Expression and Characterization of a Soluble Form of Tomato Spotted Wilt Virus Glycoprotein G _N

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.

The glycoprotein gene of Chrysanthemum stem necrosis virus and Zucchini lethal chlorosis virus and molecular relationship with other tospoviruses

“…Some studies on TSWV membrane structure supports the existence of a Gc protruding structure outside the particle with four, but not five transmembrane domains [16,37]. Other studies, though, suggest five transmembrane domains in TSWV [58,59]. In our analysis, however, five transmembrane domains as found in CSNV and ZLCV (Fig.…”

“…The real topology of CSNV and ZLCV or even other tospoviruses, especially TSWV should be urgently unraveled. The role of glycosylations in the Gn and Gc protein is still not well elucidated [58,59], but they are thought to have a positive effect on the virus uptake in animal cells since many identified viral ligants to cellular receptors are viral glycoproteins. In our study, glycosilation sites were predicted and showed to be identical in CSNV and ZLCV, except that in the first glycosilation site of CSNV was Oglicosilated while that of ZLCV was N-glycosilated.…”

Thrips and tospoviruses come of age: Mapping determinants of insect transmission

“…Modeling the structure of a bunyavirus G C protein revealed that the GPs may be class II fusion proteins, and sequence comparisons showed TSWV G N protein shares sequence similarity with another viral attachment protein, sindbis virus E2 (23). These data are supported by findings that TSWV G N likely plays a role in virus attachment to thrips midgut epithelial cells (10), and that G C is cleaved at acidic pH, consistent with it serving as a fusion protein activated at low pH (24). Determining the sequence of mature GPs and solving their crystal structure will be critical to understanding their function and importance of the amino acid at position 459.…”

Proteomic Analysis of Frankliniella occidentalis and Differentially Expressed Proteins in Response toTomato Spotted Wilt VirusInfection

“…The experiment consisted of two treatments, virus-exposed and nonexposed L1s, and each experiment was conducted five independent times, i.e., biological replicates. Four replicates were used for differential protein analysis, and one replicate was used for Western blot analysis using the methods of Whitfield et al (80).…”