Analysis of the RNA of Potato yellow vein virus: evidence for a tripartite genome and conserved 3'-terminal structures among members of the genus Crinivirus

I. C. Livieratos1,{dagger}, E. Eliasco1,{dagger}, G. Müller2, R. C. L. Olsthoorn3, L. F. Salazar2, C. W. A. Pleij3 and R. H. A. Coutts1

1 Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK
2 The International Potato Center, Apartado 1558, Lima, Peru
3 Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, 2300 RA Leiden, The Netherlands

Correspondence
R. H. A. Coutts
r.coutts{at}imperial.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Double-stranded RNA preparations produced from potato plants graft-inoculated with a Peruvian isolate of Potato yellow vein virus (PYVV; genus Crinivirus, family Closteroviridae) contain five RNA species denoted RNA 1, RNA 2, RNA 3, x and y of approximately 8, 5·3, 3·8, 2·0 and 1·8 kbp, respectively. The complete nucleotide sequences of PYVV RNAs 1, 2 and 3 and Northern hybridization analysis showed that PYVV RNA 1 contained the replication module and an additional open reading frame (p7), while two distinct species, RNAs 2 and 3, contain the Closteroviridae hallmark gene array. Pairwise comparisons and phylogeny of genome-encoded proteins showed that PYVV shares significant homology with other criniviruses but is most closely related to the Trialeurodes vaporariorum-vectored Cucumber yellows virus. Secondary structure prediction of the 3'-untranslated regions of all three PYVV RNAs revealed four conserved stem–loop structures and a 3'-terminal pseudoknot structure, also predicted for all fully characterized members of the genus Crinivirus and some members of the genera Closterovirus and Ampelovirus.

The GenBank accession numbers for the nucleotide sequences determined in this work are AJ557128 (RNA 1), AJ557129 (RNA 2) and AJ508757 (RNA 3).

{dagger}Both authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The virus family Closteroviridae contains positive-stranded, RNA plant viruses with long, flexuous virions and elongated genomes of up to 20 kb, which are transmitted by specific Homopteran vectors in a semi-persistent manner (Karasev, 2000; Martelli et al., 2002). Based on the number of genomic RNAs and mode of transmission, the closteroviruses are currently divided into three genera (Karasev, 2000). The genus Closterovirus, with Beet yellows virus (BYV) as the prototype, includes aphid-transmitted closteroviruses with unipartite genomes. The genus Ampelovirus, with Grapevine leafroll-associated virus 3 (GLRaV-3) as the prototype, includes mealybug-transmitted closteroviruses with unipartite genomes. The genus Crinivirus, with Lettuce infectious yellows virus (LIYV) as the prototype, includes whitefly-transmitted closteroviruses (WTCs) with bipartite genomes.

The genomic organization of all members of the family Closteroviridae shares two distinct features. Firstly, all closteroviruses possess genomes with a replication module that contains domains for a papain-like leader proteinase (L-Pro), a methyltransferase (MTR), a helicase (HEL) [open reading frame 1a (ORF1a)] and an RNA-dependent RNA polymerase [RdRp (ORF1b)] (Karasev, 2000). Secondly, downstream of the replication module, closteroviruses contain a hallmark gene array encoding a putative small hydrophobic protein, a heat-shock protein homologue (Hsp70h), a protein of 50–60 kDa, depending on the virus, the coat protein (CP) and the minor CP (CPm), arranged in a polar configuration (Karasev, 2000). For BYV (Alzhanova et al., 2000; Napuli et al., 2000, 2003) and Citrus tristeza virus (CTV) (Satyanarayana et al., 2000), these genes encode proteins responsible for virus assembly and movement.

