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Nucleic Acids Research Pages 420-426

In planta transcription of a second subgenomic RNA increases the complexity of the subgroup 2 luteovirus genome
Introduction
Materials And Methods
   RNA extraction and Northern analysis
   Preparation and labeling of hybridization probes
   Affinity purification of PLRV sgRNA2
   Primer extension experiment
   Synthesis of chimeric PLRV ORF-GUS constructs
   Protoplast inoculation and GUS assay
   Bacterial expression of proteins
Results
   Characterization of PLRV sgRNA2 by Northern blot analysis
   Mapping of the PLRV and CABYV sgRNA2 transcriptional start site
   Characterization of PLRV ORF6 and ORF7 by transient expression of chimeric PLRV ORF-GUS constructs and by bacterial expression
Discussion
Acknowledgements
References


In planta transcription of a second subgenomic RNA increases the complexity of the subgroup 2 luteovirus genome

In planta transcription of a second subgenomic RNA increases the complexity of the subgroup 2 luteovirus genome Ahmed Ashoub, Wolfgang Rohde and Dirk Prüfer*

Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany

Received October 23, 1997; Revised and Accepted November 17, 1997

ABSTRACT

The genetic information of potato leafroll virus (PLRV), a typical member of the subgroup 2 luteoviruses, is contained in a single-stranded (+) sense RNA of ~5.9 kb. A single subgenomic RNA (sgRNA1) of ~2.3 kb has been characterized as the mRNA for the 3' clustered viral open reading frames ORF3, ORF3/5 and ORF4. Here we demonstrate by Northern blot analyses of polysomal RNAs from PLRV-infected Solanum tuberosum and Physalis floridana plants that, as with luteoviruses belonging to subgroup 1, in planta synthesis of a second 0.8 kb subgenomic RNA (sgRNA2) increases the complexity of subgroup 2 luteoviral genomes significantly. PLRV-specific hybridization probes as well as primer extension experiments map sgRNA2 to the 3'-end of the PLRV RNA genome (positions 5190-5987). Similarly, for the closely related cucurbit aphid-borne yellows virus (CABYV) a sgRNA2 of similar size and position (positions 4888-5669) was identified. PLRV sgRNA2 may code for two viral proteins of 7.1 (ORF6) and 14 kDa (ORF7) respectively, while the CABYV proteins are 8.7 (ORF6) and 8.3 kDa (ORF7) in size, with PLRV ORF7 displaying nucleic acid binding activity. In vivo experiments by transient expression of chimeric GUS fusions in potato protoplasts demonstrated that sgRNA2 functions as a bicistronic mRNA with high expression of ORF6 and low translational efficiency for synthesis of ORF7.

INTRODUCTION

After infection of their respective hosts by viruses expression of viral genes during the viral life cycle is regulated at various levels. During multiplication of plant RNA viruses, for example, synthesis of subgenomic RNAs (sgRNAs) generally serves for expression of genes located downstream on the RNA genome. Regulation of this sgRNA transcription may depend on host factors and/or the time course of the viral life cycle. Tobacco mosaic virus (TMV) represents one example of temporal regulation of sgRNA synthesis (1). However, even sgRNAs very often code for more than one viral gene product. Non-canonical translation strategies during initiation, elongation and termination of protein biosynthesis are further mechanisms in viral gene expression by which different or partially overlapping proteins with specific functions are synthesized from a restricted region of the viral genome as represented by a sgRNA (2).

Among the plant viruses the luteoviruses are one well-studied example for such different expression strategies (3,4). The luteoviral (+) sense RNA genome is ~5.5-5.9 kb in size and contains six differently arranged open reading frames (ORFs) depending on the subgroup (3,5). Potato leafroll virus (PLRV) is a member of the subgroup 2 luteoviruses. The six major ORFs of the PLRV genome (Fig. 1) are separated by a small intergenic region into a gene cluster divergent among luteoviruses (ORF0, 1 and 2) and a conserved gene cluster (ORF3, 4 and 5). ORF0 is a factor involved in symptom development (6; Prüfer, in preparation) and ORF1 and ORF 2, with motifs characteristic of helicases (ORF1; 7) and polymerases (ORF2; 8), form part of the viral replicase complex. While ORF0, ORF1 and ORF2 are translated from genomic RNA (gRNA; unpublished results), transcription of a subgenomic RNA1 (sgRNA1; Fig. 1) provides expression of the 3'-proximal ORF3, ORF4 and ORF5 for all known luteoviruses (3,5). ORF3, the 5'-proximal ORF, encodes the capsid protein CP. Initiation at an internally located AUG codon within the CP gene, but in a different reading frame, allows for synthesis of the viral movement protein ORF4 (9-11) and suppression of the CP amber stop codon results in formation of an ORF3/ORF5 read-through protein (9,12), which is supposedly the aphid transmission factor (13,14).


