Download Identification and Characterization of the Potato Leafroll Virus

J. gen. Virol. (1989), 70, 783-788.
Printed in Great Britain
783
Key words: PLRV/coat protein/nucleotide sequence
Identification and Characterization of the Potato Leafroll Virus Putative
Coat Protein Gene
By L. M. K A W C H U K , 1. R. R. M A R T I N , 1 D. M. R O C H O N t
AND J. M c P H E R S O N 2
1Research Station, Agriculture Canada, 6660 North-West Marine Drive, Vancouver, British
Columbia V6 T IX2 and 2Department of Plant Science, 2357 Main Mall, MacMillan Building, Room
341, University of British Columbia, Vancouver, British Columbia V6T 2A2, Canada
(Accepted 9 November 1988)
SUMMARY
Complementary DNA clones representing approximately 6100 nucleotides of potato
leafroll virus (PLRV) were generated, restriction-mapped, and partially sequenced.
Within one of the cDNA clones an open reading frame (ORF) encoding a 23K protein
was identified and further characterized. Amino acid sequence comparison of this
protein showed significant homology (47.1~) with the barley yellow dwarf virus
(BYDV-PAV) coat protein. This and other observations suggested that this gene
encodes the PLRV coat protein. Other similarities were observed between PLRV and
BYDV sequences in this region of their genomes, including an ORF of 17K within the
ORF encoding the 23K putative coat protein.
Potato leafroll virus (PLRV), a member of the luteovirus group (Matthews, 1982) is difficult to
control and is responsible for significant economic losses in potato (Solanum tuberosum L.)
(Rowhani & Stace-Smith, 1979). It is a phloem-limited spherical virus transmitted by several
aphid species in a persistent manner, the most efficient vector being Myzus persicae Sulz.
(Harrison, 1984). Virions of PLRV are composed of a single-stranded RNA species of Mr
2.0 × 106 and a single coat protein of Mr 26.3K (Rowhani & Stace-Smith, 1979).
The coat protein (CP) of luteoviruses is reported to be responsible for many viral
characteristics including serological properties (Waterhouse & Murant, 1981), transmission
specificity (Massalski & Harrison, 1987; Rochow & Carmichael, 1979), cross-protection
(Harrison, 1958; Webb et al., 1952), and mutual exclusion (Jedlinski & Brown, 1965). The coat
protein genes of several viruses have been expressed in transgenic plants resulting in reduced
virus synthesis following specific exposure to virus (Abel et al., 1986; Cuozzo et al., 1988;
Hemenway et al., 1988; Loesch-Fries et al., 1987). The identification, characterization, and
comparison of PLRV and other luteovirus coat proteins should permit an understanding of their
specific roles.
An isolate of the PLRV characterized by Rowhani & Stace-Smith (1979) was purified by a
modification of their procedure. The yield of virus was increased by incubating the plant
homogenate in 0.1~ (w/v) Ultrazym 100 (Schweizerische Ferment) overnight at room
temperature. RNA was purified from virus particles by vortex-mixing in 0.38 M-Tris-HC1
pH 8.9, 20 mM-EDTA, 1~ (w/v) SDS followed by phenol-chloroform extraction.
Double-stranded cDNA copies of PLRV RNA were synthesized and cloned as follows.
Virion RNA, polyadenylated in vitro by the procedure of Sippel (1973), served as template for
oligo(dT)-primed first strand cDNA synthesis. Single-stranded cDNA was also prepared using
virion RNA as template for randomly primed cDNA synthesis (Maniatis et al., 1982). Second
strand DNA synthesis was conducted using DNA polymerase I and RNase H-generated
primers (Gubler & Hoffman, 1983). Double-stranded cDNA was dC-tailed using terminal
0000-8678 © 1989 SGM
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transferase (Bethesda Research Laboratories, BRL) and then annealed with PstI-restricted
pUC9 similarly tailed with dG. Half of the ds cDNA from the randomly primed reaction was
treated with mung bean nuclease and then ligated into EcoRV-restricted Bluescript MI 3 vector
(Stratagene) which had been treated with calf intestinal phosphatase. Following ligation the
DNA was used to transform competent DH5ct cells (BRL) which were plated on Luria-Bertani
agar containing ampicillin and X-gal. Two-hundred white, ampicillin-resistant colonies
obtained by the oligo(dT) method and 800 colonies by the random priming method were
screened by colony filter hybridization (Gergen et al., 1979) using randomly primed 32p-labelled
PLRV cDNA probes. Those colonies giving the strongest signal (approx. 100) were selected and
recombinant plasmids were isolated using the alkaline lysis method (Maniatis et al., 1982).
