Expression of the major glycoprotein G of human respiratory syncytial virus from recombinant vaccinia virus vectors.
The major glycoprotein, G, of human respiratory syncytial (RS) virus is a Mr 84,000-90,000 species that has about 60% of its mass contributed by carbohydrate, most of which is in the form of O-linked oligosaccharides. The G protein contains neither a hydrophobic N-terminal signal sequence nor a hydrophobic C-terminal anchor region. Instead, its amino acid sequence reveals only one region with significant hydrophobic character, which is between residues 38 and 66. In order to study the synthesis, processing, and functions of this unusual viral glycoprotein, full-length cDNA copies of the G protein mRNA were inserted into the DNA genome of vaccinia virus (VV) in a position that was adjacent to a strong VV promoter and within the VV gene for thymidine kinase (TK). The resulting TK- recombinant viruses were selected, plaque-purified, and characterized by Southern blot analysis of restriction enzyme digests of the viral DNA. Recombinant RNA transcripts that contained both G-specific and VV-specific sequences accumulated in cells infected with recombinant viruses having the G protein gene in the positive orientation. The translation product of these transcripts in infected cells was a Mr 84,000-90,000 glycoprotein that was indistinguishable from authentic RS virus G protein. It could be detected in cell lysates after metabolic labeling with [3H]glucosamine and was immunoprecipitated by anti-RS-virus antiserum. Immunofluorescence studies showed that the G protein accumulated intracellularly with the perinuclear distribution that is characteristic of newly synthesized glycoproteins. Furthermore, the protein was also clearly detectable on the surface of recombinant-infected cells, showing that it was transported to and inserted into the plasma membrane.
Ball
L A
LA
Young
K K
KK
Anderson
K
K
Collins
P L
PL
Wertz
G W
GW
D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics; D006168:Guinea Pigs; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics; D008970:Molecular Weight; D012136:Respiratory Syncytial Viruses; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000235:genetics*
C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins