Complete nucleotide sequence and genome organization of Grapevine leafroll-associated virus 3, type member of the genus Ampelovirus

Kai-Shu Ling{dagger}, Hai-Ying Zhu and Dennis Gonsalves{ddagger}

Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA

Correspondence
Kai-Shu Ling
KLing{at}saa.ars.usda.gov


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This study reports on the complete genome sequence of Grapevine leafroll-associated virus 3, the type member of the genus Ampelovirus. The genome is 17 919 nt in size and contains 13 open reading frames (ORFs). Previously, the sequence of 13 154 nt of the 3'-terminal of the genome was reported. The newly sequenced portion contains a 158 nt 5' UTR, a single papain-like protease and a methyltransferase-like (MT) domain. ORF1a encodes a large polypeptide with a molecular mass of 245 kDa. With a predicted +1 frameshift, the large fusion protein generated from ORF1a/1b would produce a 306 kDa polypeptide. Phylogenetic analysis using MT domains further supports the creation of the genus Ampelovirus for mealy-bug-transmitted viruses in the family Closteroviridae.

The nucleotide sequence data reported in this paper appear in GenBank under accession number AF037268.

{dagger}Present address: USDA, ARS, US Vegetable Laboratory, 2700 Savannah Highway, Charleston, SC 29414, USA.

{ddagger}Present address: Pacific Basin Agricultural Research Center, USDA, Hilo, HI 96720, USA.


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Closteroviruses cause serious diseases to a number of economically important crops worldwide, including beet, citrus, grapevine, lettuce, tomato, cherry, pineapple and sweet potato. With recent advances in molecular characterization of several closteroviruses, the taxonomic relationship of this once heterogeneous group of viruses is more defined (Karasev, 2000). However, only a few representative closterovirus genomes have been completely sequenced. These include the aphid-transmitted viruses Beet yellows virus (BYV) (Agranovsky et al., 1994) and Citrus tristeza virus (CTV) (Karasev et al., 1995) and the whitefly-transmitted Lettuce infectious yellows virus (LIYV) (Klaassen et al., 1995). Little cherry disease, previously thought to be caused by Little cherry virus 1 (Jelkmann et al., 1997), was recently shown to be caused by Little cherry virus 2 (LChV-2) (Eastwell & Bernardy, 2001; Rott & Jelkmann, 2001; Jelkmann et al., 1997), which is transmitted by mealy bugs. Another mealy-bug-transmitted virus, Pineapple mealybug wilt-associated virus 2 (PMWaV-2), has been sequenced except for the 5' terminus (Melzer et al., 2000).

Recently, the International Committee on Taxonomy of Viruses (ICTV) study group on closteroviruses and allied viruses revised the family Closteroviridae by creating a new genus, Ampelovirus (from ampelos, Greek for grapevine), with Grapevine leafroll-associated virus 3 (GLRaV-3) as its type member (Martelli et al., 2002). The initial proposal to create a new genus for mealy-bug-transmitted closteroviruses was suggested based on the analysis of the recent molecular and biological information in a review (Karasev, 2000). The revised family Closteroviridae now consists of three genera, Closterovirus, Ampelovirus and Crinivirus. The genus Closterovirus with type species BYV has a positive-sense single-stranded RNA (ssRNA) genome and contains viruses that are transmitted by aphids. The genus Ampelovirus with type member GLRaV-3 also has a positive-sense ssRNA with a larger coat protein (35–39 kDa) and is transmitted by mealy bugs (coccid or pseudococcid). The genome for viruses in the genus Crinivirus is generally divided into two ssRNA molecules, which are separately encapsidated in virions. All members of the genus Crinivirus are transmitted by whiteflies.

The objectives of the current study were to complete the genome sequence and to understand the genome organization of GLRaV-3. The NY1 isolate of GLRaV-3 (Hu et al., 1990) was used. dsRNA was extracted from phloem tissue of virus-infected grapevines collected from a central New York vineyard according to the method described by Hu et al. (1990). High-molecular-mass dsRNA (about 18 kb) was purified by electrophoresis in low-melting-point agarose gel and extracted using the phenol/chloroform method described in Sambrook et al. (1989).

cDNA synthesis and Lambda ZAPII cDNA library were carried out as described by Ling et al. (1997). Duplicate nylon membranes containing recombinant phage DNA were prepared and used for subsequent screening. A clone walking strategy was used to extend the nucleotide sequence from the previously sequenced portion of the GLRaV-3 genome (Ling et al., 1998). The clone closest to the 5' end of the contig was selected as a probe to screen the cDNA library (Ling et al., 1997) for clones that contained inserts extending further towards the 5' terminus. Using this strategy we were able to walk along the genome step by step to obtain most of the sequence for GLRaV-3. The RT-PCR gap-bridging strategy was applied to fill a sequence gap using sense primer 97-36 (5'-GGTAGAGGGGAGGAATGTGTA-3') and reverse complement primer 98-7 (5'-TAGACTGTTGGTGAAAGACA-3') derived from both ends of sequenced contigs. RT-PCRs were prepared with purified dsRNA as a template and the PCR product was either sequenced directly or cloned into pBlue T-vector (Novagen) and sequenced.