Figure 1. Schematic representation of PLRV genomic and subgenomic RNAs. The boxes represent open reading frames (ORFs). The positions of ORF6 and ORF7 (this paper) are indicated. ORF7 is in-frame with the C-terminus of ORF5, while ORF6 is located in a different reading frame.

In contrast to PLRV and other subgroup 2 luteoviruses, a second sgRNA (sgRNA2) representing the 3'-terminal 886 nt of the barley yellow dwarf virus (BYDV) genome with the coding capacity for a 6.7 kDa protein (ORF6; see also Fig. 6) has been described for BYDV (serotype PAV), a member of the subgroup 1 luteoviruses (15,16). In this paper we demonstrate the existence of a sgRNA2 with charcteristics (size, location and structural organization) similar to BYDV sgRNA2 for two members of subgroup 2 luteoviruses (PLRV and cucurbit aphid-borne yellows virus, CABYV).

MATERIALS AND METHODS

RNA extraction and Northern analysis

Polysomal RNAs from healthy and PLRV-infected Solanum tuberosum and Physalis floridana plants as well as from healthy and CABYV-infected Cucumis sativus plants were extracted according to established protocols (17). Northern analysis was carried out as follows. An aliquot of 10 µg polysomal RNA was denatured by heating at 55°C for 15 min in the presence of 50% formamide, 2.2 M formaldehyde and 1× MOPS buffer (40 mM MOPS, pH 7.0, 10 mM sodium acetate, 2 mM EDTA) and separated in a 1% agarose-formaldehyde gel. The RNA was then transferred to Nylon membranes and subsequently hybridized with specific 32P-labeled DNA fragments (Fig. 2A) for 18 h in a 50% formamide solution containing 5× SSC, 5% Denhardt's solution, 0.1% SDS and 10 µg/ml salmon sperm DNA.

Preparation and labeling of hybridization probes

For the detection of PLRV genomic and subgenomic RNAs a 705 bp EcoRI fragment [P2, positions 5282-5987; all coordinates are given with reference to the PLRV sequence first published by Mayo et al. (18), but the actual size of PLRV gRNA for various isolates is 5882 or 5883 nt] and a 1825 bp PinAI-EcoRI fragment (P1, positions 3265-5090) were isolated from a PLRV full-length cDNA clone (p35SPL-WT; 19). In order to narrow down the 5'-end of PLRV sgRNA2 a series of PCR fragments (Fig. 2A) were amplified using the following backward oligonucleotides: 5'-TCGAAGTGTTCGGCAT-3' (P3, complementary to nt 5395-5380; 18); 5'-CCAGTCTGTACCATCG-3' (P4, complementary to nt 5276-5261); 5'-CCCCGAACTCCAATCGC-3' (P5, complementary to nt 5229-5213); 5'-CCTGCGTTTGTATCGGG-3' (P6, complementary to nt 5133-5117); 5'-GTTTACCGAACCAGCA-3' (P7, complementary to nt 5189-5174); in combinition with 5'-CCAAGGTCACCCAGAA-3' (nt 4769-4784) as the forward primer.

For detection of CABYV genomic and subgenomic RNAs a 906 bp SspI-SalI fragment (C1, nt 4763-5669; 20; Fig. 3D) and a 505 bp SspI fragment (C2, nt 4257-4762; Fig 3D) were isolated from a CABYV full-length cDNA clone (pCA-WT; 21). Specific DNA probes for detection of viral RNAs were produced by mixing 100 ng appropriate cDNA (P1 and P2; C1 and C2) or PCR fragments (P3-P7) with 100 pmol either PLRV- or CABYV-specific oligonucleotides complementary to the respective 3'-end in the presence of Klenow polymerase and [[alpha]-32P]dCTP. The probe used in Figure 2B, lane e was obtained by replacing specific oligonucleotides by random primers. All reactions were carried out for 1 h at 37°C.