Plasmids were analysed by digestion with PstI or PvulI, which cleaved out the inserts, and were
then electrophoresed in agarose gels. Those colonies containing the plasmids with the largest
cDNA inserts were selected for further study.
Potato leafroll virus RNA and total RNA from healthy potato leaves were electrophoresed on
denaturing methylmercury gels and transferred to nitrocellulose (Schlei'cher & Schuell) as
described by Maniatis et al. (1982). The origin of clones B2a, LP79 and LP93 (Fig. la) was
confirmed by hybridization to PLRV RNA using Northern blot analysis. 32p-labelled probes
were synthesized from each cDNA clone using the randomly primed oligonucleotide method
(Feinberg & Vogelstein, 1983). Each probe hybridized specifically to PLRV RNA and was
negative with uninfected plant RNA (data not shown).
The clones shown in Fig. 1(a) were oriented relative to each other based on restriction enzyme
and hybridization analyses. For hybridization, end fragments were gel-purified, oligonucleotide-labelled, and used to select overlapping clones. Plasmid DNA was spotted onto Nytran
(Schleicher & Schuell) sheets and subsequently denatured by placing the Nytran on a stack of
3MM filter paper soaked in 0.5 M-NaOH.
Five clones were selected and found to cover approx. 6100 nucleotides of the virus genome
which is greater than 98~ (Fig. 1a) of the predicted size of PLRV genomic RNA (6200
nucleotides) as determined by methylmercury gel electrophoresis. Clone B2a and several other
oligo(dT)-primed clones mapped to the same location suggesting that they were derived from the
3' end of PLRV RNA. Although some differences were observed, restriction analysis revealed
overall similarities with the restriction sites of the PLRV isolates described by both Prill et al.
(1988) and Smith et al. (1988).
Recently the complete nucleotide sequence of the luteovirus barley yellow dwarf (BYDVPAV) was determined (Miller et al., 1988a) and the CP gene was shown to be located internally.
If the PLRV CP were similarly located the clone LP79 (Fig. 1a) could contain the CP coding
region of PLRV. This clone covered a large proportion of the internal region of the PLRV
genome and therefore was subcloned and sequenced in both orientations (Fig. 1b).
Clones to be sequenced were inserted into the Bluescript vector and unidirectional nested
deletions were prepared using exonuclease III and mung bean nuclease (Henikoff, 1984).
Subclones were sequenced by the dideoxynucleotide chain termination method of Sanger et al.
(1977) using double-stranded plasmid DNA as template (Korneluk et al., 1985). Sequences were
read using a Bio-Rad digitizer and analysed using Bio-Rad Gene-Master software.
The nucleotide sequence of LP79 revealed an open reading frame (ORF), nucleotides 34 to
657, as shown in Fig. 2. The predicted amino acid sequence (Fig. 2) shows extensive homology of
47.1~ (Fig. 3a) with the amino acid sequence of the CP of BYDV (Miller et al., 1988b). The
predicted Mr of the PLRV ORF protein of 208 amino acids is 23 202 (23K) which is similar to the
previously estimated Mr of 26.3K for the PLRV CP (Rowhani & Stace-Smith, 1979). The above
evidence strongly suggests that the 23K protein encoded by the 23K ORF is the coat protein of
PLRV. Like BYDV CP the amino-terminal region of the PLRV 23K ORF is highly basic,
containing many arginine residues (Fig. 2). The amino-terminal regions of many plant virus
capsid proteins have been found to be highly basic and it has been suggested that these regions
may be involved in protein-RNA interaction (Harrison, 1983). Comparison of the PLRV 23K
ORF nucleotide sequence with that of the BYDV CP gave 58.0~ similarity. This reflects the
amino acid similarity and indicates a close evolutionary relationship between these two viruses.
