Nucleotide sequence and organization of ten open reading frames in the genome of Grapevine leafroll-associated virus 1 and identification of three subgenomic RNAs

Claudia F. Fazeli1 and M. Ali Rezaian1

CSIRO Plant Industry and Cooperative Research Center for Viticulture, Adelaide Laboratory, PO Box 350, Glen Osmond, South Australia 50641

Author for correspondence: Ali Rezaian. Fax +61 8 8303 8601. e-mail ali.rezaian{at}pi.csiro.au


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The genome of Grapevine leafroll-associated virus 1 (GLRaV-1) was cloned and the sequence of 12394 nts determined. It contains 10 major open reading frames (ORFs) and a 3'-non-coding region lacking a poly(A) tract. The first ORF (ORF 1a) encodes a putative RNA helicase at the C-terminal portion of an apparently larger protein. The downstream ORF, 1b, overlaps ORF 1a and lacks an initiation codon. This ORF encodes an RNA-dependent RNA polymerase of Mr 59276. ORF 2 encodes a small hydrophobic protein of Mr 6736, and ORF 3 encodes a homologue of the HSP70 family of heat shock proteins and has an Mr of 59500. ORF 4 encodes a protein with an Mr of 54648 that shows similarity to the corresponding proteins of other closteroviruses. ORF 5 encodes the viral coat protein (CP) with an Mr of 35416. The identity of this ORF as the CP gene was confirmed by expression in Escherichia coli and testing with the viral antibody. ORFs 6 and 7 code for two CP-related products with Mr of 55805 and 50164, respectively. ORFs 8 and 9 encode proteins of Mr 21558 and 23771 with unknown functions. Using DNA probes to different regions of the GLRaV-1 sequence, three major 3'-coterminal subgenomic RNA species were identified and mapped on the GLRaV-1 genome. Phylogenetic analyses of the individual genes of GLRaV-1 demonstrated a closer relationship between GLRaV-1 and GLRaV-3 than with other closteroviruses.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Leafroll is a damaging disease of the grapevine causing yield losses of up to 40% (Woodham et al., 1984 ). Seven distinct phloem-restricted closteroviruses have been identified in various leafroll-affected material. Grapevine leafroll-associated virus 1 (GLRaV-1) is one of the most important types (Martelli et al., 1997 ). It is present in some of the major grapevine varieties grown in Australia and is associated with low crop yields in Sultana clones (Habili et al., 1996 , 1997 ). Apart from transmission by grafting, GLRaV-1 may be transmitted by the scale insects Neopulvinaria innumerabilis and Parthenolecanium corni (Fortusini et al., 1997 ).

Particles of GLRaV-1 are filamentous and contain a coat protein (CP) with an Mr of 39000 (Gugerli et al., 1984 ). A replicative form double-stranded RNA (dsRNA) species of ca. 19 kb and several smaller dsRNAs are consistently isolated from GLRaV-1-infected tissues (Habili & Rezaian, 1995 ). These smaller dsRNA species arise from infection with mixed viruses or may be subgenomic molecules. One of the smaller dsRNA species extracted from GLRaV-1-infected tissues hybridizes to a DNA probe made from the 19 kb viral genome (Habili & Rezaian, 1995 ; Habili et al., 1997 ). Subgenomic RNA species are considered to be part of gene expression strategies utilized by closteroviruses (Agranovsky, 1996 ) and have been found in Beet yellows virus (BYV) (Agranovsky et al., 1994 ), Lettuce infectious yellows virus (LIYV) (Klaassen et al., 1995 ) and Citrus tristeza virus (CTV) (Hilf et al., 1995 ).

In this paper, we report the nucleotide sequence and organization of GLRaV-1 genes in a 12·5 kb portion of the genome, and identify the viral CP gene and 3'-coterminal subgenomic RNAs associated with the virus infection.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Isolation and analysis of GLRaV-1 dsRNA.
Viral dsRNA was extracted by the method of Rezaian et al. (1991) from a GLRaV-1-infected Sultana, clone B4L (Habili et al., 1997 ; Woodham et al., 1984 ), treated with DNase I, electrophoresed in a 6% polyacrylamide gel in TAE buffer (Loening, 1967 ) and visualized by silver staining (Merril et al., 1981 ). Northern blot analysis was carried out as described previously (Habili et al., 1995 ).

