Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, Via Amendola 165/A, 70126 Bari, Italy
Correspondence
Donato Gallitelli
gallitel{at}agr.uniba.it
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ABSTRACT |
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The nucleotide sequence data reported in this paper have been deposited in the EMBL database under accession numbers AJ272327 (RNA-1), AJ272328 (RNA-2) and AJ272329 (RNA-3).
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INTRODUCTION |
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Thus, PZSV resembles ilarviruses in particle morphology, some physico-chemical properties and epidemiology, but it is serologically unrelated to Apple mosaic virus (ApMV), PDV, PNRSV, TSV and Tulare apple mosaic virus as well as to other viruses within the family Bromoviridae (Gallitelli et al., 1983). When preliminarily characterized, PZSV appeared to differ significantly from ilarviruses in having only two RNA species, nor does it require the addition of a coat protein (CP) for RNA infectivity, subgenomic RNA (RNA-4) or any of the ultrastructural modifications usually induced in host cells (Gallitelli et al., 1983
). For these reasons, the ICTV, in their last report, included PZSV in the list of unassigned viruses (Calisher et al., 2000
).
We have now determined the nucleotide sequence of the PZSV genome and show that, contrary to previous reports (Gallitelli et al., 1983), it comprises three positive-strand RNA species with an organization that is similar to that of virus species within the family Bromoviridae. These results provide the basis for which the taxonomic status of PZSV is discussed.
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METHODS |
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Viral RNA was extracted as described by Finetti-Sialer et al. (1997) and analysed by 1·2 % agarose gel electrophoresis in TBE (Sambrook et al., 1989
). For Northern blot analysis, [32P]UTP-labelled negative-sense riboprobes were generated from cloned fragments (see below) using the Sp6/T7 Transcription kit (Roche), following the manufacturer's instructions. Pre-hybridization, hybridization and detection were carried out as described previously (Gallitelli et al., 1985
).
A preparation of PZSV RNA was also subjected to one cycle of oligo(dT)cellulose chromatography, according to the protocol of Milner & Jackson (1979).
Translation of viral RNA was carried out using the Rabbit Reticulocyte Lysate system (Amersham Pharmacia) using 2 µg RNA and approximately 15 µCi [35S]methionine (Amersham Pharmacia). Translation products were analysed by PAGE, as described by Burgyan et al. (1986).
cDNA synthesis, cloning and sequencing.
PZSV RNA was fractionated by two runs of electrophoresis in low-melting-point agarose, as described by Gallitelli et al. (1985). For cDNA cloning, 1 µg RNA was polyadenylated with 4 U Escherichia coli poly(A) polymerase (Gibco-BRL), annealed to an oligo(dT)25 primer and reverse-transcribed using the cDNA Synthesis Module (Amersham Pharmacia), according to the manufacturers' protocols. The double-strand cDNA was ligated into SmaI-digested, de-phosphorylated pUC18 and was used to transform competent E. coli strain DH5
. Plasmids containing sizeable inserts were used for double-strand DNA sequencing on both strands using the Thermo Sequenase Cycle Sequencing kit (Amersham Pharmacia) and [35S]ATP as label. The 3'-terminal sequence of the genomic RNAs was determined from at least four different clones for each RNA species, each containing an artificially added poly(A) tail.
The 5'-end regions of RNA-1 and RNA-2 were cloned separately following a modified RACE protocol (Grieco et al., 1996). Primer 5'-CCATTATGAACTGATAGCTG-3', which is complementary to nt 6382 of RNA-1, and primer 5'-GCTCTGAATTTATCGATA-3', which is complementary to nt 135152 of RNA-2, were used. The 5' terminus of PZSV RNA-3 was sequenced using M-MLV RNA-dependent RNA polymerase (RdRp) (Gibco-BRL), according to the protocol described by Fichot & Girard (1990)
, using the internal primer 5'-CTATGAAATGCAAGATGCA-3', which is complementary to nt 3754. The 5'-terminal nucleotide of each genomic RNA was determined according to the method of DeBorde et al. (1986)
.