Figure 2. Characterization of PLRV sgRNA2. (A) Schematic representation of cDNA (P1, P2, grey bars) and PCR probes (P3-P7, black bars) used for molecular hybridization. The start of transcription for sgRNA2 (position 5190) is indicated by an arrow. (B) Northern blot analyses of polysomal RNA from healthy (lane a) and PLRV-infected potato plants (lanes b and e-k) as well as healthy (lane c) and PLRV-infected P.floridana plants (lane d). 32P-Labeled hybridization probes were obtained by use of either PLRV-specific (P2-P6) or random primers (P2*). In lane l affinity-purified sgRNA2 was loaded.


Figure 3. Transcriptional start mapping of PLRV and CABYV sgRNA2 by primer extension (A and C) and molecular hybridization (B and D). The primer extension products of polysomal RNAs of healthy (A, lane h) and PLRV-infected (A, lane i) S.tuberosum plants as well as healthy (C, lane h) and CABYV-infected C.sativus plants (C, lane i) were analyzed by denaturing polyacrylamide gel electrophoresis. The 5'-termini of PLRV and CABYV sgRNA2 are indicated by arrows. Corresponding sequencing reactions presented in the figure were obtained by use of the same primers. Northern blot analyses of PLRV- and CABYV-infected plants (B and D) were carried out as already described in the legend to Figure 2.

Affinity purification of PLRV sgRNA2

Aliquots of 100 µg polysomal RNA from PLRV-infected plants were mixed with 150 pmol 5'-biotin-labeled oligonucleotide (5'-ACTACACAACCCTGTAAGAGGATCCTGGCT-3', complementary to nt 5987-5960) and precipitated by addition of ethanol. The pellet was redissolved in 100 µl annealing buffer (6* SSC), denatured at 80°C for 3 min and incubated for 2 h at 63°C. After reprecipitation with EtOH the RNA-primer hybrid was resuspended in 100 µl 2 M NaCl and subsequently incubated with 100 µl Dynabeads M-280 (Dynal) suspension. In contrast to the manufacturer's recommendations for isolation of RNAs with a molecular weight <1 kb, the solution was incubated for 15 min at 25°C and subsequently for a further 30 min at 42°C. The beads were washed three times for 3 min with 1 M NaCl, resuspended in 200 µl TE buffer, pH 8.0, and phenol extracted for 30 min at 65°C for removal of the beads. After two further phenol/chloroform extractions and EtOH precipitation the RNA was redissolved in 50 µl H2O.

Primer extension experiment

Polysomal RNAs from PLRV- or CABYV-infected plants were used to detect the transcripton start of PLRV and CABYV sgRNA2s respectively. Oligonucleotides PEPLRV (5'-TAGCATCGTAGGTACTATCTGAGTTT-3', complementary to nt 5256-5231 of the PLRV genome) and PECABYV (5'-TCGGGTATATTCTCTTCCTCAAGATC-3', complementary to nt 5006-4981 of the CABYV genome) were labeled with [[gamma]-32P]ATP in the presence of T4 polynucleotide kinase. After phenol/chloroform extraction and EtOH precipitation 10 ng labeled primer were mixed with 10 µg polysomal RNA and subsequently precipitated by addition of ethanol. The pellet was redissolved in 15 µl annealing buffer (150 mM KCl, 10 mM Tris-HCl, pH 8.3, 1 mM EDTA), denatured at 80°C for 3 min and incubated for 2 h at 60°C.

cDNA synthesis was carried out at 50°C for 1 h using 200 U Superscript reverse transcriptase (Gibco-BRL). Finally, the cDNAs were precipitated and treated with RNase prior to analysis on an 8% polyacrylamide-6 M urea sequence gel. All sequence reactions were performed with a T7 sequencing kit (Pharmacia) according to the supplier's protocol.

Synthesis of chimeric PLRV ORF-GUS constructs

The construction of chimeric PLRV ORF-GUS constructs for transient expression experiments in potato protoplasts was performed as follows. In the first step a full-length cDNA copy of subgenomic RNA2 was obtained by RT/PCR using the oligonucleotides 5'-AGAAAGCTTACTACACAACCCTGTAAGAGGATCCTGG-3' (complementary to nt 5987-5960, with a newly introduced HindIII site shown in italic) and 5'-AGCGGCGGCCCAACTGAGTCGCTG-3' (nt 5190-5213). After treatment with Klenow polymerase, T4 polynucleotide kinase and HindIII the amplified PCR fragment was cloned into the StuI/HindIII restriction sites of vector pCaP35J (17), to yield p35S-sgRNA2fl.

