Biologische Bundesanstalt, Institut für Pflanzenschutz im Obstbau, Schwabenheimer Straße 101, D-69221 Dossenheim, Germany1
Plant Research International, PO Box 16, 6700 AA Wageningen, Netherlands2
Author for correspondence: Jeremy Thompson. Fax +49 6221 8680515. e-mail Jeremy.Thompson{at}urz.uni-heidelberg.de
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Abstract |
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Introduction |
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In this study we have determined the nucleotide sequence of the SMoV genome. This was in part achieved by producing a partial cDNA library derived from SMoV dsRNA extracted from N. occidentalis (Leone et al., 1995 ; Schoen & Leone, 1995
), and completed by filling in the gaps, including the 5' and 3' ends, using PCR. The results suggest that despite a general genome organization more akin to that of some nepoviruses, SMoV should probably be placed in the recently proposed SDV-like lineage of picorna-like viruses, which includes Satsuma dwarf virus (SDV), Naval orange infectious mottling virus (NIMV) and Citrus mosaic virus (CiMV) (Karasev et al., 2001
).
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Methods |
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Electron microscopy.
Leaf dip preparations were made from leaves of healthy and SMoV (isolate 1134)-infected C. quinoa, F. vesca, N. benthamiana, N. hesperis 67A and N. occidentalis 37B. Suspensions, crude plant sap, fixative and staining solution were all kept on ice. Crude plant sap was fixed in a solution of 2% glutaraldehyde and 0·2% Na2SO3 in deionized water. Negative staining was performed with a 2% potassium phosphotungstate solution, pH 6·7.
Ultrathin sections were made from new systemically infected plant material of C. quinoa and F. vesca. Samples were fixed, dehydrated, embedded in Spurrs medium, sectioned, and stained with uranyl acetate and lead citrate (Hayat, 1972 ).
Isolation of dsRNA.
Fresh N. occidentalis 37B leaf tissue (at least 5 g) was ground in liquid nitrogen with a pestle and mortar. The powder was mixed with homogenization buffer (2 ml per gram of leaf) consisting of 2x STE (STE: 50 mM Tris, 100 mM NaCl, 1 mM EDTA), pH 7·0, containing 1% (w/v) bentonite, 1% (w/v) SDS, 1% (w/v) polyvinylpyrrolidone Mr 25000 and 0·1% (v/v) -mercaptoethanol. The slurry was further extracted with 1·5 ml STE saturated-phenol and 0·75 ml chloroformisoamyl alcohol (24:1, v/v) per gram of leaf. The aqueous phase obtained after centrifugation at 10000 g for 10 min was adjusted to 15% ethanol. The supernatant was subjected to two cycles of chromatography on Whatman CF-11 cellulose suspended in STE+15% ethanol at a concentration of 10 g/100 ml. Ten ml of this suspension was poured into 20 ml disposable plastic syringes plugged with Miracloth filter paper. Each cycle of chromatography consisted of loading the columns with the sample, washing the columns with at least 150 ml of STE+15% ethanol and eluting the dsRNA from the cellulose with 10 ml of STE. After the second chromatography, dsRNA was concentrated by precipitation overnight at -20 °C with 1/10 vol. of 3 M sodium acetate, pH 5·6 and 2·5 vols of absolute ethanol. After centrifugation for 1 h at 8000 g, the pellet was resuspended in 800 µl of STE and dispensed in microfuge tubes. The content of each microfuge tube was precipitated for 15 min at -80 °C with 1/10 vol. of sodium acetate, pH 5·6 and 2·5 vols of absolute ethanol. After centrifugation the pellet was washed with 70% ethanol, centrifuged again, dried under vacuum, and stored at -80 °C. Isolated dsRNA was stored at -80 °C. Analysis of this isolated material by agarose gel electrophoresis revealed the two infection-specific dsRNA bands previously described by Leone et al. (1995)
and Schoen & Leone (1995)
.
cDNA synthesis and cloning.
Synthesis of cDNA from the dsRNAs denatured in 20 mM methylmercuric hydroxide was carried out as described by Jelkmann et al. (1989) . Blunt-end cDNA fragments were cloned into the EcoRV site of pBluescript KS (Stratagene) and transformed into E. coli DH5
as described by Sambrook et al. (1989)
.
PCR and RACE.
