United States Department of Agriculture, Agricultural Research Service1, and Department of Crop Sciences2, 1102 S. Goodwin Ave, University of Illinois at Urbana/Champaign, Urbana, IL 61801, USA
United States Department of Agriculture, Agricultural Research Service, 24106 N Bunn Rd, Prosser, WA 99350-8694, USA3
Author for correspondence: Leslie Domier. Fax +1 217 333 5251. e-mail l-domier{at}uiuc.edu
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Abstract |
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Introduction |
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The Luteoviridae family has been divided into three genera (Enamovirus, Luteovirus and Polerovirus) depending on genome organization, sequence similarity, and methods of gene expression (DArcy et al., 2000 ). The single-stranded, positive-sense genomes of the Luteoviridae contain five to six open reading frames (ORFs) designated ORF 0 through ORF 6. ORF 0 is unique to the Enamovirus and Polerovirus genera and encodes a protein of unknown function. ORFs 1 and 2 encode the replication-related proteins, which in luteoviruses are most similar to those of the Tombusviridae, while the replicases encoded by poleroviruses and enamoviruses are related to those of the Sobemovirus genus (DArcy et al., 2000
). In all three genera, ORF 2 is expressed via a translational frameshift from ORF 1. ORF 1 overlaps ORF 2 by less than 20 nt in luteoviruses, but by more than 400 nt in enamo- and poleroviruses. The intergenic region between ORFs 2 and 3 is about 100 nt in luteoviruses and about 200 nt in polero- and enamoviruses. ORFs 3 and 5 encode the coat and readthrough proteins of the viruses. Recently, parallels have been found between the coat proteins (CPs) of poleroviruses and members of the Sobemovirus genus (Terradot et al., 2001
), which suggests an even closer affiliation of the two genera. ORF 4, which is lacking in the enamoviruses, putatively encodes a movement protein.
Comparisons of the nucleotide and predicted amino acid sequences of members of the Luteoviridae with other members of the family and different virus families suggest that RNA recombination has played an important role in the generation of new species within the Luteoviridae (Miller et al., 1995 ). Recombination seems to have occurred most commonly near the site of initiation of synthesis of the subgenomic RNA (sgRNA) that encodes the capsid and movement proteins. As a consequence, the Luteoviridae contains species with similar structural proteins, but disparate RNA-dependent RNA polymerases (RdRps). For example, Cucurbit aphidborne yellows virus (CABYV) is thought to be derived from recombination between polero- and enamo-like viruses (Gibbs et al., 2000
; Gibbs & Cooper, 1995
). Similarly, Sugarcane yellow leaf virus (ScYLV) is thought to have arisen by recombination between polero-, luteo- and enamo-like viruses (Smith et al., 2000
). Finally, it has been suggested that SbDV is derived from recombination events between luteo- and polero-like viruses (Rathjen et al., 1994
; Terauchi et al., 2001
). Since one of the primary distinguishing features of the three Luteoviridae genera is the type of RdRp encoded by the virus, CP sequences alone are not sufficient to assign a virus species to a genus.
The relationship of BLRV to other members of the Luteoviridae has been examined based on biological, immunological, nucleic acid hybridization and nucleotide sequence data. Serological (DArcy et al., 1988 ; Smith et al., 1996
; van den Heuvel et al., 1990
) and nucleic acid hybridization data (Martin & DArcy, 1990
) suggest that BLRV is related most closely to Soybean dwarf virus (SbDV). Sequences of the BLRV coat protein gene have been reported (Cavileer & Berger, 1994
; Prill et al., 1990
) and predicted that BLRV was most closely related to poleroviruses. As mentioned above, SbDV has a genome organization similar to members of the genus Luteovirus, but may have arisen through recombination between a Luteovirus and a Polerovirus (Rathjen et al., 1994
; Smith et al., 2000
; Terauchi et al., 2001
). In this study we determined the complete genomic sequence of a Michigan isolate of BLRV and compared the coding and noncoding regions of the genomic sequence to determine to which of the three Luteoviridae genera BLRV was most similar.
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Methods |
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Sequence analysis.