{blacksquare} Synthesis of GLRaV-1-specific dsDNA.
First-strand cDNA synthesis was carried out as described (Fazeli et al., 1998 ), using a specific primer. The initial cloning of GLRaV-1 genomic RNA was achieved using oligonucleotide primers P3v and P5c (Table 1), which were derived from an existing DNA clone, LR34 (Habili et al., 1997 ). The virus-specificity of this clone was confirmed by PCR using GLRaV-1 particles trapped by antibody-coated magnetic beads (Karlsson & Platt, 1991 ; Fazeli et al., 1998 ).


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Table 1. Primers used for cloning GLRaV-1 RNA

 
The procedure for second-strand DNA synthesis was that of Froussard (1992) with some modifications. Random hexamer (0·1 µg/µl) linked to a known oligonucleotide at its 5'-end (P1–6N, Table 1) was added to the first-strand reaction mixture, heated at 95 °C for 3 min and cooled on ice. Primer extension was carried out for 30 min at 37 °C in a 50 µl reaction containing 50 mM Tris–HCl, pH 7·2, 10 mM MgSO4, 0·1 mM DTT, 0·3 mM each of the four dNTPs and 5 U of DNA polymerase I (Klenow fragment, Promega). The excess primer was then removed using a Centricon-30 column (Amicon). Double-stranded DNA (dsDNA) was amplified by PCR in 30 µl of 10 mM Tris–HCl, pH 9·0 at 25 °C, 50 mM KCl, 0·01% Triton X-100, 1·5 mM MgCl2, 200 µM each of the four dNTPs, 1 µM of the known oligonucleotide (P1, Table 1), 1 µM of a virus-specific primer and 2·5 U Taq DNA polymerase (Promega). The PCR protocol consisted of 4 min at 95 °C, followed by 35 cycles of 1 min at 94 °C, 2 min at 56 °C and 2 min at 72 °C, and finally an extension time of 7 min at 72 °C.

{blacksquare} cDNA cloning and sequencing.
The PCR products in a size range between 500 and 2000 nt were ligated into a pGEM-T vector (Promega) and electroporated into E. coli strain DH5{alpha}. Recombinant plasmids were digested with NcoI and SpeI and screened by Southern blot analysis (Sambrook et al., 1989 ) using a 32P-labelled probe. The template for probe synthesis was the cDNA clone from a previous round of cloning which overlapped the new clone. The DNA clones were sequenced by automated cycle sequencing at Flinders University, Adelaide.

Sequence data were analysed using the GCG package (Genetics Computer Group, USA), version 7.3. The putative translation products of major open reading frames (ORFs) were compared to other proteins in the database. Searching of the non-redundant amino acid sequence database of the National Center for Biotechnology Information (NCBI) was performed using the programs BLAST and BLASTP (Altschul et al., 1990 ).

{blacksquare} Determination of the 3'-terminal sequence of GLRaV-1 RNA.
GLRaV-1 dsRNA was 3'-poly(A)-tailed using poly(A) polymerase (Amersham). A reaction of 20 µl contained the viral dsRNA extracted from 5 g green-bark tissue, 20 mM Tris–HCl, pH 7·0, 50 mM KCl, 0·7 mM MnCl2, 0·2 mM EDTA, 100 µg/ml acetylated BSA, 10% glycerol, 3·3 µM [{alpha}-32P]ATP, 0·5 mM ATP and 1000 U poly(A) polymerase. Poly(A) tailing was carried out at 30 °C for 30 min and stopped by the addition of 80 µl TE buffer. The dsRNA was recovered using RNaid w/Spin kit (BIO 101) and eluted in 20 µl water. The tailed RNA was used in a reverse-transcription reaction (Fazeli et al., 1998 ) of 40 µl using 800 ng dT(15) primer. The second-strand DNA was synthesized by PCR as described above in 50 µl using 1 µM dT(15) primer and 1 µM of a virus-specific primer (P16v, Table 1).

{blacksquare} Expression of the GLRaV-1 ORF 5 and ORF 6 in E. coli.
The complete ORF 5 and the 3'-half of ORF 6 were amplified (Fazeli et al., 1998 ). The virion-sense primers contained a BamHI site and the complementary-sense primers contained a HindIII site close to the 5'-ends. The complete ORF 5, excluding the initiation ATG codon, was amplified using CPv and CPc primers (Table 1). The 3'-half of ORF 6 was amplified by CPdv and CPdc primers (Table 1). The amplified DNA products were fused in-frame with coding sequence for an initiation methionine and six histidine residues in the pQE-30 expression vector (Qiagen). The identity of the clones was confirmed by restriction analysis.