Total nucleic acid from N. occidentalis 37B leaves harvested 23 weeks post-inoculation with water (mock) or SMoV was extracted by the silica capture method (Rott & Jelkmann, 2001 ). For reverse transcription (RT), 5 µl of total nucleic acid extract in 6·5 µl sterile distilled water and 0·5 µl of primer (10 µM) was used. A mix of random hexamers and poly(T) primer was used for standard PCR of internal genomic regions, and sequence-specific primers (SSP) and AnchorpolydT primer (5' GCGCGAACAGAAAACGGAAATACATTTTTTTTTTTTTTTTTV 3') was used for 5' and 3' RACE. The sample was incubated at 70 °C for 10 min and then placed on ice. Four µl first strand buffer (5x) (Life Technologies), 2 µl DTT (0·1 M) and 1 µl dNTPs (10 mM) were added and incubated at 37 °C for 10 min. One µl of mMLV or Superscript II (200 U/µl) (Life Technologies) RT was added and incubated at 42 °C for 50 min. For RACE only, an incubation step of 50 °C for 10 min followed. The enzyme was inactivated by incubating at 70 °C for 10 min. For 5' RACE, the RT reaction was treated with phenol, precipitated with ethanol, resuspended in 10 µl water (Sambrook et al., 1989
), and poly(A) tailed by terminal transferase (Life Technologies) according to the manufacturers instructions. PCR was performed using Takara La Taq (Takara Shuzo Co.) according to the manufacturers instructions in a Robocycler (Stratagene). Cycling conditions consisted of an initial denaturation step at 94 °C for 2 min, followed by 35 cycles at 94 °C for 1 min, 4060 °C (depending on the primer pair used) for 1 min, 72 °C for 2 min, and a final elongation step at 72 °C for 5 min. 5' and 3' RACE involved two to three PCRs with semi-nested primers. For 5' RACE, the first PCR used an SSP with the poly(dT) primer at low stringency annealing (40 °C) followed by a second and third PCR using a nested SSP with the Anchor primer (5' GCGCGAACAGAAAACGGAAATACA 3'). For 3' RACE, the first and subsequent PCRs were done using a combination of nested SSPs with the Anchor primer. PCR products were separated by gel electrophoresis in 1% agarose, and visualized after staining with ethidium bromide under UV light. Fragments were excised from the gel by Geneclean (Bio 101) and ligated into pBluescript KS with a T-overhang (Hadjeb & Berkowitz, 1996
) at the EcoRV site. The design of primers (Life Technologies) for standard PCR, and the SSPs for RACE was based on the sequence data obtained from dsRNA-generated clones.
Sequencing and sequence comparison.
All clones were sequenced with an ABI Prism Sequence Detection System (Applied Biosystems) and analysed with the HUSAR computer program (German Cancer Research Center, Heidelberg, Germany). Initial searches for sequence similarities with other viruses was done using FASTA (Lipman & Pearson, 1988 ). Multialignments were then done with the CLUSTAL X program (Thompson et al., 1997
) after bootstrapping in 1000 replicates. Phylogenies were viewed by the NJPLOT program.
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Results |
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Molecular cloning of dsRNA of SMoV
The infection-specific dsRNA bands purified and used for molecular cloning were estimated to be about 6·3 and 7·8 kbp in size by agarose gel electrophoresis and corresponded to previously reported results (Leone et al., 1995 ; Schoen & Leone, 1995
). The difference between the real genomic sizes of RNA1 and RNA2 of SMoV (see below) is due to an extrapolation of the dsRNAs in comparison with
-HindIII markers used. Molecular cloning of dsRNA produced 120 clones, which were analysed by plasmid isolation and restriction enzyme digestion. The sizes of these clones ranged between 200 and 2000 bp. More details as well as the properties of the dsRNAs were reported by Schoen et al. (1997)
.
PCR- and RACE-derived clones
The sequences of the 5' and 3' termini of both RNA1 and 2 were verified in two separate RACE reactions from different extracts of total nucleic acid. All except one of the 5' termini clones contained one additional base (A, C or G) between the viral sequence and the terminal transferase-added poly(A) tail. Given that the viral sequence at the 5' termini of both RNA 1 and 2 was consistent between clones, the presence of an extra variable base at the extreme 5' end was assumed to be as a result of reverse transcriptase terminal transferase activity (Shi & Kaminskyj, 2000 ). Various approaches to obtain the 5' and 3' ends were employed: for example, dsRNA was used as a template for cDNA generation as in the original cloning with various additives in the PCR buffer (e.g. DMSO, formamide, PEG, glycerol etc.). Possibly because of the high melting temperature of the dsRNA (Schoen et al., 1997
), total nucleic acid extracts of infected material were always more effective as templates for cDNA synthesis. Addition of 5% formamide to the reaction buffer also improved the specificity of the amplification products (data not shown).
