Permutation of the active site of putative RNA-dependent RNA polymerase in a newly identified species of plant alpha-like virus

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Permutation of the active site of putative RNA-dependent RNA polymerase in a newly identified species of plant alpha-like virus
  Rapid Communication Permutation of the active site of putative RNA-dependent RNA polymerase in a newlyidenti fi ed species of plant alpha-like virus ☆ Sead Sabanadzovic a, ⁎ , Nina Abou Ghanem-Sabanadzovic a , Alexander E. Gorbalenya b a Department of Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA b Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands a b s t r a c ta r t i c l e i n f o  Article history: Received 4 May 2009Returned to author for revision 5 June 2009Accepted 4 August 2009Available online 29 September 2009 Keywords: VirusEvolutionRdRpPalm sub-domainInternal permutation Mara  fi virusTymoviridae GrapevineBlackberry To direct the genome synthesis, RNA viruses without a DNA stage in the replication cycle use RNA-dependentRNA polymerase (RdRp). All RdRps have conserved right hand-like shape that includes characteristicA → B → C sequence motifs forming the active site. Recently, the structural permutation of the RdRp activesite (C → A → B) has been described in few double-stranded RNA birnaviruses and a subset of positive-stranded RNA tetraviruses distantly related to Picorna-like viruses. Here we describe a permuted RdRp in thenewly identi fi ed plant alpha-like virus with 6.5 kb-long polyadenylated genome, dubbed Grapevine virus Q (GVQ). The multi-domain layout and sequence similarities place GVQ into the genus  Mara  fi virus  of thefamily  Tymoviridae . In contrast to other tymovirids, GVQ has 21 amino acid residues corresponding to themotif C relocated upstream of the motif A in the putative RdRp. This unique sequence characteristic wasextensively veri fi ed and identi fi ed in several GVQ isolates infecting wild and cultivated  Vitis  and  Rubus  spp.© 2009 Elsevier Inc. All rights reserved. Introduction Family  Tymoviridae  is currently composed of three genera( Tymovirus ,  Mara  fi virus  and  Maculavirus ), which accommodateviruses reported from cultivated and wild monocotyledonous anddicotyledonous plants as well as one entomovirus (Martelli et al.,2002;Dreheretal.,2005;Katsumaetal.,2005).Genomesofvirusesin the family  Tymoviridae  slightly differ in number and organization of cistrons, but all encode a large polyprotein essential for viralreplication (Dreher et al., 2005). This polyprotein contains signature amino acid motifs of viral methyltransferase (MTR), endopeptydase/protease (PRO), helicase (HEL) and RNA-dependent RNA-poly-merases (RdRp) as in other  “ alpha-like ”  phytoviruses (Goldbach etal., 1991).Diversed RdRps are the most conserved among virus-encodedproteins and share several conserved signature motifs in a particularorder (Poch et al., 1989; Koonin, 1991; Koonin and Dolja, 1993) that underlies remarkable structural conservation. Indeed, all viral RdRpsstudied to date have conserved  “ right hand-like ”  shape with threeconserved sub-domains referred to as  fi nger, palm and thumb(Hansen et al., 1997; van Dijk et al., 2004). The palm sub-domain is themostconservedpartofviralRdRpsandcomprisesfouroutofeightconserved motifs described by Koonin (1991) that correspond tomotifs A → B → C → D reported by Poch et al. (1989). Motifs A and Ccontain spatially juxtaposed and conserved Aspartic (Asp) residuesinvolvedin atwo-metal(Mg 2+ and/orMn 2+ )mechanism ofcatalysis(Arnold et al., 1999; Crotty et al., 2003; Ng et al., 2008). Motif B determines whether RNA or DNA will be synthesized by selectingNTPs and dNTPs (Hansen et al., 1997). Due to their functional importance, it would be expected that thecanonical order of motifs is universally conserved across the  “ RdRpuniverse. ”  Surprisingly, Gorbalenya et al .  (2002) reported the non-canonical organization of RdRp motifs in viral replicases of twoentomoviruses with +ssRNA genomes,  Thosea assigna  virus (TaV)and  Euprosterna elaeasa  virus (EeV), and in two dsRNA viruses( Infectious pancreatic necrosis  virus — IPNV and  Infectious bursal disease virus — IBDV) belonging to the family  Birnaviridae  (Table 1s, Supple- mentary material). In these RdRps, the motif C is located upstream of the motif A to form a non-canonical C → A → B arrangementassociated with a unique connectivity of major structural elementsunderneath of the active site (Gorbalenya et al., 2002). Subsequentlythe permutation was veri fi ed in two studies of the IBDV RdRp Virology 394 (2009) 1 – 7 ☆  Sequences reported in this paper have been deposited in the NCBI GenBankdatabase and have been assigned the accession numbers FJ977041-FJ977044. ⁎  Corresponding author.Department ofEntomologyand Plant Pathology, MississippiState University, 100 Twelve Lane, Mississippi State, MS 39762, USA. Fax: +1 662 3258837. E-mail address: (S. Sabanadzovic).0042-6822/$  –  see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2009.08.006 Contents lists available at ScienceDirect Virology  journal homepage:  structure (Pan et al., 2007, Garriga et al., 2007). Phylogenetic analysis of RdRps of TaV/EeV/birnaviruses and representative ssRNA viruses(nidoviruses and picorna-like viruses) with canonical polymerasesshowed that internally permuted RdRps form a monophyletic deeplyrooted clade. This clustering is due to extensive similarities outsidethe permuted motifs rather than the permutation  per se  (Gorbalenyaet al., 2002). The RdRps with the C → A → B order were referred to as “ internally permuted ”  or  “ non-canonical ”  and that terminology willbe also used in this text.Duringascreeningforvirusesinfectingnative Vitis and Rubus spp.,whichwascarriedoutin 2007/2008, wehave identi fi eda novelvirus,named Grapevine virus Q (GVQ), most resembling members of thegenus  Mara  fi virus  (family  Tymoviridae ) (Sabanadzovic and AbouGhanem-Sabanadzovic, 2009). Surprisingly, bioinformatics analysisof the newly determined GVQ genome sequence revealed theC → A → B order of RdRp motifs that is likely to have evolvedindependently from the previously reported permuted RdRp of TaV/EeV/birnaviruses. These data are detailed below. Results and discussion Identi  fi cation of GVQ  Using the  “ universal ”  tymovirid primer set RD that was designedon viral RdRp (Sabanadzovic et al., 2000) a single amplicon wasgenerated in two tested muscadine grapes. The amplicon size wasconsiderably larger than that of the PCR product ampli fi ed from thepositive controls used in tests — Grapevine  fl eck virus  (GFkV) (Saba-nadzovic et al., 2001) and  Poinsettia mosaic virus  (Bradel et al., 2000), whichpromptedfurtherstudies.Thesamespecimensdidnotgenerateamplicons with other primer sets used for the purposes of the survey.Sequence analyses of multiple clones showed that the ampliconsfrom both muscadines were of the uniform size of 450 bp and 97%identical, indicating that they were generated from very closelyrelated templates present in both samples. These amplicons were 63nucleotides larger than the sequences of known tymovirids used forprimers design (387 bp). Consistently with the primer design, Blast-mediatedcomparisonoftheampliconsequences(thatbelongtoGVQ)with the GenBank/EMBL databases showed most signi fi cant similar-ities with mara fi viruses, tymoviruses and maculaviruses of the family Tymoviridae  (E values ranging from 2e − 24 to 2e − 41 ). Accordingly,multiple sequence alignment of the analyzed region involving typicaltymoviridsidenti fi edthe21aminoacid(aa)insertionupstreamoftheRdRp motif A in the newly determined sequences. We noted that thecharacteristic 21 aa insertion in the GVQ RdRp had a high similaritywith the RdRp motif C universally located downstream in alltymovirus sequences. Combined with the lack of a counterpart tomotif C in the expected position of the GVQ genome (see below), thisobservation indicated that GVQ has the permuted C → A → B order of RdRp sequence motifs (Fig. 1).GiventhatthepermutationaffectstheRdRpactivesiteandhasnotbeen described for either plant or alpha-like viruses, we extensivelyveri fi ed the GVQ RdRp sequence. We con fi rm that the permutationwas observed independent of the type of template used (dsRNA ortotal RNA), denaturation conditions (heat, DMSO, methyl mercurichydroxide), reverse transcriptases or cloning strategies (RT-PCR based or classic