Tenuivirus utilizes its glycoprotein as a helper component to overcome insect midgut barriers for its circulative and propagative transmission
Fig 2
NSvc2 protein is required for the entrance of RSV virions into SBPH midgut.
(A) NSvc2 is absent in purified RSV virions. Extract from RSV-infected rice plants was loaded on the top of a 4 mL 20% glycerol cushion. The supernatant fractions (Sup 1 to 4), glycerol fractions (Gly 1 to 4) and the pellet (Pel) were collected separately after ultra-centrifugation. (B) The collected samples were analyzed in the SDS-PAGE gels followed by Coomassie blue staining or by immunoblotting using antibodies specific for RSV NP or NSvc2-N. NSvc2-N was enriched using protein A beads. Sizes of the protein bands are shown on the left. Asterisk indicates an RSV NP dimer band. (C) Statistic analysis of RSV acquisition rate and transmission rate by SBPHs after feeding on different fractions. Each bar represents three independent biological repeats from each experiment. *, p < 0.05 and **, p < 0.01 by the student t-test. (D) Immunofluorescence labeling of NSvc2 (red) and RSV virions (green) in the midguts of the SBPHs fed with the combined supernatant fractions, combined glycerol fractions, the resuspended pellet sample or the mixture of the combined supernatant fractions and the resuspended pellet sample. The boxed regions are enlarged and shown on the right side of the merged images. Overlapping fluorescence spectra analyses were done for the white dashed line indicated areas shown in the right panels. The overlap coefficient (OC) values were determined using the LAS X software. ML, midgut lumen; EC, epithelial cells; Bar, 25 μm. (E) Yeast two-hybrid assay for the interaction between RSV NP and NSvc2-N, or between RSV NP and NSvc2-C. RSV NP was fused to a GAL4 activation domain (AD-NP), and NSvc2-N or NSvc2-C was fused to a GAL4 binding domain (BD-NSvc2-N, BD-NSvc2-C). Yeast cells were co-transformed with the indicated plasmids and were assayed for protein-protein interactions on the synthetic dextrose -Trp/-Leu/-His/-Ade medium. Co-transformed the plasmids AD-T and BD-53 were used as a positive control while co-transformed AD-T and BD-Lam were used as negative controls. (F) Co-immunoprecipitation assay for the interaction between NSvc2-N and NP, or between NSvc2-C and NP. NSvc2-N or NSvc2-C was fused to a FLAG tag and RSV NP was fused to a 6x HIS tag. Individual recombinant proteins were expressed in Sf9 cells followed by the co-immunoprecipitation assays using a FLAG tag specific or a HIS tag specific antibody. IB:FLAG, immunoblot with a FLAG tag specific antibody; IB:HIS, immunoblot with a HIS tag specific antibody; IP:FLAG, immunoprecipitation with a FLAG tag specific antibody. The sizes of the protein bands are shown on the right side.