THIERRY WURCH, DANIEL KIRCHHERR, JEAN-MICHEL MESNARD, AND GENEVIEVELEBEURIER*. Institut de Biologie Moleculaire des Plantes du C.N.R.S., ...
Vol. 64, No. 6
JOURNAL OF VIROLOGY, June 1990, p. 2594-2598
0022-538X/90/062594-05$02.00/0 Copyright ©D 1990, American Society for Microbiology
The Cauliflower Mosaic Virus Open Reading Frame VII Product Can Be Expressed in Saccharomyces cerevisiae but Is Not Detected in Infected Plants THIERRY WURCH, DANIEL KIRCHHERR, JEAN-MICHEL MESNARD, AND GENEVIEVE LEBEURIER*
Institut de Biologie Moleculaire des Plantes du C.N.R.S., 12 rue du General Zimmer, 67084 Strasbourg Cedex, and Universite Louis Pasteur, 67000 Strasbourg, France Received 27 November 1989/Accepted 2 March 1990
Antiserum was prepared against a synthetic peptide corresponding to the N-terminal 20 amino acids of the protein encoded by cauliflower mosaic virus (CaMV) open reading frame VII (ORF VII). This antiserum was used to detect the expression of CaMV ORF VII either in Saccharomyces cerevisiae transformed by an expression vector containing CaMV ORF VII or in CaMV-infected plants. Only in S. cerevisiae has a 14-kilodalton protein been detected.
Cauliflower mosaic virus (CaMV) is a plant doublestranded DNA virus for which the complete nucleotide sequences have been determined (2, 10, 12), revealing six major open reading frames (ORFs) (I to VI) and two minor ORFs (VII and VIII). ORFs I, III, IV, V, and VI are needed for virus to infect the host plant systemically, whereas ORFs II and VII are not necessary for viral multiplication (6, 9). ORF I encodes a protein that may be involved in the cell-to-cell movement of the virus (1, 22), and ORF II encodes a protein involved in aphid transmission of the CaMV (31). ORFs III and IV encode viral coat proteins (5, 18, 24a). ORF V codes for the reverse transcriptase (20, 25), and ORF VI codes for the inclusion body matrix protein (4, 33). In vivo protein products (P1 to P6) have been detected for genes I to VI, either in inclusion body preparations or in replication complex preparations (24). It is still unknown whether ORFs VII and VIII are expressed. It was suggested that the involvement of a polycistronic mRNA in the synthesis of CaMV proteins is encoded by ORFs VII, I, II, and III (7) by a relay-race mechanism (Fig. 1): ribosomes, after passing a termination codon, reinitiate protein synthesis at the nearest AUG codon. In such a model (7), ORF VII and the intergenomic region between ORFs VII and I (60-odd nucleotides between the ORF VII stop codon and ORF I start codon) serve to establish translational continuity between the leader of polycistronic mRNA and the ORF I (Fig. 1). Translation of downstream cistrons (ORFs I, II, and III) is thus the result of reinitiation and is dependent on successful upstream translation (ORF VII; Fig. 1). In the work described here, we used a synthetic peptide corresponding to the 21st N-terminal amino acid of the product encoded by ORF VII, P7, to obtain an antiserum. To test the activity of the antiserum, a yeast expression vector containing the ORF VII sequence was constructed. The anti-P7 serum has enabled us to detect the ORF VII product in yeast extracts. Infected plant tissues were also tested with this antiserum.
