Arch Virol (2011) 156:1665–1669 DOI 10.1007/s00705-011-1015-y

BRIEF REPORT

Blackberry E: an unusual flexivirus

Sead Sabanadzovic • Nina Abou Ghanem-Sabanadzovic • Ioannis E. Tzanetakis

Received: 5 November 2010 / Accepted: 30 April 2011 / Published online: 9 June 2011 Ó Springer-Verlag 2011

Abstract A virus, named blackberry virus E (BVE), was disease (BYVD), a serious disorder observed in the south- recently discovered in blackberries and characterized. The ern United States [19]. Disease symptoms are not specific to virus genome is 7,718 nt long, excluding the poly-A tail, any given virus combination, and different virus combina- contains five open reading frames (ORFs) and resembles that tions are found associated with identical symptoms [19]. of flexiviruses. Phylogenetic analysis revealed relationships This study was initiated with four blackberry plants to allexiviruses, which are known to infect plants of the showing BYVD-like symptoms observed in northeastern family Alliaceae. BVE lacks the 3’end-proximal ORF, Mississippi (Fig. 1A). They were tested by ELISA using which encodes a nucleotide-binding protein, a putative antibodies specific for 12 and a ‘‘universal ’’ silencing suppressor in allexiviruses. The overall results of kit (Agdia Inc., USA). Additionally, they were tested by this study suggest that this virus is an atypical and as yet reverse transcription polymerase chain reaction (RT-PCR) undescribed flexivirus that is closely related to allexiviruses. using specific primers for viruses identified recently in BYVD-affected plants, or one still being characterized and Keywords Blackberry Á Virus Á dsRNA Á lacking serological diagnostics (authors, unpublished data). Alphaflexiviridae Á RT-PCR All four specimens were infected with blackberry virus Y, a virus known to be asymptomatic in single infections [17], leading to the assumption that one or more additional viruses Rubus species (blackberry, raspberry and their hybrids) are are involved in the observed symptomatology. Double- hosts to more than 40 viruses [12], with several new species stranded RNAs (dsRNAs) were isolated as described [20] identified in the last few years. Most of the new viruses have and were further purified by selective digestions with DNase, been found in association with blackberry yellow vein RNase and proteinase K. Uniform patterns of dsRNAs with multiple high-molecular-weight bands were observed in extracts from all specimens, strongly suggesting the pres- Sequence data were deposited in GenBank as accession number ence of the same or closely related virus(es) (Fig. 1B). JN053266. This pattern was reminiscent of flexivirus infections (the term ‘‘flexivirus’’ applies generally to members of the Electronic supplementary material The online version of this article (doi:10.1007/s00705-011-1015-y) contains supplementary families Alpha-, Beta- and Gammaflexiviridae), and material, which is available to authorized users. therefore, ‘‘universal’’ degenerate primers for these viruses [4] were applied in order to generate initial nucleotide & S. Sabanadzovic ( ) Á N. Abou Ghanem-Sabanadzovic sequence data. RT-PCR using dsRNA as a template yielded Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, amplicons of 366 bp from all samples (not shown). PCR Mississippi State, MS 39762, USA products from different samples showed 94-100% nucleo- e-mail: [email protected] tide identity (97-100% aa identity), confirming infection by the same virus. BLASTx searches showed that the virus I. E. Tzanetakis Division of Agriculture, Department of Plant Pathology, shares high aa identity with members of the family University of Arkansas, Fayetteville, AR 72701, USA Alphaflexiviridae. 123 1666 S. Sabanadzovic et al.

