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

Expression: Standard molecular cloning techniques were used in this work (29) . The complete sequence of genomic segment nine of Simian 11 rotavirus was obtained previously using a partial length cDNA clone that lacked 5'terminal sequences (7) . A full-length clone was isolated using a modified cloning strategy (20) . This yielded a VP7 clone inserted in the Pst I site of pBR322 which was confirmed as full length by terminal sequence analysis (30) . The insert was excised with Pst I, digested with nuclease Bal 31 to remove G:C homopolymer tails, and the blunt-ended molecule was flanked with Xho I sites by the addition of phosphorylated Xho I linkers (P-L Bioehemicals revealed that pJC 16 nevertheless contained residual homopolymeric sequences (15G residues) at the 5'-end of the gene. These were removed by replacing the 5'-terminal region of the clone proximal to the Nco I site with a fragment that lacked the residual homopolymeric tail. An Aha llI-Nco I fragment was prepared from the SAIl VP7 clone, Xho I linkers were added to the Aha III end, and after recutting, the Xho l-Nco I fragment was cloned into the SV40 vector to generate the plasmid pJC9 (Fig. 1 ).