Plant Research International BV, PO Box 16, 6700 AA Wageningen, The Netherlands1
De Ruiter Seeds, PO Box 1050, 2660 BB Bergschenhoek, The Netherlands2
Author for correspondence: Frank van der Wilk. Present address: COGEM, PO Box 578, 3720 AN Bilthoven, The Netherlands. Fax +31 30 2744461. e-mail Frank.van.der.Wilk{at}RIVM.nl
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A complex of two viruses appears to be associated with big-vein disease. Rod-shaped particles have been identified in infected plants and an antiserum has been raised against these particles (Kuwata et al., 1983 ; Vetten et al., 1987
; Huijberts et al., 1990
). They contained negative-sense single-stranded RNA and are serologically related to Tobacco stunt virus (TStV) (Kuwata et al., 1983
; Sasaya et al., 2001
). Since it was generally believed that this virus was the causal agent of big-vein disease the virus was named Lettuce big-vein virus (LBVV) (Kuwata et al., 1983
; Huijberts et al., 1990
). Based upon serological relationship, morphology and partial sequence analysis of the coat protein gene (Sasaya et al., 2001
) LBVV was classified as the type member of the genus Varicosavirus (Mayo, 2000
). However, no evidence was obtained that infection with LBVV induced big-vein symptoms in lettuce.
Recently, a second virus has been isolated from lettuce plants showing big-vein symptoms (Roggero et al., 2000 ). The particles of the virus morphologically resembled those of the ophioviruses and the virus was reported to be serologically related to the ophiovirus Ranunculus white mottle virus (RWMV) The authors tentatively named it Mirafiori lettuce virus (MiLV). Lot et al. (2002)
reported that an infection with MiLV alone was sufficient to induce big-vein-like symptoms in lettuce, while plants infected with LBVV remained symptomless, suggesting that MiLV was the causal agent of big-vein disease.
Species in the genus Ophiovirus have filamentous virus particles resembling those of the tenuiviruses. The ophiovirus ribonucleoprotein particles are pleiomorphic. Electron microscopical analysis revealed that the particles appear as thin filamentous either collapsed double-stranded forms of circular particles of 3 nm in diameter or as linear spiral filaments of 10 nm in diameter (Barthe et al., 1998 ; Milne et al., 2000
). The viruses are believed to have both small and large particles, possibly due to encapsidation of RNA molecules of different sizes (Barthe et al., 1998
; de la Torre et al., 1998
). The genome of ophioviruses is multipartite and consists of three single-stranded (ss) negative-sense RNA molecules of approximately 1·5, 1·61·8 and 7·59 kb in size (Milne et al., 2000
). The capsid proteins of the ophioviruses have reported sizes ranging between 40 and 50 kDa.
Molecular data are lacking for this virus genus. Only partial sequence data are available for RWMV and Citrus psorosis virus (CPsV; Barthe et al., 1998 ; de la Torre et al., 1998
). The absence of sequence data impedes our understanding of the molecular biology of the ophioviruses and their taxonomic relationships with the tenuiviruses or other possibly related viruses like the Bunyaviridae. In this paper, we report the nucleotide sequence of MiLV. This is the first report of a complete nucleotide sequence of an ophiovirus. Unexpectedly, the genome of MiLV consisted of four RNA molecules, ranging between 7·8 and 1·4 kb in size.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA synthesis and cloning.
Genomic RNA was purified from virus particles with a Qiagen RNA Kit (Qiagen), according to the manufacturers instructions. Synthesis of cDNA was carried out by priming with random hexanucleotide primers, using the SuperScript System II (Life Sciences) according to the suppliers instructions. The double-stranded cDNA fragments were cloned into the Lambda ZAP vector (Stratagene). Identification of recombinant clones and subsequent isolation of plasmid DNA was done following standard procedures (Sambrook et al., 1989 ).
