Journal of General Virology(1990), 71, 2251-2256. Printedin Great Britain
2251
Expression of the genome of potato leafroll virus: readthrough of the
coat protein termination codon in vit, o
I. Bahner, 1 J. Lamb, 1 M. A. M a y o 2. and R. T. H a y I
1Department of Biochemistry and Microbiology, University of St Andrews, lrvine Building, North Street, St Andrews,
Fife KY16 9AL and 2Scottish Crop Research Institute, lnvergowrie, Dundee DD2 5DA, U.K.
An antiserum was raised against a fusion protein
containing part of the 56K polypeptide (P5) encoded by
the open reading frame (ORF) at the 3' end of the
genome of potato leafroll virus (PLRV). This
antiserum reacted specifically with 80K and 90K
polypeptides in PLRV-infected protoplasts, with a 90K
polypeptide in infected potato tissue and with a 53K
polypeptide in protein extracted from purified particles
of PLRV. Monoclonal antibodies raised against
purified P L R V particles also reacted with these
polypeptides, as well as with the 23K coat protein.
Virus particles partially purified from infected
protoplasts contained some 90K polypeptide as well as
the major 23K coat protein. The ORFs of the 23K coat
protein and P5 are contiguous and in frame. The results
suggest that the P5 polypeptide of P L R V occurs in
infected cells as part of a readthrough protein
comprising the 23K coat protein joined to the P5
amino acid sequence. Moreover the readthrough
protein can be assembled into virus particles as a minor
component together with the main 23K component.
The P5 protein may thus contribute to properties of
P L R V determined by its virus particle surface.
Introduction
1989). The nucleotide sequences around the amber
termination codon of the PLRV 23K protein (Mayo et
al., 1989; van der Wilk et al., 1989), and those of BYDV
and BWYV coat proteins (Miller et al., 1988 ; Veldt et al.,
1988), resemble those around several other virus R N A
termination codons that are misread by naturally
occurring suppressor tRNAs to generate readthrough
proteins. Readthrough of the termination codon of the
gene for BWYV coat protein has been shown to occur
during translation of in vitro transcribed transcripts of
BWYV cDNA (Veldt et al., 1988). It was therefore
suggested (Mayo et al., 1989; van der Wilk et al., 1989)
that in PLRV R N A readthrough of the termination
codon of ORF 4 results in translation of the downstream
ORF to give a readthrough protein of about 80K. In this
paper we present serological evidence that the predicted
80K readthrough protein is produced in PLRV-infected
cells. Although the major protein component of purified
particles of luteoviruses is the 23K polypeptide, several
authors have reported the presence of minor, higher Mr
components (Rochow & Duffus, 1981; Waterhouse,
1981; Murant et al., 1985). We demonstrate that in
PLRV particles the 80K readthrough protein gives rise to
a minor high Mr component, presumably by the loss of
C-terminal sequences.
Potato leafroll virus (PLRV; luteovirus group) causes a
serious disease of potatoes that results in significant crop
losses in various parts of the world. It is transmitted in a
persistent manner by aphids both from infected plants
and from preparations of purified virus (Harrison, 1984).
Particles of PLRV contain a 6 kb RNA encapsidated in
a protein coat made of a 23K polypeptide. Recently, the
complete nucleotide sequences of cDNA copies of the
genome RNA of two isolates of PLRV have been
described (Mayo et al., 1989; van der Wilk et al., 1989).
Sequences have also been reported for the R N A of beet
western yellows (BWYV; Veidt et al., 1988) and barley
yellow dwarf (BYDV; Miller et al., 1988) luteoviruses.
Although little is known about the replication of
luteoviruses in infected cells, analysis of the nucleotide
sequences suggests a number of strategies f o r gene
expression. In particular, the sequence of PLRV R N A
contains six large open reading frames (ORFs) arranged
either side of a 200 nucleotide long non-coding region.
