[en] 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

[en] Infectious cDNA clones were developed for Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus, family Closteroviridae). In vitro RNA transcripts generated from cDNA clones showed replication via the production of 3′-coterminal subgenomic (sg) mRNAs in Nicotiana benthamiana protoplasts. The detection of sgRNAs and the recovery of progeny recombinant virions from N. benthamiana leaves agroinfiltrated with full-length cDNA clones confirmed RNA replication and virion formation. The 5′ non-translated region (5′ NTR) of GLRaV-3 was exchangeable between genetic variants and complement the corresponding cognate RNA functions in trans. Mutational analysis of the 5′ NTR in minireplicon cDNA clones showed that the conserved 40 nucleotides at the 5′-terminus were indispensable for replication, compared to downstream variable portion of the 5′ NTR. Some of the functional mutations in the 5′ NTR were tolerated in full-length cDNA clones and produced sgRNAs and virions in N. benthamiana leaves, whereas other mutations affected replication and virion formation.