Because of the increasing number of WTCs and their significance as potential pandemic pathogens of agriculturally important crops (Wisler et al., 1998), the molecular characterization of members of the genus is proceeding rapidly. The first WTC genome to be sequenced and characterized in its entirety was LIYV (Klaassen et al., 1995). LIYV RNA 1 (8·1 kb) encodes the replication module (Klaassen et al., 1996) and an additional p32 ORF, the product of which enhances replication of LIYV RNA 2 (Yeh et al., 2000). LIYV RNA 2 (7·2 kb) contains the Closteroviridae hallmark gene array, an additional ORF (p9) immediately upstream of the CP ORF and a putative 3'-terminal protein (p26) of unknown function (Klaassen et al., 1995).

Diversity in the genus Crinivirus was unknown until the recent publication of the complete genomic sequences of three other criniviruses, Sweet potato chlorotic stunt virus (SPCSV; Kreuze et al., 2002), Cucurbit yellow stunting disorder virus (CYSDV; Aguilar et al., 2003; Coutts & Livieratos, 2003a; Livieratos & Coutts, 2002) and Cucumber yellows virus (CuYV; Hartono et al., 2003). While LIYV, SPCSV, CYSDV RNA 2 and CuYV RNA 1 contain similarly sized and positioned ORFs, significant divergence has been reported regarding ORFs downstream of the replication module. For instance, in SPCSV RNA 1, ORFs that putatively encode an RNase III-like protein, a small hydrophobic protein (p7) and a 3'-terminal protein (p22) have been described downstream of the RdRp ORF (Kreuze et al., 2002). In CYSDV RNA 1, ORFs that putatively encode three proteins (p5, p25 and p22) with no apparent amino acid similarities to equivalent putative proteins of SPCSV or LIYV have been described (Aguilar et al., 2003; Coutts & Livieratos, 2003a). In CuYV, the replication module has been found on the smaller of the genomic RNAs, CuYV RNA 2, with no further ORFs downstream of the RdRp ORF (Hartono et al., 2003).

Potato yellow vein virus (PYVV) is a WTC vectored by Trialeurodes vaporariorum (Westwood) known to cause a yellowing disease in potato crops in South America for over 50 years (Salazar et al., 2000). The genomic sequence of PYVV is unknown except for a partial sequence of the Hsp70h gene (Salazar et al., 2000) and the complete sequence of the CPm gene (Livieratos et al., 2002). Over a period of 3 years, we have examined numerous dsRNA extracts from field isolates of PYVV from both Peru and Colombia. Single-strand conformation polymorphism analysis has strongly suggested low genetic variation within and between Peruvian and Colombian PYVV isolates (Offei et al., 2004). In this study, we have cloned the three largest PYVV RNAs present in extracts of potato plants (17·3 kbp in total) infected with a graft-maintained Peruvian PYVV isolate. Our data suggest that PYVV possesses a tripartite genome and reveals conserved secondary structures in the 3'-untranslated regions (UTRs) for all crinivirus genomes.


   METHODS
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INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Plant material and virus isolate.
The PYVV isolate used in this investigation was obtained from diseased potatoes (i.e. Solanum tuberosum ssp. tuberosumxSolanum tuberosum. ssp. andigena, cv. Yungay) originally growing in Chota, Cajamarca, Peru. The isolate was maintained in an insect-proof greenhouse in La Molina by graft inoculation of tissue culture plantlets cvs AtzimbaxR-128.6 or DTO-33 (Salazar et al., 2000). To confirm that the graft maintained PYVV isolate was biologically active, Koch's postulates for a virus were performed, which included back transmission of the isolate to potato plants using the whitefly vector.

Isolation and analysis of PYVV dsRNAs.
PYVV dsRNA was isolated from infected potato leaves as described by Valverde et al. (1990). PYVV dsRNAs were treated with DNase I, separated by agarose gel electrophoresis and individual species purified using the RNAid kit (BIO 101) as described previously (Livieratos et al., 1998). PYVV dsRNA preparations were analysed by Northern hybridization using 32P-labelled riboprobes (Bringloe et al., 1998) generated with a Riboprobe kit (Promega) from cloned RT-PCR amplification products.

