1 National Agricultural Research Center for Western Region, Shikoku Campus, Zentsuji, Kagawa 765-8508, Japan
2 National Agricultural Research Center for Western Region, Fukuyama, Hiroshima 721-8514, Japan
Correspondence
Takahide Sasaya
tsasaya{at}affrc.go.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number of the sequence reported in this article is AB114138.
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INTRODUCTION |
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LBVV is the type species of the genus Varicosavirus, which also includes Tobacco stunt virus, Camellia yellow mottle virus and Freesia leaf necrosis virus (Mayo, 2000). LBVV virions are rod-shaped with modal lengths of 320360 nm and diameters of about 18 nm. LBVV virions contain a single coat protein (CP) with an Mr of 48 000 and do not have an envelope structure (Kuwata et al., 1983
). LBVV was reported to be a two-segmented double-stranded RNA (dsRNA) virus, which contained two dsRNAs of approximately 7·0 and 6·5 kbp (Mayo, 2000
). However, we recently demonstrated that LBVV is not a dsRNA virus but a single-stranded negative-sense RNA virus with a bipartite genome. Viral (negative-sense) and virus-complementary (positive-sense) RNAs are separately encapsidated in the virions and the viral RNA is predominant (Sasaya et al., 2001
, 2002
).
LBVV was found in lettuce plants showing big-vein symptoms in the early 1980s (Kuwata et al., 1983; Vetten et al., 1987
; Huijberts et al., 1990
), but its molecular characterization has been slow because LBVV particles are refractory to standard methods for virus purification due to their instability and tendency to aggregate. After numerous efforts, we succeeded in obtaining purified LBVV virions from lettuce plants and determining the nucleotide sequence of the CP gene on RNA2 and the L polymerase gene (designated by analogy with the L polymerase of rhabdoviruses, which is a catalytic subunit of the RNA polymerase complex) on RNA1 (Sasaya et al., 2001
, 2002
). Analysis of amino acid sequences of the LBVV CP and L polymerase suggested a close relationship between LBVV and rhabdoviruses in the order Mononegavirales, even though LBVV has a two-segment negative-sense RNA genome and its particles are not enveloped. In particular, the LBVV L polymerase is most closely related to that of Northern cereal mosaic virus (NCMV) in the genus Cytorhabdovirus (Sasaya et al., 2002
). In this report, we present the nucleotide sequence of LBVV RNA2 and the predicted non-coding and coding regions. We also determined the 5' and 3' ends of LBVV mRNAs to get an insight into the possible transcription mechanism of LBVV. Our data provide further evidence for a relationship between LBVV and rhabdoviruses, as well as illustrating some unique properties of LBVV.
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METHODS |
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Cloning strategy for LBVV RNA2.
A cDNA library of LBVV RNA2 was constructed and its sequence was determined by a genome-walking method, as described by Fazeli & Rezaian (2000). The cloning strategy for LBVV RNA2 and the synthetic oligonucleotide primers used for the cloning are shown in Fig. 1
and Table 1
. Briefly, first-strand cDNA synthesis was done using 20 ng purified LBVV genomic RNAs primed with the LBVV-specific first-strand cDNA synthesis primer 1st GW, whose sequence corresponded to nt 11351151 of the previously described LBVV CP gene (Sasaya et al., 2001
). The second-strand cDNA synthesis primer P1-N6, which consisted of a random hexamer linked to a known oligonucleotide at its 5' end, was added to the first-strand reaction mixture and primer extension was carried out at 37 °C for 30 min in a 50 µl reaction volume 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 Klenow fragment (Toyobo). After excess oligonucleotide had been removed by centrifugal ultrafiltration (Centricon 100 microconcentrators; Amicon), double-strand cDNA was amplified by PCR with the known oligonucleotides P1 and the first LBVV-specific genome-walking primer, GW-1p. The PCR product was cloned and sequenced as described previously (Sasaya et al., 2002
). The next region of LBVV RNA2 was amplified by PCR using the P1 primer and a new LBVV-specific primer, which was designed on the basis of the sequence information obtained from the previous cloning step. In addition, the sequence of the entire region of LBVV RNA2 was confirmed by recloning the LBVV genomic RNAs using the LBVV-specific primers (the sequence of each primer is not shown) designed on the basis of the sequence information obtained by the genome-walking method.
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The sequences of both ends were also confirmed by 3'RACE after polyadenylation of the LBVV genomic RNAs as described previously (Sasaya et al., 2002). First-strand cDNA was synthesized from the 3'-polyadenylated LBVV RNAs using the first-strand cDNA synthesis primer P2-T17, which consisted of oligo(dT) linked to a known oligonucleotide at its 5' end. cDNA was amplified by PCR with the known oligonucleotides P2 and an LBVV-specific primer, either 5TA-R2-4n for the 3' end or 3TA-R2-2p for the 5' end of LBVV RNA2.
