Influence of N-glycans on processing and biological activity of the nipah virus fusion protein

Abstract

Nipah virus (NiV), a new member of the Paramyxoviridae, codes for a fusion (F) protein with five potential N-glycosylation sites. Because glycans are known to be important structural components affecting the conformation and function of viral glycoproteins, we analyzed the effect of the deletion of N-linked oligosaccharides on cell surface transport, proteolytic cleavage, and the biological activity of the NiV F protein. Each of the five potential glycosylation sites was removed either individually or in combination, revealing that four sites are actually utilized (g2 and g3 in the F(2) subunit and g4 and g5 in the F(1) subunit). While the removal of g2 and/or g3 had no or little effect on cleavage, surface transport, and fusion activity, the elimination of g4 or g5 reduced the surface expression by more than 80%. Similar to a mutant lacking all N-glycans, g4 deletion mutants in which the potential glycosylation site was destroyed by introducing a glycine residue were neither cleaved nor transported to the cell surface and consequently were not able to mediate cell-to-cell fusion. This finding indicates that in the absence of g4, the amino acid sequence around position 414 is important for folding and transport.

FIG. 1.

Fusion activities of F proteins with and without the HA tag. The NiV G gene was transfected either alone (A) or in combination with the gene encoding the NiV F protein with (B) or without (C) a C-terminal HA tag. At 24 h posttransfection, cell-to-cell fusion was visualized by Giemsa staining. Magnification, ×100.

FIG. 2.

Schematic diagram of the NiV F protein and amino acid sequences of mutated N-glycosylation sites. The two F protein subunits, F₁ and F₂, are indicated. Arrowheads point to the locations of the potential N-glycosylation sites. Numbers indicate the amino acid positions. Protein sequences are shown in single-letter code; boldface letters indicate exchanged amino acid residues. CD, cytoplasmic domain; FP, fusion peptide; LD, luminal domain; SP, signal peptide; TMD, transmembrane domain.

FIG. 3.

Electrophoretic mobilities and endoglycosidase digestion analysis of the NiV F protein glycosylation mutants. (A) MDCK cells expressing either parental F (lane F) or glycosylation mutants (lanes Fg1, Fg2, Fg3, Fg4, Fg5, and Fg2,3) were radiolabeled with [³⁵S]methionine (Promix) for 10 min and then incubated in chase medium for 2 h. F proteins were immunoprecipitated from cell lysates, separated on a 15% polyacrylamide gel under reducing conditions, and subjected to autoradiography. To demonstrate the migration pattern of F protein devoid of any N-glycans, the parental F protein was treated with PNGase F (lane F_deglyc). (B) MDCK cells expressing parental F (lane F) or the glycosylation mutant Fg2, Fg3, Fg4, or Fg5 were radiolabeled as described above. Immunoprecipitated samples were divided into two aliquots and either digested with endo H (+) or kept untreated (−), followed by separation on a 15% polyacrylamide gel under reducing conditions.

FIG. 4.

Cell surface expression, cleavage, and fusion activity of parental NiV F protein and glycosylation mutants. (A) At 24 h posttransfection, MDCK cells expressing either parental F or the glycosylation mutant Fg1, Fg2, Fg3, Fg4, Fg5, or Fg2,3 were surface labeled with biotin and lysed. Following immunoprecipitation, samples were subjected to SDS-PAGE under nonreducing conditions and blotted to nitrocellulose. Surface-labeled F proteins were visualized with streptavidin-peroxidase and chemiluminescence. (B) Cells were transfected with either parental F, Fg4*, or the Fg4 gene. At 24 h posttransfection, cells were radiolabeled, and F proteins were immunoprecipitated and subjected to SDS-PAGE and autoradiography as described for Fig. 3A. (C) Surface biotinylation of cells expressing either parental F or the glycosylation mutants Fg4*, Fg4, Fg5, Fg2,3, and Fg2,3,4*,5 was performed as described above. (D) The NiV G gene was cotransfected with either parental NiV F (subpanel F) or one of the glycosylation mutant genes (Fg1, Fg2, Fg3, Fg4, Fg4*, Fg5, Fg2,3, or Fg2,3,4*,5). At 24 h posttransfection, cell-to-cell fusion was visualized by Giemsa staining. Magnification, ×100.