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1.  Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission 
PLoS Pathogens  2011;7(5):e1002034.
Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.
Author Summary
Numerous pathogenic viruses from animals and plants rely on vectors such as insects, worms or other organisms for their transmission from host to host. The reasons why certain vectors transmit some viruses but not others remain poorly understood. In plants, Grapevine fanleaf virus (GFLV), a major pathogen of grapes worldwide and its specific vector, the dagger nematode Xiphinema index, provides a well-established model illustrating this specificity. Here, we determined the high-resolution structures of two GFLV isolates that differ in their transmissibility. We show that this difference is due to a single mutation in a region exposed at the outer surface of the viral particles. This mutation does not alter the conformation of the particles but modifies the distribution of charges within a positively-charged pocket at the outer surface of virions which likely affects particle retention by X. index and, thereby also transmission efficiency. Therefore, we propose that this pocket is involved in the specific recognition of GFLV by its nematode vector. This work paves the way towards the characterization of the specific compound(s) within the nematodes that trigger vector specificity and provides novel perspectives to interfere with virus transmission.
PMCID: PMC3098200  PMID: 21625570
2.  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▿ †  
Journal of Virology  2010;84(16):7924-7933.
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 βB-βC 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 βE-αB loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode transmission.
PMCID: PMC2916547  PMID: 20519403
4.  Retention of the virus-derived sequences in the nuclear genome of grapevine as a potential pathway to virus resistance 
Biology Direct  2009;4:21.
Previous studies have revealed a wide-spread occurence of the partial and complete genomes of the reverse-transcribing pararetroviruses in the nuclear genomes of herbaceous plants. Although the absence of the virus-encoded integrases attests to the random and incidental incorporation of the viral sequences, their presence could have functional implications for the virus-host interactions.
Analyses of two nuclear genomes of grapevine revealed multiple events of horizontal gene transfer from pararetroviruses. The ~200–800 bp inserts that corresponded to partial ORFs encoding reverse transcriptase apparently derived from unknown or extinct caulimoviruses and tungroviruses, were found in 11 grapevine chromosomes. In contrast to the previous reports, no reliable cases of the inserts derived from the positive-strand RNA viruses were found. Because grapevine is known to be infected by the diverse positive-strand RNA viruses, but not pararetroviruses, we hypothesize that pararetroviral inserts have conferred host resistance to these viruses. Furthermore, we propose that such resistance involves RNA interference-related mechanisms acting via small RNA-mediated methylation of pararetroviral DNAs and/or via degradation of the viral mRNAs.
The pararetroviral sequences in plant genomes may be maintained due to the benefits of virus resistance to this class of viruses conferred by their presence. Such resistance could be particularly significant for the woody plants that must withstand years- to centuries-long virus assault. Experimental research into the RNA interference pathways involving the integrated pararetroviral inserts is required to test this hypothesis.
This article was reviewed by Arcady R. Mushegian, I. King Jordan, and Eugene V. Koonin.
PMCID: PMC2714080  PMID: 19558678

Results 1-4 (4)