The recombinant plasmids were electroporated into E. coli strain M15/pREP4 (Qiagen) and expression was induced with 0·1 mM IPTG. The expressed proteins were purified using Ni2+–NTA resin (Qiagen) under denaturing conditions according to the manufacturer’s instructions. The recombinant proteins were analysed in a 10% SDS–polyacrylamide gel and detected by Western blot analysis (Fazeli et al., 1998 ) using either a virus-specific monoclonal antibody (Bioreba), a polyclonal antibody or a monoclonal antibody to the His-tag (Qiagen).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Cloning and sequencing strategy
A previously described DNA clone of GLRaV-1 (Habili et al., 1997 ) was used to extend the viral sequence towards both 5'- and 3'-ends of the viral genome. The specificity of this DNA clone, LR34, to GLRaV-1 was confirmed by a PCR test of RNA from virus particles. The GLRaV-1 particles were trapped on magnetic beads that were coated with a monoclonal antibody against the viral CP.

A 12·5 kb region at the 3'-half of the GLRaV-1 genomic RNA was cloned in 14 steps (Fig. 1A, B). In each step, a specific primer was utilized for first-strand cDNA synthesis. Second-strand DNA synthesis was achieved by the use of a primer containing a random hexamer at its 3'-end (P1-N6, Table 1). Following amplification by PCR, the dsDNA products were cloned directly and sequenced. In each cloning step, the specific primer was selected about 60 nt away from one end of the DNA clone. This allowed the use of the original clone as a probe for screening the cDNA libraries by Southern hybridization. A total of 90 DNA clones were selected and used for sequencing. In addition, the sequence of the entire 12·5 kb region was confirmed by re-cloning the viral dsRNA using specific primers. A total of 34 independent DNA cloning steps were carried out with specific primers and two clones were sequenced from each region (Fig. 1C). Sequence data from the first and second round of cloning were consistent but some of the clones from ORFs 6 and 7 showed a significant number of variations. The nature of these variations is being investigated.



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Fig. 1. A schematic presentation of the overlapping cDNA clones used in sequencing of the 3'-portion of the GLRaV-1 genome. (A) The genome organization of GLRaV-1. (B) Representatives of 90 DNA clones selected in 14 cloning steps are shown as lines. DNA clone LR34 (Habili et al., 1997 ) is the starting point for cloning and sequencing the genomic RNA. (C) DNA clones resulting from direct RT–PCR using the virus-specific primers in the second round of cloning.

 
Compilation of the data resulted in sequence information for a contiguous 12394 nt region (accession no. AF195822) from the 3'-end of the 19 kb GLRaV-1 genome.

Genome organization of GLRaV-1 RNA
ORF 1a encodes a putative helicase.
The 5' 1196 nt of the sequence represents an incomplete ORF (ORF 1a) which is likely to extend beyond the 5'-end of the sequence. The translation product of Mr 44327 from this ORF has a significant sequence similarity to the helicases (HEL) of other closteroviruses (Fig. 2), showing 60·6% similarity with GLRaV-3 HEL (Ling et al., 1998 ). The GLRaV-1 HEL contains the conserved motifs among the HELs of positive-stranded RNA viruses of superfamily I (Koonin & Dolja, 1993 ). This sequence similarity indicates that ORF 1a may be part of a long reading frame starting near the 5'-end of the GLRaV-1 genome.



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Fig. 2. Conserved motifs of closteroviruses present in the putative HEL and POL domains of GLRaV-1. (A) The amino acid sequence alignment of GLRaV-1 with the consensus sequences of HEL and POL of BYV, GLRaV-2, CTV, BYSV, LCV, LIYV and GLRaV-3 is shown. The conserved motifs I–VI in the superfamily I viral and cellular HEL (Gorbalenya & Koonin, 1993 ) and sequence elements I–VIII in polymerases (Koonin & Dolja, 1993 ) are indicated. The GDD motif in the POL conserved region VI is boxed. Dots indicate gaps and the amino acid residues which are identical or similar among the proteins are shown in upper case. The residues conserved between GLRaV-1 and other closteroviruses are shown in bold. (B) The +1 ribosomal frameshifting sites (bold) in GLRaV-1 (top) and GLRaV-3 (bottom). Identical nucleotides are shown in upper case and one-letter translation products are shown.