Determination of the 5' and 3' ends of LBVV mRNAs.
To determine the 5'-terminal nucleotide sequences of the LBVV mRNAs, including a small ORF (designated gene 6) preceding the L polymerase gene and the L polymerase gene on RNA1 (Sasaya et al., 2002), and to ascertain whether the LBVV mRNA molecules had a cap structure at the 5' end, cDNAs of the corresponding mRNAs were obtained using the FirstChoice RLM-RACE kit (Ambion), which specifically selects capped transcripts from non-capped RNAs using calf intestinal phosphatase (CIP) and tobacco acid pyrophosphatase (TAP) treatment (Liu & Gorovsky, 1993
). Briefly, isolated poly(A)+ RNA from LBVV-infected lettuce plants was treated with CIP to remove the 5' phosphate from all poly(A)+ RNA molecules except those that had a cap structure. TAP was then used to remove the cap structure from the poly(A)+ RNA, leaving a 5'-monophosphate. An RNA adapter oligonucleotide in the kit was ligated in the presence of T4 RNA ligase to the 5' end of the CIP/TAP-treated poly(A)+ RNA. The ligated RNA was reverse-transcribed using a random primer, followed by first- and second-round PCR amplification with the 5'RACE outer primer in the kit and the first LBVV-specific 5'RACE mRNA primer (sequence not shown), which was designed on the basis of the LBVV genome sequence specific to the individual gene, and the 5'RACE inner primer in the kit and the second LBVV-specific 5'RACE mRNA primer.
To determine the nucleotide sequence at the 3' ends of all seven LBVV mRNAs and to ascertain the actual size of the poly(A) tails, the 5'-end phosphorylated adapter 5'-p-Adapter was ligated to the 3' terminus of poly(A)+ RNA using T4 RNA ligase, as described by Liu & Gorovsky (1993). After excess oligonucleotide had been removed by centrifugal ultrafiltration, first-strand cDNA was synthesized using the first 3'RACE primer 1st-3'-R, whose sequence is complementary to that of 5'-p-Adapter. cDNA was amplified by PCR with 1st-3'-R and the first LBVV-specific 3'RACE mRNA primer (sequence not shown), which was designed on the basis of the LBVV genome sequence specific to the individual gene, followed by PCR amplification with the second 3'RACE primer 2nd-3'-R and the LBVV-specific 3'RACE mRNA primer.
Northern blot hybridization.
Poly(A)+ RNA samples from healthy and LBVV-infected lettuce plants were analysed by Northern blot hybridization as described previously (Kusaba et al., 1998). After heat denaturation in formaldehyde/formamide, poly(A)+ RNA was separated on 1·5 % agarose horizontal submarine gels in MOPS/EDTA buffer containing 0·22 M formaldehyde and transferred to Hybond+ nitrocellulose membranes (Amersham-Pharmacia Biotech) by capillary blotting in 20x SSC. Gel-purified inserts obtained from LBVV cDNA clones harbouring each LBVV gene were labelled with [
-32P]dCTP using a random primer labelling kit (Amersham-Pharmacia Biotech). Prehybridization, hybridization, washings and membrane exposures were carried out according to standard protocols (Sambrook & Russell, 2000
).
Primer extension analysis of the 5' end of LBVV ORF2 mRNA.
The 5' end of the ORF2 mRNA was mapped by primer extension analysis on poly(A)+ RNA from the LBVV-infected lettuce plants. Two micrograms of poly(A)+ RNA from healthy and LBVV-infected lettuce plants was reverse-transcribed with SuperScript II RNase H Reverse Transcriptase (Invitrogen) using the LBVV-specific primer 5'-PrExn and [-32P]dCTP. The radiolabelled cDNA was separated on an 8 % polyacrylamide urea gel next to the products of a sequencing reaction of single-stranded cDNA from a plasmid harbouring the gene junction region between the CP and ORF2 using the same oligonucleotide primer.
RNase H analysis of the 3' end of LBVV CP mRNA.