dscDNA synthesis employing primer walkingtechnique). One  Vitis  and one blackberry source of GVQ (see below),co-infected with typical mara fi viruses and/or maculaviruses encod-ing a canonical RdRp, were used to analyze sequences of the two PCR products. Comparison of sequences co-ampli fi ed from the samespecimen showed they were clearly different, eliminating thepossibility that the longer PCR product is an artifact produced duringmanipulation/processing(RNAextraction,reversetranscription,PCR)of the nucleic acids of the virus with  “ canonical ”  RdRps (Fig 1s;Supplementary material). Genome sequences To characterize GVQ, its complete genome sequence was deter-mined using isolate MG-02 (muscadine grape) as a template.Excluding the poly(A) tail, it is composed of 6481 nt and includestwo ORFs, a long ORF1 accounting for 96% of the whole genome and arelatively short overlapping putative ORF2 (Fig. 2A). The ORF1encodes for a 2081-amino acid-long polyprotein of an estimatedmolecular mass of ca 229.5 kDa (p230). The N-terminal three fourthsportion of this polyprotein includes signature motifs of viral MTR (Rozanovetal.,1992),PRO(Gorbalenyaetal.,1991),HEL(Gorbalenya et al., 1988) and RdRps (Koonin, 1991; Poch et al., 1989; Koonin and Dolja, 1993) implicated in virus replication. It is predicted to beautoproteolytically processed between the Hel and RdRp domains bythe Pro-mediated activity to generate two proteins, with the N-terminal one including MTR-Pro-Hel domains (Bransom and Dreher,1994; Rozanov et al., 1995). The genome region downstream of theRdRp is likely involved in formation of two carboxy co-terminal coatproteins of the estimated  M  r  of 23000 and 21000. The genomecontains the  “ tymo/mara fi box ”  signature sequences reported to be atranscription promoter for subgenomic RNA synthesis followed by aCAA  “ consensus box ”  (Ding et al., 1990). Amino acid identities of GVQ with the closest known tymovirids approximate 65% in the conservedgenome products used in the comparison (MTR, HEL, CP). Computer-assisted sequence analysis revealed the presence of an additionalputativeORF, potentiallyencodinga proline/serine rich protein (total43%) of unknown function with the calculated mass of 27 kDa (p27).We performed phylogenetic analysis using neighbor-joining,maximum likelihood and Bayesian algorithms. In trees involving theMTR-Pro-HEL and CP domains, GVQ consistently appeared as a sisterlineage to GRVFV, forming a major sub-clade within mara fi viruses(Figs. 2B, E). The clustering of GVQ with mara fi viruses is also evidentintheRdRptree(Fig.2C),althoughtherelationshipsofGVQaswellasGRVFVandMRFVarenotfullyresolvedinthistree.Thisuncertaintyisnot due to the presence of the permuted motif C in the GVQ RdRp,which along with other poorly conserved regions was removed fromthe alignment submitted to the analysis. Importantly, the trees of mara fi viruses, including GVQ, produced for the MTR-Pro-HEL proteinand for an unprocessed replicase polyprotein (including MTR-Pro-HEL and RdRp domains) were congruent (Figs. 2B, D). This resultindicates that the phylogenetic signal generated by the RdRp domainof mara fi viruses is relatively weak. In contrast, the RdRp dominatesover the MTR-Pro-HEL protein in the signal produced for somelineages of tymoviruses (Figs. 2B – D). Taken together, these observa-tions imply that the observed difference between the MTR-Pro-HEL and RdRp trees in relation to mara fi viruses is relatively minor,indicating that the RdRp motif C permutation is not likely to beassociated with another gross evolutionary event in the GVQ lineage. Virus occurrence and variability We further proceeded to study the occurrence of GVQ in othergrapevine sources. For this analysis we used new degenerate primersthat were designed for broad tymovirid detection. Speci fi cally, a pairofprimersdesignedfortheRdRpregion(TymZ-FandTymZ-R)clearlydistinguishedbetweentymoviridswiththe “ canonical ” andpermutedRdRps (Fig. 3A) by generating amplicons of 344 bp and 407 bp,respectively. Mixed infections were characterized by the presence of both bands (Fig. 3B).Amplicons of 407 bp were found in two samples of   Vitisrotundifolia , single specimens of   Vitis aestivalis  and in an