Cloning and sequencing of PYVV-specific reverse transcription products and PCR amplicons.
Specific oligonucleotides designed from the sequences of ORF 1a (L-Pro motif), the Hsp70h ORF (Salazar et al., 2000) and the CPm ORF (Livieratos et al., 2002) were used to produce clones in a genome-walking procedure as described previously (Livieratos et al., 1999) using PYVV dsRNA as template. To clone the 5' and 3' termini of the individual RNAs, a modified RNA ligase-mediated rapid amplification of cDNA ends procedure was utilized based on that of Liu & Gorovsky (1993) using methylmercuric hydroxide-denatured dsRNA as substrate (Coutts & Livieratos, 2003b). All amplicons were cloned into the pGEM-T Easy system II vector (Promega) and transformed into Escherichia coli JM109 cells and sequenced in both directions. Sequence data were assembled and compared with databases using the BLAST server on the NCBI Web library (http://www.ncbi.nlm.nih.gov/blast) (Altschul et al., 1997). Pairwise comparisons between closterovirus amino acid sequences were performed using the GAP and PILEUP programs (version 8.1; University of Madison Genetics Computer Group, Madison, USA) (Devereaux et al., 1984). Phylogenies were estimated using the neighbour-joining algorithm and amino acid sequences were aligned using CLUSTAL_X (Thompson et al., 1997). Phylograms were drawn using TREEVIEW version 1.6.1 freeware (Page, 1996). RNA secondary structures were predicted using the STAR algorithm (Gultyaev et al., 1995).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Electrophoretic analysis of dsRNA preparations from Peruvian PYVV-infected plants consistently revealed the presence of at least five dsRNA species (Salazar et al., 2000) that were absent from healthy plant extracts. The sizes of these dsRNAs (denoted dsRNA 1, dsRNA 2, dsRNA 3, x and y) were estimated to be approximately 8, 5·3, 3·8, 2·0 and 1·8 kbp, respectively (Fig. 1b). The three largest dsRNAs were tentatively assigned as double-stranded versions of single-stranded PYVV genomic RNA.



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Fig. 1. (a) Schematic representation of the genome organization of PYVV. Lines represent PYVV RNAs 1, 2 and 3, and ORFs are shown above, below or on the line, indicating which reading frame they are found in. On PYVV RNA 1, the arrow indicates the predicted autocatalytic cleavage site and +1 designates the putative ribosomal +1 frameshift site. (b) Agarose gel electrophoresis of a dsRNA preparation from PYVV-infected plants (lane 2). Lane 1 contains HindIII-digested lambda DNA molecular mass markers. (c) Northern blot hybridization analysis of PYVV dsRNAs. Positive-sense riboprobes, generated by in vitro transcription of the linearized clones shown in (a), were used to probe individually blotted dsRNAs shown in lanes 1–9.

 
RNA 1
Analysis of the determined sequence of PYVV RNA 1 (8035 nt) revealed the existence of three ORFs: the replication module, which consisted of ORF 1a and b and contained the conserved L-Pro, MTR, HEL and RdRp domains, and p7, a small hydrophobic protein (Fig. 1a). The calculated molecular mass of the polypeptide encoded by ORF 1a was 226 kDa and contained at the N terminus a putative L-Pro with a conserved papain-like domain, similar to other viruses in the family Closteroviridae (Karasev, 2000). Autoproteolysis at the predicted cleavage site between Gly-468 and Met-469 by the catalytic residue His-450 would yield a putative L-Pro of 48 kDa and a larger protein of 178 kDa. Upstream of the catalytic Cys-400, the leader proteins of PYVV RNA 1 and other viruses in the family Closteroviridae showed limited sequence similarity. In BYV, L-Pro is multifunctional where, apart from its primary role in polyprotein processing and long-distance virus transport, the 54 N-terminal amino acids of the protein also function in RNA amplification (Peng et al., 2001, 2003). Downstream of the L-Pro domain were found the conserved MTR (aa 556–730) and HEL (aa 1728–1996) domains. As with other criniviruses, the region between the MTR and HEL domains contained no significant similarity to any known proteins. The ORF 1b encoded a conserved RdRp domain that may be expressed by a +1 frameshift as proposed for other members of the Closteroviridae (Agranovsky et al., 1994). The frameshifting in PYVV RNA 1 would yield a fusion protein of 285 kDa identical in size to those predicted for the equivalent proteins of CYSDV RNA 1 (Aguilar et al., 2003; Coutts & Livieratos, 2003a) and SPCSV RNA 1 (Kreuze et al., 2002).