For the ORF6-GUS translational fusion an ~200 bp fragment carrying the CaMV 35S core promoter, the 5'-non-coding leader of sgRNA2 and the first 16 nt of the ORF6 coding sequences was amplified by PCR on the basis of p35S-sgRNA2fl using the oligonucleotides P35S (5'-CGTTCCAACCACGTCTTCAAA-3', nt 310-330 of the CaMV 35S promoter; 22) and PO6 (5'-ACTAGTCGACGTACCATCGACTG-TAGCATC-3', complementary to nt 5266-5250, with a newly introduced SalI site shown in italic). The amplified fragment was digested with EcoRV (position 350 of the CaMV 35S promoter) and subcloned together with a SalI/XbaI-digested GUS gene from p1158-GUS (Prüfer, unpublished results) in a two fragment ligation into an EcoRV/XbaI-linearized pRT100 vector (22). The resulting construct was designated pO6-GUS.

The ORF7-GUS translational fusion was obtained by the same strategy as described for the ORF6-GUS construct with the exception that the PCR reaction was carried out with oligonucleotide PO7 (5'-ACTAGTCGACCCAACATACTTCGCCCATCT-3', complementary to nt 5475-5456) instead of PO6. The resulting construct was designated pO7-GUS. The integrity of all constructs was verified by restriction and sequence analysis.

Protoplast inoculation and GUS assay

Approximately 2 * 105 S.tuberosum protoplasts were transfected with 10 µg appropriate plasmid DNAs according to established protocols (23). GUS assays were carried out as described by Jefferson et al. (24).

Bacterial expression of proteins

For construction of pGEX-O6 and pGEX-O7 appropriate DNA fragments were amplified by PCR using p35S-sgRNA2fl and the following oligonucleotides: pGEX-O6, 5'-AAGAGGTCGCGAATGCTACAGTCGATGGTACAG-3' (nt 5251-5271, with a newly introduced NruI site shown in italic) and 5'-TCTTCTAGGCCTGCCGTGCGCTATAATAGTAG-3' (complementary to nt 5447-5428, with a newly introduced StuI site shown in italic); pGEX-O7, 5'-AGATCGTCGCGAATGTTGGAGAAGAGAGAGGAA-3' (nt 5469-5489, with a newly introduced NruI site shown in italic) and 5'-AGAAAGCTTACTACACAACCCTGTAAGAGGATCCTGG-3' (complementary to nt 5987-5960, with a newly introduced HindIII site shown in italic)

The amplified fragments were digested with StuI/NruI (ORF6) and NruI/HincII (ORF7; HincII at position 5849 of the PLRV genome) and subsequently subcloned into a SmaI-linearized pGEX-3 vector (Amrad). The integrity of all constructs was verified by restriction and sequence analysis.

The purification of chimeric GST-PLRV fusion proteins was performed by established protocols (25). Nucleic acid binding experiments were carried out as described by Gramstat et al. (26).

RESULTS

Characterization of PLRV sgRNA2 by Northern blot analysis

Polysomal RNAs from healthy and PLRV-infected S.tuberosum and P.floridana plants were isolated and subjected to Northern blot analysis using a 32P-labeled cDNA probe obtained by use of either PLRV-specific (P2, Fig. 2, lanes a-d and k) or random primers (P2*; Fig. 2, lane e). In contrast to previous studies (9,27), which identified only the 2.3 kb sgRNA1, a second subgenomic RNA of ~800 nt in size could be detected with P2 in addition to gRNA and sgRNA1. However, using the same cDNA probe obtained by random primer labeling (P2*) several additional bands in the range 800-1200 nt were observed (Fig. 2B, lane e). This is explained by synthesis of short 32P-labeled DNA fragments with non-specific affinity to host RNAs due to the random primer reaction. sgRNA2 was further characterized by synthesis of successive 3'-truncated 32P-labeled PCR fragments (P3-P5) and a 32P-labeled 1825 bp EcoRI-PinAI cDNA fragment as indicated in Figure 2A. In Northern blot analysis (Fig. 2B) sgRNA2 could be identified with P3-P6 (lanes f-h), while an appropriate signal was not observed for P1 (lane j) and P6 (lane i). For identification of the putative 3'-end of sgRNA2 a 5'-biotin-labeled primer complementary to the 3'-terminal 30 nt of gRNA was used to purify sgRNA2 following the strategy for isolation of RNAs up to 1 kb (see Materials and Methods ). As is obvious from Figure 2B (lane l), sgRNA2 could be trapped by this approach, indicating that it contains the 3'-terminal sequence present in gRNA and sgRNA1.

