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University of Hamburg, Biocentre Klein Flottbek, Department of Molecular Phytopathology and Genetics, Ohnhorststrasse 18, 22609 Hamburg, Germany
Correspondence
Nicole Mielke
nicole.mielke{at}botanik.uni-hamburg.de
Hans-Peter Muehlbach
muehlbach{at}botanik.uni-hamburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY563040, AY563041, DQ831828 and DQ831831.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ebrahim-Nesbat & Izadpanah, 1992; Pol
k & Zieglerov
, 1996). In other European countries, such as Great Britain, Poland and the Czech Republic, these characteristic symptoms have also been detected (Cooper, 1979; Pol
k et al., 1990).
Many efforts were made to identify the putative infectious agent. By electron microscopic studies, Ebrahim-Nesbat & Izadpanah (1992) detected spherical, enveloped particles of approximately 80 nm diameter in leaves of European mountain ash showing mottling and ringspot symptoms. These structures resembled tomato spotted wilt virus (TSWV) particles, but in subsequent studies using ELISA, infection with TSWV could not be confirmed. On the other hand, Führling & Büttner (1995) showed that the putative pathogen could be transmitted by grafting onto seedlings of S. aucuparia. Electron microscopy, graft transmissibility and the characteristic symptoms together indicated that the ringspot disease of mountain ash could be due to a virus infection. To provide further evidence for a viral aetiology of the mountain ash ringspot disease, the promising method of searching for the presence of high-molecular-mass double-stranded (ds) RNA could be applied, as was shown for tobacco mosaic virus and citrus tristeza virus (Dodds et al., 1984), potato virus X (Valverde et al., 1990) and apple stem pitting virus (Jelkmann et al., 1992). When we used this approach in a previous investigation, we could detect four different dsRNA molecules of 7, 2.3, 1.5 and 1.3 kb in leaves and bark of symptomatic mountain ash trees (Benthack et al., 2005). No dsRNA was obtained in preparations from unaffected trees. Reverse transcription of the dsRNA fraction and amplification by degenerate oligonucleotide primer PCR followed by TA cloning resulted in a cDNA sequence of 3.8 kb in length (GenBank accession no. AY563040
[GenBank]
), which was obtained from overlapping clones. Sequence analyses showed a similarity of this sequence to the genes encoding the RNA-dependent RNA polymerase (RdRp) of members of the Bunyaviridae, a large virus family of predominantly vertebrate- and insect-infecting RNA viruses, but including the phytopathogenic genus Tospovirus. These viruses are characterized by a multipartite, single-stranded (ss) RNA genome of negative and partially ambisense polarity. Thus, our previous results strongly supported the hypothesis of a virus being associated with the mountain ash ringspot disease.
Here, we report the genomic structure of a novel plant RNA virus with four RNA molecules, which we have arbitrarily named European mountain ash ringspot-associated virus (EMARAV).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of total RNA.
Total RNA was isolated according to Boom et al. (1990) with the following slight modifications. All solutions were treated with 0.1 % (v/v) DEPC. Approximately 100 mg fresh or frozen plant tissue was ground in liquid nitrogen and homogenized in 1 ml extraction buffer [6 M guanidine hydrochloride, 0.2 M sodium acetate (pH 5.2), 1 M potassium acetate, 0.025 mM EDTA, 2.5 % PVP-40]. Aliquots of 500 µl were transferred into a new reaction tube and 100 µl 10 % SDS was added. The extract was incubated for 10 min at 70 °C with gentle shaking. After holding on ice for 5 min, the suspension was centrifuged at 12 000 g and 4 °C for 10 min (Sigma 3K20, rotor no. 12154). Three hundred microlitres of sodium iodide solution (6 M sodium iodide, 0.15 M sodium sulfite), 150 µl ethanol and 25 µl silica suspension (1 g ml–1, pH 2.0) were added to 300 µl of the supernatant, and RNA binding was performed for 10 min at room temperature with gentle shaking. Silica particles were washed twice with TEN buffer [10 mM Tris/HCl (pH 7.5), 0.5 mM EDTA, 50 mM NaCl, 50 % ethanol] and resuspended in 200 µl H2O. After stripping by heat treatment (70 °C for 5 min) and centrifugation (12 000 g, 4 °C, 5 min; Sigma 3K20, rotor no. 12154), total RNA was extracted from the supernatant with phenol/chloroform, precipitated by ethanol and resuspended in H2O. RNA was stored at –20 °C.