The 5'- and 3'-terminal ends of the MiLV genomic RNAs were determined using 5'RACE (rapid amplification of cDNA ends) and 3'RACE procedures. The RACE procedures were carried out by RTPCR using commercially available kits (Life Technologies). The tailing reaction of the cDNA in the 5'RACE procedure was performed using dCTP. In the 3'RACE procedure the genomic RNAs were tailed using ATP and yeast poly(A) polymerase (USB) according to the manufacturers instructions. The PCR fragments obtained were cloned using the TOPO TA cloning kit (Invitrogen), according to the manufacturers instructions.
DNA sequencing.
Sequence analysis was performed with an Applied Biosystems model 373 automated sequencer, using a sequencing kit with AmpliTaq DNA polymerase (Applied Biosystems), and universal and MiLV sequence-specific primers. The MiLV genomic sequence was determined from overlapping independent clones. Each nucleotide of the MiLV genome was determined by sequencing at least two different clones. The entire sequence was determined by sequencing both strands of each clone.
To determine whether the presence of two open reading frames (ORFs) on the RNA1 was not specific for isolate LS301-O and conserved among different isolates RTPCR was carried out to amplify the genomic region of the RNA1 of a Norwegian isolate spanning the intergenic region between these ORFs (position 65207273 of the MiLV genome). This isolate has been in the our virus collection since 1987 and was a kind gift of D.-R. Blystad. To confirm the observed frameshift in the reading frame of RNA segment 4, the region spanning the overlapping reading frames (position 134583 of the vRNA) was amplified by RTPCR using the genomic RNA of a Norwegian isolate as template, followed by sequencing of the obtained PCR fragments.
Computer analysis.
The nucleotide and protein sequences were compiled, analysed and compared using the Wisconsin Package software [version 9, Genetics Computer Group (GCG), Madison, WI, USA], the DNASTAR Lasergene software package and the BLAST suite (Altschul et al., 1997 ). Phylogenetic analysis was carried out using ClustalW. The phylogenetic tree was generated using the neighbour-joining method (1000 bootstrap replicates).
SDSPAGE.
Purified virus suspensions were denatured by boiling in Laemmli buffer (Laemmli, 1970 ) for 10 min. The samples were loaded onto a 12·5% SDSpolyacrylamide gel and subjected to electrophoresis. After electrophoresis the proteins were visualized by silver staining.
Northern blot analysis.
To detect MiLV-specific sequences, the purified RNA was separated on a denaturing 1% agarose gel containing formaldehyde (Sambrook et al., 1989 ) and transferred to (positively charged) nylon membranes (Roche). To detect specific strand RNA molecules the blots were probed with DIG-labelled T7 and T3 transcripts of genomic cDNA fragments. To obtain strand-specific transcripts cDNA fragments complementary to regions of the different RNA segments were cloned behind the T7 or T3 promoter: RNA1, nt 38884845; RNA2, nt 82602; RNA3, nt 481154; RNA4, nt 171188 (positions are indicated on the vRNA). Strand-specific riboprobes used to detect the presence of the different RNA polarities of a specific RNA segment spanned the same genomic region. Labelling of the probes was carried out using the DIG RNA Labelling Kit (Roche) according to the manufacturers instructions. Detection levels of the different probes were tested, compared and calibrated using cDNA clones as a control. Hybridization and subsequent detection of the trapped probes was carried out employing DIG Easy Hyb, anti-digoxigenin alkaline phosphatase-conjugated Fab fragments and CDP-Star (Roche).
Nucleotide sequence accession numbers.
The nucleotide sequences reported here appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under accession numbers AF525933, AF525934, AF525935 and AF525936.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Several other hybridization signals were observed on Northern blot, both smaller and larger in size than the genomic RNA segments (Fig. 4). The nature of these bands remains to be elucidated. Most likely, the smaller bands represent breakdown products while the larger RNAs possibly represent incompletely denatured RNA molecules.