The gene (ORF 4) for the 23K coat protein (P3) is located
on the 3' side of the non-coding sequence; ORF 6, which
is downstream from ORF 4 and is shown in Fig. 1 as
between the first A U G codon and the first termination
codon, could encode a 53K polypeptide (Mayo et al.,
0000-9544© 1990SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:02:50
2252
L Bahner and others
Methods
Viruspropagation and purification. The PLRV isolate was the Scottish
isolate of strain 1 (Tamada et al., 1984) and was purified as described by
Mayo et al. (1989). Particles were partially purified from infected
protoplasts by suspending a pellet of about 3 x 105 protoplasts in 2 ml
of 0.01 M-sodium phosphate buffer pH 7, centrifuging the extract at
about 10000g for 5min and pelleting virus particles from the
supernatant fraction by centrifugation through a 1 ml cushion of 20~
sucrose in 0-01 M-sodium phosphate pH 7 at 45 000 r.p.m, in a Beckman
SW50 rotor at 10 °C for 2.5 h.
Preparation, inoculation and culture of protoplasts. Protoplasts were
isolated from the palisade mesophyll of leaves of Nicotiana tabacum cv.
Xanthi using a two-step enzyme digestion as described by Kubo et al.
(1975). Protoplasts were inoculated with phosphate-buffered mixtures
of PLRV particles (0.2p.g/ml) and poly-L-ornithine (1 ktg/ml), as
described by Mayo & Barker (1983); mock inoculation was identical
except that virus particles were omitted. Protoplasts were then cultured
as described by Kubo et al. (1975) until they were recovered by
centrifugation and assayed either by extracting the pellets or by
staining with fluorescent antibodies (Mayo & Barker, 1983). In most
experiments 50 ~ to 80 ~ of morphologically intact protoplasts stained
2 days after inoculation were found to be infected.
Construction of expression vectors. DNA corresponding to nucleotides
4646 to 5280 of the sequence described by Mayo et al. (1989) was
inserted into pRIT21 downstream from and in frame with the Protein
A gene as follows, pRIT21 (a gift from Dr Nigel Stow) was derived
from pRIT2T (Nilsson et al., 1985) by cleavage with EcoRI, treatment
with mung bean nuclease to remove 5' extensions and religation.
pRIT21 was cleaved with PstI, treated with mung bean nuclease and
recleaved with BarnHI. Recombinant plasmid DNA containing the
PLRV P5 gene was cleaved with EcoRI, treated with mung bean
nuclease and recleaved with BamHI. The PLRV fragment corresponding to nucleotides 4646 to 5280 was isolated by electroelution from a
polyacrylamide gel and inserted into the cleaved pRIT21 to yield the
recombinant plasmid pRIT21-EP4.
Preparation of fusion protein. N4830 Escherichia coli ceils containing
pRIT21 EP4 were grown to a density of 0.6 at OD600 at 28 °C and
fusion protein expression was induced by incubation at 42 °C for 2 h.
Cells were collected, treated with 2mg/ml lysozyme, lysed in
phosphate-buffered saline (PBS; 0.15 M-NaC1, 0.01 M-sodium phosphate pH 7-0) containing 0-05~ Tween-20 and centrifuged. The
resulting protein extract was applied to a 4 ml column of IgG-Fast
Flow Sepharose (Pharmacia) and the column was washed with 50 bed
volumes of PBS containing 0.05 ~ Tween-20. Bound protein was eluted
with 0-1 M-glycine-HC1 pH 3.0, immediately neutralized with NaOH
and then concentrated and desalted by ultrafiltration.
Preparation ofantisera. Rabbits were injected with 250 ktg of fusion
protein preparation emulsified in Freund's complete adjuvant. Three
booster injections of the same amount of protein emulsified in
incomplete adjuvant were given at 20-day intervals starting 1 month
after the initial injection. The mouse monoclonal antibody (MAb) was
SCR 1 (Massalski & Harrison, 1987) which was made following'
immunization with purified particles of PLRV.
