FIG. 2.

Nef interacts with MHC-I in vivo. (A) MHC-I coimmunoprecipitates a 27-kDa protein in Nef-expressing cells. 373mg astrocytic cells were transduced with adeno-Nef or control-adeno at an estimated MOI of 300:1 (see Materials and Methods). The next day they were metabolically labeled, treated with the cross-linker DSP, and immunoprecipitated with either an antibody against β₂M or normal rabbit immunoglobulin fraction (control). Results shown are representative of three separate experiments. (B) Quantitation of binding. The relative amount of 27-kDa protein coprecipitating with a β₂M antibody versus a control antibody was determined using a phosphorimager. Results shown were normalized for the amount of total Nef immunoprecipitated with an anti-Nef antibody. Means ± standard deviations for three separate experiments are shown. (C, D, and E) MHC-I coimmunoprecipitates Nef. (C) β₂M containing complexes were immunoprecipitated as described in the legend to panel A. After the first immunoprecipitation, the agarose beads were boiled in 1% SDS to destroy the antibody and protein complexes from the first immunoprecipitation. The supernatant was then reimmunoprecipitated with either an antibody against Nef or normal serum (control). (D) A double immunoprecipitation was performed as in the experiment for which results are shown in panel C, except that complexes were first immunoprecipitated with an antibody against β₂M or with a control antibody (normal rabbit immunoglobulin fraction), followed by immunoprecipitation with an antibody against Nef. (E) Immunoprecipitations were performed as described in the legend to panel A, except that cells were not metabolically labeled and either an anti-HLA-A2 antibody (BB7.2) cross-linked to protein A/G agarose or protein A/G agarose alone was used. Immunoprecipitations were run on SDS-PAGE and Western blotted for Nef. (F) Nef coprecipitates with MHC-I in HIV-1-infected T cells. T1 T cells were transduced with an HIV molecular clone (NL-PI env frameshift mutant) pseudotyped with vesicular stomatitis virus protein G. Forty-eight hours later, the cells were metabolically labeled, treated with DSP, and immunoprecipitated as for panel A.