Fig. 1 A. Line patterns and vein yellowing/feathering symptoms observed on original BVE sources. B. Double- stranded RNAs extracted from BVE-infected blackberries (lanes 2 and 3) compared to dsRNAs of Cryphonectria 1 (lane 1) and peanut stunt virus (lane 4). Note the pattern similarity extracted from two different blackberry specimens (lanes 2 and 3)

The sequenced region had 78-83% identity to the cor- ORF 1 encodes a 1499-aa protein with a molecular mass responding genome portion of several members of the of 168.6 kDa (p169). BLASTp analysis of this protein genus . This close similarity led to the devel- identified conserved motifs for a methyl- opment of degenerate primers for members of this genus, transferase (pfam 10641), helicase 1 superfamily (pfam which were used to obtain additional sequence information 01443) and RNA-dependent RNA polymerase II super- for this virus (a list of degenerate primers is provided in family (pfam 0978). The closest orthologs of the polypro- Table S1). Degenerate primers were successfully applied tein are encoded by allexiviruses (garlic virus A, GarV-A; in RT-PCR to amplify portions of the flexivirus present in garlic virus C, GarV-C; garlic virus E, GarV-E; garlic virus diseased blackberries. Genome sequence gaps were X, GarV-X; and shalot virus X, ShVX), with 60%-62% obtained after the development of virus-specific primers, conserved amino acids, followed by the replicase of whereas the 5’ end of the genome was obtained using a Botrytis virus X (BVX, genus ) and members of RLM-RACE Kit (Ambion) [15]. The genome of the virus the genus (, plantago asiatica was then re-sequenced from numerous clones generated by , hosta virus X, etc,), with *35-40% amino RT-PCR, employing virus-specific primers in order to acid conservation. However, the BVE polyprotein shares a ensure at least 5x sequence coverage. higher level of aa identity (79%) with the partial sequences Sequence data were assembled with Lasergene soft- of helicase-polymerase domains of cassia mild mosaic ware (DNA Star Inc.) and compared to available virus (accession no. GU481094), an unassigned member of sequences present in the NCBI database using BLASTx the family Alphaflexiviridae. and BLASTp [2]. The open access resources CDD [10] ORF2 codes for a putative 246-aa protein with a predicted and Phobius [8] were used for identification and analysis Mr of 27.3 kDa (p27). The putative protein contains NTP- of conserved motifs in putative genome products. Clu- binding motifs characteristic of helicases (GxxGxGKT/ST) stalW [18] and the neighbour-joining method [16] were [5] that are present in the triple gene block protein 1 (TGBp1) used to align BVE sequences with members of the fam- of members of the families Alphaflexiviridae and Betaflexi- ilies Alphaflexiviridae and Betaflexiviridae and determine viridae [11]. The protein shares 44-46% aa identity and their relationships. Phylogenetic trees were visualized 58-63% similarity with allexivirus orthologs, whereas it is with TreeView [14]. less closely related to and potexvirus orthologs Sequence analysis confirmed that a new virus, provi- (30-34% identity and 47-50% similarity with foveaviruses sionally named blackberry virus E (BVE), infects black- and *30% identity/similarity with ). Orthologs berry. The complete genome of BVE, excluding the 3’ of the protein are known suppressors of RNA silencing poly-A tract, is 7,718 nt long, with an overall A?U content [3, 23]. of 47%. The 97-nt-long 5’untranslated region (UTR) starts ORF3 (nt 5342-5656) codes for a 104-aa-long protein with the pentanucleotide motif ‘‘GAAAA’’, which is con- with an estimated Mr of 11.5 kDa (p12) that has two served in genomes of numerous flexiviruses, followed by transmembrane domains at residues 12-31 and 70-89 [8], five open reading frames (ORFs) (Fig. S1). features that are shared with TGBp2 orthologs of allexi-,