To map the 5' end of the subgenomic RNA, the synthetic primer 5'-AGCTTAGCGAGTGCCTGCTG-3', which is complementary to nt 17041685 of PZSV RNA-3, was labelled with 10 U polynucleotide kinase and [32P]ATP (3000 Ci mM-1) and was extended using RNA-4 as template. The size of the subgenomic RNA was determined by comparison to plasmid DNA containing the primer sequence as a size ladder. The subgenomic RNA 5' termini were sequenced directly from the viral RNA, as with RNA-3, using the above primer.
Sequence data were assembled using the DNA program STRIDER (Marck, 1988). Nucleotide and deduced amino acid sequences were compared using PILEUP and CLUSTAL (GCG) (Anon., 1994
). Putative translation products were compared with the GenBank non-redundant sequence database. Tentative phylogenetic trees were constructed and bootstrap analyses were made using the programs of the PHYLIP package (Felsenstein, 1989
). The following sequences have been used in this study (EMBL accession numbers are given in parentheses): AMV, Alfalfa mosaic virus (L00163, K02702 and K02703); ApMV (AF174584, AF174585 and U15608); BSMV, Barley stripe mosaic virus (J04342 and M16576); BBMV, Broad bean mottle virus (M65138, M64713 and M60291); BMV, Brome mosaic virus (X58456, X58457 and X58458); CCMV, Cowpea chlorotic mottle virus (M65139, M28817 and M28818); CiLRV, Citrus leaf rugose virus (U23715, U17726 and U17390); CMV-Fny, Cucumber mosaic virus (D00356, D00355 and D10538); CMV-NT9 (D28778, D28779 and D28780); CMV-Q (X02733, X00985 and J02059); EMV, Elm mottle virus (U57047, U34050 and U57048); OLV-2, Olive latent virus 2 (X94346, X94347 and X76993); PDV (U57648, AF277662 and L28145); PSV, Peanut stunt virus (D11126, D11127 and U15730); RBDV, Raspberry bushy dwarf virus (S51557, S55890 and JQ1292-3); SpLV, Spinach latent virus (U93192, U93193 and U93194); TMV, Tobacco mosaic virus (AJ011933); TAV, Tomato aspermy virus (D10044, D10663 and D01015); TSV (U80934, U75538 and X00435).
Cloning and expression of the putative PZSV CP gene.
Two deoxyprimers were designed to amplify the coding region of the 23 kDa (23K) protein. The forward primer 5'-GGATCCATGCCCCCTAAGAGACAG-3' was homologous to nt 16191636 of the second ORF of RNA-3 and contained a BamHI site (underlined). The reverse primer 5'-GTCGACCTACAGAGGTATATACTCTGCTT-3' was complementary to nt 22232245 of the same ORF of RNA-3 and contained a SalI site (underlined). RT-PCR was carried out according to the method described by Finetti-Sialer et al. (1999) and the amplified product was digested and cloned into the expression vector pGEX-6P-1 (Amersham Pharmacia). Recombinant clones were transferred to E. coli strain BL21 to express the PZSV 23K product as a fusion protein with glutathione S-transferase (GST). Protein expression was induced for 3 h with 2 mM IPTG at 37 °C, after which the samples were subjected to Western blot analysis (Sambrook et al., 1989
) using a polyclonal antiserum raised against PZSV virions (Quacquarelli & Gallitelli, 1979
).
Construction of infectious PZSV transcripts and inoculation assays.
