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
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Abstract |
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Introduction |
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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.
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Methods |
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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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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. 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 ).
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 TrisHCl, pH 7·0, 50 mM KCl, 0·7 mM MnCl2, 0·2 mM EDTA, 100 µg/ml acetylated BSA, 10% glycerol, 3·3 µM [-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
).
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 manufacturers instructions. The recombinant proteins were analysed in a 10% SDSpolyacrylamide 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).
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Results |
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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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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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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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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 stemloop structures in the region. The largest structure contains 28 nts at position 1234712375 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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Discussion |
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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 5500064000, 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. 7
A). 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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Acknowledgments |
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Footnotes |
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References |
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Received 6 July 1999;
accepted 29 October 1999.