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.

Biochemical, Conformational, and Immunogenic Analysis of Soluble Trimeric Forms of Henipavirus Fusion Glycoproteins

“…The ectodomain of both NiV and HeV F is predicted to contain six N-linked glycosylation sites, three on each of F 1 and F 2 (34). On the basis of mutagenesis experiments, only four sites (two on each of F 1 and F 2 ) of NiV F have been shown to be glycosylated when expressed in human 293T cells (2) or Madin-Darby canine kidney (MDCK) cells (53). The same four sites have also been observed to be N glycosylated in HeV F expressed in Vero cells (15).…”

“…To confirm that the major species of recombinant-expressed sF is trimeric, we prepared samples of both the NiV and HeV sF GCNt glycoproteins by purification using S-agarose affinity chromatography, followed by preparative size-exclusion chromatography, and subjected these purified sF glycoprotein preparations to analytical ultracentrifugation and cross-linking analyses. A nonglycosylated sF monomer is ϳ55 kDa, thus accounting for the 3 to 4 predicted N-linked glycosylation modifications (2,15,53); a trimer would be expected to be ϳ210 to 225 kDa. Sedimentation equilibrium measurements of NiV and HeV sF GCNt revealed glycoproteins with an apparent molecular mass of ϳ221 Ϯ 26 kDa for NiV and 215 Ϯ 18 kDa for HeV, both consistent with a trimeric configuration (Fig.…”

The Nipah Virus Fusion Protein Is Cleaved within the Endosomal Compartment

“…Plasmid Vectors-cDNA fragments spanning the F gene and the G gene of the NiV genome (GenBank TM accession number AF212302) were cloned into the pczCFG5 vector as described earlier (14). Generation of the endocytosis-negative mutant NiV F YA (Fig.…”

Canine Distemper Viruses Expressing a Hemagglutinin without N-Glycans Lose Virulence but Retain Immunosuppression

“…N glycosylation has been shown to be important for the correct folding, transport, and function of other paramyxovirus fusion and attachment glycoproteins, including MeV H (17), Sendai virus hemagglutinin-neuraminidase (HN) (39), simian virus 5 HN (31), and Newcastle disease virus HN (27). In most cases, one or two N-glycans at specific locations are essential for proper folding and transport to the cell surface, and at least one or two N-glycans are required for the F or H/HN protein to function correctly (30,47). Thus, the total deletion of N-glycans is generally not tolerated.…”