1 Faculty of Agriculture, Utsunomiya University, Mine-machi 350, Utsunomiya 321-8505, Japan
2 Nihon Horticultural Production Institute, Kamishiki 207, Matsudo 270-2221, Japan
Correspondence
Tomohide Natsuaki
natsuaki{at}cc.utsunomiya-u.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The DDBJ accession numbers of the sequences reported in this paper are AB085612 and AB085613.
Present address: Department of Entomology and Phytopathology, Faculty of Agriculture, Gadjah Mada University, Sekip Unit 1, Yogyakarta 55281, Indonesia.
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
In addition to BPYV, other known whitefly-transmitted yellowing viruses are Lettuce infectious yellows virus (LIYV; Duffus et al., 1986), Tomato infectious chlorosis virus (TICV; Duffus et al., 1996a
; Wisler et al., 1996
), Tomato chlorosis virus (ToCV; Wisler et al., 1998b
), Sweet potato chlorotic stunt virus (SPCSV; Pio-Ribeiro et al., 1996
), Cucurbit yellow stunting disorder virus (CYSDV; Celix et al., 1996
), Potato yellow vein virus (PYVV; Salazar et al., 2000
) and Lettuce chlorosis virus (LCV; Duffus et al., 1996b
). With the exception of BPYV, these viruses have a bipartite genome and are classified as definitive or tentative members of the genus Crinivirus in the family Closteroviridae. Closteroviridae are single-stranded positive-sense RNA plant viruses and the family contains two genera, Closterovirus and Crinivirus (Martelli et al., 2000
). BPYV is also a member of the Closteroviridae, but is currently classified in the genus Closterovirus (Martelli et al., 2000
). Among criniviruses, only a few complete and partial genomic sequences are available in the GenBank database. In this paper we present the complete nucleotide sequence of the CuYV genome and analyse its relationship with other Closteroviridae.
To obtain the complete nucleotide sequence of CuYV, clones of a cDNA library were generated using dsRNA as a template. Two gel-purified large dsRNAs, both approximately 7 kbp in size, were denatured by methylmercuric hydroxide. First- and second-strand cDNA synthesis was performed using a cDNA synthesis kit (Amersham) according to the manufacturer's instructions. DNA sequences were obtained for both strands of the recombinant plasmids using an automatic sequencer (DSQ-1000L; Shimadzu) at least twice for each independent clone. About 95 % of the CuYV genome sequence was obtained from 29 overlapping cDNA clones for RNA1 and 26 clones for RNA2 (Fig. 1). Specific oligonucleotide primers were designed for the determination of the 5'- and 3'-terminal regions of RNA1 and RNA2 by RACE (Life Technologies). These sequence data indicated that the complete nucleotide sequences of CuYV RNA1 and RNA2 were 7899 and 7607 nucleotides, respectively.
|
RNA1 ORF1 began at AUG (nt 373) and terminated at UAA (nt 475), potentially encoding a small protein of 4·1 kDa. Designated p4, the N-terminal region of this putative protein was hydrophobic. Similar proteins are also encoded in the RNA of all Closteroviridae for which genome sequences are available. The p4 protein of CuYV showed 38 % amino acid sequence similarity to LIYV, but there was no detectable similarity to other Closteroviridae. The RNA1 ORF2 start codon was located 216 nucleotides downstream of the p4 stop codon. ORF2 encoded a putative protein of 61·7 kDa (p62) that is thought to be a heat-shock protein 70 (HSP70). HSP70 is highly conserved in the family Closteroviridae and is probably involved in ATPase activity and proteinprotein interactions (Agranovsky et al., 1991, 1997
; Peremyslov et al., 1999
). RNA1 ORF3 start codon was located 175 nucleotides downstream of the ORF2 stop codon and potentially initiated a putative protein of 59·1 kDa. Designated p59, the function of this protein is unknown and database searching with the ORF3-encoded protein did not identify similar proteins, apart from a counterpart in LIYV with 46 % similarity. RNA1 ORF4 overlapped ORF3 by 19 nucleotides and encoded a putative protein of 9·8 kDa. Designated p9, this putative protein is of unknown function and a search of databases did not show any significantly similar proteins, apart from its counterpart in LIYV RNA2 (38 % similarity). Similar genes with the same size and location were also reported for CYSDV (Livieratos et al., 1999