unknowncultivar of   Vitis vinifera . The same primer set was also applied forscreening native  Rubus  germplasm present in the Great SmokyMountain National Park. Besides the detection of a new  “ canonical ” mara fi virus in multiple blackberry accessions (Blackberry virus S,BlVS; Sabanadzovic and Abou Ghanem-Sabanadzovic, in press), two 2  Rapid Communication  distinct bands of 344 bp and 407 bp were evident in the specimenGSM-9. Sequence analyses showed that the lower band indeedsrcinated from BlVS genome (Fig. 3B and Fig. 2s; Supplementary material). Analyses of 407 bp long PCR products from all plantspecimens showed that they are (nearly) identical to the respectiveregion of the GVQ genome (muscadine isolate).Another GVQ-speci fi c primer set, designed to the CP region, wasused to determine the full sequence of viral coat proteins of severalisolatesidenti fi edintheprioranalysis.Intheseisolatestheaminoacidconservationvariedfrom93%to99%dependingonthecomparedpair(Table 3s and Fig. 3s; Supplementary material). Biochemical and biological implications of internal permutation in theGVQ RdRp Alignment of permuted RdRp sequences encoded by GVQ andTaV/EeV shows the colinearity of characteristic motifs C (GDD), A(DX 4-5 D) and B (GX 2-3 TX 3 N) (Fig. 3C) implying that the GVQ RdRp islikely to have a fold conserved in the permuted RdRps of otherviruses (Gorbalenya et al., 2002, Pan et al., 2007, Garriga et al., 2007). In this fold, major structural elements represented by motifs C, A andB form the active site at one side and are unconventionally,compared to canonical RdRps, interconnected at the opposite side.In the IBDV RdRp, this structural organization enables regulation of the active site access (Garriga et al., 2007). A multiple alignment of RdRps of mara fi viruses includes only two regions containing gaps,both of which are associated with the GVQ motif C (Fig. 1).Compared to other mara fi viruses, the motif C in the GVQ RdRp is fl anked by extra short sequences of two/three and  fi ve residues,respectively, which may be insertions accompanying the motif Cpermutation (reported in blue in Fig. 1). Interestingly,  in silico conversion of the canonical RdRp of poliovirus into the permutedform required insertion of additional residues in the proximity of motif C (Gorbalenya et al., 2002). Thus, it is tempting to speculate Fig. 1.  Alignment of the deduced amino acid sequences of RNA-dependent RNA polymerases of Grapevine virus Q and representatives of the three genera in the family  Tymoviridae : Tymovirus  ( Turnip yellow mosaic virus ; TYMV),  Maculavirus  ( Grapevine  fl eck virus ; GFkV) and  Mara  fi virus  ( Maize rayado  fi no virus — MRFV;  Oat blue dwarf virus — OBDV;  Citrus suddendeath-associated virus — CSDaV; Grapevine asteroid mosaic associated virus — GAMaV; Grapevine rupestris vein feathering virus — GRVFV). Conserved motifs A, B and C are boxed. A21-aa-long insertion located upstream of motif A containing GDD tripeptide, uniquely present in GVQ genome is reported in red. Positions of the primer sets RD and TymZ areindicated by solid- and dashed-line arrows, respectively. Direct comparison of C motifs of these viruses is presented separately.3 Rapid Communication  that the extra residues  fl anking motif C in GVQ have been acceptedto accommodate the new loop connectivity underneath the permut-ed motif C  β -hairpin.Apart of the common fold, the permuted RdRps of GVQ and otherviruses can be contrasted. First, GVQ is a plant virus while all otherviruses with permuted RdRps are of the animal srcin. All otherunique aspects of the GVQ RdRp are related to the phylogeneticpositionofGVQamongtymovirusesofthealpha-likesupergroup(seeabove) and outside of the lineage formed by TaV/EeV/birnavirusesthat has af  fi nity to Picorna-like supergroup. This deep phylogeneticseparation of the permuted RdRps, identi fi ed previously and reportedin this study, is most compatible with parallel srcin of the RdRppermutation in two lineages of RNA viruses. Although available dataare limited, RdRps in these two lineages seem to initiate the RNAsynthesis differently, either relying or not on (protein) priming (Ball,2007). Thus, the RdRp permutation of the RdRp active site iscompatible with functioning of different types of RdRps. Anintermediate position of the GVQ lineage in the tymovirus tree Fig. 2.  (A) Schematic representation of GVQ genome with nucleotide coordinates. Boxes depict ORFs and lines represent untranslated genomic regions. (B – E) Phylograms depictingthe relationships of Grapevine virus Q with the members of the family  Tymoviridae  for the MTR-Pro-Hel protein (B), RdRp (C), replicase polyprotein, (D) and coat protein (E). Thetrees weregenerated using MLalgorithm;similartrees wereproduced using neighbor-joining methodorBayesian inference. Bootstrap values larger than 50obtained in100 replicsare indicated at internal branch points. Internal branches with less than 50% support were collapsed. Congruent portions of replicase vs. MTR-Pro-Hel trees and replicase vs. RdRptrees are highlighted. Viruses used to construct trees, acronyms and RefSeq/GenBank accession numbers are:  Anagyris vein yellowing virus  (AVYV; NC_011559),  Bombyx mori macula-like latent virus (BmMLV, AB186123),  Citrus sudden death associated virus  (CSDaV, NC_006950),  Eggplant mosaic virus  (EMV, J04374), Grapevine rupestris vein featheringvirus (GRVFV, AY128949),  Grapevine  fl eck virus  (GFkV, NC_003347),  Kennedya yellow mosaic virus  (KYMV, NC_001746),  Maize rayado  fi no virus  (MRFV, AF265566), Nemesia ringnecrosisvirus (NeRNV,NC_011538), Oat blue dwarf virus (OBDV,U87832),  Ononis yellow mosaic virus (OYMV,J04375),  Physalis mottle virus (PhyMV,Y16104),  Poinsettia mosaic virus (PnMV, NC_002164) and  Turnip yellow mosaic virus  (TYMV, NC_004063). The de fi nitive/tentative members of the genera  Tymovirus ,  Mara  fi virus  and  Maculavirus  reported in black,red and blue, respectively.  Poinsettia mosaic virus , an unassigned species in the family, is reported in green.4  Rapid Communication  strongly indicates that the most recent ancestor of GVQ with thepermuted RdRp is likely to have emerged from a tymovirus with acanonical RdRp which predated the srcin of the GVQ ancestor. Thisreconstructioncontrastswiththeuncertaintyconcerningthesrcinof thepermutedRdRpsoftheTaV/EeV/birnavirusesdue todeeprootingofthislineageinatreewithmanyfamiliesofRNAviruses(Gorbalenyaet al., 2002).Mechanistically,generationofaninternalpermutationinaproteinis a complex three-point event that may partially be responsible forthe scarcity of permuted RdRps in viruses (Gorbalenya et al., 2002). ItcouldbegeneratedthroughtandemduplicationofmotifsA,BandCinan ancestral canonical RdRp to form the A → B → C → A' → B' → C'intermediate that is subsequently truncated to form the permutedC → A' → B' constellation (Gorbalenya et al., 2002). The postulatedintermediate must be short-lived to explain why it has not beenidenti fi ed so far. Alternatively, it could be envisioned that multipletemplate switching during replication of the canonical parent mightlead to the generation of permuted progeny.Regardless of the mechanism, permuted progeny is a mutant thatmust be competitive to survive. Recent study on the functionalimplications of the permuted C motif, using IBDV RdRp as a model,showed unique and strong dependency and stimulation of thisenzyme by Co 2+ ions (Letzel et al., 2007). These properties discriminate the IBDV RdRp from canonical enzymes includingthose encoded by  Poliovirus  (Arnold et al., 1999),  Hepatitis virus C  -(Ranjith-Kumaretal.,2002)orsapovirus-(Fullertonetal.,2007)allof  which are stimulated by Mn 2+ and/or Mg 2+ ions. In IBDV, permutedmotif C includes an  “ ADN ”  aa tripeptide that replaces  “ GDD ” commonly found in canonical RdRps and internally permutedpolymerases of TeV, EeV and GVQ (von Einem et al., 2004; Xu et al.,2004; Shwed et al., 2002; Letzel et al., 2007; Gorbalenya et al., 2002, present work). The precise genetic determinant, GDD-to-ADNmutation or motif permutation or both, that controls the Co2+ ionaf  fi nityisyettobemappedintheIBDVRdRp.Inthisrespect,GVQmayprovide an attractive alternative model for further studies of biological properties associated with permuted RdRps, when reversegenetics for this virus is established.The presence of two bands among PCR products in our analyseswas reminiscent of the data published by Shi et