ORF2 putatively encoded a small hydrophobic protein of 6·9 kDa (Fig. 1a) that contained a potential transmembrane helix between amino acids 31 and 51. Hydrophobic proteins of similar size but with little sequence similarity are encoded by all members of the Closteroviridae and are thought to possess membrane-binding properties. However, the number, size, genomic location and nature of these small proteins vary in all sequenced criniviruses. In BYV, a subgenomic mRNA encoding a small hydrophobic protein has been identified (Peremyslov & Dolja, 2002), whose gene product is essential for virus cell-to-cell movement (Alzhanova et al., 2000).

RNA 2
The RNA 2 (5339 nt) sequence revealed five predicted ORFs: Hsp70h, p7, p60, p10 and CP (Fig. 1a), which is part of the closterovirus gene array. PYVV RNA2 ORF 1 could be translated to produce a 62·3 kDa protein homologous to members of the Hsp70 family of molecular chaperones and closely related to those found in other closteroviruses. The putative PYVV Hsp70h protein was most closely related to the CuYV, SPCSV, CYSDV and Tomato chlorosis crinivirus (ToCV) Hsp70h genes. The Hsp70h proteins of BYV (Napuli et al., 2000, 2003) and LIYV (Tian et al., 1999) are physically associated with virus particles and are also essential for correct virion assembly and virus movement in both BYV (Alzhanova et al., 2001) and CTV (Satyanarayana et al., 2000).

ORF 2 encoded a putative protein of 6·8 kDa that was similar in size, genomic position and sequence (30 % identity) to CYSDV p6 (Aguilar et al., 2003), which was not described by Livieratos & Coutts (2002), and a putative 6·2 kDa protein predicted, but not described, from the sequence of CuYV RNA 1 (Hartono et al., 2003). Whether these putative proteins are expressed in plants is not yet known but all of the proteins contained a central conserved stretch of amino acids.

ORF 3 putatively encoded a protein of 60 kDa, which was homologous to proteins of other clostero- and criniviruses encoded at the corresponding genomic position (Karasev, 2000) with the corresponding CuYV p59 being the most similar (56 % identity) amongst criniviruses. Recently, it has been demonstrated that BYV p64 is the fourth integral component of BYV virions together with CPm and Hsp70h (Napuli et al., 2003). Napuli et al. (2003) also illustrated that ~60 kDa proteins of viruses from the genera Closterovirus and Ampelovirus contain CP-like domains and that, by analogy with BYV, these proteins may have a dual function in virion tail assembly and motility. We extended this multiple alignment of CPs, CPms and CP-like C-terminal domains of the ~60 kDa proteins to include PYVV and all sequenced criniviruses (Fig. 2). Three amino acid residues, asparagine (N), arginine (R) and aspartic acid (D), were conserved in all the crinivirus alignments and representative members of the genera Ampelovirus (GLRaV-3) and Closterovirus (CTV) (Fig. 2). These alignments suggested that the crinivirus ~60 kDa proteins may fulfil a similar role in the virus replication cycle to the BYV p64 protein and the observation that the LIYV Hsp70h and the p59 proteins are associated with virions (Tian et al., 1999) is not at variance with this notion.