) and ToCV (Wisler et al., 1998b
), i.e. characteristic of the genus Crinivirus (Agranovsky, 1996
). RNA1 ORF5 overlaps ORF4 by 11 nucleotides and encodes a putative protein of 28·4 kDa. ORF5 was identified as the coat protein (CP) gene of CuYV based on sequence comparison with other crini- and closteroviruses. Alignment of the CP amino acid sequences with other viruses revealed that the invariant consensus of amino acid residues of serine (S), arginine (R) and aspartic acid (D) (Dolja et al., 1991
) were also detected in the CP of CuYV. RNA1 ORF6 overlapped ORF5 by 11 nucleotides and encoded a putative protein of 74·4 kDa. It was identified as CPd based on the significant similarity of 38 % with CPd of Little cherry virus 1 (LChV-1), a member of the Closterovirus genus, and 31 % with LIYV CPd and moderate similarity with the CPds of other closteroviruses. The S, R and D residues conserved in CP were also recognized in the C-terminal region of CPd. The relative positions of CP and CPd were reversed compared with the orientations in the genomes of monopartite, aphid-transmitted closteroviruses (Agranovsky et al., 1994
; Karasev et al., 1995
). CuYV has a large CPd, which is comparable in size with that of ToCV (79 kDa) (Wisler et al., 1998b
) and LChV-1 (76 kDa) (Jelkmann et al., 1997
). RNA1 ORF7 overlapped ORF6 by four nucleotides and encoded a putative protein of 26·5 kDa. Designated p26, this ORF is analogous, in terms of size and location, to ORF7 of LIYV. However, the deduced amino acid sequence of CuYV did not show significant similarity to any other crini- and closteroviruses, except to its counterpart in LIYV (36 % similarity).
CuYV RNA2 includes two ORFs, ORF1a and 1b. ORF1a began at the first AUG (nt 243), terminated at UGA (nt 6054) and encoded a predicted protein of 214·6 kDa. The first AUG codon has an optimal context for translational initiation (Kozak, 1986). Analysis of the amino acid sequence of the 5' region of the ORF1a-encoded product revealed a putative papain-like protease (P-PRO) domain, which showed significant similarity (54 %) to the P-PRO of LIYV (Klaassen et al., 1995
). The predicted catalytic cysteine and histidine residues of the LIYV protease were also found in the CuYV sequence. In alignments of the LIYV P-PRO sequence with that of CuYV, the cleavage site at residues Gly-Ala (amino acid 412413) of LIYV corresponded to Gly-Val dipeptides of CuYV. Cleavage of this site would result in a leader protein of 410 amino acid residues (47 kDa). The sequence downstream of the P-PRO domain was identified as a methyltransferase domain (MTR; Rozanov et al., 1992
) based on alignment with other closterovirus proteins and shared 70 % similarity with LIYV MTR. The C-terminal region of ORF1a was identified as a helicase (HEL) domain and contained the seven characteristic conserved motifs (Gorbalenya & Koonin, 1993
). This region was similar to helicases of other Closteroviridae: most similar was LIYV HEL (62 %), followed by LChV-1 (55 %), Grapevine leafroll-associated virus 1 (GLRaV-1; 51 %) and GLRaV-3 (48 %). ORF1b overlapped the last 46 nucleotides of ORF1a and potentially encoded a putative protein of 58·3 kDa, counting from the frameshift site. This protein showed significant sequence similarity to the RNA-dependent RNA polymerases (RdRps) of Closteroviridae in the database. The CuYV RdRp contained a Gly-Asp-Asp (GDD) motif, which is a hallmark of RNA polymerases (Bruenn, 1991
). CuYV RdRp also contained eight conserved sequence motifs reported in the RNA polymerases of positive-strand RNA viruses (Koonin, 1991
; Koonin & Dolja, 1993
). This putative RdRp was most closely related to that of LIYV (75 % similarity) followed by LChV-1 (73 %), Citrus tristeza virus (CTV) and GLRaV-2 (49 %). ORF1b was in a different frame from ORF1a and may be expressed via a +1 ribosomal frameshift, like other viruses of the Closteroviridae. The sequence UUUGA was present in the CuYV ORF1a/1b overlap, as in the ORF1a/1b overlap for LIYV (Klaassen et al., 1995
). CuYV did not encode a gene for the approximately 32 kDa product located downstream of ORF1b that is present in LIYV RNA1 (ORF2; Fig. 1
), which is analogous to a 33 kDa product in CTV and a 30 kDa product in Beet yellow stunt virus (BYSV; Karasev et al., 1995
, 1996
).
