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

To examine the kinetics of secretion of A47-61 and A47-61/dhl, transfected cells were pulsed for 15 rain at 37°C in methioniue-free medium containing L[3SS]methionine at a concentration of 1 mCi/ml. Cells were rinsed after the pulse and then chased at 37°C for 1, 2, or 3 h in medium containing 2 mM unlabeled methionine. To compare the kinetics of secretion of deletions A47-61/dhl, pJC/Am and the chimera A47-61H d dhl/Am, transfected cells were pulsed for 10 min at 37°C in methionine-free medium containing L[35S]methionine at a concentration of 900 p.Ci/ml. Cells were rinsed after the pulse and then chased at 37°C for 1, 2, 3, or 6 h, and in the case of A47-61H~dhl/Am, also for 8 h, in medium containing an excess of unlabeled methionine as above. For analysis of Al-14m/Am, transfected cells were pulsed for 10 min and chased for 6 h, in the same manner. The chase media were collected at the given times and immunoprecipitated as described. The samples were applied to 11 or 12 % SDS-PAGE gels and the intensity of the bands corresponding to VP7, amylase, or chimera, on the autoradiograph obtained were quantitated at each time point by densitometry using a Quantimet 920 Image Analysis System (Cambridge Instruments, Inc., Buffalo, NY). In another experiment, to correlate the amount of intracellular wild-type amylase or Al-14m/Am product with amylase enzymatic activity, equivalent dishes of cells were labeled with L[35S]methionine for 24 h at 37°C 40 h after transfection, and lysates were either used in an amylase assay (see below) or immunoprecipitated and analyzed on SDS-PAGE, autoradiographed, and quantitated by densitometry.