Blackberry virus E: an unusual flexivirus
A virus, named blackberry virus E (BVE), was recently discovered in blackberries and characterized. The virus genome is 7,718 nt long, excluding the poly-A tail, contains five open reading frames (ORFs) and resembles that of flexiviruses. Phylogenetic analysis revealed relationships to allexiviruses, which are known to infect plants of the family Alliaceae. BVE lacks the 3’end-proximal ORF, which encodes a nucleotide-binding protein, a putative silencing suppressor in allexiviruses. The overall results of this study suggest that this virus is an atypical and as yet undescribed flexivirus that is closely related to allexiviruses.
KeywordsBlackberry Virus dsRNA Alphaflexiviridae RT-PCR
Rubus species (blackberry, raspberry and their hybrids) are hosts to more than 40 viruses , with several new species identified in the last few years. Most of the new viruses have been found in association with blackberry yellow vein disease (BYVD), a serious disorder observed in the southern United States . Disease symptoms are not specific to any given virus combination, and different virus combinations are found associated with identical symptoms .
All four specimens were infected with blackberry virus Y, a virus known to be asymptomatic in single infections , leading to the assumption that one or more additional viruses are involved in the observed symptomatology. Double-stranded RNAs (dsRNAs) were isolated as described  and were further purified by selective digestions with DNase, RNase and proteinase K. Uniform patterns of dsRNAs with multiple high-molecular-weight bands were observed in extracts from all specimens, strongly suggesting the presence of the same or closely related virus(es) (Fig. 1B).
This pattern was reminiscent of flexivirus infections (the term “flexivirus” applies generally to members of the families Alpha-, Beta- and Gammaflexiviridae), and therefore, “universal” degenerate primers for these viruses  were applied in order to generate initial nucleotide sequence data. RT-PCR using dsRNA as a template yielded amplicons of 366 bp from all samples (not shown). PCR products from different samples showed 94-100% nucleotide identity (97-100% aa identity), confirming infection by the same virus. BLASTx searches showed that the virus shares high aa identity with members of the family Alphaflexiviridae.
The sequenced region had 78-83% identity to the corresponding genome portion of several members of the genus Allexivirus. This close similarity led to the development of degenerate primers for members of this genus, which were used to obtain additional sequence information for this virus (a list of degenerate primers is provided in Table S1). Degenerate primers were successfully applied in RT-PCR to amplify portions of the flexivirus present in diseased blackberries. Genome sequence gaps were obtained after the development of virus-specific primers, whereas the 5’ end of the genome was obtained using a RLM-RACE Kit (Ambion) . The genome of the virus was then re-sequenced from numerous clones generated by RT-PCR, employing virus-specific primers in order to ensure at least 5x sequence coverage.
Sequence data were assembled with Lasergene software (DNA Star Inc.) and compared to available sequences present in the NCBI database using BLASTx and BLASTp . The open access resources CDD  and Phobius  were used for identification and analysis of conserved motifs in putative genome products. ClustalW  and the neighbour-joining method  were used to align BVE sequences with members of the families Alphaflexiviridae and Betaflexiviridae and determine their relationships. Phylogenetic trees were visualized with TreeView .
Sequence analysis confirmed that a new virus, provisionally named blackberry virus E (BVE), infects blackberry. The complete genome of BVE, excluding the 3’ poly-A tract, is 7,718 nt long, with an overall A+U content of 47%. The 97-nt-long 5’untranslated region (UTR) starts with the pentanucleotide motif “GAAAA”, which is conserved in genomes of numerous flexiviruses, followed by five open reading frames (ORFs) (Fig. S1).
ORF 1 encodes a 1499-aa protein with a molecular mass of 168.6 kDa (p169). BLASTp analysis of this protein identified conserved Tymovirales motifs for a methyltransferase (pfam 10641), helicase 1 superfamily (pfam 01443) and RNA-dependent RNA polymerase II superfamily (pfam 0978). The closest orthologs of the polyprotein are encoded by allexiviruses (garlic virus A, GarV-A; garlic virus C, GarV-C; garlic virus E, GarV-E; garlic virus X, GarV-X; and shalot virus X, ShVX), with 60%-62% conserved amino acids, followed by the replicase of Botrytis virus X (BVX, genus Botrexvirus) and members of the genus Potexvirus (potato virus X, plantago asiatica mosaic virus, hosta virus X, etc,), with ~35-40% amino acid conservation. However, the BVE polyprotein shares a higher level of aa identity (79%) with the partial sequences of helicase-polymerase domains of cassia mild mosaic virus (accession no. GU481094), an unassigned member of the family Alphaflexiviridae.
ORF2 codes for a putative 246-aa protein with a predicted Mr of 27.3 kDa (p27). The putative protein contains NTP-binding motifs characteristic of helicases (GxxGxGKT/ST)  that are present in the triple gene block protein 1 (TGBp1) of members of the families Alphaflexiviridae and Betaflexiviridae . The protein shares 44-46% aa identity and 58-63% similarity with allexivirus orthologs, whereas it is less closely related to foveavirus and potexvirus orthologs (30-34% identity and 47-50% similarity with foveaviruses and ~30% identity/similarity with potexviruses). Orthologs of the protein are known suppressors of RNA silencing [3, 23].