Rapid amplification of cDNA ends (RACE).
For amplifying cDNA ends of the partially sequenced putative viral RNA species from our previous investigation (Benthack et al., 2005), which we named RNA 1, a modified RACE protocol according to König (1997) was used.
Three to five micrograms of total RNA was reverse-transcribed by using 20 pmol of a specific, 5'-biotin-labelled primer (970 FP Bio, 5'-bio-AAACTAATGCCAACAATGAAG-3'; 1495 RP Bio, 5'-bio-GTTCTTCCATACTCATTCAC-3') and Moloney murine leukemia virus (MMuLV) H– reverse transcriptase (Promega), following the manufacturer’s instructions. First-strand synthesis was stopped by heating for 10 min at 70 °C. The DNA–RNA hybrids were bound to 30 µl streptavidin-coated magnetic particles (Roche) in TEN100 buffer [10 mM Tris/HCl (pH 7.5), 1 mM EDTA, 100 mM NaCl] for 1 h at room temperature under gentle rotation. One microlitre of RNase A (0.2 µg µl–1) was added and incubated for 10 min at 37 °C. Particles were washed twice with TEN1000 buffer [10 mM Tris/HCl (pH 7.5), 1 mM EDTA, 1 M NaCl] and resuspended in H2O. Viral cDNA was extended with an oligo(dC) tail by using terminal deoxynucleotidyl transferase (MBI Fermentas) according to the manufacturer’s instructions. Aliquots of 5–7 µl of the modified first strand were used for RT-PCR with a (nested) specific primer (970 FP, 1495 RP, 516 FP: 5'-AAGCCAGTATTGTGACGAAGT-3'; 261 RP: 5'-TGTTAGGTCATCAGTGGAAT-3') and a primer containing an oligo(dG) part (5'-GGTGGCTCGTATTTCTTCTTTAGGGXXGGGXXGGGXXG-3'). PCR was carried out following a standard protocol.
RT-PCR with primers deduced from viral RNA termini.
After identifying specific homologous ends on RNA 1, primers containing 11 nt of these sequences were deduced, which allowed subsequent RT-PCR studies: T11(3), 5'-CCGCATCCAGTAGTGTTCT-3'; T11(5), 5'-CCGCATCCAGTAGTGAACT-3'. Reverse transcription was performed with 3–5 µg total RNA and 20 pmol T11 primer. Two hundred units of MMuLV H– RT (Promega) was added and incubated for 10 min at room temperature, 30 min at 37 °C and 20 min at 42 °C. Five microlitres of the first-strand mixture was used for PCR (initial denaturation for 3 min at 94 °C; five cycles of 1 min at 94 °C, 1 min at 38 °C, 5 min at 72 °C; 30 cycles of 1 min at 94 °C, 1 min at 56 °C, 5 min at 72 °C; final elongation for 10 min at 72 °C).
For RACE analyses to verify the accurate ends of RNA 2 and RNA 3, which we obtained by RT-PCR with a viral RNA termini-specific primer, the gene-specific primers were used: for RNA 2, M91: 5'-GAAATCCATCTGAAACAAAGTCT-3'; for RNA 3, 518: 5'-CACAAAGGGAAATCAAATAAATCA-3'.