 
ORF 1b encodes a putative RNA-dependent RNA polymerase.
The second ORF of 1580 nt (ORF 1b) lacks an initiation codon and overlaps the last 50 nt of ORF 1a. ORF 1b is in a frame different from ORF 1a and may be expressed via +1 ribosomal frameshifting. The translation product of ORF 1b, a putative protein of Mr 59276, shows significant sequence similarity to the RNA-dependent RNA polymerases (POL) of closteroviruses in the database (Fig. 2). The GLRaV-1 POL contains a Gly-Asp-Asp (GDD) motif, which is a hallmark of RNA polymerases (Bruenn, 1991 ). GLRaV-1 POL also contains eight conserved sequence motifs (I-VIII, Fig. 2) reported in the RNA polymerases of positive-stranded, negative-stranded and dsRNA viruses as well as in reverse-transcriptases from retroviruses and DNA-dependent DNA polymerases (Bruenn, 1991 ; Koonin & Dolja, 1993 ).

ORF 2 encodes a small hydrophobic protein.
ORF 2 follows a large non-coding region (NCR) of 793 nt, and encodes an Mr 6736 product (p7). This protein lacks significant amino acid sequence similarity to the proteins in the current database. p7 contains a strongly hydrophobic segment at its N terminus. Direct sequence alignments showed that p7 is similar to small hydrophobic proteins reported in other closteroviruses (results not shown). The amino acid sequence of GLRaV-1 p7 shows 61·3%, 54·1% and 40% similarity to its homologues in GLRaV-3, CTV and BYV, respectively (Ling et al., 1998 ; Karasev et al., 1995 ; Agranovsky, 1996 ). The hydrophobic N-domains in these proteins suggest they may be membrane-associated proteins.

ORF 3 encodes a protein homologous to the HSP70 family of heat shock proteins.
ORF 3 (Fig. 1) encodes a product of Mr 59500. BLAST searching showed that this product is a homologue of the HSP70 family of cellular heat shock proteins as well as the corresponding proteins of other closteroviruses. The HSP70 homologue in GLRaV-1 contains the conserved motifs among cellular HSP70s (Ting & Lee, 1988 ) and shows 43·1% amino acid sequence identity (62·8% similarity) to the HSP70 homologue of GLRaV-3 (results not shown).

ORF 4.
ORF 4 (Fig. 1) overlaps ORF 3 by 1 nt and encodes a product of Mr 54648 (p55). The size and location of this ORF are similar to those of corresponding ORFs in other closteroviruses. Direct comparison of GLRaV-1 p55 with the translation products of other closteroviruses showed weak alignments with GLRaV-3 p55, with 21·3% sequence identity (44·5% similarity), and with BYV p64, an HSP90 homologue, with 18·5% sequence identity (45·4% similarity) (results not shown).

ORF 5, the putative CP gene.
ORF 5 (Fig. 1) encodes a protein of 322 amino acids with a calculated Mr of 35416. It contains the amino acid residues N, R, G and D in positions 231, 234, 264 and 275, respectively (Fig. 3). These amino acid residues are conserved among the CP and the diverged copies of CP (CPd) products of closteroviruses (Ling et al., 1997 ). In addition, two of these amino acid residues, R and D, are conserved in the CPs of all the filamentous plant viruses (Dolja et al., 1991 ). This protein shows the highest amino acid sequence similarity with the CP of GLRaV-3 (51·8%). It is therefore likely that ORF 5 encodes the GLRaV-1 CP.



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Fig. 3. Similarity among the amino acid sequences of the CP, CPd1 and CPd2 of GLRaV-1 and the CP and CPd of GLRaV-3 (Ling et al., 1997 ). Dots indicate gaps and the amino acid residues which are identical or similar among the proteins are shown in upper case. The conserved amino acid residues N, R, G and D present in all the CPs and CPds of closteroviruses are in bold.

 
ORFs 6 and 7, two diverged copies of the GLRaV-1 CP gene.
ORF 6 and ORF 7 (Fig. 1) are only three nt distant from each other and are in the same frame. They code for translation products of 500 and 440 amino acids, respectively. The amino acid sequence of the proteins encoded by ORF 6 (p56) and ORF 7 (p50) showed 27·2% identity (48% similarity) to each other. Direct amino acid sequence alignments revealed that p56 is 47·3% and p50 is 43·4% similar to the product of ORF 5, the putative CP gene. These similarities include the presence of the conserved amino acid residues N, R, G and D (Fig. 3), which are the hallmarks of the CPs and CPds of the closteroviruses. It is therefore suggested that both p56 and p50 are GLRaV-1 CPds.