Nucleotide sequence analysis
The complete nucleotide sequence of the genome of MiLV was elucidated by sequence determination of overlapping cDNA clones. Northern blot analysis using total RNA isolated from infected and non-infected plant material confirmed that the cDNA clones obtained were virus specific (data not shown). Each nucleotide of the genome was determined unequivocally by sequencing of at least two different cDNA clones. The sequences of the extreme ends of the genomic RNAs were determined by amplification of the terminal sequences employing 5' and 3'RACE procedures. The 5' ends of the different vRNAs could be amplified using both a 5'RACE procedure and a 3'RACE procedure directed against the 3' end of the vcRNAs, confirming that viral RNA molecules of both polarities were present in the purified virus preparations. On sequence analysis of the RACE clones obtained from the 5' end of the vRNA segment 1 it was shown that the sequences obtained by the 3'RACE and the 5'RACE procedures were identical, suggesting that full-length complementary RNA acted as a template and not viral mRNA-like molecules.
Both the 5' and 3' ends are conserved among the different viral RNA segments (Fig. 3A). Because the 3'-terminal sequences of the different RNA segments were determined by the 3'RACE procedure it cannot be excluded that one or more additional A residues are present at the 3' ends of the RNAs. Analysis of the terminal sequences revealed that the 5' and 3' ends are, to a certain extent, inverted repeats which potentially can form panhandle structures (Fig. 3B
). However, the 5'- and 3'-terminal sequences also contain palindromic sequences potentially able to fold into stemloop structures (Fig. 3C
).
|
|
The vcRNA4 is 1402 nt in length and comprises two overlapping ORFs in different reading frames. The first ORF putatively encodes a protein of 37261 Da (37K protein). The ORF starts with an AUG at position 1301 and ends at position 350 with an UGA stop codon. The second ORF overlaps the first ORF by 38 nucleotides, but appears to lack an initiation codon. This ORF has a theoretical coding capacity of 10618 Da (10·6K) and starts at position 388 and continues to position 91. To confirm that the observed frameshift in the reading frame was not caused by an artefact during the cloning procedure, the nucleotide sequence in this region was determined for two independent cDNA clones and three cloned RTPCR fragments. Moreover, sequence analysis of this region in the RNA segment 4 of a Norwegian isolate confirmed the presence of a frameshift in the reading frame, thus ruling out the possibility that the observed frameshift was due to a mutation or presence of a defective RNA in the Dutch isolate. We suggest that the second ORF is expressed by a +1 translational frameshift. No structures like stemloops or pseudoknots were detected in the putative frameshift region. However, a slippery sequence, GGGAAAU, known to be involved in -1 ribosomal frameshifting (Ten Dam et al., 1990 ) can be recognized on the vc strand (position 10451052) immediately in front of the UGA stop codon of the 37K ORF.
Comparison of amino acid sequences
Comparison of the deduced amino acid sequences of the putative products of the ORFs located on the different RNA segments with sequences present in computer databases revealed that the 263K protein shared a high sequence similarity (72·5%) with a partial sequence of the putative RNA-dependent RNA-polymerase (RdRp) of the ophiovirus Ranunculus white mottle virus (RWMV; AF335429). Moreover, the 263K protein showed low but significant amino acid sequence similarities with the L proteins of several rhabdoviruses [Sonchus yellow net virus (SYNV); Rice transitory yellowing virus (RTYV), Viral haemorrhagic septicaemia virus (VHSV), Infectious haematopoietic necrosis virus (IHNV); Rabies virus (RABV)]. No significant sequence similarities were detected in BLAST searches with the putative RdRps of bunyaviruses or tenuiviruses.
The 48·5K protein encoded by the RNA3 showed a high sequence similarity (44·6%) with the coat protein of Citrus psorosis virus (Barthe et al., 1998 ), indicating that the 48·5k protein is the MiLV coat protein or nucleocapsid protein. The deduced molecular mass of the putative nucleocapsid protein is consistent with the estimated size of the protein (Fig. 1
). Database searches failed to detect proteins with significant sequence similarities with the other putative MiLV products.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The genome of MiLV appears to be largely single-sense. All the ORFs were located on the vcRNA sequences, with the exception of the small 10K ORF located on the vRNA2 sequence (Fig. 5). It remains to be investigated whether the 10K ORF is functional and is expressed in vivo. The genomic organization of ophioviruses differs markedly from that of the tenuiviruses, which are ambisense.
