The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases

Abstract

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G N (N-terminal) and G C (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G N and G C. The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G C showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G N was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G C, with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G N. None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G C or G N. Treatment of the denatured virions with endoglycosidase H and peptide: N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G C to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G N. These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G C and possibly high mannose-type on G N. Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G C is likely to be more heavily N-glycosylated than G N. No evidence was observed for the presence of O-linked oligosaccharides on G N or G C.