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

lf is not sufficient. When a larger portion of the VP7 amino terminus was fused to amylase, it was found that the chimera was retained intracellularly, presumably in the ER since the asparagine-linked oligosaccharide on the VP7 portion was in the high-mannose form. The additional VP7 sequence (62-111) also was not sufficient in itself to cause retention in the ER, since the chimera A47-61m/dhl/Am was also secreted (see Fig. 9 ). One must therefore conclude that two regions near the amino terminus of VP7 cooperate in its ER retention function. We do not know if the region encompassing amino acids 62-111 is part of a binding site or whether it acts as a 'spacer' allowing the region 51-61 to function. In the chimera used to demonstrate the cooperativity of the two regions, Al-14m/Am exhibits amylase activity at levels similar to that of the wt amylase gene expressed in these cells. The specific activity of the enzyme was similar in chimeric and wild-type products. These results indicate that at least the amylase portion of the chimera was in the correct conformation and that the reason for retention was not due to denaturation of the chimeric protein.