Arch Virol (2015) 160:857–863 DOI 10.1007/s00705-015-2336-z
BRIEF REPORT
Rottboellia yellow mottle virus is a distinct species within the genus Sobemovirus Merike So˜mera • Erkki Truve
Received: 16 September 2014 / Accepted: 8 January 2015 / Published online: 23 January 2015 Ó Springer-Verlag Wien 2015
Abstract Once considered a tentative member of the genus Sobemovirus, rottboellia yellow mottle virus (RoMoV) was excluded from the latest species list of the ICTV after the discovery of imperata yellow mottle virus (IYMV), which resembles RoMoV in host range and geographic origin. Here, sequence analysis of the complete genome of RoMoV suggested that it should be considered a distinct species within the genus Sobemovirus. It has the highest sequence identity (55 %) to ryegrass mottle virus (RGMoV), whereas its sequence identity to IYMV is lower (44 %). In a phylogenetic tree, RoMoV clusters together with RGMoV and artemisia virus A (ArtVA), a dicotinfecting sobemovirus. Keywords RoMoV RGMoV IYMV ArtVA Sobemovirus Rottboellia yellow mottle virus (RoMoV; formerly abbreviated as RoYMV) was isolated from itchgrass (Rottboellia cochinchinensis) in 1981 in Nigeria [1]. A host-range study showed that it was also able to infect maize. RoMoV was identified as a positive-sense ssRNA plant virus with isometric particles measuring about 29 nm in diameter. Based on similarities in particle properties, the size of its coat protein subunits, the size and type of its genomic RNA, the distribution of virus particles within the cell, and its narrow host range, RoMoV was proposed to be a member of the genus Sobemovirus [1]. In 2005, it was entered into a list of tentative sobemovirus species [2].
M. So˜mera (&) E. Truve Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia e-mail: [email protected]
In 2000, an isolate of imperata yellow mottle virus (IYMV) was found in Burkina Faso [3]. IYMV was collected from cogongrass (Imperata cylindrica) and maize plants. Experimentally, it was also shown to infect itchgrass. Although there were similarities between IYMV and RoMoV in symptoms, virus particle size, and host range, it was not possible to classify these viruses because no sequence data on RoMoV were available [3]. Consequently, RoMoV was left out from the latest list of sobemovirus species accepted by the International Committee on Taxonomy of Viruses (ICTV) [4]. In the current study, we sequenced the complete genome of RoMoV. Sweet maize (Zea mays subsp. saccharata) cv. Golden Bantam was used as experimental host for RoMoV propagation. For mechanical inoculation onto the leaves of 2-week-old plants, virus inoculum was prepared by grinding 0.1 g of lyophilized infectious plant material (DMSZPV-0496; supplied by G. Thottappilly) in 0.5 ml of 0.1 M phosphate buffer (pH 7.0) containing 0.5 % celite. Plants were grown in a climate chamber (16 h daylight at 28 °C, 8 h darkness at 18 °C). Visible symptoms of infection (Fig. 1A–D)—chlorotic mottle and stunting—appeared at 15 days post-inoculation (dpi). In the case of fresh infectious material, the symptoms appeared already at 7 dpi. In agreement with a previous study [1], no seed transmission was detected. Also, the susceptibility of cogongrass plants to RoMoV inoculation was examined. No particular symptoms were observed (Fig. 1e). However, one of six plants tested in our experiment was positive by RT-PCR for RoMoV infection at 40 dpi (Fig. 1f), confirming that the host range of both RoMoV and IYMV includes cogongrass, itchgrass and maize. Virus particles were purified from symptomatic maize plants at 35 dpi as described [5]. Virions were dissociated with 1 % SDS, and viral RNA was isolated by phenol/
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Fig. 1 A–D Symptoms of RoMoV infection in maize plants. A Chlorotic mottling. B Severe chlorosis in older leaves. C Severe stunting and chlorosis of inflorescence potentially leading to yield reduction. D Mild infection resulting in no obvious stunting. E– F RoMoV-inoculated cogongrass plants (samples 2–7). Sample 8 represents a non-inoculated plant. E No visible symptoms occurred in an RoMoV-infected plant (sample no. 5). F A 334-bp fragment
corresponding to positions 2702 to 3036 of the RoMoV genome was amplified by RT-PCR using RoMoV-specific primers (50 -GCA TATCGCGCGACAAAATCT-30 and 50 -CCTGTGCATCTGGCA CAAAG-30 ) from systemic leaves in one of six inoculated plants. Sample 1 represents an RoMoV-infected maize plant used as a positive control; M, GeneRuler 100 bp Plus Marker (Thermo Scientific)