In additional RT-PCR studies to obtain RNA 4, primers deduced from the terminal 13 nt were used: T13(3), 5'-ATCCAGTAGTGTTCTCC-3'; T13(5), 5'-ATCCAGTAGTGAACTCC-3'.
Cloning of PCR products, sequencing and sequence analyses.
Cloning of PCR products was performed by using the pGEM-T Easy vector system (Promega) following the manufacturer’s instructions. Recombinant plasmids were transformed into Escherichia coli DH5
cells and clones were selected by blue–white screening, as well as by a standard PCR with a vector-specific T3/T7 primer.
Sequencing was carried out by using an Applied Biosystems ABI model 370A/373A automatic sequencer with a BigDye Terminator kit. For sequence editing and primer selection, we used the DNAStar software LASERGENE. Database searching was done by using BLAST (NCBI). Protein analyses were performed with TMHMM v. 2.0, SignalP v. 1.1, iPSORT prediction, TargetP and NetNGlyc v. 1.0 (http://www.expasy.org).
Northern blot hybridization.
Northern hybridization was performed as described previously (Benthack et al., 2005). A 5'-digoxigenin-labelled DNA oligonucleotide (5'-dig-ATCCAGTAGTGAACTCC-3') directed against the conserved 3' terminus of viral RNAs was used as a specific probe. Hybridization was performed overnight at 41 °C in hybridization buffer without formamide.
Isolation of viral nucleocapsids.
Enrichment of viral nucleocapsids was performed according to a protocol designed for tospoviruses (Roggero et al., 1998) with slight modifications. All steps were performed on ice or at 4 °C. Twenty grams of fresh or frozen plant tissue was ground in liquid nitrogen and extracted with 140 ml buffer [100 mM Tris/HCl (pH 8.0), 5 mM EDTA, 20 mM sodium sulfite, 10 mM DIECA, 2 % PVP-40]. After further crushing by using an UltraTurrax (Janke & Kunkel; IKA-Werk), the suspension was centrifuged for 10 min at 4000 g (Heraeus Minifuge RF, rotor no. 2150). The supernatant was treated with 2 % Triton X-100 and stirred for 30 min on ice. The nucleocapsid-containing suspension was centrifuged for 30 min at 120 000 g (Beckman L7-55, rotor SW41 Ti) and the obtained pellet was resuspended in 40 ml virus buffer [100 mM Tris/HCl (pH 7.9), 5 mM EDTA, 20 mM sodium sulfite, 10 mM DIECA, 1.5 % Triton X-100]. After stirring for 30 min on ice, the suspension was centrifuged at 12 000 g for 10 min (Centrikon H401, rotor A 8.24, Kontron Instruments). The supernatant was layered on a 30 % sucrose cushion and centrifuged for 90 min at 90 000 g (Beckman L7-55, rotor SW41 Ti). Sediment was resuspended in 200 µl TE buffer [10 mM Tris/HCl (pH 7.9), 1 mM EDTA] and centrifuged for 5 min at 12 000 g (Sigma 3K20, rotor no. 12154). The nucleocapsid-containing supernatant was stored at –20 °C. To separate viral RNA from nucleocapsid proteins, 1 % SDS (final concentration) was added to the supernatant and RNA was extracted with phenol/chloroform.
Protein extraction and gel analysis.
For total protein extraction from leaves and bark of European mountain ash and from leaves of herbaceous control plants, approximately 2 g plant tissue was ground with liquid nitrogen and homogenized in 8 ml extraction buffer [50 mM Na2HPO4, 50 mM NaH2PO4, 10 mM Na2SO3, 1 % PVP (pH 7.0)]. After centrifugation (4000 g, 10 min, 4 °C; Heraeus Minifuge RF, rotor no. 2150), the supernatant was used immediately for SDS-PAGE or stored at –20 °C. Total protein extracts and proteins from the nucleocapsid fraction were analysed by SDS-PAGE in 15 % polyacrylamide gels containing 0.1 % SDS. Gels were silver-stained according to Heukeshoven & Dernick (1988).