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Fig. 2. Multiple alignment of the CPs, CPms and CP-like C-terminal domains of the ~60 kDa proteins of all sequenced criniviruses, LChV-1 and a representative member of the genus Ampelovirus (GLRaV-3) and the genus Closterovirus (CTV). The alignment was constructed using the CLUSTAL_X algorithm (Thompson et al., 1997). The two signature residues that are conserved in the CPs of elongated plant viruses and a conserved N residue are shown in white against a black background. The ranges of the aligned regions in the corresponding protein sequences are indicated at the beginning and end of each sequence. The GenBank accession numbers are as follows: CTV, U16304; CuYV, AB085612 and AB085613; CYSDV, AJ537493 and AJ439690; GLRaV-3, AF039204; LChV-1, X93351; LIYV, U15440 and U15441; PYVV, AJ557128, AJ557129 and AJ508757; SPCSV, AJ428554 and AJ428555. Each virus is shown by its name followed by the protein.

 
ORF 4 encoded a putative protein of 9·8 kDa. This gene showed no significant similarities with any other sequences in the database, and pairwise alignment with putative proteins encoded by similar ORFs in identical positions in all sequenced criniviruses revealed moderate amino acid sequence identity. These equivalent proteins appear to be unique to the genus Crinivirus, since no similar ORFs have been detected in closteroviruses (Karasev, 2000).

ORF 5 encoded the putative CP of 28·2 kDa. Based on size and homology with the CPs of all criniviruses and the possession of the invariant consensus of amino acid residues serine (S), R, glycine (G) and D (R and D are shown in Fig. 2), as found in most filamentous virus CPs (Alzhanova et al., 2001; Dolja et al., 1991).

RNA 3
The RNA 3 (3892 nt) sequence revealed the existence of three potential ORFs: p4, CPm and p26, which represent the remainder of the closterovirus gene array (Fig. 1a). ORF1 encoded a putative protein of 4 kDa that had no counterpart with any other protein described for the Closteroviridae and no significant homology with any other sequence in the databases.

ORF2 encoded a putative protein of 77·5 kDa. The predicted protein was identified as CPm based on significant homology with other characterized closterovirus CPm proteins, especially those from criniviruses (Livieratos et al., 2002). Studies of clostero- and crinivirus particles have shown that CPm is assembled at one end of the virus (Agranovsky et al., 1995; Tian et al., 1999; Zinovkin et al., 1999) and is essential for virion assembly and cell-to-cell movement (Alzhanova et al., 2000, 2001; Napuli et al., 2003). In LIYV, CPm has been implicated in virus transmission by the whitefly vector (Tian et al., 1999).

ORF 3 encoded a putative protein of 26·4 kDa. Putative proteins of a similar size and genomic position are found in LIYV RNA 2 (Klaassen et al., 1995), CYSDV RNA 2 (Aguilar et al., 2003; Livieratos & Coutts, 2002), SPCSV RNA 2 (Kreuze et al., 2002) and CuYV RNA 1 (Hartono et al., 2003) and appear to be unique to the genus Crinivirus. However, PYVV p26 shared only limited identity with its counterpart proteins in the criniviruses (maximum 36 % identity with CuYV p26) and no significant homology with any other sequence in the databases.

Northern hybridization analysis of PYVV dsRNAs
In agreement with the single-strand conformation polymorphism analysis (Offei et al., 2004), electrophoretic and Northern hybridization analysis illustrated that the dsRNA profiles of the three largest dsRNA species from numerous field-infected Colombian PYVV isolates were virtually indistinguishable from the Peruvian isolate used in this study (Fig. 3). To distinguish between the different dsRNA species present in the PYVV dsRNA preparations, radiolabelled probes were constructed from clones of each dsRNA for Northern blot hybridization. Riboprobes generated from clones 104 and 105 only hybridized with RNA 1 (Fig. 1c, lanes 1 and 2, respectively). Riboprobes generated from clones HSP70h, 103 and CP only hybridized with RNA 2 (Fig. 1c, lanes 3–5, respectively), while riboprobes generated from clones A3-A4, 5'-CPm, CPm and p26+3'-UTR only hybridized with RNA 3 (lanes 6–9, respectively). Riboprobes A3-A4 and p26+3'-UTR also hybridized with the x and y dsRNA species (Fig. 1c, lanes 6 and 9, respectively).