chloroform extraction. The sobemovirus genus-specific primer pair SOBDF2/SOBDR2 [6] was used to amplify a conserved region in the RNA-dependent RNA polymerase (RdRp) gene. The amplified fragment was sequenced and used to design RoMoV-specific primers RoB_RpF (50 -GT CCAACATTGGGAACTGATGGAGG-30 ) and RoB_RpR
(50 -TGGCCATCAAGCAGCGGATACG-30 ). RoB_RpR was used for 50 -specific first-strand cDNA synthesis from viral RNA using Maxima Reverse Transcriptase (Thermo Scientific). The first-strand synthesis product was ligated with the self-priming anchor primer SP-ANC [7] using T4 RNA ligase. The cDNA fragment that was obtained was
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Sequence of rottboellia yellow mottle sobemovirus
amplified using RoB_RpR and the anchor-nested primer ANC [7] in the PCR step. In parallel, SP-ANC was ligated with viral RNA to create a template for genomic 30 -specific first-strand cDNA synthesis. After the first-strand synthesis, primers ANC and RoB_RpF were used for PCR. Both 50 and 30 -specific PCR products were subcloned and sequenced using a primer-walking strategy. To confirm the sequencing results, a genome-wide set of primers was designed (not shown) and utilized for direct sequencing of the RT-PCR products amplified from viral RNA. Additional 50 and 30 RACE reactions were carried out using the SP-ANC primer and virus-specific primers oriented toward the ends of the genome. Assembly of the set of genome-wide sequences revealed that the RoMoV genome consists of 4194 nt (deposited in GenBank under accession no. KC577469). The RoMoV genome organization was found to be characteristic of sobemoviruses (Fig. 2A). Open reading frames (ORFs) were identified using the NCBI ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The parameters were set to use the standard genetic code, with a minimum of 100 codons. The ORFx-encoding region [8], overlapping with ORF1 and ORF2a/2a2b was identified using EMBOSS Sixpack (http://www.ebi.ac.uk/Tools/st/ emboss_sixpack/). The sequence of the 50 end of the genome was identified as ACAAAA. The 50 ACAAA(A) motif is common for sobemo-, polero-, and polero-like dianthoviruses. It is also present in the RoMoV sequence at nt position 3,315–3,319, shortly before the beginning of ORF3, marking the possible beginning of sgRNA [9]. The 50 untranslated region (UTR) of the RoMoV genome is 96 nt long, whereas the 30 UTR has 163 nt. RoMoV ORF1 encodes a putative P1 protein with a predicted molecular mass of 17.1 kDa. Like other sobemoviral P1 proteins, the RoMoV P1 is very divergent and does not share significant similarity with any known proteins. Despite being very divergent, the P1 proteins of many sobemoviruses have been demonstrated to be involved in virus spread [10–12] and RNAi suppression [13–16]. A PSI-BLAST (http://blast.ncbi.nlm.nih.gov/) homology search against non-redundant (nr) protein sequences in the NCBI database indicated that the only similarity was to IYMV P1, with an E-value slightly lower than the threshold value (E = 0.003; score, 44.3; query coverage, 61 %; 32 % identity). The sobemovirus ORF1 is in a suboptimal context, allowing the ribosome to continue scanning and initiate translation from downstream, in ORF2a/ORF2a2b [17]. ORF2a encodes a putative polyprotein, P2a, with a calculated molecular mass of 61 kDa. A PSI-BLAST search against the NCBI nr database using the sequence of RoMoV P2a gave the best hit against ryegrass mottle sobemovirus (RGMoV) P2a (E = 2e-143; score, 436; query
859
coverage, 89 %; 48 % identity). The values for the alignment of RoMoV P2a and IYMV P2a were E = 3e-16; score, 90.5; query coverage, 89 %; 25 % identity. Like other sobemoviral P2a polyproteins [18], the N-terminus of RoMoV P2a shows no obvious conservation. Transmembrane helix prediction tests indicated that it is probably involved in the formation of a transmembrane anchor composed of two transmembrane segments (at aa positions 4–20 and 34–51). The sequence of the putative transmembrane anchor region is followed by motifs that are characteristic of serine proteases (H(X32)D(X69)TXXGXSG) and viral genome-linked protein VPg (WADMVEAEEDMWD). The non-conserved C-terminal part of P2a has been predicted to bind nucleic acids [19]. Studies on sesbania mosaic sobemovirus (SeMV) have shown that autoproteolysis of the polyprotein is needed for viable infection [20]. Amino acid sequencing of VPg proteins purified from sobemovirus particles or analysis of cleavage-site mutants has confirmed cleavage at E/S, E/T and E/N sites [21–23]. For RoMoV P2a, the putative cleavage sites at the VPg ends can be predicted for positions E319/S320 and E398/T399 based on sequence alignments (data not shown) with other sobemoviral P2a proteins. ORF2b contains characteristic RdRp motifs (SGSYCTSSSNX19GDD). The putative RdRp can be translated only as part of P2a2b via -1 ribosomal frameshifting from ORF2a to ORF2b. The frameshifting signal of sobemoviruses consists of the uniformly conserved slippery sequence UUUAAAC followed by a stem-loop [24]. The calculated molecular mass of the RoMoV P2a2b polyprotein is 104.3 kDa. A PSI-BLAST search against the NCBI nr database using the sequence of RoMoV P2a2b gave the best hit against RGMoV P2a2b (E = 0.0; score, 1009; query coverage, 94 %; 58 % identity). The values for the alignment of RoMoV P2a2b and IYMV P2a2b were E = 1e-165; score, 522; query coverage, 89 %; 39 % identity. A recent search for hidden ORFs in sobemovirus genomes resulted in the discovery of a region called ORFx, which appears to have a non-AUG initiation codon in a favourable context shortly before the end of ORF1 in the ?1 frame [8]. RoMoV has four potential non-AUG initiation codons adjacent to each other in the ORFx frame. The one that is in the best position also has the best context for initiation (here, GAGGUGG). The calculated molecular mass of RoMoV protein X is 11.9 kDa. A PSI-BLAST homology search against the nr database at NCBI identified no similarities to viral proteins having an E-value below the threshold value. The last ORF, ORF3, encodes the viral coat protein (CP), with a predicted molecular mass of 25.3 kDa. In SDS-PAGE, the estimated molecular mass of RoMoV CP
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Fig. 2 A The genome organization of RoMoV. B Phylogenetic tree showing the grouping of RoMoV within the genus Sobemovirus. A maximum-likelihood tree of predicted P2b sequences was generated using MUSCLE alignment and the LG amino acid substitution model [36] using Seaview 4.4.2 (http://doua.prabi.fr/software/seaview). The scale bar represents expected substitutions per site. Bootstrap scores (500 replicates) are shown at the nodes only when [70 %. P2b sequences (starting from the ribosomal frameshift site) were segregated from the following P2a2b or P2b sequences obtained from GenBank: artemisia virus A (ArtVA, AFI72874.1); cocksfoot mottle virus (CfMV, ABG73619.1); imperata yellow mottle virus (IYMV, CAQ48413.1); lucerne transient streak virus (LTSV, AGP50163.1);
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papaya lethal yellowing virus (PLYV, AFP67700.1); rice yellow mottle virus (RYMV, CAE81329.1); ryegrass mottle virus (RGMoV, ABN09950.1); sesbania mosaic virus (SeMV, AAG01370.2); southern bean mosaic virus (SBMV, ABI53037.2); southern cowpea mosaic virus (SCPMV, NP_042302.3); soybean yellow common mosaic virus (SYCMV, AEO16607.1); sowbane mosaic virus (SoMV, ADD64689.1); subterranean clover mottle virus (SCMoV, AAN51929.1); turnip rosette virus (TRoV, AGP50163.1); velvet tobacco mottle virus (VTMoV, ADN19015.1). The predicted RoMoV P2a2b sequence has GenBank accession no. AHB64342. The Solanum nodiflorum mottle virus (SNMoV) P2a2b sequence will be available soon as in GenBank acc. no AHB64347
Sequence of rottboellia yellow mottle sobemovirus
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Table 1 Percent identity between complete genome sequences of sobemoviruses based on MUSCLE multiple sequence alignment performed at the EMBL-EBI website RoMoV
RGMoV
ArtVA
PLYV
SoMV
SYCMV
SBMV
SeMV
SCPMV
RYMV
IYMV
CfMV
LTSV
SCMoV
TRoV
VTMoV
SNMoV
RoMoV 4194 bp RGMoV 4212 bp
55.1
ArtVA 4138 bp
50.8
48.4
PLYV 4145 bp
46.9
46.6
47.5
SoMV 3983 bp
45.0
45.1
46.7
48.1
SYCMV 4152 bp
46.6
46.7
47.7
51.0
45.7
SBMV 4132 bp
46.2
46.7
46.9
49.9
50.2
70.7
SeMV 4148 bp
46.8
45.9
47.2
49.6
50.4
70.9
71.9
SCPMV 4193 bp
46.0
46.7
47.1
48.6
49.4
59.9
59.4
58.8
RYMV 4448 bp
44.4
44.8
45.7
45.5
45.8
46.9
47.2
46.4
45.9
IYMV 4547 bp
43.9
43.8
44.9
45.8
45.0
47.3
46.4
46.3
45.2
52.3
CfMV 4082 bp
45.0
45.2
44.6
46.3
45.6
46.2
46.6
45.5
46.8
49.5
49.7
LTSV 4279 bp
46.4
45.7
46.5
50.5
47.1
47.7
47.4
47.9
47.4
45.9
44.6
45.8
SCMoV 4258 bp
44.8
45.3
47.1
49.7
59
46.9
47.8
47.7
47.9
45.5
44.5
46.1
56.7
TRoV 4035 bp
44.8
43.9
45.8
47.4
47.1
47.2
46.7
46.8
46.4
44.8
44.4
44.8
46.3
46.2
VTMoV 4247 bp
44.3
44.3
46.4
47.9
47.1
47.6
46.7
46.2
47.4
45.4
44.6
46.2
46.5
47.1
45.9
SNMoV 4297 bp
45.2
45.0
46.5
48.2
47.2
48.7
47.3
47.4
48.3
45.1
45.1
45.2
47.9
46.3
46.0
was 27 kDa [1]. ORF3 of sobemoviruses is translated from a sgRNA [25–28]. Like other sobemoviral CPs, the N-terminus of RoMoV CP is rich in arginine and lysine residues. It has been shown to be required for RNA interaction and encapsidation, association with membranes, and RNA transport, including nuclear localization [29–31]. A basic region followed by two b-strands forms a structure called a b-annulus, also known as the R (random) domain. It can be completely or partially disordered [32–34]. The subsequent S (shell) domain of the RoMoV CP is a core building block of virions that has a jellyroll b-sandwich topology, which is common in most non-enveloped icosahedral viruses. A PSI-BLAST search of RoMoV CP against the NCBI database gave the best hits against RGMoV CP (E = 8e74; score, 234; query coverage, 97 %; 53 % identity) and artemisia virus A (ArtVA) CP (E = 3e-33; score, 129; query coverage, 97 %; 37 % identity). Although several other sobemoviral CP sequences also produced alignments with the RoMoV CP with E-values below the threshold (data not shown), these were lower than the E-values and identities obtained for the alignments with the CPs of the following necroviruses: olive latent virus 1 (GenBank
57.7
accession no. BAB55656.1; E = 8e-16; score, 82.4; query coverage, 94 %; 31 % identity), beet black scorch virus (GenBank accession no. CBA34978.1; E = 7e-15; score, 79.3; query coverage, 99 %; 30 % identity) and tobacco necrosis virus A (TNV-A; GenBank accession no. NP_056828.1; E = 3e-13; score, 74.7; query coverage 99 %, 25 % identity). In conclusion, the RoMoV CP groups together with the CPs of RGMoV and ArtVA, as these CPs show slightly higher similarities to RoMoV CP than other sobemoviral and necroviral CPs (data not shown). Homology modelling of the 3D structure of the RoMoV CP using Phyre2 (http://www.sbg.bio.ic.ac.uk/ phyre2) confirms with 100 % confidence that it adopts the overall fold shown for the RGMoV CP. The 3D structure of the RGMoV CP has been determined to be a bit more similar to that of the TNV-A CP than to the other sobemoviral CPs [34]. Structure-based alignment of the RGMoV CP with other sobemoviral CPs has revealed eight invariant amino acid residues [34] that are also conserved in the RoMoV CP (P120, G128, M132, D137, D140, P198, G215, N234). According to sequence alignment (data not shown), three of these residues (D137, D140 and N234) together with S184 and S235 are most
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862
probably involved in formation of the Ca2?-binding cluster. Indirect evidence for a close relationship between the CPs of RGMoV and RoMoV was obtained earlier in serological tests, revealed by a serological cross-reaction between the RGMoV particles and an antiserum against RoMoV particles [35]. In concordance with the PSI-BLAST results for the majority of RoMoV proteins (P2a, P2a2b and CP), alignments of complete genome sequences displayed the highest identity (55 %) between RoMoV and RGMoV (Table 1). Thus, RGMoV was identified as the closest known relative of RoMoV, although they do not share a host species or geographical location, unlike RoMoV and IYMV. Interestingly, RoMoV P1 had the highest identity score with IYMV P1. Due to the divergence of the P1encoding region in the sobemovirus genome, it was not possible to detect any recombination between IYMV and RGMoV using the RDP3 software at default settings (http://web.cbio.uct.ac.za/*darren/rdp.html). However, we cannot rule out an unidentified recombination event. Therefore, instead of the complete genome sequences, we used the most conserved P2b sequences for construction of a phylogenetic tree of the sobemoviruses. In the phylogenetic tree, RoMoV clustered together with RGMoV and ArtVA, a dicot-infecting sobemovirus (Fig. 2B). The subdivision of the grass-infecting sobemoviruses (RoMoV/RGMoV versus IYMV/RYMV/CfMV) into two different lineages suggests that there is no common lineage of the Poaceae-infecting sobemoviruses and that these clades have emerged at least twice during diversification of sobemovirus species.
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8. 9. 10.
11.
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17. Acknowledgments The authors thank S. No˜u for excellent plant care, and D. Fargette and E. Hebrard for critical reading of the manuscript. I. cylindrica seeds were distributed by Kew’s Royal Botanical Gardens. This work was supported by the EU through the European Regional Development Fund (Centre of Excellence ENVIRON) and by Estonian Science Foundation Grant No. 9415.
