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

Escherichia coli (RRI) was transformed and colonies were screened for inserts as well as their orientation relative to the SP6 or T7 promoter in pGEM3. Amylase genes in the 'SP6' orientation were cut with Xho I and Sal I, and the resulting 1,640-bp fragment cloned into the Xho I site of pJCll9. Transformed colonies were screened for insert having the correct orientation relative to the SV40 late promoter and this plasmid was designated pJC/Am. portions of VP7 and a-amylase. The amino acid sequence of the first 111 residues of VP7 and the first 30 of a-amylase, are illustrated by letter code. The single glycosylation site of wild-type VP7 at residue 69 is depicted (CHO) as are the two hydrophobic domains (h/and h2). The various restriction sites used for creating the VP7 mutants lacking the first initiation codon (dhl series) or the VP7-amylase chimera are shown above the appropriate sequence. The numbers above the amino acids in the wild-type and deletions refer to the amino acid position in the wild-type sequence. The deletions A51-61, A47-61, A43-61 and A42-61 each retain the coding sequence for amino acid 1 through 50, 46, 42, or 41, respectively, and delete the amino acid coding sequence specified. The 'dhl' counterparts of these mutants each have a noncoding sequence (thin lines) preceding the initiating methionine at position 30, and the deletions of the amino acid coding sequences are shown (dotted lines). The arrow beneath the wild-type amylase sequence shows the position of the cleavage site for signal peptidase. The chimeric VP7amylase coding sequences consist of either a wildtype or altered 5' VP7 coding sequence, extending to either amino acid 63 or 111, joined to the amylase sequence that extends from amino acid 16 to its terminus (VP%3/Am, Al-1463/Am, A51-6163/ dhl/Am, Al-14.1/Am, and A47-61m/dhl/Am).