ORFs 8 and 9.
ORFs 8 and 9 potentially code for proteins of 189 and 210 amino acids with calculated Mr of 21558 and 23771, respectively. These products did not show any significant sequence similarity to other proteins in the current databases and their possible roles in virus multiplication or pathogenesis remain unknown.

The NCR at the 3'-end of the GLRaV-1 genome.
The genome of GLRaV-1 contains an NCR of 363 nt at the 3'-end, terminating with ATT. This sequence was determined by sequencing 14 independent DNA clones from this region (Fig. 1C) and sequencing clones generated from 3'-poly(A)-tailed RNA.

The sequence of the 3'-NCR of GLRaV-1 showed no significant similarity to the 3'-NCR of other closteroviruses. Computer-assisted secondary structure predictions showed several potential stem–loop structures in the region. The largest structure contains 28 nts at position 12347–12375 of the sequence. This putative structure contains a stem of 12 bp, two G:U pairs, one mismatch and a loop of 3 nts.

Identification of the GLRaV-1 CP gene
The complete 966 bp sequence of the GLRaV-1 ORF 5 was cloned in the bacterial expression vector pQE30, in-frame with the 6xHis ORF. The same procedure was also applied to ORF 6. However, due to the difficulty in expressing the complete ORF, its 5' 990 nt and 3' 933 nt were separately cloned and only the latter resulted in protein expression.

The proteins expressed from ORF 5 and ORF 6 were purified under denaturing conditions using Ni2+–NTA affinity resin and were analysed by Western blotting. The protein expressed from ORF 5 reacted with both polyclonal and monoclonal antibodies to GLRaV-1 CP (Fig. 4A and results not shown), while the ORF 6 encoded protein was not recognized by either of the antibodies (Fig. 4B). In addition, Western blot analyses showed that the protein expressed from ORF 5 had an electrophoretic mobility indistinguishable from the GLRaV-1 CP extracted from infected tissue (Fig. 4A). These results indicate that ORF 5 is the gene for GLRaV-1 CP.



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Fig. 4. Western blot analysis of the GLRaV-1 proteins expressed in E. coli. (A) Analysis of the protein expressed from ORF 5 using monoclonal antibody to the GLRaV-1 CP (mAb-1). Lanes 1, crude protein extract of E. coli containing the expressed protein; 2, the expressed protein purified by affinity matrix; 3, the viral CP partially purified from a GLRaV-1-infected tissue. (B) Analysis of the protein expressed from ORF 6. Lanes 1, the expressed protein; 2, GLRaV-1 CP extracted from infected Sultana clone B4L; 3, a mixture of the expressed protein and GLRaV-1 CP extracted from infected Sultana B4L. Antibodies to GLRaV-1 CP or to His-tag, used for detecting the proteins, are shown below each panel. The molecular masses (kDa) of the pre-stained protein markers (Novex) are shown.

 
GLRaV-1 contains 3'-coterminal sgRNAs
dsRNA isolated from GLRaV-1-infected Sultana clone B4L contained a genomic-size species of ca. 19 kbp and an array of smaller dsRNAs (Fig. 5). Using the GLRaV-1 sequence, six DNA probes were made to different locations on the viral genome. These included portions of ORF 3, ORF 4, ORF 5, ORF 6, ORF 7 and the 3'-NCR (Fig. 5). The probes were used individually in Northern blot analysis to examine their hybridization to the smaller dsRNA species. All six probes detected the genomic-size dsRNA. Probes to ORF 3 and ORF 4 showed no hybridization to any of the smaller dsRNA species (Fig. 5, lanes 1 and 2). The probe to the complete sequence of ORF 5 hybridized to a smaller dsRNA species which was estimated to be about 6 kbp (Fig. 5, lane 3). This species was also detected by probes to ORF 6 and to the DNA clone LR34 (Habili et al., 1997 ) located in ORF 7 (Fig. 5, lanes 4 and 5). Two other smaller dsRNA species of 1·7 and 1 kbp were detected using a probe to the last 497 nt at the 3'-end of the GLRaV-1 genome (Fig. 5, lane 6). These results indicate that these three small dsRNAs share the sequence of the 3'-end of the GLRaV-1 genomic RNA and are subgenomic.



