Characterization of Cestrum yellow leaf curling virus: a new member of the family Caulimoviridae

Livia Stavolone1,{dagger}, Antonio Ragozzino2 and Thomas Hohn1,{ddagger}

1 Friedrich Miescher Institute, Basel, Switzerland
2 Università di Napoli, via Università, 80055 Portici (NA), Italy

Correspondence
Thomas Hohn
thomas.hohn{at}fmi.ch


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Cestrum yellow leaf curling virus (CmYLCV) has been characterized as the aetiological agent of the Cestrum parqui mosaic disease. The virus genome was cloned and the clone was proven to be infectious to C. parqui. The presence of typical viroplasms in virus-infected plant tissue and the information obtained from the complete genomic sequence confirmed CmYLCV as a member of the Caulimoviridae family. All characteristic domains conserved in plant pararetroviruses were found in CmYLCV. Its genome is 8253 bp long and contains seven open reading frames (ORFs). Phylogenetic analysis of the relationships with other members of the Caulimoviridae revealed that CmYLCV is closely related to the Soybean chlorotic mottle virus (SbCMV)-like genus and particularly to SbCMV. However, in contrast to the other members of this genus, the primer-binding site is located in the intercistronic region following ORF Ib rather than within this ORF, and an ORF corresponding to ORF VII is missing.

{dagger}Present address: Istituto di Virologia Vegetale CNR, via Amendola 165/A, 70126 Bari, Italy.

{ddagger}Present address: University of Basel, Department of Botany, Plant Health Unit, Schönbeinstr. 6, CH-4056 Basel, Switzerland.


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The Caulimoviridae are plant pararetroviruses and replicate their genome through a process of reverse transcription similar to that of retroviruses (Rothnie et al., 1994). Viral genomes form supercoiled minichromosomes in the host cell nucleus that are transcribed into terminally redundant polyadenylated RNA. This migrates to the cytoplasm where it is used both as polycistronic mRNA to produce viral proteins (Hohn & Fütterer, 1992) and as a replication template. In the latter case, the full-length RNA, primed by the host cytosolic initiator methionine tRNA (tRNAMet), is reverse transcribed by the viral reverse transcriptase (RT)/RNase H into genomic dsDNA, which may re-enter the nucleus for amplification or be encapsidated into a virion. Virions accumulate in characteristic inclusion bodies in the cytoplasm of infected cells.

Caulimovirus genomes are single, circular, double-stranded DNA molecules ranging from 7000 to 8300 bp with between one and eight closely spaced open reading frames (ORFs). The functions of most genes are known, particularly in Cauliflower mosaic virus (CaMV), the type member of the family. ORF I encodes the cell-to-cell movement protein (MP) (Thomas & Maule, 1995) containing an RNA-binding domain conserved in the MP of most plant viruses and in the family I transport protein group (Thomas et al., 1993; Koonin et al., 1991). The ORF II product is the aphid transmission factor (ATF) (Woolston et al., 1983), whereas that of ORF III is a virion-associated protein (VAP) (Leclerc et al., 2001). VAP has been found to be an indispensable co-factor of ATF (Leh et al., 1999).

The product of ORF IV is the coat protein (CP) precursor (Kirchherr et al., 1988). It is processed by an aspartic proteinase (Torruella et al., 1989) included in the poly-functional protein (Pol) encoded by ORF V. RT and RNase H are the other known activities of Pol (Takatsuji et al., 2003). In CaMV, expression of genes from the polycistronic pregenomic RNA is efficiently transactivated by TAV, the product of ORF VI, which is translated from a subgenomic monocistronic viral mRNA (Bonneville et al., 1989). TAV is also the major constituent of the virus inclusion body matrix, and influences virus host range and symptom development (Schoelz & Shepherd, 1988). Despite the diversity of caulimovirus genome organization, most of the structural and functional protein domains described for CaMV are conserved in all members of the family, albeit not always in the same order (Calvert et al., 1995; Hohn & Fütterer, 1997).

