the complete nucleotide sequence of subterranean clover mottle virus

Arch Virol (2003) 148: 2237–2247DOI 10.1007/s00705-003-0144-3

The complete nucleotide sequence of Subterraneanclover mottle virus

G. I. Dwyer1,4, R. Njeru1, S. Williamson1, J. Fosu-Nyarko1, R. Hopkins1,R. A. C. Jones2, P. M. Waterhouse3, and M. G. K. Jones1

1Western Australian State Agricultural Biotechnology Centre, Division of Scienceand Engineering, Murdoch University, Perth, Australia

2Department of Agriculture, Plant Pathology Branch, South Perth, Australia3CSIRO, Division of Plant Industry, Canberra, Australia

4Centre for Bioinformatics and Biological Computing, Division of Business,Information Technology and Law, Murdoch University, Perth, Australia

Received December 17, 2001; accepted April 15, 2003Published online August 7, 2003 c© Springer-Verlag 2003

Summary. The complete nucleotide sequence of Subterranean clover mottlevirus (SCMoV) genomic RNA has been determined. The SCMoV genome is4,258 nucleotides in length. It shares most nucleotide and amino acid sequenceidentity with the genome of Lucerne transient streak virus (LTSV). SCMoVRNA encodes four overlapping open reading frames and has a genome organ-isation similar to that of Cocksfoot mottle virus (CfMV). ORF1 and ORF4 arepredicted to encode single proteins. ORF2 is predicted to encode two proteinsthat are derived from a −1 translational frameshift between two overlappingreading frames (ORF2a and ORF2b). A search of amino acid databases didnot find a significant match for ORF1 and the function of this protein remainsunclear. ORF2a contains a motif typical of chymotrypsin-like serine proteases andORF2b has motifs characteristically present in positive-stranded RNA-dependentRNA polymerases. ORF4 is likely to be expressed from a subgenomic RNAand encodes the viral coat protein. The ORF2a/ORF2b overlapping gene expres-sion strategy used by SCMoV and CfMV is similar to that of the polerovirusesand differ from that of other published sobemoviruses. These results suggestthat the sobemoviruses could now be divided into two distinct subgroups basedon those that express the RNA-dependent RNA polymerase from a single,

The sequence data reported in this paper has been deposited in the GenBank nucleotidedatabase under the accession number AF208001.

2238 G. I. Dwyer et al.

in-frame polyprotein, and those that express it via a −1 translational frameshiftingmechanism.

Introduction

Subterranean clover mottle virus (SCMoV) is the most widespread viral pathogenof annual pastures based on Trifolium subterraneum L. (subterranean clover) insouthern States of Australia [24, 8, 9]. It has a narrow host range and, in thefield, has only been found infecting T. subterraneum and some wild and othercultivated Trifolium species [6, 23, 5]. Diseased plants develop symptoms ofmottling, leaf distortion and stunting [23]. The virus is readily transmitted bygrazing and trampling of stock, on the wheels of vehicles and by mowing [14].No specialised vector is known. SCMoV persists readily from year to year ininfected pastures via seed transmission in clovers but is only seed-borne at lowlevels (<0.1 to 0.5%) [23, 24, 17]. Seedlings infected via the seed act as theprimary source for virus spread. The virus decreases seed and herbage productionand diminishes pasture regeneration. Over time, it results in a decline of pastureswhich become weedy and unproductive [3, 4, 8].

SCMoV is considered to belong to the genus Sobemovirus. SCMoV virionsare isometric, about 25 nm in diameter, and comprise 20% nucleic acid and 80%protein [9]. The virus genome consists of a linear single-stranded, positive-senseRNA of Mr 1.5 × 106 [6, 16]. A genome-linked protein (VPg) is covalentlyattached to the 5′ terminus of the RNA molecule and the 3′ end is not polyadenyl-ated. In addition to genomic RNA, two viroid-like satellite RNAs [2, 6] and anuncharacterised RNA species of about 700 nucleotides [16] have been isolatedfrom virus particles. The satellites replicate by a rolling circle mechanism andpossess a hammerhead type ribozyme activity [22]. Serologically, SCMoV virionsare distantly related to those of Lucerne transient streak virus (LTSV) but not tothose of other sobemoviruses [6].