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785
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Fig. 1. (a) Restriction map of the PLRV genome derived from the analysis of overlapping cDNA clones
that are indicated below the map. Although not shown, each SalI site was also restricted by HincII.
Clone B2a was from oligo(dT)-primedreactions and those of the LP series from the randomly primed
reactions. The region of the clone LP79 corresponding to the 23K ORF is represented by an open
rectangle. The line above the map corresponds to the PLRV RNA. (b) Sequencing strategy used to
determine the putative coat protein coding region of PLRV. Arrows indicate the direction and length of
sequence from unidirectional nested deletions of clone LP79.
Within the 23K O R F coding region but in a different reading frame lies another ORF,
nucleotides 59 to 526, coding for 156 amino acids. Translation of this O R F would yield a protein
of Mr 17381. BYDV and soybean dwarf virus (Miller et al., 1988b) both contain a 17K O R F
within the region encoding the CP. The similar finding of the 17K O R F within the PLRV
putative CP gene suggests that this protein is a functional gene product. Miller et al. (1988b)
suggested that the BYDV 17K protein is the genome-linked viral protein (VPg) based on its size
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being similar to isolated VPg. The PLRV VPg isolated by Mayo et al. (1982) has been estimated
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than there is between the putative coat proteins. Thus, if a functional protein is encoded by ORF
17K, it may be responsible for some shared but distinct property of each virus such as host
specificity. Other examples of plant RNA viruses which indicate extensive use of overlapping
ORFs are southern bean mosaic virus (Wu et al., 1987) and carnation mottle virus (Guilley et al.,
1985).
The 23K ORF in PLRV terminates with an amber codon (Fig. 2) which is immediately
followed by a long in-frame ORF (L. M. Kawchuk et al., unpublished data). A similar finding
was observed by Miller et al. (1988b) for BYDV and it was suggested by these authors that
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Fig. 3. (a) An alignment of the amino acid sequence of the PLRV 23K ORF (upper) and BYDV coat
protein (lower). (b) An alignment of the amino acid sequences of the PLRV 17K ORF (upper) and the
BYDV 17K protein (lower). The Needleman-Wunsch algorithm (Needleman & Wunsch, 1970) was
used to align the sequences.
readthrough of the amber codon may occur. In both cases several proline codons occur in the
region immediately following the amber codon; for P L R V seven of 13 amino acids in this region
are proline (data not shown).
Although the similarity of the predicted amino acid sequence of the P L R V O R F 23K and
BYDV proteins was high (47.1 ~), when intact virus was used as antigen and immunogen there
was a lack of serological cross-reactivity. However, extensive cross-reactivity was observed
when denatured virus was used as antigen (R. R. Martin & C. J. D'Arcy, unpublished data).
This suggests that the most variable regions of the luteovirus capsid are exposed in intact
virions.
The isolation and expression of the P L R V CP gene will provide sufficient amounts of protein
for the production of large quantities of sequence-specific antibodies. These are presently only
produced in small amounts because of difficulties in obtaining large amounts of viral coat
protein. Comparison of luteovirus coat proteins will also allow the prediction of common
antigens, i.e. regions which may be exploited in the preparation of antibodies that react with
several if not all luteoviruses. Strain-specific epitopes may also be predicted by expressing small
regions of the CP gene, producing antibodies and examining their specificity. Finally, since
earlier investigators demonstrated that different strain combinations of luteoviruses give crossprotection or mutual exclusion, it should be possible to construct chimeric genes that when
inserted into an appropriate host may permit further examination of these phenomena.
We wish to thank Dr W. A. Miller for an advance copy of the BYDV coat protein sequence and Dr B. Prill for a
copy of the submitted manuscript on the cloning of PLRV. L.M.K. acknowledges the support of the Natural
Sciences and Engineering Research Council of Canada via a postgraduate scholarship.
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