Tentative phylogenetic analysis using the putative replication-associated proteins (HEL and RdRp) of CuYV and other Closteroviridae demonstrated that CuYV is grouped in the same lineage as the whitefly-transmitted LIYV, the Crinivirus type species, but is different from the aphid- or mealybug-transmitted lineages (Fig. 2a, b). This indicates that CuYV is a member of the genus Crinivirus. Tentative phylogenetic analysis using HSP70 homologue genes among members of the Crinivirus genus, using CTV as a non-crinivirus outgroup, demonstrated that CuYV is grouped in the same lineage as PYVV, CYSDV and ToCV, with LIYV and TICV separate in another lineage (Fig. 2c
). Comparison among CPs of CuYV and criniviruses showed significant similarities of CuYV CP to the CPs of CYSDV (55 %), SPCSV (46 %) and LIYV (38 %), indicating that CuYV is closer to CYSDV than to SPCSV and LIYV (Fig. 2d
). CuYV is also more closely related to PYVV than to LIYV.
|
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Agranovsky, A. A., Boyko, V. P., Karasev, A. V., Koonin, E. V. & Dolja, V. V. (1991). Putative 65 kDa protein of beet yellows closterovirus is a homologue of HSP70 heat shock proteins. J Mol Biol 217, 603610.[CrossRef][Medline]
Agranovsky, A. A., Koonin, E. V., Boyko, V. P., Maiss, E., Frotschl, R., Lunina, N. A. & Atabekov, J. G. (1994). Beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease. Virology 198, 311324.[CrossRef][Medline]
Agranovsky, A. A., Folimonova, S. Y., Folimonov, A. S., Denisenko, O. N. & Zinovkin, R. A. (1997). The beet yellows closterovirus p65 homologue of HSP70 chaperones has ATPase activity associated with its conserved N-terminal domain but does not interact with unfolded protein chains. J Gen Virol 78, 535542.[Abstract]
Bruenn, J. A. (1991). Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases. Nucleic Acids Res 19, 217226.[Abstract]
Celix, A., Lopez-Sese, A., Almarza, N., Gomez-Guillamon, M. L. & Rodriguez-Cerezo, E. (1996). Characterization of cucurbit yellow stunting disorder virus, a Bemisia tabaci-transmitted closterovirus. Phytopathology 86, 13701376.
Coutts, R. H. & Coffin, R. S. (1996). Beet pseudo-yellows virus is an authentic closterovirus. Virus Genes 13, 179181.[Medline]
Dolja, V. V., Boyko, V. P., Agranovsky, A. A. & Koonin, E. V. (1991). Phylogeny of capsid proteins of rod-shaped and filamentous RNA plant viruses: two families with distinct patterns of sequence and probably structure conservation. Virology 184, 7986.[Medline]
Duffus, J. E. (1965). Beet pseudo-yellows virus, transmitted by the greenhouse whitefly, Trialeurodes vaporariorum. Phytopathology 55, 450453.
Duffus, J. E., Larsen, R. C. & Liu H.-Y. (1986). Lettuce infectious yellows virus a new type of whitefly-transmitted virus. Phytopathology 76, 97100.
Duffus, J. E., Liu, H.-Y. & Wisler, G. C. (1996a). Tomato infectious chlorosis virus a new clostero-like virus transmitted by Trialeurodes vaporariorum. Eur J Plant Pathol 102, 219226.
Duffus, J. E., Liu H.-Y., Wisler, G. C. & Li, R. H. (1996b). Lettuce chlorosis virus a new whitefly-transmitted closterovirus. Eur J Plant Pathol 102, 591596.
Gorbalenya, A. E. & Koonin, E. V. (1993). Helicase: amino acid sequence comparisons and structurefunction relationship. Curr Opin Struct Biol 3, 419429.