Another unique feature of the MiLV genome is the presence of two overlapping reading frames on the RNA segment 4. The 10·6K ORF is possibly expressed by a +1 ribosomal frameshift, giving rise to a fusion protein containing both 37K and 10·6K sequences. The expression of viral genes by translational frameshifting is a common expression strategy observed in many virus genera. However, ribosomal frameshift usually involves expression of the -1 reading frame and not the +1 reading frame. One of the few examples of a +1 frameshift involves the expression of ORF1b of closteroviruses (Agranovsky, 1996 ; Rennecke & Jelkmann, 1998
). Common secondary structures facilitating the +1 frameshift in the different closteroviruses have not been identified. (Fazelli & Rezaian, 2000
; Melzer et al., 2001
). It has been suggested that the occurrence of a UUUC [Grapevine leafroll-associated virus-1 (GLRaV-1); GLRaV-3; Fazelli & Rezaian, 2000
; Pineapple mealybug wilt-associated virus; Melzer et al., 2001
] or AAAG sequence (Lettuce infectious yellows virus; Klaassen et al., 1995
) in the overlap may cause frameshifting by slippage of tRNAPhe or tRNALys, respectively. In the MiLV 10·6/37K ORF overlap the slippery heptanucleotide sequence GGGAAAU, known to be involved in -1 frameshifting (Ten Dam et al., 1990
), is present. Possibly by analogy to the above the GGGA sequence provides a slippery mechanism of tRNAGly passage from one ORF to another.
It has been suggested that by analogy with the tenuiviruses the 5' and 3' end sequences of the ophiovirus RNA segments would be inverted repeats that can potentially form panhandle structures. Analysis of the MiLV terminal sequences revealed that the sequences are to a certain extent inverted repeats and that in contrast to the Tenuiviridae and Bunyaviridae the ends are not able to anneal to perfect panhandle structures. Furthermore, an absolute conservation of identical nucleotides at the genomic RNA termini as observed for the tenuiviruses and phleboviruses (family Bunyaviridae) appears to be absent in this ophiovirus. Interestingly, the terminal ends of the vRNA molecules of MiLV are able to anneal and fold into structures (Fig. 3C) faintly resembling the corkscrew conformation of the RNA termini of the Orthomyxoviridae (Flick et al., 1996
). The influenza orthomyxovirus RNA promoter consists of the genomic 5'- and 3'-terminal sequences and is folded in a symmetrically hooked or corkscrew conformation rather than a simple panhandle structure (Flick et al., 1996
; Flick & Hobom, 1999
). Interaction of the polymerase appears to be dependent on base-pairing according to the corkscrew model (Flick & Hobom, 1999
). Possibly the ophiovirus promoter consists of a rather similar conformation.
The high degrees of similarities between corresponding tenuivirus and phlebovirus proteins, the similar genomic organization of these viruses, and sequence similarities between the termini of phleboviruses, nairoviruses and tenuiviruses have prompted the suggestion that viruses in the genera Tenuivirus and Phlebovirus have evolved from a common ancestor (Falk & Tsai, 1998 ). Based upon morphology and the presence of a segmented genome the ophioviruses appear related to the tenuiviruses. However, amino acid sequence comparisons of the MiLV RdRp sequence with the RdRp sequences of other negative-strand viruses revealed that rhabdoviruses are more closely related to MiLV and that the TenuivirusPhlebovirus cluster is more distantly related. No sequence similarities were observed between the other putative MiLV ORF products, like the nucleocapsid protein, and sequences in databases, including those of tenuiviruses. The observed sequence similarities between the different RdRps are low, and phylogenetic analysis based upon such data has to be interpreted with caution. However, a phylogenetic tree can be computed in close agreement with the results of Blastp (Altschul et al., 1997
) alignments between the RdRps of the different viruses (Fig. 6A
, B
). This tree confirms the intergenic relationship between tenuiviruses and phleboviruses and shows that MiLV is closely related to RWMV but not to other viruses. These data suggest that from an evolutionary point of view ophioviruses are not closely linked to tenuiviruses.
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389-3402.