18.
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BRIEF REPORT
Rottboellia yellow mottle virus is a distinct species within the genus Sobemovirus Merike So˜mera • Erkki Truve
Received: 16 September 2014 / Accepted: 8 January 2015 / Published online: 23 January 2015 Ó Springer-Verlag Wien 2015
Abstract Once considered a tentative member of the genus Sobemovirus, rottboellia yellow mottle virus (RoMoV) was excluded from the latest species list of the ICTV after the discovery of imperata yellow mottle virus (IYMV), which resembles RoMoV in host range and geographic origin. Here, sequence analysis of the complete genome of RoMoV suggested that it should be considered a distinct species within the genus Sobemovirus. It has the highest sequence identity (55 %) to ryegrass mottle virus (RGMoV), whereas its sequence identity to IYMV is lower (44 %). In a phylogenetic tree, RoMoV clusters together with RGMoV and artemisia virus A (ArtVA), a dicotinfecting sobemovirus. Keywords RoMoV RGMoV IYMV ArtVA Sobemovirus Rottboellia yellow mottle virus (RoMoV; formerly abbreviated as RoYMV) was isolated from itchgrass (Rottboellia cochinchinensis) in 1981 in Nigeria [1]. A host-range study showed that it was also able to infect maize. RoMoV was identified as a positive-sense ssRNA plant virus with isometric particles measuring about 29 nm in diameter. Based on similarities in particle properties, the size of its coat protein subunits, the size and type of its genomic RNA, the distribution of virus particles within the cell, and its narrow host range, RoMoV was proposed to be a member of the genus Sobemovirus [1]. In 2005, it was entered into a list of tentative sobemovirus species [2].
M. So˜mera (&) E. Truve Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia e-mail: [email protected]
In 2000, an isolate of imperata yellow mottle virus (IYMV) was found in Burkina Faso [3]. IYMV was collected from cogongrass (Imperata cylindrica) and maize plants. Experimentally, it was also shown to infect itchgrass. Although there were similarities between IYMV and RoMoV in symptoms, virus particle size, and host range, it was not possible to classify these viruses because no sequence data on RoMoV were available [3]. Consequently, RoMoV was left out from the latest list of sobemovirus species accepted by the International Committee on Taxonomy of Viruses (ICTV) [4]. In the current study, we sequenced the complete genome of RoMoV. Sweet maize (Zea mays subsp. saccharata) cv. Golden Bantam was used as experimental host for RoMoV propagation. For mechanical inoculation onto the leaves of 2-week-old plants, virus inoculum was prepared by grinding 0.1 g of lyophilized infectious plant material (DMSZPV-0496; supplied by G. Thottappilly) in 0.5 ml of 0.1 M phosphate buffer (pH 7.0) containing 0.5 % celite. Plants were grown in a climate chamber (16 h daylight at 28 °C, 8 h darkness at 18 °C). Visible symptoms of infection (Fig. 1A–D)—chlorotic mottle and stunting—appeared at 15 days post-inoculation (dpi). In the case of fresh infectious material, the symptoms appeared already at 7 dpi. In agreement with a previous study [1], no seed transmission was detected. Also, the susceptibility of cogongrass plants to RoMoV inoculation was examined. No particular symptoms were observed (Fig. 1e). However, one of six plants tested in our experiment was positive by RT-PCR for RoMoV infection at 40 dpi (Fig. 1f), confirming that the host range of both RoMoV and IYMV includes cogongrass, itchgrass and maize. Virus particles were purified from symptomatic maize plants at 35 dpi as described [5]. Virions were dissociated with 1 % SDS, and viral RNA was isolated by phenol/
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Fig. 1 A–D Symptoms of RoMoV infection in maize plants. A Chlorotic mottling. B Severe chlorosis in older leaves. C Severe stunting and chlorosis of inflorescence potentially leading to yield reduction. D Mild infection resulting in no obvious stunting. E– F RoMoV-inoculated cogongrass plants (samples 2–7). Sample 8 represents a non-inoculated plant. E No visible symptoms occurred in an RoMoV-infected plant (sample no. 5). F A 334-bp fragment
corresponding to positions 2702 to 3036 of the RoMoV genome was amplified by RT-PCR using RoMoV-specific primers (50 -GCA TATCGCGCGACAAAATCT-30 and 50 -CCTGTGCATCTGGCA CAAAG-30 ) from systemic leaves in one of six inoculated plants. Sample 1 represents an RoMoV-infected maize plant used as a positive control; M, GeneRuler 100 bp Plus Marker (Thermo Scientific)
