Description

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 GN (N-terminal) and GC (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 GN and GC. 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. GC showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. GN was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than GC, with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of GN. None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to GC or GN. Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of GC to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with GN. These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on GC and possibly high mannose-type on GN. Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that GC is likely to be more heavily N-glycosylated than GN. No evidence was observed for the presence of O-linked oligosaccharides on GN or GC.