Total RNA was extracted from leaf and lyophilized broad bean tissue using Trizol reagent according to the manufacturers protocols (Invitrogen). To amplify regions of the BLRV genome upstream and downstream of the previously published CP gene (nt 30933683; Cavileer & Berger, 1994 ; Prill et al., 1990
), degenerate primers were designed from conserved sequences in the SbDV and BYDV-PAV genomes. A DNA fragment representing nt 12763151 was amplified using 5' GGKTTTTTAGAGGGGCTCTG 3' (nt 12761295) and a primer complementary to nt 31313151. A fragment representing nt 3171355 was amplified with 5' CAYGAYGCYTTTGTSGACATGTGCT 3' (nt 317341) and a primer complementary to nt 13401355. A fragment containing nt 35744401 was amplified using 5' TTCTAARTGCTCATCAAG 3' (nt 43844401) and a primer complementary to nt 35743594. Fragments representing the 5' and 3' termini were cloned as described below. PCR products were either cloned (Topo Cloning vector, Invitrogen) and sequenced, or sequenced directly using an ABI 3700 sequencer and Big-Dye terminator sequencing reagents (ABI). When cloned fragments were sequenced, at least three independently isolated clones were analysed. Sequences were edited and assembled with Sequencher version 4.0 (Genecodes, Ann Arbor, MI, USA).
Analysis of RNA termini.
The 5' terminus of the BLRV RNA was determined by rapid amplification of cDNA ends (RACE) as described by Dieffenbach & Dveksler (1995) . First-strand cDNA was primed with an oligonucleotide complementary to nt 520544 and tailed separately with dATP and dGTP. For the dATP-tailed cDNA, fragments containing the 5' terminus of the BLRV genome were amplified using an oligo(dT) primer [5' GGCGTTGGGGTACC(T)10 3'] and a primer complementary to nt 456472. For the dGTP-tailed cDNA, fragments containing the 5' terminus of the BLRV genome were amplified in nested PCR reactions using an oligo(dC) primer [5' CGACTGGTCTAGAATT(C)11 3'] and a primer complementary to nt 322345. To determine the sequence of the 3'-terminal region of the BLRV genome (nt 43245965), total RNA extracted from BLRV-infected broad bean tissue was polyadenylated using poly(A) polymerase according to the manufacturers recommendations (Promega). Because the genomic sequences of SbDV and BYDV-PAV contain at least three 3'-terminal C residues, an anchored oligo(dT) primer [5' GGCGTTGGGGTACC(T)10GGG 3'] was used to synthesize cDNA and amplify the 3'-terminal fragment in combination with a primer corresponding to positions nt 43244349.
The termini of the BLRV sgRNAs were predicted based on nucleotide sequence similarity to the 5' terminus of genomic RNA. To determine the 5' terminus of the largest sgRNA, cDNA was primed with an oligonucleotide complementary to nt 31253160, tailed with dGTP and amplified in nested PCR reactions with the oligo(dC) primer and oligonucleotides complementary to 30273061 and 29622995. To determine the 5' terminus of the intermediate sgRNA, cDNA was primed with an oligonucleotide complementary to nt 53955422, tailed with dGTP and amplified in nested PCR reactions with the oligo(dC) primer and oligonucleotides complementary to 52815304 and 51975233. To determine the 5' terminus of the smallest sgRNA, cDNA was primed with an oligonucleotide complementary to nt 59505965, tailed with dGTP and amplified in nested PCR reactions with the oligo(dC) primer and oligonucleotides complementary to 58905922 and 58355858.
Phylogenetic analysis.
Phylogenetic relationships of BLRV were inferred by comparing the predicted amino acid sequences of ORF 2 and ORF 3 to those of members of the Luteoviridae. Sequences were aligned with ClustalX (Thompson et al., 1997 ). Trees were constructed using the NEIGHBOR-joining method in PAUP version 4.0B8 (Sinauer Associates, Sunderland, MA, USA). Sister scanning analysis was performed using SISCAN version 2.0 (Gibbs et al., 2000
) and the BYDV-PAV (AF235167) and PLRV (NC 001747) genomic sequences. A window of 100 nt was shifted 50 nt at each step. To allow scoring of the 5' and 3' noncoding regions, gapped positions were evaluated.
