Fig 1.
NSvc2 associated with RSV virions in SBPH midgut.
(A) NSvc2 (red) and RSV virions (green) were co-localized in the midgut lumen and on the surface of intestinal microvillus (blue) at 4 h post feeding on the RSV-infected rice plants. The presence of RSV virions, NSvc2, and actin were detected with antibodies specific for RSV NP, NSvc2-N, or actin. The boxed region in each image was enlarged and shown in three different panels on the right side. The detection signal for NSvc2 is in red, the detection single for virions is in green, and the merged detection signals is in yellow. (B) Co-localization of NSvc2 and RSV virions in vesicle-like structures in epithelial cells at 8 h post feeding. (C) Co-localization of NSvc2 and RSV virions in the epithelial cells at 16 h post feeding. (D) NSvc2 and RSV virions complexes were detected in the epithelial cells of a midgut at 24 h post feeding. (E–H) Analyses of overlapped fluorescence spectra from NSvc2 (red) and RSV virions (green) at different stages. Fluorescence signals were from the white dashed line. The overlap coefficient (OC) values were determined using the LAS X software. ML, midgut lumen; EC, epithelial cells; Bar, 25 μm.
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.
Fig 3.
Electron Microscopy and immunogold labelling of purified RSV virions.
(A) An electron microscopic image of RSV virions from the resuspended pellet sample. (B) Immunogold labeling of purified RSV virions using an RSV NP specific antibody followed by a goat anti-mouse IgG conjugated with 15 nm gold particles. (C) A negative control of immunogold labeling assay using only the goat anti-mouse IgG conjugated with 15 nm gold particles. (D-F) Immunogold labeling of purified RSV virions with an RSV NSvc2-N specific antibody followed by a goat anti-rabbit IgG conjugated with 8 nm gold particles. Arrows indicate filamentous RSV virions. Bar, 50 nm.
Fig 4.
N-glycosylation of NSvc2-N is essential for the recognition of midgut surface receptors.
(A) Binding of purified NSvc2-N:S to microvillus surface in SBPH midgut. Arrows indicate the accumulation of NSvc2-N:S (green, left panel) in the midgut lumen. Purified TSWV Gn:S was used as a negative control in this study (right panel). ML, midgut lumen; EC, epithelial cells; Bar, 25 μm. (B) Enzymatic de-glycosylation of NSvc2-N:S. Purified NSvc2-N:S was incubated with PNGaseF or O-Glycosidase + Neuraminidase (O-Gly + Neur) to determine the types of glycans. PNGaseF was used to remove the N-linked glycans, and O-Gly + Neur were used to remove the O-linked glycans. (C) The NSvc2-N:SN114A/N199A/N232A mutant was treated with PNGaseF. (D) The NSvc2-N:SS38A/S128A/S183A mutant was treated with O-Gly + Neur. (E) The NSvc2-N:SN114A/N199A/N232A mutant failed to bind SBPH midgut (left) but the O-glycosylated NSvc2-N:SS38A/S128A/S183A mutant did (right). ML, midgut lumen; EC, epithelial cells; Bar, 25 μm. (F) Percentages of RSV acquisition and transmission by SBPHs pre-fed with NSvc2-N:S, NSvc2-N:SN114A/N199A/N232A mutant, NSvc2-N:SS38A/S128A/S183A mutant, TSWV Gn:S, or sucrose only. The experiment was performed three times with 100 SBPHs per treatment. *, p < 0.05 and ** p < 0.01 by the student t-test. (G) Quantitative RT-PCR analysis of RSV acquisition by SBPHs at 6, 9- or 12-days post RSV feeding. The SBPHs were pre-fed with NSvc2-N:S, NSvc2-N:SN114A/N199A/N232A mutant, NSvc2-N:SS38A/S128A/S183A mutant, TSWV Gn:S, or sucrose only for 24 h, then were allowed to feed on the RSV-infected rice plants for 48 h. The experiment was performed three times. *, p < 0.05 by the student t-test.
Fig 5.
Localizations of RSV virions, NSvc2-N and NSvc2-C in SBPH midgut epithelial cells.
(A and B) RSV virions were detected in the early endosomes inside midgut epithelial cells by labeling using the Rab5 antibody (A) or in the late endosomes by labeling using the Rab7 antibody (B). Localization of RSV virions in the Rab5 antibody-labeled early endosomes or in the Rab7 antibody-labeled late endosomes are indicated with arrows. Actin antibody was used to visualize actin filaments inside the midgut epithelial cells. Bar, 25 μm. (C and D) NSvc2-N and NSvc2-C were detected inside the early endosomes labeled with the EEA1 antibody. Co-localizations of NSvc2-N or NSvc2-C with EEA1 or actin in different endosomes are indicated with arrows. Bar, 25 μm. (E) RSV virions:NSvc2-N complexes were released from the actin labelled late endosomes into the cytosol of epithelial cells. The white dashed cycles indicate the regions where the RSV virions:NSvc2-N complexes were detected in the cytosol. Bar, 25 μm. (F) RSV virions was released into the cytosol of epithelial cells but not NSvc2-C. The white dashed boxes indicate a region where RSV virions were released from the endosomes while NSvc2-C was still associated with endosomes. Bar, 25 μm. (G) RSV virions were released from the Rab7 antibody-labeled late endosomes into the cytosol of epithelial cells. The released RSV virions are indicated with arrows. ML, midgut lumen; EC, epithelial cells; Bar, 25μm.