Mapping of the PLRV and CABYV sgRNA2 transcriptional start site

According to the approximate size of sgRNA2 (~800 nt) and the results obtained from Northern analysis (Fig. 2), the sgRNA2 transcription start point was expected at a position between nt 5133 and 5213 of the PLRV RNA genome. To characterize this transcription start a primer extension experiment was carried out using 32P-labeled primer PEPLRV (see Materials and Methods). Gel electrophoresis revealed an elongation product of 67 nt terminating at an adenosine residue at position 5190 as the start of transcription by comparison with a sequence reaction with the identical primer but using p35SPL-WT as template (Fig. 3A). Thus sgRNA2 corresponds to an RNA of 797 nt in length. In order to verify this observation polysomal RNA of PLRV-infected S.tuberosum plants was subjected to Northern analysis and hybridized with P2 and P7, a PCR fragment (positions 5174-5189) ending 1 nt upstream of the putative transcription start for sgRNA2. As expected, no signal was observed for sgRNA2 with P7 as the probe (Fig. 3B), confirming the result of the primer extension experiment.

Expression of sgRNA2 was also examined for CABYV, as a further representative of subgroup 2 luteoviruses. Polysomal RNAs from healthy and CABYV-infected C.sativus plants were isolated and used in a primer extension experiment with 32P-labeled oligonucleotide PECABYV (see Materials and Methods). Sequence gel electrophoresis (Fig. 3C) revealed a strong stop signal which, with reference to the control sequence obtained from pCA-WT with the same primer, placed the CABYV sgRNA2 transcription start at an adenosine residue at position 4888 of the CABYV genome. Thus CABYV sgRNA2 is 782 nt in size. To further characterize the start of transcription by molecular hybridization two cDNA fragments were isolated from pCA-WT (see Materials and Methods), 32P-labeled and hybridized to polysomal RNA from CABYV-infected C.sativus plants. As expected, with hybridization probe C1, representing the last 900 nt of the CABYV genome, all three RNA species (gRNA, sgRNA1 and sgRNA2) were detected (Fig. 3D). In contrast, only signals for gRNA and sgRNA1 were observed with a hybridization probe located upstream of the sgRNA2 start of transcription (C2, Fig. 3D)

Characterization of PLRV ORF6 and ORF7 by transient expression of chimeric PLRV ORF-GUS constructs and by bacterial expression

Computer analysis of PLRV sgRNA2 sequences revealed the presence of two ORFs (Fig. 1) with the capacity to encode two proteins of 7.1 (ORF6) and 14 kDa (ORF7) respectively. Both ORFs are located within the 3'-proximal half of the ORF5 coding region, with ORF6 located in a different reading frame, while ORF7 is in the ORF5 frame and, therefore, represents the C-terminus of ORF5. The start of sgRNA2 transcription is located 61 nt upstream of the first ORF (ORF6) and 268 nt upstream of ORF7. In order to determine the relative frequency of translation initiation for both ORFs on the identical mRNA ORF6 and ORF7 were separately fused in-frame to the [beta]-glucuronidase gene of Escherichia coli. In the resulting constructs only a few additional amino acids (pO6-GUS, nine amino acids for the first AUG and five amino acids for the second AUG; pO7-GUS, five amino acids; Fig. 4) were fused to the GUS enzyme in order to minimize an inhibitory effect of long N-terminal fusions on enzyme activity. An out-of-frame construct (9) and the vector pRT104GUS (encoding GUS wild-type protein; 28) served as negative and positive controls respectively.


Figure 4. Translational fusions of ORF6 and ORF7 with the GUS reporter gene. Viral (italic) and non-viral amino acids fused to the second amino acid of the GUS enzyme (V) are indicated. 35S P and T are cauliflower mosaic virus (CaMV) promoter and terminator sequences respectively. Homologies of the different translation initiation codons to the consensus sequence in plants (29) are indicated by dots.