A virus disease of Cestrum parqui was first described in the Campania region of Italy (Ragozzino, 1974). C. parqui is a member of the Solanaceae family, and is ubiquitous in the southern part of Italy, where it is used mainly for surrounding cultivated fields because it repels goats and has strong resistance to bacterial and cryptogrammic diseases. Infected plants show leaf mosaic, curling and stunting (Fig. 1a). Isometric virus particles of about 50 nm in diameter prevalently located in cytoplasmic inclusion bodies and very similar to those typical of caulimoviruses have been found in infected tissues (Fig. 1b).



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Fig. 1. Pathological alteration induced by CmYLCV on Cestrum parqui. (a) In infected plants symptoms develop from leaf yellow mosaic and curling (left panel) to shoot stunting (right panel). (b) Electron micrograph of ultra-thin section of infected C. parqui leaf showing virus inclusion body (ib). In the lower right corner a magnified image shows single virions within the inclusion body. White bars in the upper right corners of the figures represent 200 nm.

 
The isolated infectious virus (formerly Cestrum virus) was named Cestrum yellow leaf curling virus (CmYLCV) and its biological and serological properties were studied. CmYLCV was mechanically inoculated by leaf rubbing (55 plants) and grafting (7 plants) to test and crop species (Table 1). Symptoms were observed only on Cestrum elegans and C. parqui (4 weeks after inoculation, 5 to 6 weeks after grafting) and N. clevelandii (6 to 7 weeks after grafting), while the other plants remained symptom-free. CmYLCV infection and non-infection was confirmed by PCR analysis with virus-specific oligonucleotides and by back-inoculation to C. parqui plants.


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Table 1. List of test and crop plants inoculated with CmYLCV

Plant species are grouped by family. Plants susceptible to CmYLCV infection are indicated in bold; plants indicated by * were also grafted with CmYLCV-infected bark tissue.

 
Serological affinity among members of the Caulimoviridae family is commonly used for classification (van Regenmortel et al., 2000). We tested any potential CmYLCV serological relationship with CaMV, Dahlia mosaic virus (DaMV) and Carnation etched ring virus (CERV) by heterologous DAS-ELISA of infected C. parqui leaf extracts with specific antibodies (Agdia Inc.) against the three viruses. The absence of any reaction indicates distinct serological properties of CmYLCV.

CmYLCV was partially purified from infected C. parqui leaves and DNA was extracted according to a conventional procedure (Gardner & Shepherd, 1980). Electrophoretic separation in an agarose gel revealed two DNA components, corresponding to the linear and circular forms of caulimoviral DNAs (Menissier et al., 1983). pCmYLCV, a clone of the entire CmYLCV genome flanked by 141 bp terminal repeats (Fig. 2a), was constructed in the pBluescript vector (Stratagene). To determine whether this sequence was infectious, C. parqui plants were mechanically inoculated with viral DNA excised from pCmYLCV at the unique KasI restriction site present in the terminal repeats. Six weeks after inoculation, the first systemic symptoms appeared on plant leaves. The presence of complete CmYLCV genome molecules in non-inoculated symptomatic leaves was proven by PCR amplification using different pairs of virus-sequence-specific oligonucleotides spanning the entire sequence. Back-inoculation of healthy C. parqui also resulted in infection. The sequence of both strands of the cloned viral DNA was determined (GenBank acc. no. AF364175). Several clones of distinct fragments spanning the whole virus genome were also obtained and their sequences aligned with that of pCmYLCV. DNA sequencing reactions were performed using BigDye Terminators with a GeneAmp PCR system 9700 thermocycler, and analysed on an ABI PRISM 3700 DNA Analyser (all from Applied Biosystems). SeqWeb, the web-based version of the Wisconsin Package version 10.2 [Genetics Computer Group (GCG), Madison, WI, USA] and the VectorNTI package from Informax Inc. (Frederick, MD, USA) were used for all DNA and deduced amino acid sequence analyses.