Nucleotide sequence and coding regions
RNA1.
RNA1 and RNA2 are, respectively, 7036 and 5619 nt long, excluding the 3'-terminal poly(A) tail. RNA1 has a single open reading frame (ORF) beginning at AUG (154156) and terminating at UAA (58995901). It encodes a polyprotein of molecular mass 215828 Da (1916 aa, 215K), which contains regions encoding a putative protease cofactor (Pro-C), helicase (HEL), viral genome-linked protein (VPg), protease (Pro) and RNA-dependent RNA polymerase (RdRp) (Fig. 2). The sequence context of the first in-frame initiation codon, CAGGAUGGGA, is favourable for translation initiation in plants with an A in position -3 and a G in positions +4 and +5 (Lütcke et al., 1987
), and also in animals with a C and a G in the -4 and +4 positions, respectively (Kozak, 1986
). All other ORFs in the negative and positive sense were less than 594 nt. A conserved amino acid motif (Fx27Wx11Lx21LxE) found at the N-terminal end of the protease cofactor domain of several members of the Comoviridae was not detected in the putative Pro-C region (Ritzenthaler et al., 1991
). Further upstream, the 215K protein contains the HEL motifs A, B and C, and the RdRp motifs IVIII (Gorbalenya et al., 1990
; Koonin, 1991
).
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The most plausible locations for putative cleavage sites based on alignments with sequences of Comoviridae are at amino acids 465/6, 964/5, 989/90 and 1221/2, and are all of the type Q/G. Interestingly, the residue at position -2 in the first three sites is a glutamic acid (E), and in the last a glutamine (Q). Other motifs or conserved residues up- and downstream of these putative cleavage sites (Fig. 4a) were not found. There was only one other EQG tripeptide (residues 146/7), and three other QG dipeptides [1013/4 (AQG), 1248/9 (AQG) and 1675/6 (GQG)] (Fig. 4b
). Assuming the positions of the cleavage sites are correct the subsequent molecular mass of the cleaved proteins would be: 52031 Da, Pro-C; 57049 Da, HEL; 2838 Da, VPg; 25606 Da, PRO; 78629 Da, RdRp.
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Discussion |
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The genomic organization of SMoV, accepting at present the unknown organization of the RNA2-encoded polyprotein, is typical of Comoviridae. The striking characteristics are the large ORF of RNA2, and the long (1150nt), and highly homologous (95·7%) 3' NCRs of RNA1 and RNA2, both being unique (within the Comoviridae) to a handful of nepoviruses. Sequences of a PCR-amplified region of the 3' NCR of a dozen SMoV isolates gathered to date by us have also shown high levels of homology (results unpublished). A more favourable context for translation initiation at the second AUG of RNA2 has been similarly reported for ToRSV (Rott et al., 1991
), GFLV (Serghini et al., 1990
) and ALSV (Li et al., 2000
). A suboptimal AUG at the beginning of the large ORF is a common feature to all comoviruses sequenced so far (Goldbach & Wellink, 1996
). The nature of the putative cleavage sites in SMoV RNA1 is of particular interest. All the putative ALSV cleavage sites but one (EG) are QG with either the G or A a preceding residue (Li et al., 2000
). All plausible cleavage sites for RNA1 of SMoV are of the type QG, and this consistency appears to agree somewhat with that found with ALSV, particularly with regards to the amino acid in the -2 position. This apparent uniformity is not the case for Comoviridae and tentative members, where the number of possible sites is large (Wellink & van Kammen, 1988
; Mayo & Robinson, 1996
) and continues to grow; TS and AA dipeptides being recently identified as the cleavage sites between the large and small CPs of SDV and NIMV, respectively (Iwanami et al., 1998
). In RNA2 of SMoV, alignment with the CPs of SDV and related viruses (Fig. 6
) shows that there is a certain degree of homology between residues 529 and 741. QG dipeptides are found at 122/2, 219/20, 854/5, 1078/9, 1108/9 and 1320/1. These cleavage sites would not yield proteins of the expected sizes. In addition, they do not have an E or Q in the -2 position. Another alternative is the dipeptide EG, the best candidate of which is EEG (residues 357/8). If EG were the cleavage site the resulting CP would have an unusually large size (molecular mass 112 kDa). Unfortunately, attempts to experimentally determine the cleavage sites have not been successful due to an inability to purify the CP or CPs of SMoV.