Antibody production and Western blot.
For serological detection of the putative viral glycoprotein G1 in plant extracts, a polyclonal antiserum directed against a 15 aa synthetic peptide of the deduced protein sequence (p2) with the following sequence was used: NH2-CH424EPKIRDTFTHDRE437-COOH. Design of this antigen was made in consideration of the antigen index of Jameson & Wolf (1988) (LASERGENE; DNAStar). The peptide was extended by an additional N-terminal cysteine residue, as was recommended by the manufacturer (SeqLab). Immunization was done in rabbits.
A second polyclonal antiserum directed against the N-terminal part of the recombinant protein p3 (aa 1–93) was also produced in rabbits.
In Western blot analyses, proteins from various plant tissues and from fractions enriched for nucleocapsids were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond-C extra; Amersham Biosciences) by electroblotting.
Immunodetection was carried out by a colorimetric procedure with a secondary antibody conjugated with alkaline phosphatase (anti-rabbit–AP; Promega) and NBT/BCIP as substrate according to the instruction of Roche.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Primers were derived from this so-called ‘starter cDNA’ in order to perform further RACE experiments with total RNA from affected mountain ash trees. This strategy allowed the extension of the previous sequence in several steps up to a total length of 7040 nt. The corresponding RNA was named RNA 1 (Fig. 1).
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), two genera predominantly infecting mammals. No similarity of the terminal sequences of RNA 1 with those of the phytopathogenic genus Tospovirus could be observed, nor with those of the genera Nairovirus, Phlebovirus or Tenuivirus.
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) and 1560 nt (RNA 3, Fig. 1).
Comparison of the 3' and 5' ends of these three characterized RNAs showed that the terminal sequence identity was 13 nt. With further RT-PCR studies carried out with a primer pair deduced from these 13 nt long conserved terminal sequences, a fourth RNA (RNA 4) of 1348 nt could be obtained as a full-length cDNA clone (Fig. 1).
These four published sequences, obtained by the described cloning strategy, were verified by further RT-PCR and RACE assays with at least four resulting cDNA clones for each RNA region (data not shown). The ends of the four identified RNAs are almost fully complementary over a sequence of between 19 and 23 nt. Base pairing is only interrupted by a dinucleotide in either strand of RNA 1, 2 and 3 (5' U8–U9) or two dinucleotides in RNA 4 (5' U8–U9 and 5' G17–A18). All four RNA ends are fully conserved in a stretch of 13 nt. The conserved terminal sequence shows high similarity to that of orthobunyaviruses, but not with the ends of tospoviruses, members of the only plant-infecting genus among the Bunyaviridae.
In order to assess whether the virus genome consists of only four RNAs, Northern blot hybridization with a digoxigenin-labelled DNA oligonucleotide directed against the 3' terminus was carried out (Fig. 2). Four clearly visible hybridization signals of the expected sizes were only obtained with total RNA of symptomatic mountain ash. No EMARAV-specific RNA could be detected in trees without characteristic symptoms.
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). Sequence analyses in the database confirmed our earlier finding that the deduced amino acid sequence of p1 shows similarity to the RdRp of members of the family Bunyaviridae (Benthack et al., 2005). We were able to detect all conserved motifs, A–E, of the RdRp. Additionally, we could now identify by sequence identity a domain of the bunyaviral RdRp that is known as the endonucleolytic centre (Fig. 3) and is involved in the ‘cap-snatching’ mechanism (Duijsings et al., 2001; Li et al., 2001; Aquino et al., 2003).