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Fig. 3. (a) Agarose gel electrophoresis (1 %) of dsRNA preparations from three different PYVV Colombian field isolates (lanes 1–3). (b) Northern blot hybridization analysis of dsRNA extracts from a Colombian field PYVV isolate. The blots were hybridized with positive-sense riboprobes derived from the HSP70h and p26 ORFs (lane1), the HSP70h ORF (lane 2) and the HEL motif (lane 3). A molecular mass marker (Hyperladder I; Bioline) was included in lane 4 of both analyses and plant rRNA is indicated with an arrow.

 
Using probes constructed from cloned RT-PCR amplification products (detailed in Fig. 1a), Northern hybridization analysis confirmed that the PYVV dsRNAs contained the typical crinivirus Hsp70h, p60, p9, CP, CPm and p26 ORFs distributed on two dsRNA molecules, RNA 2 and RNA 3. In addition, the two smallest PYVV dsRNA species (designated x and y) contained sequences derived from the 5' and 3' termini of PYVV dsRNA 3 with extensive internal deletions. These observations suggest that these species are unlikely to represent 3' co-terminal subgenomic dsRNAs, as they do not contain contiguous sequences and are more likely to be defective (D) dsRNAs. D RNAs are a common feature of closterovirus infections and in the most extensively studied crinivirus example, LIYV, the D RNAs were derived exclusively from LIYV RNA 2 and included both the 5' and 3' termini of LIYV RNA 2, but had one extensive internal deletion. In LIYV-infected plants, these D RNAs exist as a heterogeneous population, differing in size and deletion location (Rubio et al., 2000).

Phylogeny
Phylogenetic analyses were performed on the amino acid sequences of the conserved proteins RdRp, Hsp70h, p60 and CP of all sequenced members of the family Closteroviridae. The data revealed that PYVV sequences consistently cluster together with the known members of the genus Crinivirus and the unassigned Little cherry virus 1 (LChV-1), whose vector is not known (Fig. 4). PYVV is distinct from the members of the aphid-transmissible genus Closterovirus and the mealybug-transmissible genus Ampelovirus, and is most closely related to CuYV, a T. vaporariorum-transmitted crinivirus denoted as a strain of Beet pseudoyellows virus (Hartono et al., 2003).



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Fig. 4. Dendrograms showing phylogenetic trees of closteroviruses as determined from homologous putative genes. These relationships were determined from the amino acid sequences deduced for the RdRp (a), Hsp70h (b), p60 and equivalent proteins (c), and CP (d) of the respective closteroviruses. For RdRp analysis, the corresponding domain of Maize dwarf mosaic virus (MDMV; GenBank accession no. NP569138) was used as an outgroup. For Hsp70h analysis, the corresponding domain of the tomato Hsp70h (Lycopersicon esculentum; GenBank accession no. P34935) was used as an outgroup. For CP analysis, the corresponding sequence from Tobacco mosaic virus (TMV; GenBank accession no. P03576) was used as an outgroup. The phylograms were generated by the CLUSTAL_X program (Thompson et al., 1997) as described in the text. The scale box represents distances scaled as substitutions per amino acid residue. For each internal node, bootstrap numbers (runs out of a thousand in which the given node appeared) are included. The closterovirus GenBank accession numbers are as shown in Fig. 2, plus: BYV, AF190581; Beet yellow stunt virus (BYSV), U51931; GLRaV-1, -2, -3 and -5, AF195822, AF039204, AF037268 and AF039552, respectively; LChV-2, AF333237; Pineapple mealybug wilt-associated virus 1 (PMWaV-1), AF414119; PMWaV-2, AF28313; ToCV, AF024630.

