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

Additional VP7-amylase chimera were constructed to determine whether the VP7 sequence spanning amino acid residues 62-111, including its single glycosylation site at residue 69, could provide an ER retention function whose presence would be necessary to supplement the role of the region spanning residues 51-61. The chimera constructed comprised the 5' coding region of deletion Al-14 extending to the coding region of the 11 lth amino acid in the wild-type VP7 open reading frame, or the equivalent region of A47-61/dhl extending to the same amino acid, each attached to the coding sequence of amylase corresponding to the precise amino terminus of the mature molecule to form Al-14m/Am and A47--611tl/dhl/Am. When these chimeric genes were expressed, it was observed that the products for each were glycosylated, since the single VP7 glycosylation site was now present in the fusion protein (Fig. 5 ). The chimera with the mature wt VP7 amino terminus extending to amino acid 111 (Al-14m/Am) was retained intracellularly (Fig. 5) , probably in the ER since it was endo-H sensitive (Fig. 5) , whereas the chimera lacking the VP7 amino terminal region amino acids 51-61 but retaining amino acids 62-111 (A47-61 m/ (1) and secreted (M) products were immunoprecipitated from cells transfected with either the Al-14m/Am or A47-61m/dhl/Am constructs, labeled for 10 min with LpSS]methionine and chased in medium containing excess unlabeled methionine for 1, 2, 3, 6, or 8 h (6 h for Al-14m/Am only). The proteins of the virus infected cell lysate serve as markers.