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

A pulse-chase experiment was performed to examine the kinetics of secretion of the VP7 amylase chimera and to compare it to wild-type amylase and the secreted mutant of VP7. When transfected cells were pulsed for 10 min with L[35S]methionine and followed by a 1-, 2-, 3-, 6-, or 8-h chase, the secretion kinetics of A47-61 m/dhl/Am, A47-61/dhl and wt amylase were compared (Figs. 6 and 7). As expected, amylase was secreted the most rapidly and efficiently. Secretion of the chimera was less rapid and complete (Fig. 7) . The half-time for secretion of amylase was about 1.5 h, whereas A47-61 m/dhl/Am and A47-61/dhl were each about 2.25 h, consistent with the presence of one of the two necessary regions for ER retention thus slowing down exit from the ER. However, even after 6 h of chase, none of the complete VP7 amino terminal/amylase chimera (Al-14m/Am) was secreted (Fig. 6) , underscoring that the amino terminal region contains the VP7 retention domain.