Electrophoresis. Protein extracts were prepared from pellets of
protoplasts or from fresh leaf tissue by suspension in 2 ~ SDS,
0.15 M-dithiothreitol, 10 ~ glycerol pH 6.8. Samples equivalent to about
3 x 104 protoplasts or 50 mg fresh weight of leaf tissue were separated
by electrophoresis in 10~ SDS-polyacrylamide gels essentially as
described by Laemmli (1970). Polypeptide Mr values were estimated
using as markers polypeptides from virions of adenovirus type 2 (Mr
108000, 85000, 66000, 62000, 48500, 23400) and fl-galactosidase
(113000), phosphorylase b (95000), bovine serum albumin (67000),
ovalbumin (45000), glyceraldehyde 3-phosphate dehydrogenase
(36000), carbonic anhydrase (29000), trypsinogen (24000) and trypsin
inhibitor (20100). Samples of PLRV particle protein contained about
100 ng for immunoblotting and several Ixg for staining with Coomassie
blue.
Immunoblotting. Polypeptides were transferred from gels to nitrocellulose (Randall & Young, 1988) by using a 'semi-dry blotter' (LKB)
using a discontinuous buffer system according to the manufacturer's
instructions. Alternatively, extracts of protoplasts were applied to
nitrocellulose as 5 ~tl dots. Nitrocellulose sheets were blocked by
incubation in PBSMN [PBS containing 10~ non-fat milk powder
(Marvel; Cadburys) and 0.1 ~ Nonidet P-40] and incubated with a
1/ 100 dilution of polyclonal anti-P5 or a 1/ 1000 dilution of MAb SCR 1
(anti-P3) in PBSMN. After the blots had been washed, antibodies were
detected by incubation with either (i) 0-5 ktCi/ml of lzSI-labelled
Protein A (Amersham) in PBSMN followed by autoradiography or (ii)
a 1/1000 dilution of an alkaline phosphatase conjugate of goat antirabbit or goat anti-mouse serum (Sigma) in PBS containing 10~
Marvel followed by reaction with NBT/BCIP substrate (Bio-Rad).
Results
Antibodies directed against the P5 gene product
To generate antisera directed against the protein
e n c o d e d by O R F 6, c D N A to t h i s r e g i o n o f t h e g e n o m e
(Fig. 1) w a s i n s e r t e d i n t o t h e p r o k a r y o t i c e x p r e s s i o n
v e c t o r p R I T 2 1 in s u c h a w a y t h a t a n i n - f r a m e f u s i o n w a s
c o n s t r u c t e d w i t h t h e p r o t e i n A g e n e o f Staphylococcus
aureus ( N i l s s o n et al., 1985). T h e a n t i s e r u m r a i s e d in
r a b b i t s a g a i n s t t h i s p r o t e i n ( a n t i - P 5 ) r e a c t e d in d o t b l o t s
at d i l u t i o n s o f 1/512 o r less w i t h e x t r a c t s o f P L R V i n f e c t e d p r o t o p l a s t s b u t d i d n o t r e a c t at d i l u t i o n s o f m o r e
t h a n 1/4 w i t h e x t r a c t s o f m o c k - i n o c u l a t e d p r o t o p l a s t s
(data not shown).
Detection o f P5 product in extracts o f P L R V-infected
protoplasts
T o d e t e c t p o l y p e p t i d e s e n c o d e d b y t h e P5 r e g i o n o f t h e
P L R V g e n o m e , p r o t e i n s p r e s e n t in P L R V - i n f e c t e d a n d
mock-inoculated protoplasts were analysed by immunob l o t t i n g . S a m p l e s w e r e t a k e n 1, 2 a n d 3 d a y s a f t e r
1000
2000
3000
4000
5000
5987
I
I
I
I
I
I
ORF 1 (P1)
ORF 3 (P2b)
. . . . . . . . . . . . . . . . . . . . . . . . . .