In this paper we report the cloning and sequencing of the full-length genomicRNA molecule of Western Australian SCMoV isolate P23. Nucleic acid and de-duced amino acid sequence comparisons with published sobemovirus sequencesconfirmed that SCMoV is a sobemovirus. However, a comparison of predictedmajor open reading frames (ORF) indicate that its genome organisation is similarto that of Cocksfoot mottle virus (CfMV) and differs from those of other publishedsobemoviruses.

Materials and methods

SCMoV isolate P23 [23] was maintained in T. subterraneum cv. Woogenellup plants ina temperature-controlled glasshouse at 20(±5) ◦C. The virus was subcultured at regularintervals by sap inoculation onto T. subterraneum plants using extracts from SCMoV-infectedleaves ground in 100 mM potassium phosphate buffer, pH 7.4, mixed with diatomaceous earthbefore gently rubbing the mixture on the upper leaf surface. SCMoV-P23 virions were purifiedfrom leaves of systemically infected T. subterraneum cv. Woogenellup plants by sucrosedensity gradient centrifugation and the purified particles stored at 4 ◦C in 20 mM phosphate

The complete nucleotide sequence of SCMoV 2239

buffer (pH 7.4) [16]. Genomic RNA was extracted from purified viral particles with phenol–chloroform, size fractionated in a 1% low melting point formaldehyde–agarose gel and RNApurified from the gel using phenol/chloroform (pH 8) extraction, ethanol precipitated andstored at −80 ◦C in absolute ethanol.

Four different cDNA synthesis methods were used to amplify SCMoV-P23 genomic RNAfor cloning according to the manufacturer’s procedures. 1. Purified genomic RNA was usedas template for first-strand cDNA synthesis using a Riboclone cDNA synthesis kit (Promega).2. The 5′ terminal sequence was amplified and cloned using a 5′ RACE system (Life Technolo-gies). First-strand cDNA was synthesised using a downstream gene-specific primer (Gsp1,5′-GACTATATGCAGCTCGTGTG) designed from known sequence data. Second-strandcDNA was amplified using the nested downstream primer (Gsp2, 5′-CACACAGAATTCTAGCCTTCTGGGCCACTGTCTCTG) and an upstream abridged anchor primer (AAP) comple-mentary to the homopolymeric dC-anchor sequence added to the 5′ terminus of the first-strandcDNA (AAP, 5′-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG). 3. The 3′ terminalsequence was polyadenylated using poly(A) polymerase (Pharmacia). First-strand cDNA syn-thesis was done using Superscript II RNase H− Reverse Transcriptase (Life Technologies) andthe primer 5′-CTCCAGAATTCGAGCTC(dT25), which includes EcoRI and SacI restrictionsites at the 5′ end for cloning purposes. An upstream SCMoV-specific primer containingHindIII and SphI restriction sites (5′-CAGGAAAGCTTGCATGCGTCCTGCAGCAGAGG)was used for second-strand cDNA synthesis. 4. The intervening regions of the SCMoV genomewere amplified by RT-PCR using primers designed from sequenced clones. All RT-PCRfragments were cloned into pGEM-T (Promega).

Cloned cDNAs were sequenced from both strands using the dideoxynucleotide chain-termination method [20]. Sequencing was done with an Applied Biosystems Model 373automated sequencer using ABI PRISM Dye Terminator Cycle Sequencing Ready ReactionKit, With AmpliTaq DNA Polymerase, FS (Applied Biosystems). Sequence ambiguities wereresolved by sequencing RT-PCR fragments directly amplified from genomic RNA. Rawsequence data was analysed using the SeqEd Version 1.0.3 software (Applied Biosystems).