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Results and Discussion |
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Even though the 3'NCR of BYDV-PAV does not encode protein products, it is very important for the expression of viral proteins. The BYDV-PAV 3' NCR contains a translational enhancer (3'TE) that mediates the cap-independent translation initiation of its genomic and subgenomic RNAs. The 3'TE binds sequences near the 5' termini of translated BYDV-PAV RNAs and eukaryotic initiation factors eIF4F to 40S ribosomal subunits for translation initiation (Wang et al., 1997 ). The BYDV-PAV 3' NCR also contains sequences that are required for the frameshifting between ORFs 1 and 2 (Paul et al., 2001
). The 3'NCR of BLRV (and that of SbDV) contained sequence elements similar to both of the BYDV-PAV control sequences. A region (nt 52935387) just downstream of ORF 5 in the BLRV nucleotide sequence showed a high degree of sequence identity and conserved secondary structure to the BYDV-PAV 3'TE (Fig. 2A
). The longest consecutive set of conserved nucleotides (5' AUCCUGGGAAACAGG 3') was shared by BLRV, SbDV and the BYDVs and was predicted to form the first stem-and-loop of the enhancer. The second and probably more interesting conserved structure was the third stem and loop which, like BYDV-PAV, contained a sequence complementary to regions in the 5'NCR that were also capable of forming two small stem-and-loops structures (nt 220 and 3051). These structures were similar to those recently proposed for SbDV (Guo et al., 2001
). In BYDV-PAV, the interaction of these two regions is thought to mediate translation enhancement. These observations suggest that, like BYDV-PAV, the BLRV genes are expressed through cap-independent processes that are facilitated by the long-distance interaction of the 5' and 3'NCRs.
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Intergenic region
Even though the predicted amino acid sequences of proteins derived from ORFs 1 and 2 were very similar to those of BYDV-PAV, the length of the intergenic region between ORFs 2 and 3 of BLRV (211 nt) and the predicted amino acid sequences of the protein products of ORFs 3 and 5 were more similar to those of the poleroviruses. Other studies have mapped recombination events to this area (Chalhoub & Lapierre, 1995 ; DArcy et al., 2000
; Gibbs & Cooper, 1995
; Smith et al., 2000
). It has been proposed that the mechanism underlying the initiation of transcription of the sgRNA from this position might favour template switching for RNA recombination (Miller et al., 1995
). This size difference would be consistent with those proposals. To generate the BLRV genome organization, a second recombination event near the end of ORF 5 would have had to occur to place a luteovirus-like 3' NCR downstream of ORF 5. As proposed for the recombination event between ORFs 2 and 3, the recombination could have been facilitated by the initiation of RNA synthesis at the sgRNA promoter located in this region in Luteovirus genomes.
ORFs 1 and 2
ORF 1 (nt 1791285) and ORF 2 (nt 12552883) were predicted to encode proteins of 42 kDa and 62 kDa, respectively. Their predicted amino acid sequences were most similar to those of SbDV, followed by BYDV-PAV. BLAST comparisons with BLRV ORF 1 yielded E values of e-123 (62% identity) with SbDV and 4e-25 (27% identity) with BYDV-PAV. ORF 2 was more conserved and yields E values of 0·0 with both SbDV (82% identity) and BYDV-PAV (62 %). There were no significant alignments with members of the Polerovirus or Enamovirus genera. As with other members of the Luteoviridae, ORF 2 is predicted to be translated as a translational fusion with the product of ORF 1 through a frameshift. In many systems, frameshifting is promoted by a slippery sequence that allows frameshifting ribosomes to retain the tRNAs in the peptidyl and aminoacyl sites during the -1 transition (Jacks et al., 1988 ). A sequence which fits the canonical slippery sequence was found at the junction of BLRV ORFs 1 and 2 (GGUUUUU); this is identical to that of SbDV and similar to that of BYDV-PAV (GGGUUUU) and Red clover necrotic mosaic virus (RCNMV; GGAUUUU), a Dianthovirus in the Tombusviridae (Kim & Lommel, 1998
). Like BYDV-PAV, the shifty sequence was flanked by sequences that could form two bulged stem-and-loop structures. Comparisons of the sequences and predicted secondary structures showed that the upstream structure was highly variable. The upstream structure was not required for frameshifting of RCNMV (Kim & Lommel, 1998
). In contrast, the downstream structure was highly conserved among BLRV, BYDVs, SbDV and RCNMV (Fig. 2C
). Thirty-one nucleotides downstream of the GGDUUUU slippery sequence and UAG termination codon was the sequence CCCVUWYYCUAUUYUCSG, which was conserved in all the viruses and formed a loop at the top of the second stem. All of the predicted structures also contained bulges with the sequence UUGA unpaired on the upstream side of the stem. It has been suggested that two features, a shifty sequence and a complex secondary structure downstream of the termination codon, are needed to promote efficient frame shifting. All of the viruses here have both elements. However, the primary sequence conservation in the unpaired bulge and loop of the downstream structure suggests that these sequences may interact with a second factor, possibly conserved host proteins or RNAs.