ORF 4
(P3)
LX~*~-',~XXl
ixx~xxxxxxxxxxxxxx~
ORF 2 (P2a)
ORF 6 (P5)
hxxxxxxxxx~.~l
i~xxxl
ORF 5
(P4)
a-P5
Fig. I. Diagram of the PLRV genome. The upper line represents the
RNA genome; the numbers and the positions of the six ORFs (ORF 1
to ORF 6) are taken from Mayo et al. (1989). P1 to P5 represent the
polypeptide translation products of the ORFs. The stippled block
(a-P5) shows the position of the coding sequence used to construct the
Protein A fusion protein vector pRIT21-EP4.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:02:50
Readthrough in PLRV genome expression
1
1 234
5
6 7
2
3
4
5
6
2253
7
90K80K-
8 91011 12 13 14
-108K
90K
80K
-67K
-66K
-48-5K
-45K
23.4K
CPM
•
• ....
-29K
CP-
iii(
Fig. 2
Fig 3
Fig. 2. Immunoblots of extracts of PLRV-infected protoplasts after reaction with 125I-labelled Protein A. Protoplasts were mockinoculated (lanes 1, 2, 3, 8, 9, 10) or PLRV-inoculated (lanes 5, 6, 7, 12, 13, 14) and extracts were immunoblotted 1 day (lanes 1, 5, 8, 12),
2 days (lanes 2, 6, 9, 13) or 3 days (lanes 3, 7, 10, 14) post-inoculation. Protein from purified PLRV was used as a marker (lanes 4 and 11).
Immunoblotting was with M A b SCR 1 (lanes 1 to 7) or polyclonal anti-P5 (lanes 8 to 14). The positions of the 90K and 80K proteins and
the 23K coat protein (CP) are shown. The bar indicates the top of the gel and the positions of marker proteins are shown on the right.
Fig. 3. Immunoblots of extracts of PLRV-infected protoplasts after reaction with antibodies conjugated to alkaline phosphatase.
Samples were protein from protoplasts taken 2 days after inoculation with buffer (lanes 1 and 4) or PLRV (lanes 2 and 5) or protein from
purified PLRV (lanes 3, 6, 7). Immunoblotting was with MAb SCR l (lanes 1 to 3) or polyclonal anti-P5 (lanes 4 to 6). Lane 7 is protein
from purified PLRV stained with Coomassie blue. Symbols are as in Fig. 2.
inoculation and immunoblotted with anti-P5 or MAb
SCR 1. Anti-P5 detected polypeptides of 66K, 80K and
90K in PLRV-infected protoplasts, particularly 2 and
3 days after inoculation (Fig. 2, lanes 13 and 14), but did
not react with polypeptides present in mock-inoculated
protoplasts (Fig. 2, lanes 8 to 10). MAb SCR 1 did not
react with proteins from mock-inoculated protoplasts
(Fig. 2, lanes 1 to 3) but reacted strongly with a 23K
polypeptide which comigrated with the major polypeptide component of purified virus particles (CP; Fig.
2, lane 4) and also reacted with polypeptides of 90K and
80K which comigrated with products detected by anti-P5
(Fig. 2, lanes 5 to 7). In addition, SCR 1 detected a
protein of 53K in purified preparations of virions (Fig. 2,
lane 4) which was not detected in infected protoplasts
(see below). A faint band corresponding to the 53K
protein was visible in the original autoradiograph in lane
11. These data suggest that the 80K and 90K polypeptides represent the predicted readthrough protein
which consists of the 23K polypeptide at the N terminus
(detected by SCR 1) and polypeptide P5 at the C
terminus (detected by anti-P5).