The 3' end of the CP mRNA was analysed by electrophoretic mobility shifts of Northern blot hybridizations following RNase H digestion, as described by Masters & Samuel (1984). Five micrograms of poly(A)+ RNA from LBVV-infected lettuce plants was incubated for 20 min at 25 °C in a volume of 30 µl reaction mixture containing 20 mM Tris/HCl, pH 7·5, 10 mM MgCl2, 100 mM KCl and 0·1 mM DTT, with or without 1·0 µg of appropriate oligonucleotides primers. RNase H digestions were performed by the addition of 2·0 U RNase H (Gibco-BRL) and incubation for 30 min at 37 °C. Digested RNAs were recovered by phenol/chloroform extraction and ethanol precipitation prior to Northern blot hybridization using the LBVV CP-specific probe. The oligonucleotide primers used were as follows: RNase dig1n, which annealed to the virus-complementary RNA from nt 1407 to 1426 of the LBVV RNA2; RNase dig1p, which annealed to the viral RNA from nt 1389 to 1408; RNase dig2n, which annealed to the virus-complementary RNA from nt 1435 to 1454 and presumably does not exist in the CP mRNA; and oligo(dT)1218, which annealed to the poly(A) sequence.
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RESULTS AND DISCUSSION |
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The overall genomic organization of LBVV resembled that of plant rhabdoviruses (genera Nucleorhabdovirus and Cytorhabdovirus). Plant rhabdoviruses contain at least six genes encoding the nucleocapsid protein, phosphoprotein, non-structural protein, matrix protein, glycoprotein and L polymerase (Scholthof et al., 1994; Wetzel et al., 1994
; Chen et al., 1998
; Tanno et al., 2000
). The matrix protein and glycoprotein form the viral envelope and are involved in the movement of virus into and out of host cells. The nucleocapsid protein, phosphoprotein and L polymerase form the viral nucleocapsids and are involved in expressing and replicating the virus genome (Conzelmann, 1998
; Walker et al., 2000
; Rose & Whitt, 2001
; Barr et al., 2002
). In the case of LBVV, its genome appeared to be split between the glycoprotein and L polymerase genes, with the L polymerase gene present on RNA1 and the other five genes present on RNA2 (see Fig. 3A
). Although previous analysis of the LBVV CP and L polymerase revealed sequence similarity to the nucleocapsid proteins and the L polymerases of rhabdoviruses, respectively (Sasaya et al., 2001
, 2002
), FASTA and BLAST analyses of translated sequences from the four other LBVV genes showed no apparent similarity to other known viral sequences. However, this was not surprising since the primary sequences of the phospho-, non-structural, matrix and glycoproteins are poorly conserved among plant rhabdoviruses. The overall amino acid sequence identities of corresponding genes are less than 20 % (Heaton et al., 1987
; Hillman et al., 1990
; Scholthof et al., 1994
; Chen et al., 1998
; Luo & Fang, 1998
; Luo et al., 1998
; Tanno et al., 2000
).
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Identification of LBVV mRNAs
Poly(A)+ RNA from healthy and LBVV-infected lettuce plants was analysed by Northern hybridization with probes prepared from cDNA clones of all LBVV genes, including the L polymerase gene on RNA1 and a small ORF (designated gene 6) preceding it (Sasaya et al., 2002). As shown in Fig. 3
, seven monocistronic transcripts of the expected sizes were detected with cDNA probes corresponding to each LBVV gene. None of the probes reacted with poly(A)+ RNA prepared from healthy lettuce plants. The results clearly showed that each monocistronic mRNA of LBVV appeared to represent a full-length polyadenylated transcript of the corresponding gene in the LBVV-infected lettuce plants.
Analysis of LBVV mRNAs
We determined the 5'-terminal sequences of all LBVV mRNAs corresponding to the seven genes (Fig. 3A) by an RNA ligase-mediated RACE system, which specifically selects for capped transcripts and determines only their 5' ends (Liu & Gorovsky, 1993
). After nested PCR amplification, the sizes of the PCR fragments for the individual LBVV mRNAs were obtained, which suggested that LBVV mRNAs carry a cap structure on their 5' ends. To obtain a representative set of sequences, 610 clones were randomly selected and their sequences analysed. Almost all of the cDNAs initiated with the sequence 5'-GAGA-3', which was the exact complement of the corresponding LBVV genomic templates, 3'-CUCU-5' (Fig. 4
). In addition, the 5' end of ORF2 mRNA was analysed by primer extension (Fig. 5
). Autoradiography of the primer extension products using poly(A)+ RNA from LBVV-infected lettuce plants revealed doublet bands corresponding to nt 1434 and 1435 of LBVV RNA2. No similar bands were detected in the primer extension products using poly(A)+ RNA from healthy lettuce plants (Fig. 5
). The more slowly migrating band (nt 1434) most likely resulted from copying of the cap structure at the 5' end of ORF2 mRNA, as described in other viral mRNAs carrying a cap structure (Gupta & Kingsbury, 1984
; Heaton et al., 1987
; Zuidema et al., 1987
; Hillman et al., 1990
; Scholthof et al., 1994
). The more rapidly migrating band (nt 1435) probably represented extension up to, but not including, the putative cap structure. These results thus indicated that ORF2 mRNA began at nt 1435 of LBVV RNA2 and that the LBVV mRNA presumably had a cap structure.
