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

The data presented in this paper more accurately define the region of rotavirus VP7 responsible for its retention in the ER. In our previously published work (31) we presented evidence that deletions in portions of the amino terminus of VP7 resulted in the secretion of altered molecules, apparently via the normal secretory pathway, including passage through the Golgi apparatus (see Fig. 9 ; wt VP7, Al-14, and A47-61). A major conclusion arising from our previous work was that the secretory pathway may be the default pathway in the absence of specific overriding signals which target membrane or lumenal proteins to destinations other than the extracellular milieu. Support for this hypothesis derived from observations of Wieland et al. (41) who showed that a tripeptide, comprising an asparagine-linked glycosylation site, could be glycosylated in the ER, further processed in the Golgi apparatus and rapidly secreted. Likewise, ER lumenal proteins characterized by the sequence 'KDEIg at their carboxy terminus were also efficiently secreted when this sequence was removed (25) . A review of the subject (30) also reaches the conclusion that intracellular targeting may be the result of specific retention signals. As described above, VP7 has two amino terminal hydrophobic domains (hl and h2) in the open reading frame. It appears from the work of others (37) , and is confirmed by the present study, that hi is not translated in the bulk of VP7 in vitro or in transfected cells. The preferred site of initiation is at M3o immediately preceding the h2 domain. h2 functions as a cleavable signal sequence (37) , an observation confirmed in the present work by the decrease in size of the product processed in the presence of membranes for the wt VP7, and may also in A47-61, A47-61/dhl (Fig. 2 ) and the previously described deletion mutants A42-61 and A43-61 (data not shown). Our previous conclusion (31) that the h2 domain itself contains the ER retention information must be modified, refocussing attention on the sequence Qs~ to G6t, lying immediately to the carboxy terminus of h2, which also had been deleted in the secreted ver- sions of VP7. To test its role in retention of VP7 in the ER, this sequence and the appropriate sequences of the amino terminus of VP7 were joined to the exact mature amino terminus of mouse salivary a-amylase, normally a secreted protein. As shown in the present work, the presence of this sequence was not sufficient to prevent the secretion of amylase ( Fig. 4 ; Al-1463/Am). As expected, the chimera A51-6163/ dhl/Am was also secreted but, curiously, its signal sequence h2 domain was apparently cleaved (data not shown) whereas in A51-61/dhl (Fig. 3 ) the h2 domain appeared not to be cleaved. The VP7 h2 cleavage site As0-Qst is not present in A51-61/dhl nor in A51-6163/dhl/Am. However, at the deletion junction, R49AsoA62M63 forms a high probability signal peptidase cleavage site which may not be used in A51-61/dhl because of the proximity of the glycosylation site at N69 in VP7. The absence of any nearby glycosylation site in the chimera may allow peptidase activity for A51-6163/dhl/Am thereby generating a cleaved product. The data obtained when the expressed chimeric products were immunoprecipitated with either VP7 or amylase antiserum, add support to the conclusion that the signal of each chimera is cleaved. It was apparent from the aforementioned that the presence of the sequence Q51-G61 in VP7 is required for retention but in itse