Despite the obvious relationship between SMoV and SDV, SMoV is apparently more nepovirus-like due to the long 3' NCR, which is a characteristic of some, but not all, nepoviruses, and as yet unobserved in como- and fabaviruses, or in SDV. What may clarify the phylogenetic relationship of SMoV with other Comoviridae is a better understanding of the genomic organization of RNA2, the polyprotein of which is larger than that of como- or fabaviruses. In view of its apparent relationship with SDV and because of the points already discussed, SMoV RNA2 may code for two CPs, a feature present in all definitive como- and fabaviruses, and absent in definitive nepoviruses. The latter point does not taxonomically exclude SMoV from the Nepovirus genus, as there are numerous tentative species, including SDV, that code for multiple CPs (Mayo & Robinson, 1996 ). The most outstanding feature of SMoV, based its nepovirus-like organization, is that it is aphid-transmissible in a semi-persistent manner. Transmission by nematodes has only been demonstrated for around one-third of all nepoviruses. For the remaining two-thirds, except for one species, the mode of transmission either is unknown or has been shown to be via seed and pollen (Brown et al., 1996
). Interestingly, all fabavirus isolates are aphid transmissible in a non-persistent manner. As far as we know, there is only one example in the literature of a nepovirus being transmitted by aphids, that of TRSV by Myzus persicae Sulz. and Aphis gossyppi Glov. (Rani et al., 1969
). However, the virus in this study was not unequivocally identified as TRSV. There are nevertheless further reports of TRSV being transmitted by several arthropod vectors, namely, the grasshopper Melanoplus spp. (Dunleavy, 1957
), flea beetle Epitrix hirtipennis (Schuster, 1963
), and thrips Thrips tabaci (Messieha, 1969
). For SDV the mode of transmission is not clear, though is assumed, based on the spread of infection, to be soil-borne (Iwanami et al., 1999
). As for SMoV, there is no experimental evidence to show that it is nematode-transmissible; however the 162K polyprotein encoded by RNA2 contains the motif VQV at two positions, one at 212214 in the hypothetical MP, and the other at 727729 in the CP region. This motif was suggested to be involved in virus transmission by the nematode Xiphinema diversicaudatum (Micoletzky) Thorpe. (Kreiah et al., 1994
).
Relationships of the RdRps of SMoV and other viruses, different than Comoviridae, were most significantly with other viruses in the picornavirus superfamily, in particular the aphid- and leafhopper-transmitted RTSV from the family Sequiviridae. The polymerase of SMoV, SDV, ALSV and NIMV has a greater similarity to that of RTSV than to Comoviridae. The RdRp of Cricket paralysis virus (CrPV) from the novel genus of picorna-like viruses Cricket paralysis-like viruses (Wilson et al., 2000 ) and the unassigned Acute bee paralysis virus (ABPV) (Govan et al., 2000
), also showed a low but significant similarity with that of SMoV.
As to the classification of SMoV, the phylogenetic data seem to suggest that it probably belongs to the recently proposed SDV-like lineage of picorna-like viruses. In an evolutionary context the evidence so far gathered implies that the bipartite Comoviridae were formed as a result of a splitting of a Sequiviridae-like ancestral monopartite genome. This splitting event has probably occurred more than once so giving rise to separate lineages; the Comoviridae being one, and SDV-like viruses being another (Karasev et al., 2001 ). ALSV and other tentative Comoviridae may also have come into existence in the same way. If SMoV were to be included in the SDV-like lineage, it would add diversity to what is, at present, a very homogeneous group. Significant differences between SMoV and SDV, such as the more apparent nepovirus-like organization of SMoV RNA1 than in SDV, the long and conserved 3' NCR of SMoV, and the close relationship of the MP of SDV with como- and fabaviruses, all probably point to a horizontal transfer of genetic material as a result of adaptation to a particular host or vector. The unusual mode of transmission of SMoV when compared to Comoviridae might, for example, explain the lack of homology observed between the putative MP of SMoV, and the MPs of Comoviridae, assuming this protein has a role in vector specificity (Blok et al., 1992
).
In conclusion, based on its similarity with SDV, we suggest that SMoV be, for the present, included as a tentative member of the SDV-like lineage of picorna-like viruses. The close identity of SMoV and SDV-like viruses with the aphid-transmissible RTSV suggests that these viruses form a new group either between the Sequiviridae and the Comoviridae, or within the Comoviridae. The aphid transmissibility of SMoV may also have implications for SDV and ALSV, the transmission of which is still unknown.
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Acknowledgments |
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Footnotes |
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Received 27 July 2001;
accepted 3 October 2001.