To determine the exact 5' end sequence, poly(A) was added to purified GLRaV-3-specific dsRNA by yeast poly(A) polymerase (US Biochemical) and reverse transcribed using oligo(dT) primer [KSL95-7; 5'-GGTCTCGAG(T)15-3'] and Moloney murine leukemia virus reverse transcriptase, similar to the method that was used to obtain the 3'-terminal sequence (Ling et al., 1998). Using this newly synthesized cDNA as the template, an RT-PCR product was amplified with oligo(dT) primer and the GLRaV-3 specific primer 97-47 (5'-AGGAAGTGGTACGTGGACGC-3'). The RT-PCR product was then cloned into pBlue T-vector for sequencing.

pBluescript SK(+) DNA inserts of selected clones were initially sequenced using T3 or T7 primers. Internal nucleotide sequences were obtained with virus-specific primers. Plasmid DNA was prepared according to the manufacturer's instruction for mini alkaline-lysis/PEG precipitation (Applied Biosystems); the sequencing reaction of the cloned DNA was prepared using ABI Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems) and sequenced by using an ABI 373 automatic sequencer at Cornell University, Geneva, NY.

Nucleotide sequences were assembled and analysed using the DNASTAR sequence analysis package (Madison, WI). Nucleotide sequences were entered into EditSeq and assembled using the SeqMan program. The MegAlign program was used to depict amino acid sequence similarity of GLRaV-3 with respect to other closteroviruses. The CLUSTAL W method of MegAlign was used to compare multiple sequences, to identify consensus sequences and to generate putative phylogenetic relationships. The 272 aa residue region starting from aa residue 460 of GLRaV-3 ORF1a that contains the MT conserved motifs was used for multiple alignment. The tentative phylogenetic tree was generated using CLUSTAL W method, followed by the neighbour-joining method in the MegAlign program with PAM250 residue weight table. To assess further the phylogenetic relationship, bootstrap was used to obtain a consensus tree and Tobacco mosaic virus (TMV) tomato strain (L) with protein ID CAA26085.1 (GenBank accession no. X02144) was selected as an outgroup member. Other sequences that were taken for alignment were derived from GenBank for BYV with protein ID CAA51871.1 (X73476), CTV with protein ID AAC59623.1 (U16304), LIYV with protein ID AAA61797.1 (U15440), LChV with protein ID CAA71285.1 (Y10237), GLRaV-2 with protein ID AAC40855.1 (AF039204) and PMWaV-2 with protein ID AAG13938.1 (AF283103).

The GLRaV-3 genome encompasses 13 open reading frames (ORFs) with 5' and 3' UTRs of 158 and 277 nt, respectively. Designations of ORF1a, 1b and ORF2–13 were done using the convention for closteroviruses (Dolja et al., 1994). Previously, we reported on the 13 154 nt sequence of the 3'-terminal two-thirds of the genome (Ling et al., 1998). In this work, the 5'-terminal 4765 nt sequence was completed to make a total of 17 919 nt for the entire genome of GLRaV-3, NY1 isolate (Fig. 1). Ten representative cDNA clones were selected for this sequencing project. The criteria used for clone selection were based on each clone's sequence overlapping with the existing sequence contig and extending toward the 5'-terminal region of the genome. Their cDNA insertion sizes (ranging from 370 to 1940 bp) and location relative to the genome are presented in Fig. 1.



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Fig. 1. Complete genome organization of GLRaV-3 and the sequencing strategy. Lines below the genome represent the cDNA or RT-PCR clones used to determine the nucleotide sequences. Scale represents nucleotide coordinates (kb). Identified domains are represented by boxes and designated PRO, papain-like protease; MT, methyl-transferase; HEL, helicase; RdRp, RNA-dependent RNA polymerase; HSP70, heat shock protein 70; 55K, unknown function; CP, coat protein; and CPd, diverged coat protein; along with five other ORFs with unknown functions.

