DEFINITION AND FUNCTIONAL-ANALYSIS OF THE SIGNAL ANCHOR DOMAIN OF THEHUMAN RESPIRATORY SYNCYTIAL VIRUS GLYCOPROTEIN-G

The attachment protein G of human respiratory syncytial (RS) virus is a type II transmembrane glycoprotein. A secreted form of the G protein is also produced. To examine the two distinct hydrophobic regions in the N-terminal 63 amino acids of G protein for their role(s) in membra ne insertion and anchoring, transport to the cell surface, and secreti on, G proteins that contained point mutations or deletions were synthe sized by cell-free transcription-translation and in cells by expressio n from recombinant vaccinia virus vectors. A mutant protein lacking th e entire major hydrophobic region (amino acids 38-63) was not glycosyl ated, not expressed on the cell surface, and not secreted, because it was not inserted into membranes. In contrast, deletion of the minor hy drophobic region (amino acids 23-31) had no detectable effect on membr ane insertion or anchoring. These data provided direct evidence that a mino acids 38-63 were necessary for membrane insertion and contained t he signal/anchor domain of RS virus G protein. Mutant proteins that la cked either the N-terminal or the C-terminal half of this 26 residue h ydrophobic region were inserted into membranes and processed to maturi ty, showing that either half of this region was sufficient for membran e insertion. However, these two mutant proteins were secreted more abu ndantly than wild-type G protein. We propose that their truncated hydr ophobic domains interacted with membranes in a way that mimicked the N -terminal signal sequence of naturally secreted proteins, allowing pro teolytic cleavage of the mutant proteins.