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). Database analyses of the deduced amino acid sequence revealed a conserved motif of the glycoprotein precursor of phleboviruses (GCY.C..G), another vertebrate-infecting genus of the family Bunyaviridae. By computer-aided analyses, all essential sequence elements of such glycoprotein precursors could be identified (Fig. 4a). There are three putative transmembrane helices, six putative glycosylation sites, a possible N-terminal signal sequence and a putative processing site, THA
DD, at which the precursor may be cleaved proteolytically into two single glycoproteins, G1 (52 kDa) and G2 (23 kDa). Comparison with the glycoprotein precursors of various members of the family Bunyaviridae and with that of the unassigned genus Tenuivirus revealed similar organization of the typical sequence elements (Fig. 4b). To detect the putative glycoprotein G1 that might be integrated into the viral envelope, we raised polyclonal antibodies against a synthetic oligopeptide (15-mer) from the G1-specific domain of the deduced p2 sequence. The antigen was designed by using the antigen index of Jameson & Wolf (1988) (Fig. 5a). In Western blot analyses with approximately 2 µg total protein from leaves of European mountain ash and from leaf tissue of two herbaceous control plants (Abutilon sp., Euphorbia pulcherrima), we could detect a protein of the expected molecular mass of 52 kDa exclusively in leaves of symptomatic S. aucuparia (Fig. 5b). This peptide may represent the putative glycoprotein G1. In the same sample, an additional protein of about 75 kDa was recognized by the antiserum, corresponding in size to the putative glycoprotein precursor p2 (Fig. 5b). Close to each of these two protein bands, other, somewhat larger peptides could be detected.
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), which shows similarity to two viral proteins (Fig. 6a). One of them is the putative nucleocapsid protein of high plains virus (HPV) (GenBank accession no. U60141
[GenBank]
). The other, only partially known sequence belongs to a hypothetical protein of pigeonpea sterility mosaic virus (PPSMV) (GenBank accession no. AJ439561
[GenBank]
; Kumar et al., 2003). Neither plant-pathogenic RNA virus has yet been classified. Interestingly, we found no identity of the deduced amino acid sequence of p3 to any known protein of the family Bunyaviridae. To determine whether p3 could be involved in the formation of viral nucleocapsids, we prepared fractions enriched for putative nucleocapsids from mountain ash leaves by using a protocol that was developed for the isolation of tospovirus nucleocapsids (Roggero et al., 1998). SDS-PAGE analyses of the ‘nucleocapsid’ fraction in a 15 % gel and a further Western blot assay with a polyclonal antiserum directed against the N-terminal part of p3 supported our assumption that the 35 kDa protein encoded by RNA 3 could be a viral nucleocapsid protein. A substantial quantity of a protein of approximately 35 kDa could be detected in preparations from symptomatic mountain ash trees, but not in protein extracts from unaffected trees (Fig. 6b, c). Several attempts to purify intact viral RNA from these ‘nucleocapsid’ fractions failed. In gel electrophoresis, RNA always showed a certain degree of degradation, but the presence of fragmented viral RNA of both polarities could be verified reproducibly by RT-PCR (data not shown).
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). Database research of the deduced polypeptide revealed similarity neither to a viral peptide nor to any other known protein. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The four identified RNAs contain complementary terminal sequences that are typical for segmented ss(–)RNA virus families, such as Arenaviridae, Orthomyxoviridae and Bunyaviridae, although members of the family Rhabdoviridae with its monopartite ss(–)RNA genome also possess these characteristic ends. The complementary termini consist of a conserved domain of typically 8–11 nt within different virus families and, by base pairing, cause the formation of a ‘panhandle structure’. Such a characteristic structure for nucleocapsids has been shown already by atomic force microscopy (Kellmann et al., 2001). The role of these characteristic termini has been discussed. They seem to function as promoters for transcription and replication (Fodor et al., 1994, 1995; Prehaud et al., 1997; Flick et al., 2002; Barr et al., 2003; Barr & Wertz, 2004; Kohl et al., 2006). Normally, these cis-acting sequences contain a few nucleotides that do not base pair and affect short loops that may have regulatory functions (Hellendoorn et al., 1997; Ito & Lai, 1997; Koev et al., 2002; Satyanarayana et al., 2002; Qi et al., 2003; Kohl et al., 2006). The four viral RNAs that we analysed in our studies also contain nucleotides within the complementary stretches that cannot form base pairs. Furthermore, the 5' end of the viral RNA is thought to be a signal sequence for encapsidation (Osborne & Elliott, 2000; Severson et al., 2001). Comparison of the identified terminal sequences of RNAs 1–4 shows high similarity to the viral RNA ends of the orthobunyaviruses and hantaviruses, both belonging to the family Bunyaviridae. Interestingly, the phytopathogenic, unassigned genus Tenuivirus also shares the same termini nucleotide sequence as the vertebrate-infecting bunyaviral genus Phlebovirus (Estabrook et al., 1996; Falk & Tsai, 1998). As the deduced polypeptides of RNA 1 and RNA 2 also indicate a significant relationship between EMARAV and several negative-strand RNA viruses, it is very likely that all four RNAs also show negative-strand orientation.
