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

Full length VP7, A47-61 or A51-61 were digested with Nhe I/Xho I and the resulting 1,000-bp fragment was cloned into Xba I/Sal I site ofpGEM3, with an orientation compatible with the T7 promoter. The A47-61/dhl and A51-61/dhl variations, (which served to remove the first initiation codon for each of these VP7 deletions), were made by digesting A47-61/pGEM3 and A51-61/pGEM3 with Sma I and Cla I, treating with Klenow enzyme to fill in the overhangs and ligating the DNA closed. Mutant Al-14 was digested with Xho I and the 1,000-bp fragment isolated and cloned into the Sal I site of pGEM3. Restriction analysis was used to select the construct with the insert compatible with the T7 promoter. For the construction of VP76flAm, Al-1463/Am or A51-6163/dhl/Am chimera in the pGEM3 transcription vectors, the respective plasmids were each restricted with Barn HI, the 1,800-bp piece was isolated and ligated to Barn HI linearized pGEM3. Miniprep DNA restriction analysis was performed to determine if the inserts were in the correct orientation for utilization of either the T7 or SP6 promoter. For transcription of wild-type amylase, VP763/Am or A51-6163/ dhl/Am, the SP6 promoter orientation was used. For AI-14JAm the T7 promoter orientation was used.