 
Except for the PCR gap-bridging region, where 237 bp were covered only by GAP1, all other sequences were confirmed by at least two overlapping cDNA clones. In some regions, as many as 10 independent sequences were determined. Overall, we experienced more heterogeneity in the 5'-terminal region than in other parts of genome. Of the 5'-terminal 4765 nt sequenced, 86 nt or 1·8 % were ambiguous nucleotide changes, compared with 0·8 % in the 3'-terminal, 13 154 nt. To our knowledge, the GLRaV-3 genome (17 919 nt) is the second largest genome after CTV (19 296 nt) among sequenced closteroviruses.

The 5'-terminal sequence was determined with sequences derived from two cDNA clones (namely #24 and #31) and the RT-PCR product amplified from dsRNA after 3' polyadenylation (PCR 9-22). cDNA sequences matched one another and were confirmed by the sequence generated from the RT-PCR product. GLRaV-3 had the longest 5' UTR (158 nt) among closteroviruses sequenced to date, followed by BYV and CTV (107 nt), LIYV (97 nt) and LChV-1 (76 nt). The 5'-terminal nucleotide was assumed to be a single C after removal of an extra nucleotide (C) from the dsRNA-minus strand. The assumption of an extra nucleotide on the dsRNA-minus strand was based upon the report for CTV (Karasev et al., 1995), where an additional nucleotide (G) was removed from its dsRNA-minus strand. Similar to other ssRNA viruses, the 5' UTR of GLRaV-3 had low G+C content (30 %, 54 out of 158 nt). The resulting low degree of secondary structure would facilitate ribosome binding in the 5' UTR and initiate an efficient translation process. Pairwise comparison of the GLRaV-3 5' UTR with that of BYV or CTV showed significant nucleotide sequence similarity, with 50 % to BYV and 41 % to CTV (data not shown). However, the three viruses did not show significant consensus sequences in the 5' UTR. High sequence similarities in the 5' UTR regions may be due to AT-rich stretches.

ORF1a started from the first ATG at positions 159–161, which had a favourable context for translational initiation with G at the +4 and –3 positions. The ORF1a terminated at nt 6870–6872 and encoded a large polypeptide of 245 277 Da. In a multi-sequence alignment of GLRaV-3 with another ampelovirus (PMWaV-2), using 92 aa residues located from amino acid position 280 to 371 of GLRaV-3, ORF1a revealed a papain-like protease characteristic signature similar to catalytic cysteine and histidine residues (Fig. 2). The GLRaV-3 p-protease was predicted to cleave ORF1a polypeptide between residues Gly371 and Gly372, similar to the BYV papain-like protease. This would generate an N-terminal peptide of 371 aa of 41 963 Da. Unlike GLRaV-2 (Zhu et al., 1998) and CTV (Karasev et al., 1995), which possess two papain-like proteases, GLRaV-3 only had a single papain-like protease.



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Fig. 2. Identification and comparison of the papain-like protease between two ampeloviruses (GLRaV-3 and PMWaV-2). Exclamation marks indicate the predicted catalytic residues of the leader papain-like protease and the slash indicates the predicted cleavage sites.

 
Following the p-protease processing site of the leader protein, ORF1a also contained the newly identified methyltransferase-like domain (MT) and the previously characterized helicase (Hel) domain (Ling et al., 1998). A BLAST search of GenBank with the MT sequence region of 272 aa residues produced a significant similarity to those of other closteroviruses. A phylogenetic analysis using MT sequences of closteroviruses generated three distinct branches (Fig. 3). GLRaV-3 and PMWaV-2, both mealy-bug-transmitted viruses, were quite unique and formed a separate branch from that of aphid-transmitted BYV and CTV or whitefly-transmitted LIYV. This evidence further supports the establishment of a new genus for mealy-bug-transmitted closteroviruses, Ampelovirus, with GLRaV-3 as the type representative (Martelli et al., 2002; Karasev, 2000). To our knowledge, this is the first member of the genus Amplerovirus that has been completely sequenced.



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Fig. 3. Tentative phylogenetic relationship generated from the MT sequence regions (272 aa). Sequence alignment and phylogenetic tree were constructed by CLUSTAL W in the megalign program of DNASTAR. The scale beneath the phylogenetic tree represents the distance between sequences. Potential vector transmissibility of each group of viruses is indicated.

 


   ACKNOWLEDGEMENTS
 
This research was supported in part by the USDA/ARS Cooperative Agreement no. 58-2349-01 with the USDA Clonal Repository at Geneva, NY, USA. We thank Dr A. V. Karasev for critical review of the manuscript.


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Received 3 February 2004; accepted 7 April 2004.



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