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Fig. 2. CmYLCV genome and predicted proteins properties. (a) Predicted genome organization of CmYLCV based upon the nucleotide sequence of cloned viral DNA. The circle represents the double-stranded DNA genome. The outer arrows depict the position of the ORFs (I through VI) in the three reading frames. The black and white stars indicate the position of the potential full-length transcript promoter TATA-box (TATAAATA) and the tRNAMet binding site, respectively. The dashed arc represents the virus DNA fragment cloned in pCmYLCV. The position of the KasI restriction enzyme is marked. (b) Alignment of biologically significant homologous regions of proteins encoded by one member per each genus of the Caulimoviridae family. The virus acronym, ORF designation and the number of residues separating the protein N termini from the aligned segment are indicated in front of each sequence. Identical amino acids are shown in italics below the alignments. The spacing between amino acid blocks is given in angle brackets. (c) CmYLCV domains of biological significance conserved in the Caulimoviridae family. Numbers indicate the distance from the N termini of the proteins. Position a and d of the coiled–coil heptad repeats, prevalently occupied by hydrophobic amino acids that form the helix interface, are underlined. (d) Phylogenetic trees. Phylogeny was inferred from reverse transcriptase (RT) and movement protein (MP) domains as aligned above (second block in the MP alignment, first block in RT). Genetic distances represented by the branches of the trees are indicated by bars corresponding to 10 (RT tree) and 100 (MP tree) substitutions per 100 residues.

 
The viral genome comprises 8253 nucleotides and has a GC content of 37 %. According to the convention for caulimovirus sequences, numbering begins at the 5'-end of the putative minus-strand primer-binding site. This sequence is highly conserved in all members of the Caulimoviridae family and, in the case of CmYLCV, has 12 nucleotides complementary to the consensus tRNAMet sequence (Fig. 2c). The virus shows the highest overall nucleotide sequence similarity with members of the Soybean chlorotic mottle virus (SbCMV)-like genus, e.g. SbCMV (47·8 %) and Peanut chlorotic streak virus (PCSV, 45·5 %); 45 % to CaMV and less than 41 % to all other genus members.

Two non-coding regions are found in the viral DNA (Fig. 2a). The larger (6085–6755) contains a TATA box at nt 6395–6402 (TATAAATA) that is part of the promoter directing transcription of the (pre)genomic RNA (L. Stavolone and others, unpublished) followed by a polyadenylation signal (AATAAA; nt 6687–6693). As in other caulimoviruses, a long pregenomic RNA 5'-leader, folding into a typical stem–loop structure, precedes the first viral ORF (Pooggin et al., 1999). Unusually, in CmYLCV, the predicted stem–loop structure starts immediately after the TATA-box and constitutes almost the entire intergenic region. This particular position of the stem–loop, limiting the space necessary to accommodate a ribosome on the RNA molecule, and the absence of a short ORF proximal to the RNA 5'-end (Ryabova et al., 2000), suggest a mechanism of translation distinct from the ‘ribosome shunting’ typical of other members of the Caulimoviridae family (Ryabova & Hohn, 2000). The second non-coding region is 326 bp long (7961–0033) and includes the primer (tRNAMet) binding site.

Computer analysis predicts seven putative ORFs encoding proteins of more than 10 kDa on the positive strand of the virus genome sequence (Fig. 2a) and none in the minus strand. All motifs characteristic of the Caulimoviridae and those shared with retroid elements are conserved in the CmYLCV sequence (Hohn & Fütterer, 1997). Genome organization (number and distribution of the ORFs) of CmYLCV resembles that of SbCMV-like genus members (Hasegawa et al., 1989). Indeed, the SbCMV-like genus is characterized by the presence of three ORFs between ORF I and ORF IV instead of the two typical of CaMV. The additional ORF (‘Ib’ in SbCMV and ‘A’ in PCSV) following gene I contains the tRNAMet binding site but, excluding this domain, it is dispensable for virus infectivity (Mushegian et al., 1995; Takemoto & Hibi, 2001). In CmYLCV, ORF I terminates the larger intergenic region and is followed by a small ORF Ib (Fig. 2a) separated by a single ATGA start/stop codon characteristic of the SbCMV-like genus. However, CmYLCV ORF Ib is smaller and followed by an intergenic region (bp 7961–0033) including the tRNAMet binding site.