chloroform extraction. The sobemovirus genus-specific primer pair SOBDF2/SOBDR2 [6] was used to amplify a conserved region in the RNA-dependent RNA polymerase (RdRp) gene. The amplified fragment was sequenced and used to design RoMoV-specific primers RoB_RpF (50 -GT CCAACATTGGGAACTGATGGAGG-30 ) and RoB_RpR
(50 -TGGCCATCAAGCAGCGGATACG-30 ). RoB_RpR was used for 50 -specific first-strand cDNA synthesis from viral RNA using Maxima Reverse Transcriptase (Thermo Scientific). The first-strand synthesis product was ligated with the self-priming anchor primer SP-ANC [7] using T4 RNA ligase. The cDNA fragment that was obtained was
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Sequence of rottboellia yellow mottle sobemovirus
amplified using RoB_RpR and the anchor-nested primer ANC [7] in the PCR step. In parallel, SP-ANC was ligated with viral RNA to create a template for genomic 30 -specific first-strand cDNA synthesis. After the first-strand synthesis, primers ANC and RoB_RpF were used for PCR. Both 50 and 30 -specific PCR products were subcloned and sequenced using a primer-walking strategy. To confirm the sequencing results, a genome-wide set of primers was designed (not shown) and utilized for direct sequencing of the RT-PCR products amplified from viral RNA. Additional 50 and 30 RACE reactions were carried out using the SP-ANC primer and virus-specific primers oriented toward the ends of the genome. Assembly of the set of genome-wide sequences revealed that the RoMoV genome consists of 4194 nt (deposited in GenBank under accession no. KC577469). The RoMoV genome organization was found to be characteristic of sobemoviruses (Fig. 2A). Open reading frames (ORFs) were identified using the NCBI ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The parameters were set to use the standard genetic code, with a minimum of 100 codons. The ORFx-encoding region [8], overlapping with ORF1 and ORF2a/2a2b was identified using EMBOSS Sixpack (http://www.ebi.ac.uk/Tools/st/ emboss_sixpack/). The sequence of the 50 end of the genome was identified as ACAAAA. The 50 ACAAA(A) motif is common for sobemo-, polero-, and polero-like dianthoviruses. It is also present in the RoMoV sequence at nt position 3,315–3,319, shortly before the beginning of ORF3, marking the possible beginning of sgRNA [9]. The 50 untranslated region (UTR) of the RoMoV genome is 96 nt long, whereas the 30 UTR has 163 nt. RoMoV ORF1 encodes a putative P1 protein with a predicted molecular mass of 17.1 kDa. Like other sobemoviral P1 proteins, the RoMoV P1 is very divergent and does not share significant similarity with any known proteins. Despite being very divergent, the P1 proteins of many sobemoviruses have been demonstrated to be involved in virus spread [10–12] and RNAi suppression [13–16]. A PSI-BLAST (http://blast.ncbi.nlm.nih.gov/) homology search against non-redundant (nr) protein sequences in the NCBI database indicated that the only similarity was to IYMV P1, with an E-value slightly lower than the threshold value (E = 0.003; score, 44.3; query coverage, 61 %; 32 % identity). The sobemovirus ORF1 is in a suboptimal context, allowing the ribosome to continue scanning and initiate translation from downstream, in ORF2a/ORF2a2b [17]. ORF2a encodes a putative polyprotein, P2a, with a calculated molecular mass of 61 kDa. A PSI-BLAST search against the NCBI nr database using the sequence of RoMoV P2a gave the best hit against ryegrass mottle sobemovirus (RGMoV) P2a (E = 2e-143; score, 436; query
859
coverage, 89 %; 48 % identity). The values for the alignment of RoMoV P2a and IYMV P2a were E = 3e-16; score, 90.5; query coverage, 89 %; 25 % identity. Like other sobemoviral P2a polyproteins [18], the N-terminus of RoMoV P2a shows no obvious conservation. Transmembrane helix prediction tests indicated that it is probably involved in the formation of a transmembrane anchor composed of two transmembrane segments (at aa positions 4–20 and 34–51). The sequence of the putative transmembrane anchor region is followed by motifs that are characteristic of serine proteases (H(X32)D(X69)TXXGXSG) and viral genome-linked protein VPg (WADMVEAEEDMWD). The non-conserved C-terminal part of P2a has been predicted to bind nucleic acids [19]. Studies on sesbania mosaic sobemovirus (SeMV) have shown that autoproteolysis of the polyprotein is needed for viable infection [20]. Amino acid sequencing of VPg proteins purified from sobemovirus particles or analysis of cleavage-site mutants has confirmed cleavage at E/S, E/T and E/N sites [21–23]. For