123 Blackberry virus E characterization 1667 fovea-, potex-, and carlaviruses (plant viral movement observed when analyses were performed on other putative proteins pfam_01307). genome products, including the CP (data not shown). Instead of the ‘‘classic’’ TGBp3-encoding ORF, BVE BVE-encoded proteins shared identities ranging from has a 110-codon-long putative ORF (designated ORFx) *25% (ORF4) to *60% (ORF1) with the corresponding between nt 5454 and 5786, which lacks an AUG initiation products of members of recognized species in the genus codon. In silico translation of this ORF showed that the Allexivirus (Table S2). Regardless of the gene used for C-terminal half of the putative protein shares 30-35% comparisons, the levels of identity between BVE and any identical amino acids with the TGBp3 proteins encoded by recognized allexivirus were always significantly lower than the potexviruses mint virus X (MVX), (LVX) the identities among allexiviruses. Furthermore, unlike all and alstroemeria virus X (AlVX). Furthermore, this puta- extant allexiviruses, which have a uniform genomic orga- tive protein contains a hallmark motif, CX(5)GX(6-9)C, that nization, BVE lacks the 3’-end-proximal ORF (p15), which is present in TGBp3s of viruses belonging to different encodes a nucleic-acid-binding protein (NABP). BVE also genera in the families Alphaflexiviridae and Betaflexiviri- differs from extant allexiviruses in its natural host range. dae (Fig. 2A and B). Allexiviruses have been reported exclusively from mono-

ORF 4 encodes a protein of 356 aa with a Mr of 40 kDa, cots and have a very restricted natural host range consisting which is the least conserved protein in the BVE genome. of members in the genus Allium, whereas BVE was found The serine-rich 40K protein contains conserved regions of in blackberry, a dicot. the Allexi_40K protein superfamily (pfam05549) and is Nevertheless, BVE shares significant affinities with the probably involved in virion assembly [22]. Overall aa allexiviruses. First and foremost, it contains an ORF identity levels with orthologs in allexiviruses ranged from encoding a serine-rich p40 protein, a ‘‘hallmark’’ of the 26% (ShVX) to 22% (GarV-D, GarV-X). extant allexiviruses. This type of genome product has not The 25K protein encoded by ORF5 was identified as the been reported in members of any other virus taxon. Addi- coat protein and contains conserved motifs of the Flexi_CP tionally, the overall aa conservation in putative BVE rep- superfamily (pfam00286) (aa 56-190). It shares 42-44% aa lication-associated protein allexivirus orthologs is about identity with allexivirus CPs. BVE does not encode an 60% (Table S2). According to the original description of allexivirus ORF6 ortholog, which is present in all currently the family [1], these levels of identity are recognized members of the genus. characteristic of members of the same genus. The genome terminates with a 109-nt 3’UTR, which Furthermore, similar to all known allexiviruses and precedes a poly-A tail and contains the hexanucleotide LVX (a potexvirus), BVE contains a putative coding region ‘‘ACUUAA’’ at nt 7676-7681, a motif that may represent a between ORFs 3 and 4 that lacks an AUG initiation codon. cis-acting element involved in RNA synthesis [24] and is When compared to the genomes of related viruses (pote- present in the 3’UTRs of all sequenced potexviruses. xviruses, mandariviruses, carlaviruses, etc.), this region Phylogenetic analysis performed on the entire product of corresponds to the genome portion coding for TGBp3 ORF1 (replication-associated protein) grouped BVE with (Fig. S1). allexiviruses as the most distant member of the clade It is assumed that in the case of ‘‘potexvirus-like viru- (bootstrap value of 1000) (Fig. 3). Very similar topology was ses’’, coordinated action of TGB proteins, together with the

Fig. 2 A. Amino acid alignment of the C-terminal portions of the Alphaflexiviridae and Betaflexiviridae. Three hallmark amino acids putative product encoded by BVE ORFx and TGBp-3 of mint virus are indicated by asterisks. Amino acid residues that are conserved in X. Identical amino acids are indicated by asterisks. B. Conservation of all proteins used in the analysis are indicated on a black background, the motif Cx(5)Gx(7)C in a TGBp-3-like protein putatively expressed while those conserved in at least 50% of the sequences analyzed are by BVE ORFx and orthologs in several genera in the families shaded in grey 123 1668 S. Sabanadzovic et al.

