FIG. 7.

Identification of minimal requirements for the association of US3 with MHC class I molecules and retention of US3 in the ER. (A) Calnexin-, tapasin-, or TAP-deficient cells were infected with US3-expressing recombinant vaccinia virus for 1 h, incubated for 2 h, and then labeled with [³⁵S]methionine for 30 min. The labeled cells were lysed with digitonin, and the lysate was subjected to coimmunoprecipitation using MAb W6/32. (B) Proteins were in vitro transcribed and translated in the presence of [³⁵S]methionine using a rabbit reticulocyte lysate supplemented with canine pancreatic microsomes. Immunoprecipitation (IP) was done as described in Materials and methods. (C) The labeled cells were chased for 90 min and lysed with detergent. The lysates were then treated with anti-US3 antibody, and the immunoprecipitates were digested with endo H and analyzed by SDS-PAGE.