Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

To confirm and extend the above results, we also examined VP7 expression from wild-type and mutant genes by immunoprecipitating radiolabeled proteins from cell lysates (see Materials and Methods). Cells were transfected with plasmids containing the wild-type gene (pJC9) or deletions 1-14, 2-8, 42-61, 43-6 l, 47-6 l, or 51-61 (Fig. 2) . VP7 proteins were immunoprecipitated and displayed by gel electrophoresis before or after digestion with endo-H and compared on the same gel alongside L-[35S]methionine-labeled SAI l-infected MA104 cell lysate. No significant products were seen when either no DNA (Fig. 6 ) or the vector pJC119 was used as a control (see below, Fig. 8) . However, the wild-type and mutant genes all expressed a protein(s) precipitable with anti-VP7 antiserum (Fig. 6, -lanes) . In the case of the intact VP7 gene (pJC9) and the deletion 1-14, two products were seen in the absence of endo-H, but the lower band was relatively minor, not always reproducible, and its origin is uncertain. For mutants 42-61 and 51-6 l, two products were expressed and it is very likely that the lower molecular weight species was the nonglycosylated version of each protein. In the case of mutant 51-61, it is possible that the lower molecular weight band arises from use of the third in-frame AUG as a start The immunofluorescent localization of VP7 (left column) compared with that of R-WGA (right column) in transfected COS 7 cells. Fluorescein coupled to goat anti-rabbit IgG was used secondary to primary rabbit polyclonal antiserum to immunolocalize VP7. All pairs were photographed in the same plane of focus. Cells transfected with pJC9 (a and b); mutants 1-14 (c and d); 2-8 (e and/); 42-61 (g and h); 43-61 (i and j); 47-61 (k and/); or 51-61 (m and n). VP7 is localized to an arborizing structure probably corresponding to ER, as well as to a perinuclear reticular structure, possibly transitional ER elements, which are distinct from but close to that of the perinuclear punctate stain for WGA and the Golgi apparatus as displayed in a-f, and m and n. In some cases, a negative image of the staining region of VP7 is seen for WGA (c and d). By contrast, the three deletions shown in g-I not only exhibit the ER reticular localization for VP7, but also a prominent perinuclear staining region that is precisely coincident with, or a subset of, the staining pattern for WGA. × 126. codon. This possibility is made less likely because for mutant 42-6l a similar unglycosylated band of the same size is secreted, implying the use of a functional signal which would not be translated if the third AUG was used as an initiation codon. The wild-type and mutant genes each expressed a product which was sensitive to endo-H (Fig. 6, + lanes) , indicating that they were glycosylated with N-linked, highmannose oligosaccharides. There was no significant intracellular pool of VP7 products that were resistant to endo-H (Fig. 6 ). Cells were transfected and then incubated in the presence or absence of tunicamycin and the resulting unglycosylated products were slightly smaller in size than those treated with endo-H (data not shown), again indicating the N-linked nature of the oligosaccharide. Taken together, these results imply that each mutant codes for proteins which retain a functional signal for translocation into the ER. For the 1-14 deletion, where only the second ATG and hydrophobic domain are retained, the latter probably constitutes the signal peptide. For the wild-type and ot