A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

Cells and Transfection COS-7 and HeLa cells were maintained in DME with 5 % FBS . COS-7 cells min and an aliquot of each cell lysate was immunoprecipitated with antibody to the ectodomain of G protein (aVSV), the cytoplasmic tail (ceCTG), or one of two conformation-specific mAbs (II and 114) . Microsomal membranes from transfected HeLa cells labeled for 10 min were incubated with (lanes 10 and 12) or without (lanes 9 and 11) trypsin, solubilized, and immunoprecipitated with polyclonal anti VSV serum . Samples were electrophoresed and the gel was fluorographed . plated in 35-mm dishes (70% confluent) were transfected with an SV-40based expression vector using DEAE-dextran as described (28) . El expression was analyzed 44 h posttransfection. For expression using the vacciniaT7 system, HeLa cells (70% confluent) were infected with the recombinant vaccinia virus vTF7-3 encoding T7 RNA polymerase (10) at a multiplicity of infection of 20. After adsorption for 30 min at 37°C, the inoculum was replaced with 0.75 nil of serum free medium containing 4 Wg of a vector (pAR2529) encoding the appropriate gene behind the T7 promoter and 10 pl of the cationic lipid "TransfectACE" (Bethesda Research Laboratories, Gaithersburg, MD ; and reference 37) . Expression was analyzed by metabolic labeling starting at 4 h postinfection .