Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum

The message transcribed for each construct was translated using a rabbit reticulocyte lysate prepared as described by Pelham and Jackson (27) . Rotavirus total mRNA was synthesized in an in vitro reaction taking advantage of endogenous transcription in viral cores (23) . Dog pancreas microsomal membranes were isolated according to the procedure of Shields and Blobel (35) and in vitro protein synthesis was carried out as described (35) . Each 50-0`1 worth of reaction mixture contained 1-5 0`1 of transcribed message, or 0.04 A260 U of mRNA for rotavirus total message, 0.25 A280 U of microsomal membranes, 40 0`Ci of L[35S]methionine and 10 0`g of calf liver tRNA (Boehringer Mannheim Diagnostics, Inc., Indianapolis, IN). The samples were incubated for 90 min at 30°C in the presence or absence of membranes and at the end of the incubation the samples were immunoprecipitated with polyclonal antiserum to VP7, described previously (31), or by rabbit anti-human salivary ct-amylase (Sigma Chemical Co., St. Louis, MO). Samples containing membranes were layered in an airfuge tube above a 50-0.1 cushion of 0.5 M sucrose, 20 mM Tris-HC1, pH 7.4, 500 mM KCI, 2 mM CaCI2 and 5 mM MgCI2. and spun using an air pressure of 24 psi for 5 rain at 4°C. The supernatant was carefully removed and the membrane pellet was washed in 120 0`1 20 mM Tris-HC1, pH 7.4, 100 mM KCI, 2 mM CaCI2, 5 mM MgCI2, and respun in the airfuge. The supernatant was carefuly removed and the membrane pellet resuspended in 150 0`1 of lysis buffer containing protease inhibitors, before immunoprecipitation as described previously (31) . For samples that were translated in the absence of membranes, the reaction mixture was diluted with an equal volume of 2× lysis buffer, the volume adjusted to 150 0`1 with 1x lysis buffer and the sample immunoprecipitated as above. Antigen antibody complexes were precipitated with protein A-Sepharose CIAB (Pharmacia Fine Chemicals) and eluted from the beads by boiling in 1% SDS 0.05 M Tris, pH 6.7, as described previously (31) . Samples were adjusted to pH 5.0 by the addition of 0.2 M citrate phosphate buffer, pH 5.0, and half of the samples which had undergone translation in the presence of membranes had 0.041 U of endo-I~-N-acetylglucosaminidase H (endo-H) (38) added. All samples were incubated at 37°C for 1 h and then processed for analysis by SDS-PAGE on 10 or 11% gels (18) .