A Stretch of 11 Amino Acids in the βB-βC Loop of the Coat Protein of Grapevine Fanleaf Virus Is Essential for Transmission by the Nematode Xiphinema index

Abstract

Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) from the genus Nepovirus, family Secoviridae, cause a severe degeneration of grapevines. GFLV and ArMV have a bipartite RNA genome and are transmitted specifically by the ectoparasitic nematodes Xiphinema index and Xiphinema diversicaudatum, respectively. The transmission specificity of both viruses maps to their respective RNA2-encoded coat protein (CP). To further delineate the GFLV CP determinants of transmission specificity, three-dimensional (3D) homology structure models of virions and CP subunits were constructed based on the crystal structure of Tobacco ringspot virus, the type member of the genus Nepovirus. The 3D models were examined to predict amino acids that are exposed at the external virion surface, highly conserved among GFLV isolates but divergent between GFLV and ArMV. Five short amino acid stretches that matched these topographical and sequence conservation criteria were selected and substituted in single and multiple combinations by their ArMV counterparts in a GFLV RNA2 cDNA clone. Among the 21 chimeric RNA2 molecules engineered, transcripts of only three of them induced systemic plant infection in the presence of GFLV RNA1. Nematode transmission assays of the three viable recombinant viruses showed that swapping a stretch of (i) 11 residues in the ␤B-␤C loop near the icosahedral 3-fold axis abolished transmission by X. index but was insufficient to restore transmission by X. diversicaudatum and (ii) 7 residues in the ␤E-␣B loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode transmission.

Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission

“…Gly 297 and R2 are located at the edge of a positively charged pocket within the B-domain, whereas most of the GFLV outer surface is negatively charged ( Figure 6A ). The walls of this pocket are formed essentially by the GH, BC and C′C″ loops encompassing Gly 297 , R2 and the previously defined region R3 [32], respectively ( Figure 6B ). The base of the pocket ( Figure 6B , purple residues) is formed by at least 11 residues deeply embedded in the capsid shell but still accessible to the solvent ( Figure 3 , stars ).…”

Insight into the three-dimensional structure of maize chlorotic mottle virus revealed by Cryo-EM single particle analysis

“…7D). The location of βB-βC loop in MCMV corresponds to the loop in GFLV that is essential for virus transmission (Schellenberger et al, 2010), while the βH-βI loop in MCMV corresponds to the transmission determinant loop in CMV (Liu et al, 2002).The characteristic of all three loops is that they are exposed on the surface of the virus and are accessible to the insect vectors. In GFLV, the single site mutation Gly297Asp located in the βB-βC loop seems to be sufficient to affect the transmission of the virus by nematodes (Schellenberger et al, 2011).…”

“…site for their insect vectors (Andret-Link and Fuchs, 2005). Studies on the transmission of Grape fan leaf virus (GFLV) suggest that the exposed loop (defined as the βB-βC loop in the GFLV capsid structure) is essential for the transmission of GFLV by nematodes (Schellenberger et al, 2010). In the case of Cucumber mosaic virus (CMV), disruption of the charged amino acid residues in the βH-βI loop was found to be related to virus transmissibility (Liu et al, 2002).…”

“…For the viruses in the second group, the subunit is composed of only one β-barrel structure that contributes to the surface domain (Oda et al, 2000;Makino et al, 2013). The exposed residues on the viral surface most likely are the potential target of their transmission vectors (Liu et al, 2002;Andret-Link and Fuchs, 2005;Schellenberger et al, 2010). Thus, it is of great significance to understand the surface features prior to initiating an investigation into MCMV transmission by the insect vector.…”

Strategies for the crystallization of viruses: Using phase diagrams and gels to produce 3D crystals of Grapevine fanleaf virus

“…After several years of mechanical inoculation in this systemic host, it produced a variant during this study. Its capsid protein gene was characterized by IC/RT/PCR and sequenced as described (Schellenberger et al, 2010) and the variant was subsequently named GFLV-TD.…”