cDNAs of PZSV RNAs were obtained by RT-PCR using Thermoscript (Invitrogen) and Expand Long (Roche) enzymes, according to the manufacturers' protocols. Two overlapping RT-PCR products encompassing RNA-1 were generated using two sets of oligonucleotide pairs. One set was constituted by a primer (5'-end primer) homologous to nt 120 (5'-GGTTTGAGTGCATTTTGTGTA-3') and a primer complementary to nt 16091588 (5'-TGTCTAGAGACATCTTAATAGC-3'), whereas the second set contained a primer homologous to nt 12981313 (5'-TGTTATCAGCACGACG-3') and a primer (3'-end primer) complementary to nt 33583383 (5'-CTTTTTGGTCTCTCTTAGAGAAACCG-3'). After digestion with EcoRI, the two amplicons were ligated with Ready-to-go T4 DNA ligase (Amersham Pharmacia) and were used as template for full-length cDNA synthesis by PCR in the presence of the 5'-end primer of the first primer set, anchored with the T7 promoter, and the 3'-end primer of the second set. Amplicons of full-length cDNA of RNA-2 were obtained by RT-PCR using an oligonucleotide complementary to nt 24122435 (5'-TGGTCTCTCTTAGAGAAACCGAAG-3') and the same 5'-end primer anchored with T7 RNA promoter sequence used for RNA-1. A primer complementary to nt 26342659 (5'-CTTTTTGGTCTCTCTTAGAGAAACCG-3') and a primer homologous to nt 122 (5'-GTTTGAACTTAGTAATTGCATG-3'), anchored with the T7 promoter, were used to generate RT-PCR products corresponding to full-length cDNAs of RNA-3.
All full-length PCR products were gel-purified using the QIAquick Gel Extraction kit (Qiagen) and used for transcription. For long-term storage, full-length cDNAs were ligated into the plasmid vector pCR-XL-TOPO (Invitrogen) and used to transform E. coli strain TOP10, following the manufacturer's instructions. Capped transcripts representing RNA-1, -2 and -3 were obtained directly with the mMessage mMachine (Ambion) and, after treatment with RQ1 RNase-free DNase (Promega), were used in different combinations for plant inoculation. Two leaves from each of two N. glutinosa plants were rubbed with 20 µl of inoculum containing 5 µg of each transcript in 10 mM Tris/HCl, pH 7·4, and 1 mM EDTA in the following combinations: RNA-1+RNA-2+RNA-3; RNA-1+RNA-2; RNA-1+RNA-3; and RNA-2+RNA-3. Another two plants for each inoculum combination were mock-inoculated with buffer alone.
Total RNA was extracted from 100 mg of inoculated and systemic leaves of the different tests using TriPure Isolation reagent (Roche), according to the manufacturer's instructions, and subjected to treatment with RNase-free DNase for 1 h at 37 °C, phenol-extracted and ethanol-precipitated. The dried pellet was resuspended in RNase-free water and used in RT-PCR. RNA-1 was amplified with a primer complementary to nt 6382 (5'-CCATTATGAACTGATAGCTG-3') and the 5'-end primer (see above). RNA-2 was amplified with an oligonucleotide complementary to positions 135152 (5'-GCTCTGAATTTATCGATA-3') and the 5'-end primer used for RNA-1. RNA-3 was amplified with an oligonucleotide complementary to nt 26402659 (5'-CTCTAAGAGAGACCAAAAAG-3') and a primer homologous to nt 15421562 (5'-GTTAGTAATTCAAAGTATTTC-3').
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RESULTS AND DISCUSSION |
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In rabbit reticulocyte lysates, unfractionated PZSV RNA stimulated incorporation of [35S]methionine up to 10 times the amount of the control to which no RNA was added. Analysis of the translation products yielded four major protein species with estimated relative molecular masses of 110, 82, 34 and 24 kDa, respectively (Fig. 2, lane 1). The endogenous activity of the lysate was limited to the formation of a polypeptide of 46 kDa (Fig. 2
, lane 2).
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As shown in Fig. 3, the genome of PZSV consists of three major RNA species, RNA-1, RNA-2 and RNA-3, whose nucleotide sequence was completely determined and deposited in the EMBL database under the following accession numbers: AJ272327 (RNA-1), AJ272328 (RNA-2) and AJ272329 (RNA-3). RNA-1 is 3383 nt long and contains a major open reading frame (ORF-1) (Fig. 4
A) beginning at AUG (nt 7981) and ending at UGA (nt 29652967). The sequence context of the first in-frame initiation codon (CAUAAUGGCUGC) seems in a favourable translation context with an A at position -3 and a G at position +4 (Lütcke et al., 1987
). ORF-1 encodes a putative polypeptide of 962 aa with a molecular mass of 108 419 Da (108K), denoted protein 1a, which has a size comparable to that of the 110K translation product. The N-terminal domain (aa 78270) of protein 1a contains conserved sequence motifs IIII of type I methyltransferases of positive-strand RNA viruses (Koonin & Dolja, 1993
). The C-terminal domain contains the tobamovirus lineage (Koonin & Dolja, 1993
) of the seven consensus motifs of the helicases of superfamily 1, including the motifs AB of the purine NTP-binding pattern (aa 691728) (Koonin & Dolja, 1993
). Pairwise alignments of the conserved sequence motifs of protein 1a with the comparable signatures of other members of the family Bromoviridae are shown in Table 1
. A second putative ORF of about 300 nt is present in the +2 position of ORF-1.
