N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus.
The human respiratory syncytial virus (Long strain) fusion protein contains six potential N-glycosylation sites: N27, N70, N116, N120, N126, and N500. Site-directed mutagenesis of these positions revealed that the mature fusion protein contains three N-linked oligosaccharides, attached to N27, N70, and N500. By introducing these mutations into the F gene in different combinations, four more mutants were generated. All mutants, including a triple mutant devoid of any N-linked oligosaccharide, were efficiently transported to the plasma membrane, as determined by flow cytometry and cell surface biotinylation. None of the glycosylation mutations interfered with proteolytic activation of the fusion protein. Despite similar levels of cell surface expression, the glycosylation mutants affected fusion activity in different ways. While the N27Q mutation did not have an effect on syncytium formation, loss of the N70-glycan caused a fusion activity increase of 40%. Elimination of both N-glycans (N27/70Q mutant) reduced the fusion activity by about 50%. A more pronounced reduction of the fusion activity of about 90% was observed with the mutants N500Q, N27/500Q, and N70/500Q. Almost no fusion activity was detected with the triple mutant N27/70/500Q. These data indicate that N-glycosylation of the F2 subunit at N27 and N70 is of minor importance for the fusion activity of the F protein. The single N-glycan of the F1 subunit attached to N500, however, is required for efficient syncytium formation.
Zimmer
G
G
Institut für Virologie, Tierärztliche Hochschule Hannover, D-30559 Hannover, Germany.
Trotz
I
I
Herrler
G
G
D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D002522:Chlorocebus aethiops; D015726:Giant Cells; D006031:Glycosylation; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology*; D016296:Mutagenesis; D011134:Polysaccharides / Q000378:metabolism*; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D011134:Polysaccharides; D014764:Viral Proteins