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Fig. 5. Northern blot analysis of dsRNA extracted from GLRaV-1-infected tissue. The GLRaV-1-specific probes were to ORF 3 (lane 1), ORF 4 (lane 2), ORF 5 (lane 3), ORF 6 (lane 4), ORF 7 (lane 5) and the 497 nts at the 3'-end of the sequence (lane 6). The subgenomic RNAs of GLRaV-1 are shown by arrows on the right. The sizes of the dsRNA species present in a GLRaV-1-infected grapevine given on the left are as estimated by Habili & Rezaian (1995) .

 
The 5' extremity of the 6 kb sgRNA was investigated further. Northern blot analysis showed that this sgRNA can be detected by a probe to ORF 5 but not with a probe from the 3' 900 nt of ORF 4 (Fig. 5, lane 2 and 3). This sgRNA was also detected using another probe to the sequence of the 103 nt intergenic region located between ORF 4 and ORF 5 plus the 5' 120 nt of ORF 5 (results not shown). It is therefore possible that the 5'-end of the 6 kb sgRNA species terminates in the 103 nt intergenic region. The sequence of this region was compared to that of the sgRNA promoters. The comparison showed that this region in GLRaV-1 contains the conserved nucleotides of the core promoter of alphavirus sgRNAs (Siegel et al., 1997 ). These conserved nucleotides (AGACGAA), which are located between positions -18 to +1 in the alphavirus-like superfamily, are located in the 103 nt intergenic region upstream of ORF 5 between positions 6805 and 6834 of the sequence.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The 3' 12394 nts of the GLRaV-1 genomic RNA contain 10 major ORFs (Fig. 6). These ORFs in the 5' to 3' direction encode a HEL, a POL, a small hydrophobic protein, an HSP70 homologue, a protein with weak similarity to the HSP90 homologues of closteroviruses, a CP, two diverged copies of the CP, and two GLRaV-1-specific proteins of unknown function. Interestingly, duplication of the CP gene has occurred in two ORFs in GLRaV-1. The translation products of both of these ORFs contain high amino acid sequence similarity with the viral CP and contain the four N, R, G and D residues (Fig. 3) which are the hallmarks of the CPs and CPds of closteroviruses (Ling et al., 1997 ). Dual duplication of CP in two different ORFs has not been reported in other closteroviruses. The existence of apparently large duplications indicates that recombination events may have been involved (Mawassi et al., 1995 ). The biological significance of these gene repeats in the GLRaV-1 genome remains unknown. The CPd genes in GLRaV-1 are located downstream of the gene encoding the viral CP gene (Fig. 6). This arrangement is similar to that of GLRaV-3 (Ling et al., 1998 ), LIYV (Klaassen et al., 1994 ) and Little cherry virus (LCV) (Keim-Konrad & Jelkmann, 1996 ).



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Fig. 6. Comparison of the genome organization of GLRaV-1 with that of other known closteroviruses. Rectangles represent ORFs. Homologous genes are shaded similarly. Open boxes indicate genes with no statistical similarity to other proteins in existing databases. P-Pro, papain-like protease; MTR, methyltransferase; HEL, helicase; POL, polymerase; HSP70, homologue of HSP70 proteins; CP, coat protein; CPd, diverged copy of coat protein.

 
In BYV, CTV and LIYV, CPd packages a segment of 75–85 nm at one end of the viral RNA and may be involved in insect-transmission of the virus (Agranovsky, 1995 ; Febres et al., 1996 ; Tian et al., 1999 ). If the putative CPds in GLRaV-1 have a similar role, the sequence variation observed may provide an advantage in interaction of the virus with its host and vectors.

The presence of an HSP70-related gene in GLRaV-1 confirms the relationship of this virus with closteroviruses. The translation product of the HSP70 homologue of GLRaV-1 shows 62·8% amino acid sequence similarity to that of GLRaV-3. It also has 49·4% amino acid sequence similarity to the BYV HSP70 homologue, mostly in the N terminus (results not shown). The N-terminal motifs of the BYV HSP70 homologue show ATPase activity (Agranovsky et al., 1997 ), which is characteristic of the N termini of cellular HSP70s (Bork et al., 1992 ). It has been suggested that these protein homologues participate in the cell-to-cell movement of closteroviruses (Karasev et al., 1992 ; Agranovsky et al., 1998 ). In the case of Pea seed-borne mosaic virus, which does not encode this protein, the virus selectively induces the host HSP70 expression (Aranda et al., 1996 ). In some animal RNA viruses HSP70-related proteins may enhance the viral polymerase activity (Oglesbee et al., 1996 ).