As shown previously, highest similarity of the protein encoded by RNA 1 was found to replicases of members of the virus family Bunyaviridae, especially to those of the vertebrate-infecting genus Orthobunyavirus and the phytopathogenic tospoviruses (Benthack et al., 2005). With the complete RNA sequence, we could now also identify a region similar to the so-called ‘endonucleolytic centre’, which is involved in the mechanism of ‘cap snatching’ (Duijsings et al., 2001; Li et al., 2001; Aquino et al., 2003). This finding provides further evidence in favour of the hypothesis that the newly characterized EMARAV belongs to the negative-strand RNA viruses.
The single gene of RNA 2 is likely to encode a glycoprotein precursor (p2), which contains a short conserved motif of the glycoprotein of phleboviruses. However, the further three homologous domains of this protein (Liu et al., 2003) could not be detected in the p2 sequence. There is also no sequence similarity to the glycoprotein precursors of the orthobunyaviruses and tospoviruses, genera that were shown to have the highest phylogenetic relationship to EMARAV based on their RdRp motifs. Nevertheless, the protein p2 could exert the function of a glycoprotein precursor because all essential elements were detected in the deduced protein sequence. Viral glycoproteins are characteristic for viruses that are enveloped by a host-derived lipid membrane, in which they are inserted specifically to form so-called spikes. Among plant viruses, only members of the genera Cytorhabdovirus, Nucleorhabdovirus, Tenuivirus and Tospovirus are known for the presence of glycoprotein-encoding ORFs. Viral glycoproteins play a role in endocytosis of virus particles in human and animal cells (Hase et al., 1989; Gavrilovskaya et al., 1998; Rajcani & Vojvodova, 1998; Germi et al., 2002) by interacting with cellular receptors. In the case of plant-infecting tospoviruses, which are transmitted by thrips, these proteins may be important for virus–vector interaction (Whitfield et al., 2005). Interestingly, in symptomatic leaves of mountain ash, enveloped virus-like particles could be detected by electron microscopy (Ebrahim-Nesbat & Izadpanah, 1992). Therefore, the immunological detection of two proteins of approximately 52 kDa, the expected size of the processed G1, and 75 kDa, which matches perfectly the size of the putative precursor p2, indicates that the ORF of RNA 2 is expressed in the cells of mountain ash leaves. The extra peptides that were detected by the antiserum may represent glycosylated forms, although glycostaining assays did not give convincing results (data not shown). The fact that we could detect the putative glycoprotein precursor may indicate that it is processed post-translationally, as has been shown for the genus Nairovirus (Sanchez et al., 2002). All other members of the family Bunyaviridae exhibit cotranslational cleavage, therefore no precursor protein is detectable (Andersson et al., 1997; Schmaljohn & Hooper, 2001).