RoMoV P2a, the putative cleavage sites at the VPg ends can be predicted for positions E319/S320 and E398/T399 based on sequence alignments (data not shown) with other sobemoviral P2a proteins. ORF2b contains characteristic RdRp motifs (SGSYCTSSSNX19GDD). The putative RdRp can be translated only as part of P2a2b via -1 ribosomal frameshifting from ORF2a to ORF2b. The frameshifting signal of sobemoviruses consists of the uniformly conserved slippery sequence UUUAAAC followed by a stem-loop [24]. The calculated molecular mass of the RoMoV P2a2b polyprotein is 104.3 kDa. A PSI-BLAST search against the NCBI nr database using the sequence of RoMoV P2a2b gave the best hit against RGMoV P2a2b (E = 0.0; score, 1009; query coverage, 94 %; 58 % identity). The values for the alignment of RoMoV P2a2b and IYMV P2a2b were E = 1e-165; score, 522; query coverage, 89 %; 39 % identity. A recent search for hidden ORFs in sobemovirus genomes resulted in the discovery of a region called ORFx, which appears to have a non-AUG initiation codon in a favourable context shortly before the end of ORF1 in the ?1 frame [8]. RoMoV has four potential non-AUG initiation codons adjacent to each other in the ORFx frame. The one that is in the best position also has the best context for initiation (here, GAGGUGG). The calculated molecular mass of RoMoV protein X is 11.9 kDa. A PSI-BLAST homology search against the nr database at NCBI identified no similarities to viral proteins having an E-value below the threshold value. The last ORF, ORF3, encodes the viral coat protein (CP), with a predicted molecular mass of 25.3 kDa. In SDS-PAGE, the estimated molecular mass of RoMoV CP
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Fig. 2 A The genome organization of RoMoV. B Phylogenetic tree showing the grouping of RoMoV within the genus Sobemovirus. A maximum-likelihood tree of predicted P2b sequences was generated using MUSCLE alignment and the LG amino acid substitution model [36] using Seaview 4.4.2 (http://doua.prabi.fr/software/seaview). The scale bar represents expected substitutions per site. Bootstrap scores (500 replicates) are shown at the nodes only when [70 %. P2b sequences (starting from the ribosomal frameshift site) were segregated from the following P2a2b or P2b sequences obtained from GenBank: artemisia virus A (ArtVA, AFI72874.1); cocksfoot mottle virus (CfMV, ABG73619.1); imperata yellow mottle virus (IYMV, CAQ48413.1); lucerne transient streak virus (LTSV, AGP50163.1);
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papaya lethal yellowing virus (PLYV, AFP67700.1); rice yellow mottle virus (RYMV, CAE81329.1); ryegrass mottle virus (RGMoV, ABN09950.1); sesbania mosaic virus (SeMV, AAG01370.2); southern bean mosaic virus (SBMV, ABI53037.2); southern cowpea mosaic virus (SCPMV, NP_042302.3); soybean yellow common mosaic virus (SYCMV, AEO16607.1); sowbane mosaic virus (SoMV, ADD64689.1); subterranean clover mottle virus (SCMoV, AAN51929.1); turnip rosette virus (TRoV, AGP50163.1); velvet tobacco mottle virus (VTMoV, ADN19015.1). The predicted RoMoV P2a2b sequence has GenBank accession no. AHB64342. The Solanum nodiflorum mottle virus (SNMoV) P2a2b sequence will be available soon as in GenBank acc. no AHB64347
Sequence of rottboellia yellow mottle sobemovirus
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Table 1 Percent identity between complete genome sequences of sobemoviruses based on MUSCLE multiple sequence alignment performed at the EMBL-EBI website RoMoV
RGMoV
ArtVA
PLYV
SoMV
SYCMV
SBMV
SeMV
SCPMV
RYMV
IYMV
CfMV
LTSV
SCMoV
TRoV
VTMoV
SNMoV
RoMoV 4194 bp RGMoV 4212 bp
55.1
ArtVA 4138 bp
50.8
48.4
PLYV 4145 bp
46.9
46.6
47.5
SoMV 3983 bp
45.0
45.1
46.7
48.1
SYCMV 4152 bp
46.6
46.7
47.7
51.0
45.7
SBMV 4132 bp
46.2
46.7
46.9
49.9
50.2
70.7
SeMV 4148 bp
46.8
45.9
47.2
49.6
50.4
70.9
71.9
SCPMV 4193 bp
46.0
46.7
47.1
48.6
49.4
59.9
59.4
58.8
RYMV 4448 bp
44.4
44.8
45.7
45.5
45.8
46.9
47.2
46.4
45.9
IYMV 4547 bp
43.9
43.8
44.9
45.8
45.0
47.3
46.4
46.3
45.2
52.3
CfMV 4082 bp
45.0
45.2
44.6
46.3
45.6
46.2
46.6
45.5
46.8
49.5
49.7
LTSV 4279 bp
46.4
45.7
46.5
50.5
47.1
47.7
47.4
47.9
47.4
45.9
44.6
45.8
SCMoV 4258 bp
44.8
45.3
47.1
49.7
59
46.9
47.8
47.7
47.9
45.5
44.5
46.1
56.7
TRoV 4035 bp
44.8
43.9
45.8
47.4
47.1
47.2
46.7
46.8
46.4
44.8
44.4
44.8
46.3
46.2
VTMoV 4247 bp
44.3
44.3
46.4
47.9
47.1
47.6
46.7
46.2
47.4
45.4
44.6
46.2
46.5
47.1
45.9
SNMoV 4297 bp
45.2
45.0
46.5
48.2
47.2
48.7
47.3
47.4
48.3
45.1
45.1
45.2
47.9
46.3
46.0