ORF I shows significant homology with caulimovirus MPs, particularly in the conserved RNA-binding domain (Fig. 2b). It has been suggested for CaMV that MPs mediate virus movement by modifying plasmodesmata, forming tubules that can accommodate virus particles in the channel (Linstead et al., 1998). Indeed, in the course of our electron microscopy investigations we observed such tubules containing CmYLCV particles in C. parqui-infected leaf tissue. This suggests that CmYLCV could employ a similar mechanism for cell-to-cell movement.

The amino acid sequence of ORF II shows 41 % similarity with SbCMV ORF II and contains a putative double {alpha}-helix domain (Fig. 2c). A similar motif in CaMV ORFII is responsible for ATF trimerization (Hebrard et al., 2001) and interaction with the product of ORFIII (Leh et al., 1999). Although no aphid (or other insect) transmission test was performed, we observed that healthy C. parqui transplanted under field conditions in the vicinity of a plant naturally infected with CmYLCV acquired the virus and displayed symptoms during the spring season. This suggests possible insect-mediated CmYLCV transmission.

CmYLCV ORF III also contains a coiled–coil domain (Fig. 2c) responsible for tetramerization, a conserved feature of caulimovirus VAPs (Stavolone et al., 2001).

ORF IV encodes the viral coat protein precursor as suggested by the presence of an RNA-binding domain (zinc finger; Fig. 2c) arranged as CX2CX4HX4C and preceded by a lysine-rich domain (aa 380–431; 44 % lysine content) (Covey, 1986). An acidic domain within its C-terminal part resembles the PEST-related degradation signal of CaMV responsible for protein destabilization (Karsies et al., 2001).

The putative protein encoded by ORF V contains all motifs conserved in caulimovirus replicases: aspartic protease, RT and RNase H (Fig. 2b). All domains conserved in ORFs IV and V follow the typical ‘gagpol’ arrangement of most retro- and pararetroviruses (Rothnie et al., 1994) and show the highest homology with SbCMV, confirming that CmYLCV is most appropriately classified with this genus.

The product of CmYLCV ORF VI conserves the key domains of the CaMV TAV (Fig. 2b) and hence probably also protein function. Although there is some indication for the absence of a shunting mechanism in SbCMV (Takemoto & Hibi, 2001) and CmYLCV, a possible alternative strategy of gene expression involving TAV-mediated reinitiation of translation (Park et al., 2001) is not excluded.

Predicted relationships among representative members of the Caulimoviridae family were determined using SeqWeb. One member per each genus of the family was included in the phylogenetic analyses. These were based on conserved amino acid regions in MP and RT, aligned as in Fig. 2(b). A matrix of the pairwise evolutionary distances between aligned sequences, corrected using the Kimura method, was generated. Unrooted phylogenetic trees were reconstructed from the generated matrixes using the neighbour-joining method, and displayed as phylograms. Both trees identify CmYLCV as member of the SbCMV-like genus of the Caulimoviridae family with the closest homology to SbCMV (Fig. 2d).

However, CmYLCV differs from the two other members of the SbCMV-like genus in the absence of the nonessential ORF VII (Mushegian et al., 1995; Takemoto & Hibi, 2001). The lack of ORF VII and the exclusion of the tRNAMet binding site from ORF Ib, the other dispensable gene in the genome, could represent an evolutionary strategy of CmYLCV to increase efficiency.

Analysis of the CmYLCV sequence, and comparison with the other available sequences of members of the Caulimoviridae, confirms that despite the many conserved features within this virus family, single virus properties can differ significantly, even among members of the same genus. The most intriguing question deriving from this study is whether ribosome shunting does indeed not occur in CmYLCV and what kind of translation mechanism is used instead.

Finally, further investigations on the activity of the potential full-length transcript promoter could uncover a new biotechnological tool for expression of exogenous genes in plants.


   ACKNOWLEDGEMENTS
 
We thank Maciej Pietrzak and Herbert Angliker for DNA sequencing and Andreas Gisel for valuable help with bioinformatic analysis. We are also grateful to Antonio Peluso and Sjoerd van Eeden for expert technical assistance and to Helen Rothnie for critical reading of the manuscript. L. S. was partially supported by an EMBO short-term fellowship.


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Received 10 June 2003; accepted 12 September 2003.



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