TMV (outgroup) strawberry mild yellow edge-associated virus [7] and peach SLV chlorotic mottle virus [6], both of which are taxonomically BlScV 1000 PVM related to BVE. Under a less likely scenario, BVE and ASPV 726 Foveavirus allexiviruses might have developed an alternative strategy 943 GRSPaV for cell-to-cell movement that does not require a functional ACLSV 1000 ASGV TGBp3. Both hypotheses have yet to be tested in vivo. CLBV Considering all of the above, it appears that BVE, which Fam. GVA was identified and characterized in this work, is a member SsDRV LoLV of a novel virus species in the family Alphaflexiviridae that 953 ICRSV is closely related to members of the genus Allexivirus. Its 1000 CVX 1000 Potexvirus unique genome organization, phylogeny and dicot host 997 FoMV 999 BVX Botrexvirus make BVE an unusual member of the family that may be GarV-E an atypical allexivirus or the type member of an as yet 1000 1000 GarV-X undescribed genus. Its involvement and possible etiological 1000 999 GarV-C Allexivirus GarV-A role in blackberry yellow vein disease, as well as the

1000 Fam. 1000 ShVX identification of natural vectors, are the focus on ongoing 0.05 BVE research.

Fig. 3 Phylogenetic tree constructed with amino acid sequences of Acknowledgments Deep thanks to M.C. and Melvin Ellis for their the complete replicase gene of BVE and members of genera kind hospitality and free access to original blackberry specimens. This belonging to the families Alphaflexiviridae and Betaflexiviridae. work was partially supported by NIFA-SCRI grant 2009-51181- Bootstrap percentage values are shown at the nodes. The amino acid 06022 and by the Special Research Initiative Program of the Mis- sequence of tobacco mosaic virus replicase (TMV, NC_001367) was sissippi Agricultural and Forestry Experiment Station (MAFES), used as an outgroup. Complete names, abbreviations, and accession Mississippi State University. Approved for publication as Journal numbers of viruses used in phylogenetic analysis are listed in Table Article No. J-12015 of the Mississippi Agricultural and Forestry S3 Experiment Station, Mississippi State University. 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, References i.e., white clover mosaic virus and bamboo mosaic virus [9], has been demonstrated experimentally. 1. Adams MJ, Antoniw JF, Bar-Joseph M, Brunt AA, Candresse T, Comparative analysis of the in silico-translated product Foster GD, Martelli GP, Milne RG, Zavriev SK, Fauquet CM (2004) The new plant virus family Flexiviridae and assessment of of ORFx showed the presence of a conserved ‘‘7-kDa viral molecular criteria for species demarcation. Arch Virol 149: coat protein domain’’ (pfam02495) present in TGBp3s of 1045–1060 many potex-, carla- and foveaviruses (E-value=6.9e-09). 2. Altschul SF, Madden TL, Scha¨ffer AA, Zhang J, Zhang Z, Miller Additional computer-assisted analysis showed the clear W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res presence of both structural elements in functional TGB-p3s 25:3389–3402 of members of several viral genera: a hydrophobic, 3. Bayne EH, Rakitina DV, Morozov SY, Baulcombe DC (2005) ‘‘transmembrane’’ motif between aa 46 and 64, followed by Cell-to-cell movement of Potato potexvirus X is dependent on a C-terminal ‘‘cytoplasmic segment’’ including the hall- suppression of RNA silencing. Plant J 44:471–482 4. Dovas CI, Katis NI (2003) A spot nested RT-PCR method for the mark motif CX(5)GX(6-9)C (posterior probability for both simultaneous detection of members of the Vitivirus and Fovea- segments higher than 80%). Similar results were obtained virus genera in grapevine. J. Virol. Meth 107:99–106 with translated TGBp3-like proteins of allexiviruses (not 5. Gorbalenya AE, Blinov VM, Donchenko AP, Koonin EV (1989) shown). An NTP-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive Nevertheless, taking into consideration that TGBp3 is strand RNA viral replication. J Mol Evol 28:256–268 the least conserved among the TGBs [13], it is intriguing 6. James D, Varga A, Croft H (2007) Analysis of the complete that BVE and allexiviruses maintain relatively high con- genome of peach chlorotic mottle virus: identification of non- servation of this portion of the genome if not functionally AUG start codons, in vitro coat protein expression, and elucida- tion of serological cross-reactions. Arch Virol 152:2207–2215 important and evolutionary advantageous. Our results 7. Jelkmann W, Maiss E, Martin RR (1992) The nucleotide suggest that BVE and allexiviruses adopt a ‘‘non-AUG’’ sequence and genome organization of strawberry mild yellow initiation strategy for expression of TGBp3-like protein edge associated potexvirus. J Gen Virol 73:457–479 rather than ceasing its functionality. Translation initiation 8. Ka¨ll L, Krogh A, Sonnhammer ELL (2007) Advantages of combined transmembrane topology and signal peptide predic- at codons differing from AUG in one base has been tion—the Phobius web server. Nucleic Acids Res. 35:W429– described for other ORFs in other plant viruses, i.e., W432 123 Blackberry virus E characterization 1669