Table 1. Sobemoviruses that have been completely sequenced, including public databaseaccession numbers and references where applicable

Sobemovirus Acronym Acc. No. Reference No.

Cocksfoot mottle virus CfMV-JP AB040447 Zhang and Toriyama,unpublished

CfMV-NO Z48630 Makinen et al., 1995 [13]CfMV-RU L40905 Ryabov et al., 1996 [19]

Lucerne transient streak virus LTSV U31286 Jeffries et al., unpublishedRice yellow mottle virus RYMV-CI L20893 Yassi et al., 1994 [25]

RYMV-NG U23142 Pinto and Baulcombe,unpublished

Rygrass mottle virus RGMoV AB040446 Zhang et al., 2001 [26]Sesbania mosaic virus SeMV AY004291 Lokesh et al., 2001 [12]Southern bean mosaic virus SBMV-BARK AF055887 Lee and Anderson, 1998 [11]

SBMV-S AF055888 Lee and Anderson, 1998 [11]SBMV-B L34672 Othman and Hull, 1995 [18]

Southern cowpea mosaic virus SCPMV M23021 Wu et al., 1987Subterranean clover mottle virus SCMoV AF208001 this paper


Sequence comparisons were done using the Wisconsin GCG software suite version 9.1made available through the Australian National Genomic Information Service (ANGIS,http://www.angis.org.au/). The complete SCMoV cDNA sequence was translated in threeforward reading frames using MAP. Nucleotide and amino acid sequence multiple alignmentswere compiled using PILEUP and alignment consensus sequences generated using eitherPRETTY or PRETTYPLOT. Percent nucleotide identity and amino acid similarities werederived from pairwise alignments using GAP (note: PILEUP and GAP both use the algorithmof Needleman and Wunsch [15]). Predicted protein molecular weights (Mr) were calculatedusing PEPSTATS. The BLASTn program [1] was used to determine the sense strand inoverlapping SCMoV clones and the position of clones along comparative sobemovirus RNAgenomes.

The complete genome sequences for seven sobemoviruses have been submitted to thepublic databases (Table 1). Lee andAnderson [11] reported considerable sequence differencesbetween the SBMV-BARK and SBMV-B [18] and concluded that the differences probablyresult from sequencing errors in SBMV-B. Our sequence analyses also indicate considerablediscrepancies. For this reason, sequence comparisons against SBMV-B were only done withthe SBMV-BARK isolate.

Results and discussion

The complete SCMoV-P23 genomic RNA was 4258 nucleotides in length. SC-MoV had a base composition of adenine (28%), guanine (25%), uracil (24%) andcytosine (23%) with an overall G + C content of 48%. The SCMoV 5′ and 3′untranslatable regions (UTR) were 68 and 177 nucleotides in length, respectively.The first AUG codon in the SCMoV genomic RNA molecule occurs at nucleotideposition 69–71 and is preceded by an A + U rich (68%) 5′ UTR of 68 nucleotides.The SCMoV 3′ UTR began at position 4082 and was 174 nucleotides in length.Nucleotide and amino acid comparisons of SCMoV-p23 genomic sequences con-firmed SCMoV as a sobemovirus (Table 2) that was most closely related toLTSV. Except with LTSV, individual SCMoV ORF sequences were not closelyrelated to those of other sobemoviruses. There was little conservation for ORF1.

