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

Rotavirus VP7 is a glycoprotein of particular interest as a potential model for the study of protein transport (21) . This protein associates with viral cores that bud into the lumen of the rough endoplasmic reticulum (RER) j (1) from cytoplasmic structures, called viroplasms. Mature virus remains within the lumen of the RER until release by cell lysis. VP7 is an integral membrane glycoprotein with a lumenal orientation (21) and is located in the endoplasmic reticulum (ER) (10, 32). The Golgi apparatus is not involved in processing the mature form of VPT, a fact confirmed by the presence on the molecule of the high-mannose form of carbohydrate (9) . VP7 therefore constitutes an example of a membrane glycoprotein that is targeted to the ER and is not subsequently directed further along the secretory pathway. The cloning and sequencing of the SA 11 VP7 gene revealed the presence of two tandem NH2-terminal hydrophobic domains in the protein, and the absence of a COOH-terminal hydrophobic domain (7) . Each of the hydrophobic domains is preceded by an AUG codon and could ostensibly serve as a signal peptide for the translocation of VP7, depending on which codon is read for initiation. However, the specific role of each of these codons and hydrophobic segments in VP7 synthesis is not clear. In studying the function of the hydrophobic domains, we constructed a series of deletions in the VP7 gene. Three mutants were obtained for which the VP7 proteins acquired complex carbohydrate, as distinct from the high-mannose type exhibited by wild-type VP7, showing that they traversed the secretory pathway and reached the Golgi apparatus. These proteins were also secreted. A priori, the transport of VP7 to more distal locations along the secretory route should require either the modification of a positive signal specifying ER location, or alternatively, the addition of a signal enabling vectorial transport to occur. The induction of VP7 secretion by creating various deletions in the protein is more easily explained by the disruption of a positive signal specifying ER location. We conclude that there must be a positive signal for retention of a protein in the ER. Since wild-type VP7 is naive to the secretory pathway, it is also implied that in the absence of overriding sorting signals, secretion is constitutive.