Jelkmann, W., Fechtner, B. & Agranovsky, A. A. (1997). Complete genome structure and phylogenetic analysis of little cherry virus, a mealybug-transmissible closterovirus. J Gen Virol 78, 20672071.[Abstract]
Karasev, A. V., Boyko, V. P., Gowda, S. & 10 other authors (1995). Complete sequence of the citrus tristeza virus RNA genome. Virology 208, 511520.[CrossRef][Medline]
Karasev, A. V., Nikolaeva, O. V., Mushegian, A. R., Lee, R. F. & Dawson, W. O. (1996). Organization of the 3-terminal half of beet yellow stunt virus genome and implications for the evolution of closteroviruses. Virology 221, 199207.[CrossRef][Medline]
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, 99110.[CrossRef][Medline]
Koonin, E. V. (1991). The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. J Gen Virol 72, 21972206.[Abstract]
Koonin, E. V. & Dolja, V. V. (1993). Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28, 375430.[Abstract]
Kozak, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283292.[Medline]
Liu, H.-Y. & Duffus, J. E. (1990). Beet pseudo-yellows virus: purification and serology. Phytopathology 55, 866869.
Liu, H.-Y., Wisler, G. C. & Duffus, J. E. (2000). Particle lengths of whitefly-transmitted criniviruses. Plant Dis 84, 803805.
Livieratos, I. C., Avgelis, A. D. & Coutts, R. H. A. (1999). Molecular characterization of the cucurbit yellow stunting disorder virus coat protein gene. Phytopathology 89, 10501055.
Martelli, G. P., Agranovsky, A. A., Bar-Joseph, M. & 15 other authors (2000). Closteroviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 943964. 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.
Peremyslov, V. V., Hagiwara, Y. & Dolja, V. V. (1999). HSP70 homolog functions in cell-to-cell movement of a plant virus. Proc Natl Acad Sci U S A 96, 1477114776.
Pio-Ribeiro, G., Winter, S., Jarrett, T. L., Demski, J. W. & Hamilton, R. I. (1996). Detection of sweet potato virus diseased-associated closterovirus in a sweet potato accession in the United States. Plant Dis 80, 551554.
Roossinck, M. J., Bujarski, J., Ding, S. W., Hajimorad, R., Hanada, K., Scott, S. & Tousignant, M. (2000). Bromoviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 923935. 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.
Rozanov, M. N., Koonin, E. V. & Gorbalenya, A. E. (1992). Conservation of the putative methyltransferase domain: a hallmark of the Sindbis-like supergroup of positive-strand RNA viruses. J Gen Virol 73, 21292134.[Abstract]
Salazar, L. F., Mller, G., Lazarte, V., Querci, M., Zapata, J. L. & Owen, R. A. (2000). Potato yellow vein virus: its host range, distribution in South America and identification as a crinivirus transmitted by Trialeurodes vaporariorum. Ann Appl Biol 137, 719.
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 Res 22, 46734680.[Abstract]
Tian, T., Soong, J., Wisler, G. C., Duffus, J. E. & Falk, B. W. (1996). Generation and cloning of specific cDNAs corresponding to four whitefly-transmitted viruses using RT-PCR and degenerate oligonucleotide primers corresponding to the closterovirus gene encoding the heat shock protein 70 homolog. Phytopathology 86, 11671173.
Wisler, G. C., Liu, H.-Y., Klaassen, V. A., Duffus, J. E. & Falk, B. W. (1996). Tomato infectious chlorosis virus has a bipartite genome and induces phloem-limited inclusions characteristic of the closteroviruses. Phytopathology 86, 622626.
Wisler, G. C., Duffus, J. E., Liu, H.-Y. & Li, R. H. (1998a). Ecology and epidemiology of whitefly-transmitted closteroviruses. Plant Dis 82, 270280.
Wisler, G. C., Li, R. H., Liu, H.-Y., Lowry, D. S. & Duffus, J. E. (1998b). Tomato chlorosis virus: a new whitefly-transmitted, phloem-limited, bipartite closterovirus of tomato. Phytopathology 88, 402409.
Yamashita, S., Doi, Y., Yora, K. & Yoshino, M. (1979). Cucumber yellows virus: its transmission by the greenhouse whitefly, Trialeurodes vaporariorum (Westwood), and the yellowing disease of cucumber and muskmelon caused by the virus. Ann Phytopathol Soc Jpn 45, 484496.
Zenbayashi, R., Shimazaki, Y. & Shibukawa, S. (1988). Some properties of cucumber yellows virus occurred on cucurbitaceous crops in Japan. Abstr Intl Congr Plant Pathol 5th, Kyoto, Japan 50, I-124.
Received 24 May 2002;
accepted 2 January 2003.