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

To test whether the VP7-amylase chimeric product made and retained by cells transfected with the construct Al-14m/Am was indeed in a conformation which displayed amylase activity, two enzymatic assays were performed. Lysates of COS7 cells that were transfected with either the plasmid containing the gene for wild-type amylase or the chimera Al-14m/Am were assayed for the release of maltose, or dextrins, due to the enzymatic action of a-amylase and compared with a maltose standard curve. Amylase activity was proportional to the amount of lysate from cells producing either wild-type amylase or the Al-14ttt/Am chimera, whereas lysate from cells treated with only the transfection reagents (DEAE/dextran) did not display any activity (Fig. 8) . A second assay, where the amount of It-amylase activity of lysates was measured by determining the amount of soluble chromogen liberated by enzymatic hydrolysis from a Procion Yellow starch substrate also confirmed these results (data not shown). To check the relative specific activity of chimeric and wild-type amylase, transfected cells were steady state labeled for 24 h with L[35S]methionine and the amount of Al-14m/Am or wt amylase in equal cell numbers was determined by densitometry of autoradiographs after immunoprecipitated products were analyzed by SDS-PAGE. Amylase activity was determined in a separate duplicate sample of transfected cells for both wt amylase or the Al-14m/Am chimera. The specific activity, expressed as a ratio of enzyme activity to gel band intensity, was 3.9 x 10 -4 for Al-14m/Am compared with 4.2 x 10 -4 for wt amylase. The similarity in specific activities shows that amylase activity was proportional to the amount of enzyme present, whether in the chimeric or in the wild-type forms. We conclude that the retained chimeric protein was probably not in a denatured form in the ER and exhibits a con- . Amylase enzymatic activity of lysates from cells transfected with wildtype amylase (pJC/Am) or the VP7-amylase chimera (Al-14m/Am). Lysates were assayed for the release of maltose from a starch substrate due to the enzymatic action of a-amylase. This release was compared with a control amylase standard and a maltose standard curve (squares, Al-14m/Am; triangles, pJC/ Am; circles, control, transfection reagents only).