was 27 kDa [1]. ORF3 of sobemoviruses is translated from a sgRNA [25–28]. Like other sobemoviral CPs, the N-terminus of RoMoV CP is rich in arginine and lysine residues. It has been shown to be required for RNA interaction and encapsidation, association with membranes, and RNA transport, including nuclear localization [29–31]. A basic region followed by two b-strands forms a structure called a b-annulus, also known as the R (random) domain. It can be completely or partially disordered [32–34]. The subsequent S (shell) domain of the RoMoV CP is a core building block of virions that has a jellyroll b-sandwich topology, which is common in most non-enveloped icosahedral viruses. A PSI-BLAST search of RoMoV CP against the NCBI database gave the best hits against RGMoV CP (E = 8e74; score, 234; query coverage, 97 %; 53 % identity) and artemisia virus A (ArtVA) CP (E = 3e-33; score, 129; query coverage, 97 %; 37 % identity). Although several other sobemoviral CP sequences also produced alignments with the RoMoV CP with E-values below the threshold (data not shown), these were lower than the E-values and identities obtained for the alignments with the CPs of the following necroviruses: olive latent virus 1 (GenBank
57.7
accession no. BAB55656.1; E = 8e-16; score, 82.4; query coverage, 94 %; 31 % identity), beet black scorch virus (GenBank accession no. CBA34978.1; E = 7e-15; score, 79.3; query coverage, 99 %; 30 % identity) and tobacco necrosis virus A (TNV-A; GenBank accession no. NP_056828.1; E = 3e-13; score, 74.7; query coverage 99 %, 25 % identity). In conclusion, the RoMoV CP groups together with the CPs of RGMoV and ArtVA, as these CPs show slightly higher similarities to RoMoV CP than other sobemoviral and necroviral CPs (data not shown). Homology modelling of the 3D structure of the RoMoV CP using Phyre2 (http://www.sbg.bio.ic.ac.uk/ phyre2) confirms with 100 % confidence that it adopts the overall fold shown for the RGMoV CP. The 3D structure of the RGMoV CP has been determined to be a bit more similar to that of the TNV-A CP than to the other sobemoviral CPs [34]. Structure-based alignment of the RGMoV CP with other sobemoviral CPs has revealed eight invariant amino acid residues [34] that are also conserved in the RoMoV CP (P120, G128, M132, D137, D140, P198, G215, N234). According to sequence alignment (data not shown), three of these residues (D137, D140 and N234) together with S184 and S235 are most
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862
probably involved in formation of the Ca2?-binding cluster. Indirect evidence for a close relationship between the CPs of RGMoV and RoMoV was obtained earlier in serological tests, revealed by a serological cross-reaction between the RGMoV particles and an antiserum against RoMoV particles [35]. In concordance with the PSI-BLAST results for the majority of RoMoV proteins (P2a, P2a2b and CP), alignments of complete genome sequences displayed the highest identity (55 %) between RoMoV and RGMoV (Table 1). Thus, RGMoV was identified as the closest known relative of RoMoV, although they do not share a host species or geographical location, unlike RoMoV and IYMV. Interestingly, RoMoV P1 had the highest identity score with IYMV P1. Due to the divergence of the P1encoding region in the sobemovirus genome, it was not possible to detect any recombination between IYMV and RGMoV using the RDP3 software at default settings (http://web.cbio.uct.ac.za/*darren/rdp.html). However, we cannot rule out an unidentified recombination event. Therefore, instead of the complete genome sequences, we used the most conserved P2b sequences for construction of a phylogenetic tree of the sobemoviruses. In the phylogenetic tree, RoMoV clustered together with RGMoV and ArtVA, a dicot-infecting sobemovirus (Fig. 2B). The subdivision of the grass-infecting sobemoviruses (RoMoV/RGMoV versus IYMV/RYMV/CfMV) into two different lineages suggests that there is no common lineage of the Poaceae-infecting sobemoviruses and that these clades have emerged at least twice during diversification of sobemovirus species.
5.
6.
7.
8. 9. 10.
11.
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15.
16.
17. Acknowledgments The authors thank S. No˜u for excellent plant care, and D. Fargette and E. Hebrard for critical reading of the manuscript. I. cylindrica seeds were distributed by Kew’s Royal Botanical Gardens. This work was supported by the EU through the European Regional Development Fund (Centre of Excellence ENVIRON) and by Estonian Science Foundation Grant No. 9415.
18.
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