Role of N-linked glycosylation of the Hendra virus fusion protein

Abstract

The Hendra virus fusion (F) protein contains five potential sites for N-linked glycosylation in the ectodomain. Examination of F protein mutants with single asparagine-to-alanine mutations indicated that two sites in the F(2) subunit (N67 and N99) and two sites in the F(1) subunit (N414 and N464) normally undergo N-linked glycosylation. While N-linked modification at N414 is critical for protein folding and transport, F proteins lacking carbohydrates at N67, N99, or N464 remained fusogenically active. As N464 lies within heptad repeat B, these results contrast with those seen for several paramyxovirus F proteins.

FIG. 1.

Schematic of the Hendra virus fusion protein. FP, fusion peptide; HR, heptad repeat; TM, transmembrane domain; S-S, disulfide bond. The positions of the potential N-linked glycosylation sites and mutations produced are shown.

FIG. 2.

Examination the wt Hendra virus F protein and the six mutated forms for expression, mobility, and digestion by N-glycosidase F. (A) Hendra virus wt and mutant F proteins were expressed in Vero cells, starved, labeled with Tran-[³⁵S] for 30 min, and chased for 3 h. Samples were prepared as described previously (13), separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed using the STORM imaging system. (B) Effect of N-glycosidase F treatment. Hendra virus wt and mutant F proteins were expressed in Vero cells as described previously. Following immunoprecipitation, samples were digested overnight with N-glycosidase F (0.2 U) (14), separated by 15% SDS-PAGE, and analyzed via the STORM PhosphorImager system.

FIG. 3.

Membrane fusion promoted by the Hendra virus F wt and mutant proteins. (A) Syncytium formation. Hendra virus wt and mutant F proteins were coexpressed in BHK-21F cells with the Hendra virus G attachment protein. Approximately 18 h posttransfection, photographs were taken at a magnification of ×10 using the Nikon Eclipse TS100 microscope outfitted with a Nikon Coolpix 995 digital camera. (B) Luciferase fusion assay of Hendra virus wt and mutant F proteins. pCAGGS Hendra virus F wt or the indicated mutants, along with pCAGGS Hendra virus G and a plasmid containing the luciferase gene under the control of the T7 promoter, were transfected into Vero cells. BSR cells, which stably express the T7 polymerase (4), were overlaid onto the F- and G-expressing cells, and the mixed cell populations were incubated at 37°C for 3 h. Cells were lysed and analyzed for luciferase activity on a luminometer (Molecular Devices). Samples used were averages of triplicates and are representative of five separate experiments. G, Hendra virus G protein alone; F, Hendra virus F protein alone; V, the pCAGGS vector alone.

FIG. 4.

Cell surface expression and examination of possible additional modifications to the Hendra virus F protein. (A) Biotinylation assay of wt and mutant Hendra virus proteins for cell surface expression. Vero cells were transfected and metabolically labeled, and biotinylation on an identical numbers of cells was performed as described in the text. The biotinylated proteins were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed via the STORM PhosphorImager system. (B) Comparison of mobilities of Hendra virus F and SV5 F proteins transiently expressed in Vero cells after tunicamycin treatment (T) or N-glycosidase F digestion (P). For tunicamycin samples, cells were preincubated in 1 μg/ml tunicamycin, labeled for 30 min, and chased for 3 h. N-glycosidase F digestions were performed as described previously (14). Samples were separated on a 10% acrylamide gel and analyzed using the STORM PhosphorImager system.