Barthe, G. A., Ceccardi, T. L., Manjunath, K. L. & Derrick, K. S. (1998). Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridization and RTPCR. Journal of General Virology 79, 1531-1537.[Abstract]
Campbell, R. N. (1962). Relationship between the lettuce big-vein virus and its vector, Olpidium brassicae. Nature 195, 675-677.[Medline]
De la Torre, S., Riva, O., Zandomeni, R., Grau, O. & Garcia, M. L. (1998). The top component of citrus psorosis virus contains two ssRNAs, the smaller encodes the coat protein. Molecular Plant Pathology On-Line http://www.bspp.org.uk/mppol/1998/1019sanchez.
Falk, B. W. & Tsai, J. H. (1998). Biology and molecular biology of viruses in the genus Tenuivirus. Annual Review of Phytopathology 36, 139-163.[Medline]
Fazelli, C. F. & Rezaian, M. A. (2000). Nucleotide sequence and organization of ten open reading frames in the genome of grapevine leafroll-associated virus 1 and identification of three subgenomic RNAs. Journal of General Virology 81, 605-615.
Flick, R. & Hobom, G. (1999). Interaction of influenza virus polymerase with viral RNA in the corkscrew conformation. Journal of General Virology 80, 2565-2572.
Flick, R., Neumann, G., Hoffmann, E., Neumeier, E. & Hobom, G. (1996). Promoter elements in the influenza vRNA terminal structure. RNA 2, 1046-1057.[Abstract]
Huijberts, N., Blystad, D.-R. & Bos, L. (1990). Lettuce big-vein virus: mechanical transmission and relationships to tobacco stunt virus. Annals of Applied Biology 116, 463-475.
Klaassen, V. A., Boeshore, M. L., Koonin, E. V., Tian, T. & Falk, B. W. (1995). Genome structure and phylogenetic analysis of lettuce infectious yellows virus, a whitefly-transmitted, bipartite closterovirus. Virology 208, 99-110.[Medline]
Kuwata, S., Kubo, S., Yamashita, S. & Doi, Y. (1983). Rod-shaped particles, a probable entity of lettuce big-vein virus. Annals of the Phytopathological Society of Japan 49, 246-251.
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277, 680-685.
Lot, H., Campbell, R. N., Souche, S., Milne, R. G. & Roggero, P. (2002). Transmission by Olpidium brassicae of Mirafiori lettuce virus and Lettuce big-vein virus, and their roles in lettuce big-vein etiology. Phytopathology 92, 288-293.
Mayo, M. A. (2000). Genus Varicosavirus. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses , pp. 521-523. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego:Academic Press.
Melzer, M. J., Karasev, A. V., Sether, D. M. & Hu, S. J. (2001). Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2. Journal of General Virology 82, 1-7.
Milne, R. G., Garcia, M. L. & Grau, O. (2000). Genus Ophiovirus. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses , pp. 627-631. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego:Academic Press.
Rennecke, B. & Jelkmann, W. (1998). Expression of the little cherry closterovirus RNA-polymerase by a +1 ribosomal frameshift. Arbeitskreis Viruskrankheiten der Pflanzen, November 1213, Wageningen, The Netherlands.
Roggero, P., Ciuffo, M., Vaira, A. M., Accotto, G. P., Masenga, V. & Milne, R. G. (2000). An Ophiovirus isolated from lettuce with big-vein symptoms. Archives of Virology 145, 2629-2642.[Medline]
Roossinck, M. J. (1997). Mechanisms of plant virus evolution. Annual Review of Phytopathology 35, 191-209.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sasaya, T., Ishikawa, K. & Koganezawa, H. (2001). Nucleotide sequence of the coat protein gene of Lettuce big-vein virus. Journal of General Virology 82, 1509-1515.
Ten Dam, E. B., Pleij, C. W. A. & Bosch, L. (1990). RNA pseudoknots: translational frameshifting and readthrough on viral RNAs. Virus Genes 4, 121-136.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673-4680.[Abstract]
Vetten, H. J., Lesemann, D.-E. & Dalchow, J. (1987). Electron microscopical and serological detection of virus-like particles associated with lettuce big-vein disease. Journal of Phytopathology 120, 53-59.
Received 23 May 2002;
accepted 9 July 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |