Nucleotide sequence and genome organization of Cucumber yellows virus, a member of the genus Crinivirus

Sedyo Hartono1,{dagger}, Tomohide Natsuaki1, Yoshikatsu Genda2 and Seiichi Okuda1

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


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The genome of Cucumber yellows virus (CuYV), isolated in Japan from cucumber (Cucumis sativus L.), was completely sequenced and shown to be bipartite. CuYV RNA1 consisted of 7889 nucleotides and encompassed seven open reading frames (ORFs), which is typical of the Closteroviridae, including a heat-shock protein 70 homologue, a coat protein and a diverged coat protein (CPd). CuYV RNA2 consisted of 7607 nucleotides and included two ORFs: ORF1a potentially encoded a polyprotein containing putative papain-like protease, methyltransferase and helicase domains, and ORF 1b potentially encoded an RNA-dependent RNA polymerase, which is probably expressed via a +1 ribosomal frameshift. The size and organization of the CuYV genome are similar to those of Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, indicating that CuYV is a member of that genus, although CuYV differed in several points from LIYV.

The DDBJ accession numbers of the sequences reported in this paper are AB085612 and AB085613.

{dagger}Present address: Department of Entomology and Phytopathology, Faculty of Agriculture, Gadjah Mada University, Sekip Unit 1, Yogyakarta 55281, Indonesia.


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Cucumber yellows virus (CuYV), which causes a yellowing disease on cucumber (Cucumis sativus L.) and melon (C. melo L.) in Japan (Yamashita et al., 1979), is transmitted by the greenhouse whitefly (Trialeurodes vaporariorum Westwood). CuYV isolated in Saitama Prefecture, Japan, has a broad host range and was able to infect 30 plant species from nine families following experimental transmission by whiteflies (Zenbayashi et al., 1988). Our field surveys done in Ibaraki and Tochigi Prefectures, Japan, found a high incidence of symptoms of yellowing disease in cucumber plants previously described as resulting from CuYV infection. T. vaporariorum, the vector of CuYV, was also observed to infest cucurbit crops in these areas. Recently, CuYV was postulated to be a strain of Beet pseudo-yellows virus (BPYV) based on the host range and vector specificity (Wisler et al., 1998a). BPYV, a whitefly-transmitted virus first identified in California (Duffus, 1965), is known worldwide as a destructive pathogen of many plant species.

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.



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Fig. 1. Genome organization of CuYV RNA1 and RNA2, and cloning strategy. Boxes represent open reading frames (ORFs) with encoded proteins designated above. The scale gives approximate sizes in kb. Horizontal bars below represent sequenced clones from the cDNA library and from PCR products of RACE. The genome organization of LIYV RNA2 and RNA1 are illustrated above the genome organization of CuYV RNA1 and RNA2, respectively.

 
The genome organization of CuYV is similar to that of LIYV, the type member of the genus Crinivirus, although CuYV RNA1 corresponded to RNA2 of LIYV and vice versa (Fig. 1). In CuYV, the Closteroviridae hallmark gene array was contained in RNA1 (the longer genomic RNA) and potentially encompassed seven ORFs (designated ORF1–7), whereas the putative replication-associated proteins were encoded in RNA2 (the shorter genomic RNA) and comprised two ORFs (designated ORF1a and 1b). The difference in the relative sizes of the RNAs is probably because: (i) CuYV RNA2 lacks the 32 kDa protein located downstream of ORF1b in LIYV RNA1; and (ii) CuYV RNA1 contains a larger coat protein duplicate (CPd) than LIYV CPd (Fig. 1).

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 protein–protein 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 412–413) 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.



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Fig. 2. Phylogenetic relationship of CuYV. HEL (a) and RdRp (b) proteins compared with corresponding proteins of the Closteroviridae and HSP70 (c) and CP (d) proteins compared with corresponding proteins of the characterized members of the genus Crinivirus. EMBL or GenBank accession numbers of the sequences used in this phylogenetic analysis are: BYV (AF056575), BYSV (U51931, L20761), CTV (AF001623), CYSDV (AJ223619, AJ243000), GLRaV-1 (AF195822), GLRaV-2 (AF039204), GLRaV-3 (AF037268), LChV-1 (Y10237), LChV-2 (AF333237), LIYV (U15440, U15441), PYVV (AF150984), SPCSV (X80995), ToCV (AF024630) and TICV (U67449). For comparisons of HEL and RdRp, corresponding sequences of Tobacco mosaic virus (TMV; Z29370) and Tobacco rattle virus (TRV; AF034622), respectively, were used as non-closterovirus outgroups. For comparisons of HSP70 and CP, the CTV HSP70 and CP sequences, respectively, were used as non-crinivirus outgroups. The number above each node shows the percentage of bootstrap values in which a given node was recovered. The type of vectors are indicated on the right: aphids (A), mealybugs (M), whiteflies (W) and unknown (?). Phylogenetic trees were constructed using the CLUSTAL W program (Thompson et al., 1994) in the MacVector package (Oxford Molecular Ltd).

 
The 5' non-coding regions (NCRs) of RNA1 and RNA2 of CuYV were 372 and 242 nucleotides in length, respectively. Analysis of this region did not show any homology to other viruses of the Closteroviridae, nor between the 5'NCRs of CuYV RNA1 and RNA2, except that the first ten nucleotides (5'-AUAAAUUUAU-3') were identical. Comparison of the 3'NCRs of CuYV RNA1 (186 nucleotides) and RNA2 (188 nucleotides) showed that they were highly conserved (Fig. 3), sharing 89 % nucleotide sequence identity, whereas the 3' NCRs of LIYV RNA1 and RNA2 were distinct (Klaassen et al., 1995). Furthermore, analysis of this region did not show any homology with other Closteroviridae. Usually, multipartite RNA viruses have conserved or shared 3' nucleotide sequences on the genomic segments (Roossinck et al., 2000), and CuYV has this property.



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Fig. 3. Nucleotide sequence alignment of the 3' non-coding region of CuYV RNA1 and RNA2. The common sequence is shown above the alignment. Stop codons of RNA1 and RNA2 are boxed.

 
Recently, at least eight whitefly-transmitted closteroviruses have been reported and classified as members of the genus Crinivirus; the exception is BPYV, which is classified in the genus Closterovirus (Martelli et al., 2000), based on the particle length of BPYV with a range from 1500 to 1800 nm (Liu & Duffus, 1990), which is similar to the lengths of other monopartite closteroviruses. Recently, however, Liu et al. (2000) suggested that the particles of BPYV range in length from 600 to 850 nm, which is similar to the particle lengths of other criniviruses. We have compared the HSP70 homologue gene of CuYV with that of BPYV HSP70 from an American isolate (accession no. U67447; Tian et al., 1996) and a Greek isolate (accession no. Y15568; Coutts & Coffin, 1996), sharing 99 % and 98 % amino acid sequence identity, respectively. This evidence strongly suggests that CuYV is a strain of BPYV, because the vector transmission and host range characteristics of CuYV were very close to those of BPYV (Wisler et al., 1998a). Taken together, this indicates that BPYV should also be classified as a member of the genus Crinivirus, of the family Closteroviridae, in addition to CuYV.


   ACKNOWLEDGEMENTS
 
The authors gratefully acknowledge Dr M. A. Mayo for critically reading this manuscript. This work was supported in part by Grant-in-Aid for Scientific Research (12660041 & 14560036) from Japan Society for the Promotion of Science.


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Received 24 May 2002; accepted 2 January 2003.