ORFs 3, 4, and 5
BLRV ORFs 3 (nt 30933683), 4 (nt 32383664) and 5 (nt 36845262) were predicted to produce proteins of 22, 16 and 59 kDa, respectively. In contrast to ORFs 1 and 2, the predicted amino acid sequences of BLRV ORFs 3, 4 and 5, after SbDV, were most similar to members of the Polerovirus genus. BLAST comparisons using the BLRV CP amino acid sequence, encoded by ORF 3, produced their highest E values with SbDV (e-53, 72% identity) followed by Groundnut rosette assistor virus (GRAV; 8e-46, 60% identity) and Potato leafroll virus (PLRV; 4e-45, 52% identity). ORF 4 was the least conserved of the five BLRV ORFs, and yielded E values with SbDV of 2e-13 (37% identity), with Beet western yellows virus of e-10 (34% identity) and with PLRV of 2e-08 (32% identity). Finally, comparisons with ORF 5 yielded E values with SbDV of 2e-99 (57% identity), with Beet mild yellows virus of 2e-83 (62% identity) and with BYDV-RPV of 4e-82 (54% identity). For comparison, E values for alignments with BYDV-PAV for ORFs 3 and 5 produced E values of 4e-43 and 8e-48, respectively. No significant homology was reported between ORF 4s of BLRV and BYDV-PAV. These results showed that the E values in the CP regions of BLRV, PLRV and BYDV-PAV were similar. However the E values derived from the ORF 5 comparisons showed a much closer relationship between BLRV and the poleroviruses.
The above results are consistent with observations from immunosorbent electron microscopy that showed strong interactions between polyclonal antisera for BLRV and GRAV (Casper et al., 1983 ; Reddy et al., 1985
). Recently the three-dimensional structure of the PLRV CP was predicted based on homology modelling by aligning the PLRV sequence with that of Rice yellow mottle virus, a member of the Sobemovirus genus (Terradot et al., 2001
). The analysis predicted that five acidic amino acids (E109, E170, D173, E176 and D177) are present on the surface of PLRV particles at the trimer axis. These five residues are conserved among the poleroviruses (Fig. 3
). However, all of the monocot-infecting members of the Luteovirus genus (BYDVs PAV, PAV129, MAV and SGV) and ScYLV have hydrophobic or polar substitutions at E176 (Fig. 3
). The BLRV sequence, like that of SbDV, contained the conserved E109, D173, E176 and D177, but contained a hydrophobic substitution (A) at the E170 position. The amino acids downstream of E109 were conserved in the polerovirus and luteovirus lineages of the Luteoviridae. However, these residues were much less conserved in the CP sequences of BLRV, SbDV, Sweet potato leaf speckling virus (SPLSV), SCYLV and Pea enation mosaic virus 1 (PEMV-1). The viruses in this group did not fit neatly into either the Luteovirus or Polerovirus genera. PEMV-1 is the sole member of the Enamovirus genus. As mentioned above, BLRV, SbDV and SCYLV seem to have arisen through interspecific recombination. (Smith et al., 2000
; Rathjen et al., 1994
; Terauchi et al., 2001
). Because only the CP sequence has been reported for SCYLV (Fuentes et al., 1996
), its genomic organization is not known. While recombination may have played a role in the generation of these genomes, the lack of conservation in the amino acids thought to be on the surface of the virions may reflect unique selection pressures placed on the viruses by their hosts and/or aphid vectors.
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Received 15 January 2002;
accepted 26 February 2002.
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