Table 2. Comparisons of SCMoV ORFs with those of published sobemovirus sequences.The ORF columns show percent amino acid identity and similarity (in parentheses). ORF3/4

refers to the coat protein gene

Virus ORF1 ORF2a ORF2b ORF4

CfMV-NO 19.4 (40.7) 30.8 (48.9) 52.4 (68.0) 23.9 (44.5)CFMV-RU 20.4 (38.5) 30.6 (49.5) 52.2 (67.9) 24.0 (44.7)CFMV-JP 12.6 (38.8) 30.5 (49.0) 52.2 (67.4) 24.0 (44.7)RGMoV 21.1 (41.3) 31.3 (48.8) 46.7 (63.4) 22.6 (42.6)RYMV-CI 16.4 (37.3) 28.0 (47.6) 49.7 (64.1) 24.3 (46.8)RYMV-NG 17.1 (36.3) 27.4 (48.6) 50.2 (64.0) 27.4 (47.6)SBMV-BARK 16.0 (39.7) 32.8 (52.8) 45.6 (61.2) 29.0 (50.8)SeMV 16.8 (39.6) 33.5 (53.7) 45.7 (59.8) 30.3 (52.2)SCPMV 17.3 (37.6) 34.5 (53.9) 42.5 (61.1) 30.6 (50.0)LTSV 28.3 (46.0) 48.9 (65.8) 54.1 (67.4) 49.2 (66.5)


For ORF2a and ORF4, those of SCMoV were more closely related to virusesthat infect dicotyledonous plants. In contrast, its ORF2b shares slightly higherhomology with those of viruses that infect monocotyledonous plants.

SCMoV coding regions

Translation of the RNA sense strand in three forward reading frames revealedfour potential ORFs (Fig. 1). ORF1 and ORF4 were predicted to encode singlepolypeptides while ORF2 comprised two overlapping reading frames termedORF2a and ORF2b (based on the convention given for CfMV). There was alsoa large stretch of 134 amino acids (nucleotides 451 to 858 in the +1 frame) thatoverlapped ORF1 and ORF2a but did not contain an initiating AUG codon. Thepolypeptide encoded by SCMoV ORF1 was predicted to begin at nucleotides69–71, end at nucleotides 602–605, and encode a protein of 179 amino acidswith a calculated Mr of 20.32 kDa. Comparison of the SCMoV ORF1 amino acidsequence indicated that, except with LTSV which shares 28% identity and 46%similarity, there was little similarity with other sobemoviruses (Table 2). A searchof the nucleotide and protein databases did not reveal any entry with significantidentity and/or similarity to the SCMoV ORF1 sequence, so it was not possibleto assign a putative function for this ORF. SCMoV and CfMV have an ORF2a/2bgenome organisation whilst those of the other published sobemoviruses containa single ORF2 polyprotein with a small overlapping ORF3 gene. Except withCfMV, it was difficult to make direct comparisons between the SCMoV ORF2aand ORF2b coding sequences that span the overlap region and those of othersobemoviruses. To simplify this process, separate pairwise alignments were donebetween the SCMoV ORF2a (nucleotides 605 to 2344) and ORF2b (nucleotides1849 to 3501) amino acid sequences and the entire ORF2 coding regions ofeach sobemovirus (see Table 2). These comparisons revealed that the SCMoV

Fig. 1. SCMoV genome organisation. The ORFs are labelled and shown as dark boxes. VPgattached to the 5′ terminus is shown as a filled ellipse. The position and length of 5′ and3′ UTRs are indicated. Location of the chymotrypsin-like serine protease domain, RNA-

