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

To further assess signal cleavage on the wt amylase or VP7-amylase chimera, products were immunoprecipitated from transfected cells or the media with anti-amylase antibody or with anti-VP7. Wild-type amylase was only immunoprecipitable with anti-amylase antibody and not with anti-VP7 as expected (data not shown). However, for the chimeric products VP763/Am and Al-1463/Am, not only were the intracellular or secreted products immunoprecipitable with anti-amylase but they were also immunoprecipitable, although to a lesser degree, with anti-VP7 (data not shown). For the chimera A51-6163/dhl/Am, intracellular and secreted products were only observed when anti-amylase antibody SAil-infected MA104 ceil lysates (lanes 9, 10, 21, and 22) display marker glycosylated VP7 (white arrowhead) and endo-H treated VP7 (black arrowhead). The arrow marks the mobility of wild-type amylase and VP7amylase chimera alongside the virus protein NCVP2 (54 kD). was used. No product of that size was obtained when anti-VP7 antibody was used, since it is likely that only two amino acids of the VP7 sequence remain following signal cleavage and are insufficient for recognition by anti-VP7 antibody. Taken together with the in vitro data, these results imply that the amino terminal hydrophobic domain of each of the three VP7-amylase chimera, VP763/Am, Al-1463/Am or A51-6163/ dhl/Am, is probably removed by signal peptidase. Though the protein sequence of VP7 from amino acid 51-61 is necessary for ER retention, it is apparent that it is not sufficient since its presence on amylase does not prevent secretion of this molecule.