9. Lin MK, Hu CC, Lin NS, Chang BY, Hsu YH (2006) Movement 17. Susaimuthu J, Tzanetakis IE, Gergerich RC, Martin RR (2008) A of potexviruses requires species-specific interactions among the member of a new genus in the infects Rubus. Virus cognate triple gene block proteins, as revealed by a trans-com- Res 131:145–151 plementation assay based on the bamboo mosaic virus satellite 18. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: RNA-mediated expression system. J Gen Virol 87:1357–1367 improving the sensitivity of progressive multiple alignment 10. Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese- through sequence weighting, position-specific gap penalties and Scott C, Gonzales NR, Gwadz M, Hao L, He S, Hurwitz DI, weight matrix choice. Nucleic Acids Res 22:4673–4680 Jackson JD, Ke Z, Krylov D, Lanczycki CJ, Liebert CA, Liu C, 19. Tzanetakis IE, Susaimuthu J, Sabanadzovic S, Martin RR (2011) Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Thanki N, Blackberry yellow vein disease complex (BYVD). In: Martin RR, Yamashita RA, Yin JJ, Zhang D, Bryant SH (2007) CDD: a Ellis MA, Williams RN, Williamson B (eds) Compendium of conserved domain database for interactive domain family anal- raspberry and blackberry diseases and insects, 2nd edn. APS ysis. Nucleic Acids Res 35:237–240 Press, St Paul (in press) 11. Martelli GP, Adams MJ, Kreuze JF, Dolja VV (2007) Family 20. Valverde RA, Dodds JA, Heick JA (1986) Double-stranded Flexiviridae: a case study in virion and genome plasticity. Ann ribonucleic acid from plants infected with viruses having elon- Rev Phytopathol 45:73–100 gated particles and undivided genomes. Phytopathology 76: 12. Martin RR, Ellis MA, Williams RN, Williamson B (eds) Com- 459–465 pendium of raspberry and blackberry diseases and insects, 2nd 21. Verchot-Lubicz J, Ye CM, Bamunusinghe D (2007) Molecular edn. APS Press, St Paul (in press) biology of potexviruses: recent advances. J Gen Virol 13. Morozov SYu, Solovyev AG (2003) Triple gene block: modular 88:1643–1655 design of a multifunctional machine for plant virus movement. 22. Vishnichenko VK, Stel’mashchuk VY, Zavriev SK (2002) The J Gen Virol 84:1351–1366 42 K protein of Shallot virus X participates in formation of virus 14. Page RDM (1996) TreeView: an application to display phylo- particles. Mol Biol 36:1080–1084 genetic trees on personal computers. Comput App Biol Sci 23. Voinnet O, Lederer C, Baulcombe DC (2000) A viral movement 12:357–358 protein prevents spread of gene silencing signal in Nicotiana 15. Sabanadzovic S, Abou Ghanem-Sabanadzovic N (2009) Identi- benthamiana. Cell 103:157–167 fication and molecular characterization of a marafivirus in Rubus 24. White KA, Bancroft JB, Mackie GA (1992) Mutagenesis of a spp. Arch Virol 154:1729–1735 hexanucleotide sequence conserved in potexvirus RNAs. Virol- 16. Saitou N, Nei M (1987) The neighbor-joining method: a new ogy 189:817–820 method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

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