N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus

Abstract

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.

FIG. 1

Schematic diagram of the HRSV (strain Long) fusion protein. The two disulfide-linked F protein subunits, F₁ and F₂, are indicated; the arrows point to proteolytic cleavage sites. The black boxes represent the signal peptide, the fusion peptide, and the membrane anchor. Heptad repeats A and B are shown as hatched boxes. The locations of the six potential N-glycosylation sites are indicated by arrowheads. Closed arrowheads point to sites that in this study were shown to contain oligosaccharides.

FIG. 2

Electrophoretic mobilities of the F protein mutants. MVA-T7-infected BSR-T7/5 cells were transfected with recombinant pTM1 plasmids (lane a, parental F; lane b, N27Q; lane c, N70Q; lane d, N27/70Q; lane e, N116Q; lane f, N120Q; lane g, N116/120Q; lane h, N126Q; lane i, N500Q; lane j, N27/500Q; lane k, N70/500Q; lane l, N27/70/500Q; and lane m, pTM1). The cells were metabolically labeled with [³⁵S]methionine-[³⁵S]cysteine, F protein was immunoprecipitated from the cell lysates, and the immunoprecipitates were separated by Tricine–SDS–10% polyacrylamide gel electrophoresis under reducing conditions. The relative positions of standard proteins of the indicated molecular masses (in kilodaltons) are shown on the left.

FIG. 3

Detection of carbohydrates by periodate oxidation. Parental F protein (lane a) and mutants N500Q (lane b), N27/500Q (lane c), and N27/70/500Q (lane d) were expressed in BSR-T7/5 cells, immunoprecipitated with a monoclonal antibody, and separated by SDS-polyacrylamide gel electrophoresis. Cells transfected with a nonrecombinant vector plasmid (lane e) were used as a control. (Upper panel) The F proteins were transferred to a polyvinylidene difluoride membrane and treated with sodium metaperiodate. The oxidized carbohydrates were conjugated with biotin-hydrazide and detected with streptavidin-peroxidase. (Lower panel) The F proteins were transferred to a nitrocellulose membrane and detected by sequential incubation with a monoclonal antibody (Mab) directed against the HRSV F protein, a biotinylated anti-mouse immunoglobulin serum, and streptavidin-peroxidase.

FIG. 4

Detection of cell surface-biotinylated F protein. Transfected BSR-T7/5 cells were labeled with sulfo-NHS-biotin at 4°C, and F protein was immunoprecipitated from the cell lysates by using a monoclonal antibody. The immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to nitrocellulose membranes, and probed with streptavidin-peroxidase. (A) Lanes a and m, parental F; lane b, N27/70/500Q; lane c, N27/500Q; lane d, N70/500Q; lane e, 27/70Q; lane f, N500Q; lane g, N27Q; lane h, N70Q; lane i, N116Q; lane j, N120Q; lane k, N116/120Q; lane l, N126Q). (B) Lane a, N500A; lane b, S502A; lane c, N500Q; lane d, parental F. The relative positions of standard proteins of the indicated molecular masses (in kilodaltons) are shown on the left.

FIG. 5

Indirect surface immunofluorescence analysis of HRSV F protein mutants. Recombinant vaccinia virus MVA-T7-infected BSR-T7/5 cells were transfected with pTM1 plasmid encoding the parental or mutant HRSV F protein as indicated. Twenty hours after transfection, the cells were fixed with 3% paraformaldehyde. F protein was visualized using a monoclonal antibody directed against the HRSV F protein and an FITC-conjugated anti-mouse immunoglobulin serum. The cells were examined at 200× magnification with a Zeiss Axioplan 2 microscope equipped for epifluorescence (left panels) and phase-contrast (right panels) studies.