dependent RNA polymerase domain (RdRp) and coat protein are indicated


ORF2a sequence is more closely related to that of LTSV with 49% identity(66% similarity) for ORF2a and 54% identity (67% similarity) for ORF2b. Incontrast, the SCMoV ORF2a sequence shared only 27 to 31% identity (48 to50% similarity) with those of viruses that infect monocots and 33 to 35% identity(53 and 54% similarity) with those of viruses infecting dicots. Conversely, theSCMoV ORF2b sequence shared 43 to 46% identity (63 to 69% similarity) withthose of viruses infecting dicots and 47 to 53% identity (60 to 61 similarity)with those of viruses infecting monocots. Beginning at amino acid residues wherethe frameshift commences (KLPA. . .), the small overlapping ORF3 sequences ofother sobemoviruses were then compared with the SCMoV ORF2b sequence.Except with RYMV, there was considerable identity over the first 90 to 100amino acids with good conservation over the next 50 to 60 amino acids but nosimilarity beyond this point (Fig. 2). The polypeptide encoded by the SCMoVORF4 was predicted to begin at nucleotides 3323–3325, end at nucleotides 4079–4081, and encode a protein of 253 amino acids with a calculated Mr of 27.3 kDa.The SCMoV ORF4 overlaps ORF2b by 218 nucleotides. This ORF shares mostidentity (49%) and similarity (66%) with the LTSV ORF4, followed by those ofother dicot infecting viruses (29 to 31% identity and 50 to 52% similarity), andthen those of the monocot infecting viruses (23 to 27% identity and 43 to 48%similarity).

Expression of SCMoV proteins

Comparative analyses of the SCMoV genome with genomic sequence and struc-tural information reported for the other sobemoviruses was used to construct anoverview of the likely strategies that SCMoV uses for gene expression (Fig. 3).For example, a comparison of the sequence context surrounding the AUG codonsof different sobemoviruses is shown in Table 3. In a bioinformatics study of 5074plant genes, Joshi et al. [10] found that purines (A or G) were present at the −3 and+4 position 80% of the time. The authors generated a translation initiation contextfor dicots (Aa/caAUGGCu) and monocots (A/Ga/ccAUGGCG). For dicots, an Aat postion −3 occurred 60% of the time, a G at +4 occurred 67% of the time anda C at +5 occurred 53% of the time. The GC at +4 and +5 form the first twobases of an alanine codon. Pyrimidines (C/U) at −3 were regarded as unfavorableor poor context. Based on these observations, SCMoV ORF1 has an unfavorablecontext, as with all sobemoviruses (Table 3). This fits translation initiation ofsobemovirus ORF1 and ORF2 proteins occuring via a leaky ribosomal scanningmechanism [21].

The SCMoV ORF2 coding region is comprised of two overlappingreading frames, ORF2a and ORF2b (Fig. 1). ORF2a contains a characteristicchymotrypsin-like serine protease motif [H(x25)[D/E](x70−80)T[R/K]xGxSG]and ORF2b a characteristic RNA-dependent RNA polymerase motif [GxxxTxxxN(x19)GDD]. The genome arrangement and mode of expression for theprotease and polymerase genes is similar to that of the Poleroviruses, Potatoleafroll virus (PLRV) and Beet western yellows virus (BWYV) (previously known


Fig. 2. Partial SCMoV and CfMV ORF2b sequence alignment with complete ORF3sequences of six sobemoviruses. The alignment begins at the point of the frameshift. Amino

acids with similar properties are shown in the same colour

as subgroup II luteoviruses). Three possible methionine residues could be usedto initiate translation of the SCMoV ORF2a gene product. Translation from thefirst AUG codon at position 605–607 in an ORF2a product of 580 amino acidswith a calculated Mr of 63.76 kDa, translation from the second at position 644–646 in a product of 567 amino acids with a calculated Mr of 62.13 kDa, andtranslation from the third at position 662–664 would result in an ORF2a productof 561 amino acids with a calculated Mr of 61.53 kDa. For SCMoV, the thirdcodon had an optimal context. Possibly, it is the actual one used to initiatetranslation. However, for this to occur the ribosome would have to ignore the