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RNA-3 is 2659 nt long and contains two major ORFs (Fig. 4C), the first of which (ORF-3) begins at AUG (nt 335337, in the context GAAAAUGUCUC) and ends at UGA (nt 12621264). The putative polypeptide encoded by ORF-3 (protein 3a) consists of 310 aa with an estimated molecular mass of 33 735 Da (34K) and is comparable in size to the 34K translation product. This protein contains the LXDX50-70G motif and the SIS tail (aa 233235) found near the C termini of most members of the 30K superfamily of virus movement proteins (MPs) (Melcher, 2000
). Amino acid sequence comparison revealed 3648 % similarity with the putative MPs of virus species of the family Bromoviridae (Table 1
).
The second ORF (ORF-4) starts at AUG (nt 16191621, in the context GCAUAAUGCCCCC) and ends at UGA (nt 22432245). ORF-4 encodes a putative product of 209 aa with a predicted molecular mass of 23 070 Da (23K), which presumably corresponds to the 24K translation product. This putative CP showed 3649 % similarity with the CPs of other species of the family Bromoviridae (Table 1). The ORF-4 product was expressed as a GST fusion protein in E. coli and was recognized specifically in Western blots analyses by an antiserum to PZSV (Fig. 5
, lanes 1 and 2), thus confirming that ORF-4 encodes the putative PZSV CP. There was no cross-reaction with the wild-type pGEX-6P-1 plasmid, which expresses GST alone (Fig. 5
, lane 3).
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The two major ORFs of RNA-3 are separated by an intergenic region (IR) of 354 nt, which includes an 11 nt sequence (GGUUCAAUUCC) resembling the internal control region (ICR-2) of eukaryotic tRNA gene promoters. This sequence starts at nt 1406 and ends at nt 1416 and is identical to that found in the IR of BMV RNA-3 (Pogue et al., 1992).
RNA-4 is 1118 nt long and is monocistronic. It has 100 % sequence identity with the 3'-proximal half of RNA-3 on which it maps, downstream of the ICR-2-like sequence (nt 1542) in the IR of this RNA and was, therefore, identified as a likely subgenomic RNA for the expression of the PZSV CP. By analogy with other viruses of the family Bromoviridae, the sequences required for the synthesis of subgenomic RNA are likely contained upstream and downstream of the transcription initiation site (TIS). The region between the ICR-2-like motif and downstream of the TIS is particularly rich in A and U residues, thus resembling the sequences thought to be important as promoters of subgenomic mRNA production (Marsh et al., 1988). This tract includes an UUAgUUAUU block, 58 nt upstream of the TIS, which is similar to the UUAUUAUU block thought to act as an enhancer for the transcription of subgenomic RNAs. A GC
GUUA block (the symbol
precedes the first nucleotide of RNA-4) identical to one of the four sequence blocks of promoter sequences of plant viruses is also evident (Marsh et al., 1988
; Buck, 1996
).
RNA-5 is 712 nt long and has 100 % sequence identity with the 3'-proximal half of RNA-4. Thus, it may represent a degradation product derived from subgenomic RNA-4.