Table 1 summarizes the results of transient expression experiments in potato protoplasts. The results demonstrated that translation initiation at the appropriate AUGs of ORF6 and ORF7 occurs at a ratio of ~16:1 (ORF6:ORF7) with respect to the two control constructs. Interestingly, transient expression of the pO6-GUS construct in potato protoplats reached 93% of wild-type GUS activity under control of the strong constitutive CaMV 35S promoter (pRT104GUS), although the sequences flanking AUG do not share significiant homology with the consensus sequence reported for optimal initiation in plants (Fig. 4; 29).

Table 1. Results of transient expression experiments in potato protoplasts using the constructs described in Figure 4
ORF expressed GUS activity(pmol MU/mg protein/min) Ratio ORF6:ORF7
6 12 123 16:1
7 747  
6 14 715 15.6:1
7 941  
6 13 628 15.2:1
7 879  

The putative viral gene products ORF6 and ORF 7 are basic proteins with isoelectric points of 10.4 (PLRV ORF6) and 10.8 (PLRV ORF7), suggesting that they might interact with nucleic acids. To test for this property PLRV ORF6 and ORF7 were expressed in the bacterial pGEX expression vector system, purified (Fig. 5A) and incubated with radioactively labeled PLRV RNA after gel electrophoresis and blotting to nitrocellulose membrane. As shown in Figure 5B, only PLRV ORF7 displayed nucleic acid binding properties.


Figure 5. Nucleic acid binding capacity of the PLRV ORF7 protein. (A) Polyacrylamide gel electrophoresis of affinity-purified ORF6 and ORF7 proteins. (B) Autoradiogram of (A) after blotting and incubation with 32P-labeled PLRV sense RNA.

DISCUSSION

By Northern analysis of polysomal RNAs and of affinity-purified RNA from different host plants infected with the subgroup 2 luteoviruses PLRV and/or CABYV we have demonstrated the in planta presence of a second sgRNA (sgRNA2) with a size of ~800 nt. sgRNA2 has the capacity to code for two viral proteins, ORF6 and ORF7 respectively, one of which (ORF7) represents the C-terminus of ORF5 and displays nucleic acid binding properties. Several precautions were taken in order to identify the newly characterized luteoviral RNA as a true sgRNA and not a specific breakdown product. Firstly, RNA was isolated from polysomes as an indication of the biological activity of sgRNA2 as mRNA. In addition, PLRV viral RNAs (gRNA, sgRNA1 and sgRNA2) were further characterized by hybridization and affinity purification with a biotin-labeled oligonucleotide representing the 3'-terminus of genomic RNA. Furthermore, care was taken to avoid non-specific cross-hybridization to cellular RNAs, which was reported earlier for PLRV (9) using primer-labeled rather than randomly labeled probes. And, finally, primer extension experiments as well as molecular hybridization with probes representing defined regions of the luteoviral genome further supported the unique origin of sgRNA2 as an RNA synthesized from a specific promoter site on gRNA.

For subgroup 1 luteoviruses a sgRNA2 has been known for some time. With the PAV serotype of barley yellow dwarf virus (BYDV-PAV; 15,16,30) sgRNA2 represents the 3'-terminal 868 nt of the BYDV genome. Its start was mapped to position 4809 of the BYDV genome, some 111 nt upstream of BYDV ORF6 (16). Comparison of the sequences flanking the BYDV sgRNA2 transcription start site with those of PLRV and CABYV sgRNA2 did not show any significant homologies except that the sgRNAs all start with AG. However, a previous BYDV sequence comparison by Dinesh-Kumar et al. (15), in an attempt to identify the putative start of sgRNA2 transcription, demonstrated significant homologies exactly in that region of the BYDV genome where PLRV and CABYV sgRNA2 start sites have been mapped on the corresponding genomes (Fig. 6A): PLRV and BYDV-PAV (both the Illinois and Australian isolates) are significantly homologous immediately upstream of the transcription start, with six out of seven nucleotides being identical. Furthermore, as with CABYV and PLRV sgRNA2, BYDV sgRNA2 would also start with AG if the transcription start site were placed in this region. However, the identified start site (Australian BYDV isolate; 16) is 35 nt upstream of this sequence.


Figure 6. (A) Comparison of sequences flanking the start of transcription of sgRNA2. Subgenomic promotor sequences for BYDV-PAV sgRNA2 (1) were taken from Dinesh-Kumar et al. (15). The starts of transcription of PLRV and CABYV sgRNA2s are indicated by arrows. (B) Possible organization of sgRNA2 for different luteoviruses. Structures were the results of this work (PLRV and CABYV), of other published work (BYDV-PAV; 16) and computer analysis (BMYV and BWYV; 39,40).