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A conserved sequence for the initiation of LBVV genes, 3'-CUCU-5', was found. This conserved sequence is also found in the gene junction regions of LNYV and NCMV in the genus Cytorhabdovirus (Wetzel et al., 1994; Tanno et al., 2000
). Although the transcription initiation consensus signal sequence of most rhabdoviruses, including genus Nucleorhabdovirus, is 3'-UUGU-5' (Heaton et al., 1989
; Walker et al., 2000
), the 3'-CUCU-5' sequence in the gene junction regions was considered to act as a transcription initiation signal in both LBVV and rhabdoviruses in the genus Cytorhabdovirus.
In all rhabdoviruses, gene-end sequences comprise an AU-rich region and a cytidylate and a poly(U) tract (Conzelmann, 1998). The poly(U) tract is thought to be a template for the mRNA poly(A) tail by reiterative transcription or slippage (Barr et al., 2002
). The gene-end sequences of LBVV were either 3'-UAUNCAUUUUUUU-5' (type A) or 3'-AUAAUCUUUUUU-5' (type B), reminiscent of those observed in rhabdoviruses (Fig. 4
). Interestingly, when the transcription termination/polyadenylation signal of LBVV was 3'-UAUNCAUUUUUUU-5', the transcription of the next gene initiated immediately after the mononucleotide G (IGR), while for the type B sequence, the two genes (genes 5 and L) were located at the 5'-terminal ends of each RNA (viral RNA) or the transcription was re-initiated after a long 42 nt IGR (gene 4). These gene-end sequences of LBVV might play a role not only in the termination of transcription and polyadenylation but also in re-initiating transcription of the downstream genes. The role of gene-end sequences in signalling initiation of the downstream mRNA synthesis was recently reported for Vesicular stomatitis Indiana virus (VSIV) (Hinzman et al., 2002
).
The IGRs of LBVV, except for the gene junction between genes 4 and 5, were highly conserved, consisting of the mononucleotide G (Fig. 4). The IGR of a fish rhabdovirus in the genus Novirhabdovirus also consists of a single nucleotide, G or A (Walker et al., 2000
). The IGRs of rhabdoviruses either are highly conserved in sequence and length (genera Vesiculovirus and Nucleorhabdovirus) or are not conserved in either sequence or length (genera Lyssavirus, Ephemerovirus and Cytorhabdovirus), whereas the first residue of the IGRs in the most rhabdoviruses is G (Conzelmann, 1998
; Walker et al., 2000
). The G residue also followed immediately downstream of the last genes, gene 5 on RNA2 and the L polymerase gene on RNA1. The G residue might play an important role in the termination of LBVV mRNA transcripts, as reported for VSIV in which the first G residue in the IGR is required for efficient termination of the upstream transcript (Barr et al., 1997
; Stillman & Whitt, 1997
, 1998
). The IGR between genes 4 and 5 differed from the other LBVV IGRs and consisted of 42 nt. Such a long IGR is also found in Rabies virus (RABV) (Tordo et al., 1986
), Bovine ephemeral fever virus (Walker et al., 2000
) and LNYV (Wetzel et al., 1994
). The long IGR of RABV was reported to correlate with transcriptional attenuation (Finke et al., 2000
). However, because of the varying specific activities of our probes, Northern blot analysis failed to show a different degree of transcription attenuation between genes 4 and 5 (Fig. 3
).
Conclusion
This study completes the sequence analysis of the entire LBVV genome, which is the first report of the entire sequence of a member of the genus Varicosavirus. The LBVV genome is composed of 12 878 nt, divided into two segments. LBVV RNA1 consists of 6797 nt and contains a small gene 6 and L polymerase gene (Sasaya et al., 2002). LBVV RNA2 consists of 6081 nt and contains five genes. The genomic organization of LBVV and the amino acid sequences of LBVV CP and L polymerase were similar to those in rhabdovirus genomes. At the LBVV gene junctions, there were transcription termination/polyadenylation and initiation signals comparable with those of rhabdoviruses, and LBVV transcribed capped and polyadenylated monocistronic RNAs. LBVV and rhabdoviruses may utilize a similar mechanism to express individual genes differentially from a contiguous viral genome. These observations provide further evidence that LBVV is most closely related to viruses in the family Rhabdoviridae of the order Mononegavirales, even though LBVV has a genome that is divided into two segments and does not have enveloped virions. Furthermore, compared with viruses in the family Rhabdoviridae, purified LBVV contains a relatively large amount of positive-sense RNA (Sasaya et al., 2001), which is also a unique property of LBVV.
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ACKNOWLEDGEMENTS |
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Received 26 February 2004;
accepted 30 April 2004.