RNA 3 encodes a protein, p3, that reveals similarity to proteins of unknown function of two RNA viruses that are not yet classified. One of these peptides is the putative 32 kDa envelope protein of HPV; the other is an unidentified peptide that is only partially sequenced and encoded by the 1.4 kb RNA 5 of PPSMV. Interestingly, the dsRNA pattern of PPSMV is comparable to that correlating with the ringspot disease of European mountain ash (Kumar et al., 2003; Benthack et al., 2005). It shows five to seven dsRNA bands in roughly the same size range as EMARAV. Partially purified preparations of HPV and PPSMV are visualized under the electron microscope as low-contrasted, filamentous structures that resemble tenuiviruses, ophioviruses and nucleocapsids of tospoviruses. Further, both viruses were detected as spherical, enveloped particles (Kumar et al., 2002; Skare et al., 2006), which are similar to those observed previously in mountain ash showing chlorotic ringspots and mottling (Ebrahim-Nesbat & Izadpanah, 1992). However, the phylogenetic relationship of these three viruses is not known, because neither the RdRp gene of HPV nor that of PPSMV has been sequenced.
The potential function of the 35 kDa p3 as nucleocapsid protein (N protein) is supported by the detection of a protein of that size in a fraction from affected mountain ash leaves, obtained by a method for the enrichment of nucleocapsids. No sequence similarity was found to N proteins of members of the family Bunyaviridae. On the other hand, this is not uncommon because N proteins are known to be highly variable, even in the same genus. The finding that viral RNAs of both polarities could be detected in these nucleocapsid-enriched fractions is not surprising. Severson et al. (1999) showed for the genus Hantaanvirus that not only the genomic RNA, but also the antigenomic RNA and even mRNA, are encapsidated by the N protein, although only the genomic RNA is found in virions. Thus, there is still no unambiguous experimental proof that EMARAV is a negative-strand RNA virus, but sequence similarities strongly suggest this.
Peptide p4, encoded by the fourth identified RNA, RNA 4, shows no similarity to any known viral or plant-specific protein. It may have the function of a movement protein, as is typical for all phytopathogenic viruses serving for systemic distribution within the plant (Seron & Haenni, 1996; Santa Cruz, 1999; Lucas, 2006). However, comparison with conserved domains within movement proteins (Melcher, 1990, 2000; Koonin et al., 1991) did not show clear results for either p4 or p3. It is possible that p4 could instead, or in addition, represent a gene-silencing suppressor, which has been shown for several phytopathogenic viruses, as well as for the genera Tospovirus and Tenuivirus (Brigneti et al., 1998; Kasschau & Carrington, 1998; Pfeffer et al., 2002; Takeda et al., 2002; Bucher et al., 2003). However, silencing suppressors are insufficiently conserved and no intergenus-specific motifs are known, making similarity searches in databases impossible. Thus, the role of p4 in the infection cycle of EMARAV is still completely unclear.
In conclusion, we could characterize the genome of a novel virus associated with the ringspot disease of European mountain ash (S. aucuparia L.). Although we are fully aware that Koch’s postulates still have to be fulfilled to identify this virus as the causative agent of the disease, the strong association of the presence of viral RNAs with the characteristic symptoms justifies our suggestion to name it European mountain ash ringspot-associated virus (EMARAV).
Based on the now-available sequence data of the four identified RNAs of EMARAV, there is a noticeable relationship to the family Bunyaviridae, especially to the genera Tospovirus and Orthobunyavirus. However, there is strong evidence that it could not be classified into one of these, because EMARAV possesses more than three genomic RNAs. Furthermore, except for the RdRp gene, there is insufficient identity of the deduced peptide sequences to already known bunyaviral peptides. However, the existing similarities of EMARAV to the family Bunyaviridae and also to the unassigned genus Tenuivirus point to a common origin. Further studies concentrating on protein function and virus distribution should give more information about this new pathogen and its relationship to already described viruses.
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ACKNOWLEDGEMENTS |
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Received 14 November 2006;
accepted 20 December 2006.
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