 
UTRs
As with the first 5, 8, 10 and 6 nt of the respective 5'-UTRs of LIYV, SPCSV, CuYV and CYSDV RNA 1 and 2, which are identical for each virus (Klaassen et al., 1995; Kreuze et al., 2002; Hartono et al., 2003; Aguilar et al., 2003), the first 5 nt of all three PYVV RNAs were also identical. The putative 5'-UTRs of PYVV RNAs 1, 2 and 3, which were 182, 620 and 626 nt in length, respectively, were analysed for secondary structure using the STAR program. Interestingly, RNA 1, having by far the shortest 5'-UTR, revealed a repeated sequence (nt 61–87 and nt 95–121) just upstream of a long (31 nt) single-stranded region of C and A residues only. These repeats were folded into a hairpin with a conserved symmetrical internal loop having two conserved C–U mismatches, while the bottom stem was supported by one covariation. The only other difference was a single substitution in the hairpin loop (Fig. 5a). Interestingly, an identical hairpin was present in RNA 3 (nt 77–101) as part or top of a longer hairpin, which in turn was also immediately adjacent to a downstream A/C-rich region (Fig. 5a). In this case, three covariations strongly supported the existence of the top part of this hairpin. However, no equivalent repeated structure was found upstream in RNA 3, except for possibly two similar C residues in an internal loop (nt 18–19, Fig. 5a). For RNA 2, two hairpins having a stem region in common (nt 25–57 and nt 76–120) were also proposed. Again, both hairpins were followed downstream by a C/A-rich region. The occurrence of two single-stranded C and U residues, respectively, at the bottom of the repeated stem may be reminiscent of the situation in the two repeated hairpins of RNA 1. So far, no common features have been detected for PYVV RNAs 2 and 3 for the regions downstream of the C/A-rich regions, nor when compared with the sequences of the other crinivirus RNAs. Also, no (repeated) hairpins have been found so far in either RNA 1 or RNA 2 of the other criniviruses.



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Fig. 5. (a) Structure alignment of the 5' part of the 5'-UTR of PYVV RNAs 1, 2 and 3. Pairs of rightward- and leftward-pointing arrows below the sequence indicate stem–loop structures. Nucleotides involved in base pairing are shown in highlighted upper case; unpaired bases are shown in lower case. (A/C)n denotes the A/C-rich region in the 5'-UTRs (see text). The underlined regions in PYVV RNA 2 represent a repeated stem region. Dots have been introduced to maximize the alignment. (b) Structure alignment of the 3'-terminal region of the 3'-UTRs of RNAs of the genus Crinivirus. For comparison, the 3' termini of two representative members each of the genera Closterovirus and Ampelovirus are shown in the bottom panel. Note that LIYV RNA 2 is set apart to emphasize its divergent structure with respect to hpI and PK1. Pairs of rightward- and leftward-pointing arrows above the sequence indicate stem–loop structures. The putative stem region A1 is shown in bold upper case. See (a) for definition of stem regions. Abbreviations of closteroviruses are as shown in Figs 2 and 4.

 
The putative 3'-UTRs of PYVV RNAs 1, 2 and 3 are 241, 291 and 232 nt in length, respectively. Using the STAR algorithm (Gultyaev et al., 1995) and sequence comparisons, four stem–loop structures, hpI–hpIV, were identified in the 3'-terminal 180 nt of RNAs 1, 2 and 3 (Fig. 5b; Fig. 6). HpI and hpII were flanked by a putative interaction, designated A1. Of these stem–loops, hpII, hpIII and hpIV were supported by covariations in RNAs 1 and 2 (Fig. 5b). HpI was strongly conserved both in primary and secondary structure and showed the possibility of pseudoknot formation with the G/C-rich sequence near the 3' end (not predicted by STAR). To obtain phylogenetic support for hpI and the pseudoknot PK1, the 3'-UTRs of RNAs from other criniviruses were folded. HpII, III and IV could also be identified in RNAs from SPCSV, CYSDV, LIYV and CuYV, yielding many covariations that supported their existence (Fig. 5b). HpI was conserved in RNAs 1 and 2 of SPCSV, CYSDV and CuYV. In LIYV RNA 1, hpI was missing stem region B2 but was otherwise almost identical to hpI of the SPCSV RNAs. In LIYV RNA 2, whose 3'-UTR shares only 30 % sequence identity with that of RNA 1, the equivalents of hpI and PK1 could not be formed. Interestingly, the possibility of pseudoknot formation is conserved in the other crinivirus RNAs and is supported by covariations in SPCSV and CYSDV RNAs (Fig. 7). This type of pseudoknot is quite unusual in that it has a small stem–loop inside loop L1 (Pleij et al., 1985). Comparison with other members of the family Closteroviridae showed that the pseudoknot is conserved in the genus Closterovirus as well (Fig. 5b; Fig. 7). It is noteworthy that the 3'-UTR of LChV-1 RNA, which is an unassigned species in the family Closteroviridae (Martelli et al., 2002), shared many features with the 3'-UTR of criniviruses, including the formation of PK1 (Fig. 5b; Fig. 7).



