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 membrane insertion and anchoring, transport to the cell surface, and secretion, G proteins that contained point mutations or deletions were synthesized by cell-free transcription-translation and in cells by expression from recombinant vaccinia virus vectors. A mutant protein lacking the entire major hydrophobic region (amino acids 38–63) was not glycosylated, not expressed on the cell surface, and not secreted, because it was not inserted into membranes. In contrast, deletion of the minor hydrophobic region (amino acids 23–31) had no detectable effect on membrane insertion or anchoring. These data provided direct evidence that amino acids 38–63 were necessary for membrane insertion and contained the signal/anchor domain of RS virus G protein. Mutant proteins that lacked either the N-terminal or the C-terminal half of this 26 residue hydrophobic region were inserted into membranes and processed to maturity, showing that either half of this region was sufficient for membrane insertion. However, these two mutant proteins were secreted more abundantly than wild-type G protein. We propose that their truncated hydrophobic domains interacted with membranes in a way that mimicked the N-terminal signal sequence of naturally secreted proteins, allowing proteolytic cleavage of the mutant proteins.


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