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

In our previous studies (31) , cells transfected with the deletion mutants A42-61, A43-61, or A47-61, secreted endo-H resistant VP7 products. However, since the coding regions for both initiation codons and hydrophobic domains were present, it was not known from which codon initiation began nor which hydrophobic domain was used for translocation. Deletion mutants A42-61/dhl, A43-61/dhl, and A 4 7 -61/dhl, each lacking the first initiation codon and thus hi, were constructed in the present study to compare the products of both types of mutants. COS7 cells transfected with mutants containing either both initiation codons or those with only the second initiation codon secreted products of identical size, indicating that the first hydrophobic domain is probably not normally translated (Fig. 2, lanes 1-12, upper and lower arrows, glycosylated and unglycosylated VP7, respectively). Intracellular VP7 products also exhibited the same mobility whether or not the first A U G was present and were endo-H sensitive (data not shown). It is apparent that the mutant VP7 proteins of the 'dhl' series were each able to use their shortened h2 hydrophobic domains to target to and translocate into the ER. Secreted VP7 products which were glycosylated (upper arrow in Fig. 2) , were all endo-H resistant, consistent with their passage out of the E R through the Golgi apparatus and their modification to complex type of carbohydrate. As noted previously (31), wild-type VP7 was not secreted when cells were transfected with pJC9 (lanes 13 and 14) . Likewise, nonspecific bands in the position of VP7 were not observed in the media of control cells transfected with vector that did not contain insert, p J C l l 9 (lanes 15 and 16) . Roughly equivalent amounts of material were obtained from either the original deletion or its 'dhl' counterpart (i.e., A42-61 and A42-61/dhl, Fig. 2, lanes 1-4) . When the kinetics of secreted VP7 for deletions A47-61 and A47-61/dhl were examined in transfected cells after a 15-min pulse with L[asS]methionine followed by a 1-, 2-, or 3-h chase in medium containing 2 mM unlabeled methionine, a lag time of ~ 30 rain was seen for A47-61 and A47-61/dhl before secretion occurred in a linear fashion (inset in Fig. 2) .