Recently it was observed that an ACAAAA sequence element is present at the 5'-end of gRNA and sgRNA1 for several subgroup 2 luteoviruses (for reviews see 3,5). The same motif was also found at the 5'-end of several other viruses, like red clover necrotic mosaic dianthovirus (RCNMV) RNA-1 (31,32) and RCNMV sgRNA (33), and for a number of sobemoviruses (34). Although a similar sequence ACAAA is located further upstream (positions 5124-5128; 18) of the mapped start of PLRV sgRNA2 transcription (position 5190), this element is absent from PLRV sgRNA2 as deduced from Northern and primer extension analyses. For CABYV ACAAAA-like sequences are not present upstream of CABYV sgRNA2. This observation is supported by experiments with BYDV sgRNA1, in which the authors mapped the start of transcription further downstream of an ACAAAA element. (15). Miller and co-workers (3) could identify at least six copies of ACAAAA and 19 copies of ACAAA in the PLRV genomic sequence and more experiments are needed to analyze possible functions of these motifs during replication and/or recognition of subgenomic promoters.

Two ORFs, with the capacity to code for 7.1 (ORF6) and 14 kDa (ORF7) proteins respectively, are located on PLRV sgRNA2, although the presence of an ORF6-orthologous protein in subgroup 2 luteoviral genomes was previously dismissed (3,35). Both ORFs are present in all PLRV isolates sequenced so far [PLRV-Scotland (18), PLRV-Netherlands (36), PLRV-Australia and Canada (37) and PLRV-Germany (Prüfer unpublished)], with the single exception that the ORF6 protein is truncated to 4.5 kDa in the Australian PLRV isolate (36) due to conversion of a UGG codon for tryptophan to a UAG amber stop codon at positions 5263-5265. A similar observation was made for the Australian and Illinois islolates of BYDV-PAV, in which the ORF6 protein is truncated from a 6.7 (Illinois isolate; 3) to a 4.5 kDa protein (Australian isolate; 16). The physiological function of the BYDV ORF6 protein during the infection cycle in the host plant is not yet clearly determined. Young and co-workers (30) were able to show that mutations in BYDV-PAV ORF6 resulted in a non-infectious virus. More recent studies by Mohan et al. (35) indicated that translation of BYDV-PAV genomic RNA and its replication were affected by these mutations.

With respect to PLRV ORF7 we could demonstrate by transient expression of chimeric GUS fusions in potato protoplasts that it is expressed at a low level as compared with ORF6 from sgRNA2. ORF7 corresponds to the C-terminus of the read-through protein ORF5 and is present in all PLRV isolates sequenced so far. The only significant homology detected to recorded proteins other than ORF5 is to an ARAC-like trancription regulator (38). With the existence of three different luteoviral RNAs (gRNA, sgRNA1 and sgRNA2), it is envisioned that their time course of transcription from the identical template (gRNA) may be regulated during different stages of virus multiplication in the host cell. Its nucleic acid binding properties together with its low level expression from sgRNA2 make ORF7 a good candidate for participating in luteoviral transcription regulation, possibly at late stages during the viral life cycle. Studies with mutant transcripts of an infectious PLRV full-length cDNA copy are underway to analyze its function.

A comparison of the PLRV sgRNA2 structure with that of other luteoviruses is shown in Figure 6B. Except for the size of the single ORFs, an almost identical structural organization is evident for PLRV, CABYV and BYDV-PAV sgRNA2, with the two ORFs ORF6 and ORF7 being separated by small non-coding regions. For beet mild yellows virus (BMYV; 39) and beet western yellows virus (BWYV; 40), members of the subgroup 2 luteoviruses with an as yet uncharacterized sgRNA2, only a single ORF (referred to as ORF7 because of its location within the ORF5 reading frame) for a 25 kDa protein could be detected by computer analysis. Additional studies are needed to identify the synthesis of sgRNA 2 for other subgroup 2 luteovirues and their biological role in the viral life cycle.

ACKNOWLEDGEMENTS

This work was supported by a DAAD fellowship to A.A. and by the BMBF (Förderkennzeichen 0311186). The technical assistance of Alice Kaufmann is gratefully acknowledged.

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*To whom correspondence should be addressed. Tel: +49 221 5062 423; Fax: +49 221 5062 413; Email: pruefer@mpiz-koeln.mpg.de

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