in lanes 3 and 4. Lanes 5 and 6 used a combination of SIV and HIV-1 antisera, and lanes 7 and 8 used anti-Gag (mouse p28) antiserum. For another set of controls, we transfected HeLa T4 cells with Env-expressing plasmids, treated with anti-p28 antibody, and lysed in CHAPS buffer. To one portion of the lysate, SIV antiserum was added to detect the Env protein (lane 9) and the other portion (lane 10) was processed as described in above for panel A. Lane 11 shows that the Gag protein reacted with anti-p28 antibody after saponin permeabilization.