ORF3 (nt 5342-5656) codes for a 104-aa-long protein with an estimated Mr of 11.5 kDa (p12) that has two transmembrane domains at residues 12-31 and 70-89 , features that are shared with TGBp2 orthologs of allexi-, fovea-, potex-, and carlaviruses (plant viral movement proteins pfam_01307).
ORF 4 encodes a protein of 356 aa with a Mr of 40 kDa, which is the least conserved protein in the BVE genome. The serine-rich 40K protein contains conserved regions of the Allexi_40K protein superfamily (pfam05549) and is probably involved in virion assembly . Overall aa identity levels with orthologs in allexiviruses ranged from 26% (ShVX) to 22% (GarV-D, GarV-X).
The 25K protein encoded by ORF5 was identified as the coat protein and contains conserved motifs of the Flexi_CP superfamily (pfam00286) (aa 56-190). It shares 42-44% aa identity with allexivirus CPs. BVE does not encode an allexivirus ORF6 ortholog, which is present in all currently recognized members of the genus.
The genome terminates with a 109-nt 3’UTR, which precedes a poly-A tail and contains the hexanucleotide “ACUUAA” at nt 7676-7681, a motif that may represent a cis-acting element involved in RNA synthesis  and is present in the 3’UTRs of all sequenced potexviruses.
BVE-encoded proteins shared identities ranging from ~25% (ORF4) to ~60% (ORF1) with the corresponding products of members of recognized species in the genus Allexivirus (Table S2). Regardless of the gene used for comparisons, the levels of identity between BVE and any recognized allexivirus were always significantly lower than the identities among allexiviruses. Furthermore, unlike all extant allexiviruses, which have a uniform genomic organization, BVE lacks the 3’-end-proximal ORF (p15), which encodes a nucleic-acid-binding protein (NABP). BVE also differs from extant allexiviruses in its natural host range. Allexiviruses have been reported exclusively from monocots and have a very restricted natural host range consisting of members in the genus Allium, whereas BVE was found in blackberry, a dicot.
Nevertheless, BVE shares significant affinities with the allexiviruses. First and foremost, it contains an ORF encoding a serine-rich p40 protein, a “hallmark” of the extant allexiviruses. This type of genome product has not been reported in members of any other virus taxon. Additionally, the overall aa conservation in putative BVE replication-associated protein allexivirus orthologs is about 60% (Table S2). According to the original description of the family Flexiviridae , these levels of identity are characteristic of members of the same genus.
Furthermore, similar to all known allexiviruses and LVX (a potexvirus), BVE contains a putative coding region between ORFs 3 and 4 that lacks an AUG initiation codon. When compared to the genomes of related viruses (potexviruses, mandariviruses, carlaviruses, etc.), this region corresponds to the genome portion coding for TGBp3 (Fig. S1).
It is assumed that in the case of “potexvirus-like viruses”, coordinated action of TGB proteins, together with the viral CP, is required for successful cell-to-cell transport of the virus [9, 13, 21]. Furthermore, the essential role of TGBp3 in intercellular movement of several potexviruses, i.e., white clover mosaic virus and bamboo mosaic virus , has been demonstrated experimentally.
Comparative analysis of the in silico-translated product of ORFx showed the presence of a conserved “7-kDa viral coat protein domain” (pfam02495) present in TGBp3s of many potex-, carla- and foveaviruses (E-value=6.9e−09). Additional computer-assisted analysis showed the clear presence of both structural elements in functional TGB-p3s of members of several viral genera: a hydrophobic, “transmembrane” motif between aa 46 and 64, followed by a C-terminal “cytoplasmic segment” including the hallmark motif CX(5)GX(6-9)C (posterior probability for both segments higher than 80%). Similar results were obtained with translated TGBp3-like proteins of allexiviruses (not shown).
Nevertheless, taking into consideration that TGBp3 is the least conserved among the TGBs , it is intriguing that BVE and allexiviruses maintain relatively high conservation of this portion of the genome if not functionally important and evolutionary advantageous. Our results suggest that BVE and allexiviruses adopt a “non-AUG” initiation strategy for expression of TGBp3-like protein rather than ceasing its functionality. Translation initiation at codons differing from AUG in one base has been described for other ORFs in other plant viruses, i.e., strawberry mild yellow edge-associated virus  and peach chlorotic mottle virus , both of which are taxonomically related to BVE. Under a less likely scenario, BVE and allexiviruses might have developed an alternative strategy for cell-to-cell movement that does not require a functional TGBp3. Both hypotheses have yet to be tested in vivo.
Considering all of the above, it appears that BVE, which was identified and characterized in this work, is a member of a novel virus species in the family Alphaflexiviridae that is closely related to members of the genus Allexivirus. Its unique genome organization, phylogeny and dicot host make BVE an unusual member of the family that may be an atypical allexivirus or the type member of an as yet undescribed genus. Its involvement and possible etiological role in blackberry yellow vein disease, as well as the identification of natural vectors, are the focus on ongoing research.
Deep thanks to M.C. and Melvin Ellis for their kind hospitality and free access to original blackberry specimens. This work was partially supported by NIFA-SCRI grant 2009-51181-06022 and by the Special Research Initiative Program of the Mississippi Agricultural and Forestry Experiment Station (MAFES), Mississippi State University. Approved for publication as Journal Article No. J-12015 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University.
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