Fig 6.
NSvc2-C hydrophobic fusion-loop motifs are required for cell-cell membrane fusion.
(A) Schematic diagrams showing recombinant NSvc2-N and NSvc2-C. Baculovirus gp64 signal peptide was used to replace the original signal peptide at the N-terminus of NSvc2-N or NSvc2-C. (B) Immunofluorescence labeling of the recombinant NSvc2-N or NSvc2-C in Sf9 cells. The Sf9 cells were infected with baculoviruses expressing the recombinant NSvc2-N or NSvc2-C (MOI = 5). The infected cells were probed with the NSvc2-N or NSvc2-C specific antibody at 48 h post infection followed by a TRITC fluorescence-labeled secondary antibody. Bar, 10 μm. (C–F) Fusogenic activity assays using Sf9 cells expressing the recombinant NSvc2-N, NSvc2-C or both (NSvc2-N + NSvc2-C). The Sf9 cells were infected with individual recombinant baculoviruses (MOI = 5). At 48 h post transfection, the growth medium was replaced with a PBS, pH 5.0, for 2 min and then changed back to the normal growth medium for 4 h. Formation of syncytium was observed under a microscope. (G) The number of cells per syncytium was counted under the inverted microscope. The experiment was repeated three times. **, p < 0.01 by the student t-test. (H) Sequence alignment using the two fusion loop sequences (FL1 and FL2) from the NSvc2-C of five different tenuiviruses [RSV (NC_003754.1), maize stripe virus (MSV; U53224.1), rice hoja blanca virus (RHBV; L54073.1), Iranian wheat stripe virus (IWSV; AY312434.1) and rice grassy stunt virus (RGSV; KF438676.1)]. Red residues are highly conserved and yellow residues are semi-conserved. Residues indicated with asterisks are hydrophobic residues and were selected for site-directed mutagenesis. The resulting mutants were tested for their abilities to induce cell-cell membrane fusion. (I) Two fusion loops (FL1 and FL2, green) were found at the top of the 3D-structure model of NSvc2-C. Three different domains are shown in three different colors, and the boxed region of the 3D-structure is enlarged and shown on the right side. Locations of the hydrophobic residues are shown. (J and K) Analyses of fusogenic activities caused by the WT or mutant NSvc2-C. Sf9 cells were infected with the recombinant baculoviruses expressing the WT or mutant NSvc2-C for membrane fusion assays (J). The number of cells per syncytium was counted and analyzed (K). The experiment was repeated three times. **, p < 0.01 by the student t-test. Fig 6C–6F and 6J are bright field light microscopic images.
Fig 7.
NSvc2 functioned as a helper factor to mediate the entrance of RSV virions into SBPH midgut cells.
(A) Crude extract of Sf9 cells expressing NSvc2, NSvc2N114A/N199A/N232A or NSvc2F460A/F489A/Y498A was incubated with purified RSV virions for 3 h at 4°C, and then used to feed SBPHs for 24 h. Insect midguts were dissected and detected for the presence of RSV virions and NSvc2-N using specific antibodies. The boxed regions inside the left column images were enlarged and shown in the second to fifth columns on the right. The white circled areas show the release of RSV virions (green) and NSvc2-N (red) from endosomes into the cytosol. The actin labeling signal is shown in blue. ML, midgut lumen; EC, epithelial cells; Bar, 25 μm. (B) Percentages of SBPHs acquired or transmitted RSV. SBPHs were fed with the mixtures containing purified RSV virions and NSvc2, purified RSV virions and NSvc2N114A/N199A/N232A mutant or purified RSV virions and NSvc2F460A/F489A/Y498A mutant prior to virus transmission. (C) Percentages of SBPHs acquired or transmitted RSV. SBPHs were first fed with NSvc2, NSvc2N114A/N199A/N232A mutant or NSvc2F460A/F489A/Y498A mutant, and then fed with purified RSV virions prior to virus transmission. **, p < 0.01 by the student t-test.
Fig 8.
A helper component model for RSV virion entrance into SBPH midgut cells.
During SBPH feeding on the RSV-infected rice plants, RSV virions, NSvc2-N and NSvc2-C are acquired into the midgut lumen (Step I). Inside the midgut lumen, NSvc2-N directs the virions:NSvc2-N:NSvc2-C complexes to microvillus surface through recognition of unidentified microvillus surface receptor(s) (Steps II and III). The complexes-attached midgut lumen membrane undergoes endocytosis and then compartmentalizes RSV virions:NSvc2-N:NSvc2-C complexes in vesicles (Steps IV and V). These vesicles further develop into early endosomes (Step VI) and then late endosomes (Step VII). The acidic condition inside the late endosomes causes the conformation change of NSvc2-C, leading to cell-cell membrane fusion (Step VII). Finally, the RSV virions:NSvc2-N complexes are released from the late endosomes into the cytosol of epithelial cells for further RSV replication and spread between midgut cells (Step VIII).