Genomic and biological analysis of Grapevine leafroll-associated virus 7 reveals a possible new genus within the family Closteroviridae

Abstract

Deep sequencing analysis of an asymptomatic grapevine revealed a virome containing five RNA viruses and a viroid. Of these, Grapevine leafroll-associated virus 7 (GLRaV-7), an unassigned closterovirus, was by far the most prominently represented sequence in the analysis. Graft-inoculation of the infection to another grape variety confirmed the lack of the leafroll disease symptoms, even though GLRaV-7 could be detected in the inoculated indicator plants. A 16,496 nucleotide-long genomic sequence of this virus was determined from the deep sequencing data. Its genome architecture and the sequences encoding its nine predicted proteins were compared with those of other closteroviruses. The comparison revealed that two other viruses, Little cherry virus-1 and Cordyline virus-1 formed a well supported phylogenetic cluster with GLRaV-7.

Fig.1

Genome organizations of GLRAV-7 in comparison to CoV-1, LIYV, GLRaV-2 and GLRaV-3 approximately to scale. The different segments represent open reading frames; their vertical heights represent the different frame registers. Shared colors represent conserved sequences. L-Pro, leader protease; MET, methyltransferase domain; HEL, RNA helicase domain; RdRp, RNA-dependent RNA polymerase; HSP70h, heat shock protein 70 homolog; CP, coat protein; CPm, minor coat protein.

Fig. 2

(a) Phylogenetic analysis of the heat shock protein 70 homolog sequences of GLRaV-7 and other species in Closteroviridae family. Bootstrap values are shown as percentages; percentages less than 70% are not show. Hsp70 of Anabaena variabilis (ABA20196) was used as an outgroup. The Velarivirus lineage is shaded blue, and all other leafroll viruses are shaded pink. (b) Phylogenetic analysis of the coat protein sequences of GLRaV-7 and other species in Closteroviridae family. Bootstrap values are shown as percentages; percentages less than 70% are not shown. The CP sequence of Bean calico mosaic virus (BCMoV) NC 003504 was used as an outgroup. The Velarivirus lineage is shaded blue, and all other leafroll viruses are shaded pink. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2

(a) Phylogenetic analysis of the heat shock protein 70 homolog sequences of GLRaV-7 and other species in Closteroviridae family. Bootstrap values are shown as percentages; percentages less than 70% are not show. Hsp70 of Anabaena variabilis (ABA20196) was used as an outgroup. The Velarivirus lineage is shaded blue, and all other leafroll viruses are shaded pink. (b) Phylogenetic analysis of the coat protein sequences of GLRaV-7 and other species in Closteroviridae family. Bootstrap values are shown as percentages; percentages less than 70% are not shown. The CP sequence of Bean calico mosaic virus (BCMoV) NC 003504 was used as an outgroup. The Velarivirus lineage is shaded blue, and all other leafroll viruses are shaded pink. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Alignment of the leader regions of GLRaV-7 with those of criniviruses (positions in the polyprotein are indicated in parentheses). Asterisks denote amino acid residues identical in the aligned sequences; colons and dots show alignment columns occupied by similar residues depending on the degree of the similarity.

Fig. 4

Alignment of the penultimate gene products of the three Velariviruses. Conserved blocks of predicted secondary structure are shown as spans of α helix (h) and β strand (e).