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Fig. 6. Secondary structure model for the 3'-UTR of PYVV RNAs (the sequence of RNA 3 is shown). Stem–loop structures are denoted as hpI, hpII, hpIII and hpIV, and are composed of stem elements B1 and B2, C1 and C2, D1–D3, and E1, respectively. Sequences involved in the putative interaction A1 are underlined. Dotted lines indicate the pseudoknot interaction PK1. Lines between E1 and D1 and between C1 and B1 have been introduced for the sake of clarity.

 


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Fig. 7. Schematic representation of the pseudoknot in the three PYVV RNAs. Only the relevant bases are shown. The inset shows a schematic diagram of a classic H-type pseudoknot. S1 and S2 denote stems 1 and 2; L1 and L2 denote loops 1 and 2. Covariations occurring in S2 of other viral RNAs from the family Closteroviridae are indicated by the boxed base pairs below S2. {Delta} denotes the absence of the hairpin in L1 from LIYV RNA 1 and BYV RNA.

 
Conclusions
Due to the lack of a successful protocol for the purification of PYVV virions (Salazar et al., 2000) and, subsequently, genomic ssRNA, we used dsRNA preparations to clone and sequence the PYVV genome of 17 kb. Both our sequence and Northern hybridization analysis showed that RNA 1 encodes the replication module, while two distinct RNA molecules contain the remainder of the closterovirus hallmark gene array. These data suggest that PYVV could possess a tripartite genome and are supported by the observation that all three PYVV RNA 3'-UTRs are very similar but different from each other. To confirm this, an analysis of virion ssRNA and the detection of subgenomic RNAs in infected plants are ultimately required, bearing in mind the well-documented formation of D RNAs for several closteroviruses (Che et al., 2003; Rubio et al., 2000). Irrespective of this, crinivirus genomes appear to be particularly diverse immediately downstream of the RdRp ORF and the presence of a sole ORF (p7) in PYVV RNA 1 is in agreement with this notion. Also, the p7 ORF, which is positioned between the Hsp70h and p60 ORFs in PYVV (this study), CuYV (Hartono et al., 2003) and CYSDV (Aguilar et al., 2003), is absent from LIYV and SPCSV RNA 2 (Klaassen et al., 1995; Kreuze et al., 2002). The 3'-UTRs of all crinivirus and some closterovirus RNAs are predicted to contain a very similar secondary structure that includes four stem–loops followed by a pseudoknot structure. One of the major functions of the 3' cis elements is the initiation of negative-strand synthesis, which involves higher-order structures concerned with motif presentation or promoter replicase binding functions (Buck, 1996). In addition, 3' structures can be involved in RNA stability, cellular targeting, packaging and regulation of RNA synthesis and translation (Dreher, 1999). Studies on the importance of 3' structures in closterovirus RNA replication have been described for CTV (Satyanarayana et al., 2002) but similar investigations with criniviruses have yet to be addressed.


   ACKNOWLEDGEMENTS
 
We thank The Central Research Fund of the University of London and the Department for International Development (DFID-UK) for financial support.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 16 December 2003; accepted 17 February 2004.



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