5' and 3' non-coding regions (NCRs)
The 5'-terminal NCRs of PZSV RNA-1, -2 and -3 are 78, 81 and 334 nt long, respectively. The 5'-proximal 78 nt displayed the following sequence identities: RNA-1/RNA-2, 93·6 %; RNA-1/RNA-3, 48 %; and RNA-2/RNA-3, 46 %. The 5' ends of PZSV RNA extracted from virions showed two major run-off products when analysed by oligonucleotide-primed run-off reverse transcription, as indicated by two intense bands across the four lanes (data not shown). The upper band is likely to reflect that it is capped, whereas the lower band corresponds to the initiation of transcription site.
ICR-2-like sequences (GGUUCAAUUCC) are present in the 5' NCRs of both RNA-1 and RNA-2 (nt 2536) but ICR-like motifs were not readily identified in the 5' NCR of RNA-3.
The 3'-terminal NCRs of RNA-1, -2 and -3 were 416, 289 and 414 nt long, respectively, with the following identities in the 3'-proximal 260 nt: RNA-1/RNA-2, 88·5 %; RNA-1/RNA-3, 89·5 %; and RNA-2/RNA-3, 94 %. In the last 80 nt, the match was 100 %. All three RNAs contained a terminal CCA-box, which may serve as an RNA initiation signal for the viral RdRp (Buck, 1996; Yoshinari & Dreher, 2000
) and RNA-1 and RNA-3 also contain an AAAAG extension after the CCA-box. Computer-assisted analyses showed that the 3' end of each RNA has the potential to form tRNA-like structures (data not shown).
Infectivity of in vitro transcripts representing PZSV RNA-1, -2 and -3
Within 2 weeks after inoculation, the two plants challenged with a mixture of T7 polymerase run-off transcripts of PZSV RNA-1, -2 and -3 showed chlorotic/necrotic local lesions, followed by clear symptoms of systemic infection that were similar to those induced by the wild-type virus, although they developed less rapidly. RT-PCR from total RNA extracted from inoculated and systemic leaves of the two plants yielded amplicons of the expected size for RNA-1, -2 and -3 (Fig. 6, lanes 1, 3 and 5). Sap from these plants was infectious to healthy N. glutinosa plants from which the virus was purified and was shown to contain all PZSV RNAs, as determined by gel electrophoresis (data not shown). Plants inoculated with RNA-1+RNA-2, RNA-1+RNA-3 and RNA-2+RNA-3 or mock-inoculated did not show either local or systemic symptoms. RT-PCR from total RNA extracted from the two plants challenged with a mixture of RNA-1+RNA-2 transcripts yielded RNA-1- and RNA-2-specific amplicons from inoculated leaves only (Fig. 6
, lanes 7 and 9). The two RNAs were still detectable 22 days after inoculation, suggesting that they probably replicate in inoculated leaves but cannot move systemically. The observed survival of RNA-1 and RNA-2 in the inoculated leaves is now the subject of further investigation. Total RNA from plants inoculated with RNA-1+RNA-3 and RNA-2+RNA-3 or mock-inoculated did not yield any amplification products.
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It is also noteworthy that the PZSV genome does not seem to encode a 2b gene similar to cucumoviruses and ilarviruses; however, there are small ORFs in RNA-1 and RNA-3 (Fig. 4) which could potentially encode a protein with a similar role to the cucumovirus 2b protein. These small ORFs are being analysed in our laboratory.
The similarities found between PZSV and the other known members of the Bromoviridae in the overall organization and expression of genome products warrant the inclusion of this virus in this family. However, PZSV differs enough from other bromoviruses to substantiate the suggestion that it may be the representative of a novel genus of the family. With the information available, the evolutionary pattern of PZSV is difficult to establish. As a plant pathogen, PZSV is a quite recent discovery and there are no sequences available for other strains or isolates, as the virus has been detected only in Europe where its natural host range seems also to be restricted to few species in four different botanical families. While the trees obtained from ORFs establish a clear cut between the CP encoded by PZSV and that of the other genera within the Bromoviridae, the remaining gene products of the virus seem less similar to AMV and ilarviruses than to members of the genera Bromovirus, Cucumovirus, Oleavirus and Idaeovirus. Therefore, it is possible that PZSV originated from ancestors common to these viruses but acquired its CP gene from another source.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 June 2003;
accepted 17 July 2003.
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