An intriguing feature of the gene expression of closteroviruses is the presence of the unusually long ORF 1a encoding the viral protease, methyltransferase and RNA HEL. The downstream ORF 1b appears to lack an initiation codon and exists as an overlapping frame with ORF 1a. It has been proposed that polymerases in BYV (Agranovsky et al., 1994 ), CTV (Karasev et al., 1995 ), Beet yellow stunt virus (BYSV) (Karasev et al., 1996 ), GLRaV-2 (Zhu et al., 1998 ), LCV (Jelkmann et al., 1997 ), GLRaV-3 (Ling et al., 1998 ) and LIYV (Klaassen et al., 1995 ) are expressed via a +1 ribosomal frameshift. In this process, a translational frameshift takes place at some point prior to the termination of ORF 1a, resulting in continued translation in the frame containing ORF 1b and producing a fusion protein. It has been suggested that frameshifting involves a slippery sequence and a UAG stop codon in BYV (Agranovsky et al., 1994 ), a rare CGG arginine codon in CTV (Karasev et al., 1995 ) and a UAG stop codon in BYSV and GLRaV-2 (Karasev et al., 1996 ; Zhu et al., 1998 ). None of these features was found in GLRaV-1 using GCG-FOLD and squiggles sequence analysis softwares. In addition, no special sequence or stable secondary structure which may be indicative of frameshifting (Farabaugh, 1993 ) was found in GLRaV-1. The ORF 1a/1b overlapping region in GLRaV-1, however, is similar to LIYV in which frameshifting may be caused by slippage of tRNALys on an AAAG sequence (Klaassen et al., 1995 ). This overlapping region also shows significant similarity to that of GLRaV-3 (Fig. 2B). In both viruses, a UUUC is present which encodes phenylalanine in two adjacent frames, i.e. UUU and UUC. This may provide a slippage mechanism of tRNAPhe from one ORF to the other.

The overall organization of the GLRaV-1 genome (Fig. 6) is similar to those of other closteroviruses. The five-gene module of this family of viruses, consisting of genes encoding a small hydrophobic protein, an HSP70 homologue, a product of Mr 55000–64000, a CP and a CPd, is present in GLRaV-1. The phylogenetic proximity of this virus to other closteroviruses was evident from the sequence comparison between the individual genes of GLRaV-1 and those available in the database. The relationship of GLRaV-1 with closteroviruses was confirmed by the amino acid sequence similarity of their POL domain, which is considered to be a reliable region for phylogeny analysis (Koonin & Dolja, 1993 ). More than 66% sequence similarity between the POL domains of GLRaV-1 and GLRaV-3 has placed these two viruses in one branch in a phylogenetic tree (Fig. 7A). This phylogenetic proximity is also evident when comparing their HSP70 homologues and CPs with 43·1% and 32·9% amino acid sequence identity, respectively (Fig. 7B, C).



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Fig. 7. Phylogenetic analyses of closteroviruses. The viruses are compared based on the similarity between their POL domains (A), their HSP70 homologues (B) and their CPs and CPds (C). The amino acid sequences were obtained from the database. The trees were constructed by Pileup analysis software in the GCG package (University of Wisconsin, Madison, WI, 1991).

 

   Acknowledgments
 
We thank Giovanni Martelli for supplying polyclonal antibody to GLRaV-1, Nuredin Habili for providing the original DNA clone for GLRaV-1, Nigel Scott and Bob Symons for useful discussions, and Jamus Stonor for technical assistance.


   Footnotes
 
The GenBank accession number of the nucleotide sequence reported in this paper is AF195822.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Agranovsky, A. A. (1995). Structure and expression of RNA genomes of closteroviruses.Molecular Biology 29, 751-754.

Agranovsky, A. A. (1996). Principles of molecular organization, expression and evolution of closteroviruses: over the barriers.Advances in Virus Research 47, 119-159.[Medline]

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Received 6 July 1999; accepted 29 October 1999.