Fig. 3. Overview of possible mechanisms used by SCMoV for gene expression. Translationinitiation from genomic (gRNA) and subgenomic RNA (sgRNA) occurs via a 5′ end-dependentribosomal scanning mechanism. The ORF1 initiation codon has suboptimal context allowingtranslation of ORF2 to occur via a leaky ribosomal scanning mechanism. ORF2b is expressedvia a −1 ribosomal frameshifting mechanism and the individual mature proteins are releasedthrough proteolytic digestion by the protease encoded by ORF2a. ORF 4 is expressed from a

subgenomic RNA molecule

Table 3. Nucleotide context surrounding sobemovirus translation initiation codons. Translationinitiation codons are shown in bold, the −3 and +4 nucleotides are underlined, the AUG columnrefers to the number of times a methionine residue is present in the first 80 amino acids of ORF2/2a.The ORF2.1, ORF2.2 and ORF2.3 columns show the context surrounding the first 3 AUG codons in

ORF2 sequences

Virus ORF1 AUG ORF2.1 ORF2.2 ORF2.3 ORF4

CfMV-NO UAGAUGU 7 AGAAUGG GUGAUGU UUGAUGA GAAAUGACfMV-RU UAGAUGU 7 AGAAUGG GUGAUGU UUGAUGA GAAAUGACfMV-JP UAGAUGU 7 AGAAUGG UUGAUGA AGGAUGA GAAAUGARYMV-CI UGUAUGA 3 GGGAUGG CCUAUGU UCCAUGC AAGAUGGRYMV-NG UGUAUGA 2 GGGAUGG CCCAUGU none AAGAUGGRGMoV UUUAUGC 2 AAGAUGU AGCAUGG none AAGAUGGSCPMV UUCAUGA 3 AGAAUGU AACAUGU UUGAUGA GUAAUGGSBMV-BARK UGCAUGA 2 ACAAUGU UGGAUGA none GCUAUGGSeMV UUGAUGC 5 AGAAUGU AATAUGG CUUAUGG CCUAUGGLTSV UGUAUGC 5 AAGAUGC CCGAUGU UCGAUGC AAAAUGGSCMoV UAUAUGC 4 AGAAUGA UGGAUGA AGCAUGG AAAAUGA

two upstream AUG codons that fall less than 20 residues away. The real initiationcodon will therefore only be identified through N-terminal sequencing of theexpressed ORF2a protein.


Ribosomal frameshifting is a strategy to produce more than one protein fromoverlapping reading frames that is frequently employed by viruses. The presenceof a consensus heptanucleotide frameshifting sequence two thirds of the wayalong ORF2a (nucleotides 1846–1852) suggests that ORF2b is expressed via a−1 ribosomal frameshifting mechanism. This gene expression strategy is similarto those of CfMV and of some poleroviruses but differs from those of othersobemoviruses (Fig. 3). Thus, at least two individual protein products are likelyto be expressed from the ORF2a/2b genes, the first a product of the ORF2a geneand the second a large polyprotein comprised of the ORF2a gene, as far as the −1frameshifting site, and the entire ORF2b coding region.

The sobemovirus coat protein, encoded by the last ORF in the genome, isexpressed from a subgenomic RNA molecule via a 5′ end-dependent ribo-somal scanning mechanism [7]. For SCMoV, the translation context of ORF3 is not optimal, however, because there are no other methionine residues inthe N-terminal region, this codon is probably the one used for translationinitiation.

The results from this study clearly show that SCMoV is a member of thegenus Sobemovirus. However, its genome organisation and putative mode of geneexpression differs from those of other sobemoviruses with the exception of CfMV.The presence of a large overlapping region suggest that the sobemoviruses couldnow be divided into two distinct subgroups based on those that express the RNA-dependent RNA polymerase from a single, in-frame polyprotein, and those thatexpress it via a −1 translational frameshifting mechanism.

Acknowledgments

This research was supported in part by theAustralian Research Council Small Grants Scheme,CSIRO Division of Plant Industry, Canberra and Murdoch University, Perth.

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Author’s address: Dr. Geoffrey Dwyer, Plant Biotechnology Research Group (PBRG),Murdoch University, Perth, 6150, WA, Australia; e-mail: [email protected]

the complete nucleotide sequence of subterranean clover mottle virus

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