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

Immunoprecipitated VP7 products translated in vitro, using the transcription vector pGEM3 were also compared in size. Translation of total viral message, in the presence of membranes, yielded a band which was similar in size to that from infected cells (Fig. 3, compare lanes 1 and 4) . In the present study, the VP7 messages did not appear to be bicistronic as previously described by others (6) . The primary translation product (Fig. 3, lane 3) was slightly larger than the membrane modified product which had carbohydrate removed with endo-H (lane 2), likely indicating signal sequence cleavage. Wild-type VP7 made from the in vitro transcription/ translation system was the same size as VP7 made from viral message in the case of the primary translation product (Fig. 3 , lane 3 vs. lane 7), the membrane modified product (Fig. 3 , lane 4 vs. lane 5), or the latter treated with endo-H (Fig. 3 , lane 2 vs. lane 6). When Al-14 products were also compared, it was observed that the glycosylated species had the same mobility as VP7 in infected cells and the in vitro translated wild-type VP7 product. The primary translation product of Al-14 (Fig. 3, lane 9 ) was slightly larger than the membrane modified product treated with endo-H (Fig. 3 , lane 8), and was identical to primary products translated from wild-type VP7 mRNA and that of viral message. This also implies signal sequence cleavage of the Al-14 product in the presence of membranes. Since A1-14 lacks the first AUG, one can conclude that VP7 produced in infected cells, VP7 translated from either total viral message, wild-type VP7 mRNA, or Al-14 mRNA, each initiate at the second AUG immediately preceding h2. A similar comparison of the size of deglycosylated products made in transfected cells or in the in vitro translation system showed that initiation and translation were occurring identically in both systems (data not shown). Within these VP7 sequences, Alaso-Glns~ occurs in a context of amino acid residues which form a site with a high probability of signal peptidase cleavage, as the predictive rules regarding signal cleavage are applied (39) . Significantly, this site is absent in the mutant product of A51-61/dhl. Its primary translation product (Fig. 3, lane 12 , upper band) is the same size as the membrane modified product treated with endo-H (Fig. 3, lane 11) . Evidently the h2 domain is not cleaved in A51-61/dhl. The size of the glycosylated molecule (Fig. 3, lane 13) is larger than VP7 found in infected cells, or that translated in vitro from wildtype VP7 mRNA, consistent with this conclusion. The primary translation product showing a faster gel mobility (Fig. 3 , lane 12, lower band) probably corresponds to a product resulting from downstream initiation of Met63 and represents a species probably not translocated into the lumen of the ER.