al. (2003) whoreported two PCR bands of 353 bp and 416 bp in selected grapevinesamples using a single primer pair srcinally designed for the speci fi cdetection of GFkV. The authors tentatively assigned bands as variantsGFkV  353  and GFkV  416  of   Grapevine  fl eck virus  and did not furtherinvestigate the genetic basis of the variability. Comparison of limitedamino acid sequences reported in the paper (see Shi et al., 2003; Fig. 2B) with our data showed that GFkV  416  is indeed an isolate of GVQ (94% aa identity).Finally,whilereviewingthismanuscriptbefore fi nalsubmissiontothe publisher, we noticed newly deposited sequences of a mara fi virusdenominated Grapevine Syrah virus 1 (GSyV-1; Al Rwahnih et al.,2009). Comparison of GVQ and GSyrV-1 revealed over 98% sequencesimilarity including conservation of the permuted RdRp, which wasnot observed/reported in the srcinal publication describing GSyV-1(Al Rwahnih et al., 2009). Thus, we propose that GVQ, GSyrV-1 and,likely, GFkV  416  are different isolates of a single mara fi  virus speciesdistinguished by the signature organization of RdRp motifs. Materials and methods Screening for viruses Plant materials initially collected for the purpose of screening forpossible viruses in native  Vitis  spp present in the Southeastern UnitedStatesweretestedinRT-PCRwithdegenerateprimersformembersof the family  Closteroviridae  (Tian et al., 1996),  Flexiviridae  (Dovas andKatis, 2003),  Tymoviridae  (Sabanadzovic et al., 2000) and members of the genus  Nepovirus  (Digiaro et al., 2007) which covered the most common taxa of viruses reported in grapevines. Virus sources The primary plant material for this investigation was collected in2007/2008 from an apparently healthy muscadine ( Vitis rotundifolia Michx.)accessionMG-02,whichtested positivefor tymovirids duringinitial assessment of the viruses infecting  Vitis  spp, and was used forthe complete molecular characterization of the virus. Mature vines of  fi eld-grown muscadine were collected and used for double strandedRNA analyses and/or leaf petioles early in the spring for total RNAextractions.Additionally, a total of 47 samples belonging to native andcultivated grapevines and blackberries were analyzed in this study.Samples positive for GVQ were used to partially sequence viral RdRpand coat protein (CP). Cloning, sequencing and data analyses Double-stranded RNAs were extracted from phloem scrapings of the accession MG-02 by selective chromatography through CF-11columns in the presence of an appropriate buffer containing 16%ethyl alcohol as described (Valverde et al., 1990). In addition,extracted nucleic acids were treated with RQ DNase and RNase A(in high salt conditions) in order to eliminate possible traces of DNAand ssRNA molecules prior to use as a template for further molecularwork.Complementary DNA (cDNA) was synthesized applying a slightlymodi fi ed protocol of  Froussard (1992) involving cDNA synthesis andits enrichment via PCR, size selection and directional cloning into anEcoRI-cutpUC119/EcoRIplasmid(TaKaRa,SouthKorea).Ligationmixwas transferred into  Escherichia coli  DH5 α cells and selectedplasmidswere custom sequenced at MWG Biotech facility (Huntsville, AL, Fig. 3.  (A) Diagrammatic representation of canonical and internally permuted activesites of the palm sub-domains of viral RdRps and differences in sizes of PCR productsgeneratedwithprimerssetTymZ.IP=internallypermuted,Can=canonical.(B)Agarose gel electrophoresis of PCR products generated by TymZ primers. Canonicalmara fi virusesgaveuniformproductof344bp,whileGVQinfectedsamplesgenerated407-bp-long amplicons. Viruses used in this gel are:  Poinsettia mosaic virus  (PnMV, lane 1),Blackberry virus S (BlVS, lane 2),  Grapevine  fl eck virus  (GFkV, lane 3). Grapevine virusQ-infected samples are in lanes 5 and 6. Blackberry samples GSM-9 with mixedinfection (BlVS+GVQ) is in lane 4. Negative control (tymovirids-free muscadine) is inlane 7. (C) Comparison of internally permuted regions in +ssRNA viruses: EeV and TaV and GVQ. All three motifs appear conserved among phylogenetically distant viruses.Inter-motif distances are indicated in brackets.5 Rapid Communication
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