T4 DNA ligase, and calf intestinal phosphatase were from Boehringer (Mannheim, Federal Republic of Germany). The enzymes were used according to the recommendations of the manufacturers. Protocols for bacterial transformation and DNA preparations have been described previously (23). Yeast transformations have already been described (16). Preparation of antiserum. A peptide corresponding to the N-terminal sequence of the CaMV ORF VII expression product was purchased from Neosystem. The peptide was coupled to ovalbumin, mixed with Freund incomplete adjuvant, and injected intramuscularly into a rabbit every 21 days. Antiserum titers were determined by enzyme-linked immunosorbent assay. Strains and plasmids. Escherichia coli C600-5K and Saccharomyces cerevisiae GRF18 (ot His3 Leu2) have already been described (16, 17). Cultures of plasmid-carrying E. coli were grown at 37°C in LB containing 100 ,ug of ampicillin per ml. Cultures of plasmid-carrying S. cerevisiae were grown at 28°C in yeast-nitrogen base containing 20 ,ug of L-histidine per ml. The plasmid pVTL-102 has already been described (29). It enabled selection in strain GRF18 by complementation of Leu2. Electrophoresis analysis of yeast proteins. Aliquots (1 ml) of cultures were harvested by centrifugation and were lysed in 150 ,ul of 2 M NaOH-7% 3-mercaptoethanol. The proteins were precipitated by 150 ,ul of 50% trichloroacetic acid, pelleted by centrifugation, and washed with acetone. This method has already been described (34). Proteins were dissolved in sample buffer by the method of Laemmli (19), boiled for 5 min, and separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparations of CaMV-infected turnip extracts. The CaMV isolate used in this study was Cabb-S (21). Turnip extracts were prepared from healthy or 21-day CaMV-infected leaves. Inclusion body preparation has already been described (4). After the grinding of turnip leaf tissue, the slurry was filtered and centrifuged. The pellet was washed twice with extraction buffer (4) containing 5 mM EDTA and 1% Triton X-100. The washed pellet contained inclusion bodies. Proteins in the supernatant were also precipitated (24). Replication complexes were prepared as described previously (26). Briefly, leaf tissue was homogenized, filtered, and centrifuged. After this centrifugation, we obtained a first
MATERIALS AND METHODS Enzymes and reagents. Restriction enzymes were purchased from New England BioLabs, Inc. DNA polymerase, *
Corresponding author. 2594
EXPRESSION OF CaMV ORF VII
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FIG. 1. Map of the CaMV genome. The circular Cabb-S genome (8,024 base pairs) is represented in the map as a linearized DNA form. The double line represents the double-stranded DNA. The arrangement of the open reading frames I to VII and of the polycistronic mRNA (below DNA) are shown. The hatched box represents the 600-nucleotide-long 5' leader sequence of the mRNA, and the arrowhead represents the 3' end of the mRNA. Since the ORF VIII (3264 to 3587) was not studied in this paper, we did not represent it in this figure. The numbers above the DNA correspond to its size in kilobase pairs.
pellet, which was retained. The supernatant was layered over a sucrose cushion and centrifuged. This second pellet contains replication complexes and polyribosomes (1, 24). The proteins present in the supernatant were also precipitated (24). Nuclei, virus particles, and cell wall extractions were carried out as already described (1). All the preparations were denatured for SDS-PAGE as described above. Immunoblotting. Proteins were fractionated by SDSPAGE and then transferred to nitrocellulose (28). After reaction with the antiserum, the specific proteins were visualized by using goat antirabbit immunoglobulin G alkaline phosphatase conjugate. For preabsorption experiments, the synthetic peptide (1 ,ug of peptide per ,ul of nondiluted serum) was added to the antiserum. In these experiments, antisera were used diluted 1,000-fold. RESULTS Antiserum against CaMV ORF VII protein. The amino acid sequence of CaMV ORF VII protein was deduced from the corresponding nucleotide sequence of the Cabb-S strain (10). The peptide contains the 20 amino acids at the N terminus of the gene VII product. Enzyme-linked immunosorbent assay showed that the antiserum so prepared was immunogenic and could detect 10 ng of the peptide per ml at an antiserum dilution of 50,000. Construction of an expression vector for the synthesis of CaMV P7 in S. cerevisiae. To synthesize the CaMV P7, coded by ORF VII, we cloned the coding sequence (13 to 303) into plasmid pVTL-102 (29). This plasmid has the following features: (i) plasmid replication and selection (Ap') in E. coli, (ii) replication in S. cerevisiae based on the 2,um plasmid origin of replication, (iii) selection in yeast by complementation of Leu2, and (iv) seven unique restriction sites (polylinker) for cloning within an "expression cassette" which includes the promoter and 3' sequence of the ADH1 gene
(alcohol dehydrogenase). CaMV DNA was digested with ClaI, and the 864-base-pair fragment with nucleotide coordinates 7980 to 820 was isolated (the genome is circular; Fig. 2). The ClaI extremities were filled in with DNA polymerase (Klenow fragment), and the CaMV DNA was cloned in the PvuII site of pVTL-102. This strategy enabled the control of the transcription of the ORF VII by the ADH1 gene promoter (Fig. 2). Bacterial recombinants were selected for the presence of plasmids with the viral sequence inserted in the proper orientation. This final construction will be referred to as pMW701. Expression of CaMV P7 in S. cerevisiae. Expression in S. cerevisiae was achieved by transforming the strain GRF18
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