Expression and characterization of a soluble form of tomato spotted wilt virus glycoprotein GN

Abstract

Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, G(N) and G(C), are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play important roles in the entry of TSWV into the insect midgut, the first site of infection. To directly test the hypothesis that G(N) plays a role in TSWV acquisition by thrips, we expressed and purified a soluble, recombinant form of the G(N) protein (G(N)-S). The expression of G(N)-S allowed us to examine the function of G(N) in the absence of other viral proteins. We detected specific binding to thrips midguts when purified G(N)-S was fed to thrips in an in vivo binding assay. The TSWV nucleocapsid protein and human cytomegalovirus glycoprotein B did not bind to thrips midguts, indicating that the G(N)-S-thrips midgut interaction is specific. TSWV acquisition inhibition assays revealed that thrips that were concomitantly fed purified TSWV and G(N)-S had reduced amounts of virus in their midguts compared to thrips that were fed TSWV only. Our findings that G(N)-S binds to larval thrips guts and decreases TSWV acquisition provide evidence that G(N) may serve as a viral ligand that mediates the attachment of TSWV to receptors displayed on the epithelial cells of the thrips midgut.

FIG. 1.

Schematic of the TSWV glycoprotein ORF and of soluble, truncated G_N (G_N-S). The top figure represents the precursor polyprotein, with putative signal sequences, signal peptidase cleavages sites, N- and O-linked glycosylation sites, and transmembrane domains indicated. The bottom figure is a schematic of G_N-S, from amino acids 35 to 309, expressed from a baculovirus. Note that the putative hydrophobic domains were removed and six-His tags were added. The figure is not drawn to scale.

FIG. 2.

Purification of soluble G_N (G_N-S) by nickel affinity chromatography. Culture supernatants were harvested at 72 h postinfection, purified, and dialyzed against PBS. The samples were analyzed by SDS-PAGE, one gel was stained with Coomassie brilliant blue (lanes 3 and 5), and another gel was analyzed by Western blotting (lanes 1, 2, and 4). Equal volumes were added to each well. Lane 1, six-His-tag molecular weight marker; lanes 2 and 3, cell culture medium added to column; and lanes 4 and 5, G_N-S protein eluate from the 200 mM imidazole wash.

FIG. 3.

Immunoprecipitation of soluble G_N (G_N-S) with G_N MAb. Antibodies were cross-linked to protein A gel, poured into a column, and incubated with G_N-S. The columns were washed extensively, and the protein was eluted with a low-pH buffer. Fractions were analyzed by SDS-PAGE, and Western blots were probed with a six-His MAb. (A) G_N-S incubated with G_N MAb column. Lane 1, six-His marker; lane 2, G_N-S added to the column; lanes 3 and 4, washes 1 and 5, respectively; lane 5, eluant 1; and lane 6, eluant 2. (B) G_N-S incubated with G_C MAb column. Lane 1, G_N-S added to the column; lanes 2 and 3, washes 1 and 5, respectively; lane 4, eluant 1; lane 5, eluant 2; and lane 6, six-His molecular weight marker.

FIG. 4.

Analysis of wild-type G_N glycosylation. Purified TSWV was incubated with glycosidases to remove oligosaccharides and then separated by SDS-PAGE. The proteins were transferred to a nitrocellulose membrane, and G_N was detected with a G_N MAb. G_N was incubated with enzymes to remove the oligosaccharides. Lane 1, O-linked glycans; lane 2, N-linked glycans; lane 3, mock digestion, no glycosidases; lane 4, N- and O-linked glycans; and lane 5, endoglycosidase A and N- and O-linked glycans.

FIG. 5.

Analysis of soluble G_N (G_N-S) glycosylation. Purified G_N-S was incubated with glycosidases to remove oligosaccharides and then separated by SDS-PAGE. The proteins were transferred to nitrocellulose membranes, and G_N-S was detected with a six-His MAb. (A) G_N-S incubated with enzymes to remove oligosaccharides. Lane 1, O-linked glycans; lane 2, N-linked glycans; lane 3, mock digestion, no glycosidases; and lane 4, N- and O-linked glycans. (B) Short exposure of panel A showing the differences in size of G_N-S incubated with enzymes to remove N-linked glycans (lane 2) and with no enzymes for a mock digestion (lane 3).

FIG. 6.

Analysis of G_N and G_N-S dimerization. Increasing amounts of β-ME were added to TSWV purified from infected plants (G_N) or to G_N-S. Proteins were detected by Western blotting. (A) Purified TSWV detected with G_N MAb. Lane 1, no β-ME and sample was not boiled; lane 2, no β-ME; lane 3, 0.1% β-ME; lane 4, 1.0% β-ME; lane 5, 2.5% β-ME; and lane 6, 5% β-ME. (B) Purified G_N-S protein was detected with a six-His MAb. Lane 1, no β-ME and sample was not boiled; lane 2, no β-ME; lane 3, 0.1% β-ME; lane 4, 1.0% β-ME; lane 5, 2.5% β-ME; and lane 6, six-His-tagged molecular weight marker.

FIG. 7.

In vivo binding assay. Larval thrips were fed BSA, TSWV N protein, HCMV glycoprotein gB, soluble G_N (G_N-S), or purified TSWV. After the feeding, thrips guts were cleared for 2 h in a 7% sucrose solution. Thrips were then dissected, fixed in 4% paraformaldehyde, and permeabilized. The guts were immunolabeled with a six-His MAb conjugated to Alexa fluor 488 (green), except for panels F and G, for which the samples were labeled with a G_N MAb and a fluorescein isothiocyanate-conjugated goat anti-mouse antibody. Actin was stained with Texas red phalloidin (red). Staining was visualized by confocal microscopy. (A) Thrips fed BSA; (B) thrips fed six-His-tagged nucleocapsid (N) protein; (C) thrips fed purified, six-His-tagged HCMV gB protein; (D) thrips fed G_N-S; (E) exterior of a gut from a thrips that was fed G_N-S showing that labeling was associated with midgut epithelial cell layers and not with other tissues; (F) thrips fed purified TSWV; and (G) thrips fed purified G_N-S. Bar, 50 μm.

FIG. 8.

Effect in vivo of purified, recombinant TSWV G_N (G_N-S) on thrips acquisition of TSWV in feeding experiments. Thrips were given 2-h acquisition access periods to BSA, TSWV alone, TSWV plus G_N-S, and TSWV plus gB. All treatments contained the same concentrations of virus and/or buffers. Thrips were then allowed to feed on a sucrose solution to clear their guts. Acquisition was measured by immunolabeling with a TSWV nucleocapsid polyclonal antibody. The amount of immunolabeled TSWV was quantified by measuring the average amount of fluorescence (647 nm) in an optical section of a thrips gut, using Adobe Photoshop 7.0. Each bar represents a mean of three or two replicates for experiment, A or B, respectively. Bars headed by different letters are significantly different, with P values of <0.05. (A) Thrips were fed BSA in buffer, TSWV, and a combination of TSWV and G_N-S. In a second set of experiments (B), thrips were also fed recombinant HCMV gB and TSWV, which served as another negative control. Thrips guts were imaged with a laser scanning confocal microscope.