Similar results were obtained when antisera conjugated to alkaline phosphatase were used to detect
antibodies bound to blots (Fig. 3) although with this
method several polypeptides present both in PLRVinfected and in mock-inoculated protoplasts bound
anti-P5 (Fig. 3, lanes 4 and 5). Attempts to decrease this
background reaction by using purified gamma globulin
were unsuccessful.
Immunoblots of protein from purified PLR V particles
If, as predicted, the readthrough proteins of 80K and
90K detected in infected protoplasts contain the 23K
protein it seems probable that they could be assembled
with the 23K protein into virus particles. This was tested
by immunoblotting proteins extracted from purified
virus preparations. The major protein component
detected by staining with Coomassie blue was the 23K
protein but small amounts of a 53K protein were always
present (Fig. 3, lane 7). Both proteins reacted with SCR 1
(Fig. 3, lane 3) but only the 53K protein reacted with
anti,P5 antiserum (Fig. 3, lane 6). This result suggests
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:02:50
2254
I. Bahner and others
1
2
3
4
5
6
7
90K80K-
2
3
4
5
53K- ~
90K-
53K-
CP- W
CPFig. 4
Fig. 5
Fig. 4. Immunoblots of protein from partially purified PLRV after reaction with antibodies conjugated to alkaline phosphatase. In one
experiment (lanes 1 to 4) immunoblotting was with M A b SCR 1, in the other (lanes 5 to 7) immunoblotting was with either MAb SCR 1
(lane 5) or polyclonal anti-P5 (lanes 6 and 7). Lane 1, protein from purified PLRV. Lanes 2 and 7, extracts of mock-inoculated
protoplasts treated as those in lane 3. Lanes 3, 5 and 6, protein in a pellet obtained by sedimenting an extract of PLRV-infected
protoplasts 2 days post-inoculation through a sucrose cushion. Lane 4, an equivalent extract of unfractionated protoplasts. Symbols are
as in Fig. 2.
Fig. 5. Immunoblots of extracts of PLRV-infected plants after reaction with antibodies conjugated to alkaline phosphatase.
Immunoblotting was with MAb SCR 1. Samples were protein extracted from : leaf tissue from PLRV-infected N. clevelandii (lane 1),
leaf tissue from a potato plant grown from a PLRV-infected potato tuber (lane 2), leaf tissue from a potato plant infected earlier in its
development by viruliferous aphids (lane 3), PLRV-infected protoplasts (lane 4) and mock-inoculated protoplasts (lane 5). Symbols are
as in Fig. 2.
that the 53K protein is a readthrough protein derived
from the 80K and 90K polypeptides found in infected
protoplasts. A 53K polypeptide has been detected in
protein from particles of PLRV in preparations purified
from potato tissue and Physalis floridana using either
Celluclast (as in this work) or Driselase (as used by
Waterhouse, 1981) to macerate the tissue prior to
extracting the virus particles.
To determine the relation between the 53K polypeptide and the 90K/80K polypeptides detected in
infected protoplasts, protein was extracted from PLRV
particles that had been isolated rapidly without enzyme
treatment from protoplasts 2 days after infection and
immunoblotted using SCR 1. The main polypeptides
detected by SCR 1 in these partially purified virus
particles were the same species (CP, 80K and 90K; Fig.
4, lane 3) as were detected in unfractionated protoplasts
(Fig. 4, lane 4) and little reactive polypeptide comigrated
with the 53K component present in protein from virus
particles purified from whole plants by the standard
enzyme procedure (Fig. 4, lane 1). Polypeptides in a
similar preparation from mock-inoculated protoplasts
did not react with SCR 1 (Fig. 4, lane 2). In a second
experiment, in which samples were immunoblotted with
anti-P5 or SCR 1 (Fig. 4, lanes 5 to 7), anti-P5 reacted
with several polypeptides in extracts of mock-inoculated
protoplasts (Fig. 4, lane 7; as in the experiment shown in
Fig. 3, lane 4). However both antisera reacted with
several polypeptides in virus purified from PLRVinfected protoplasts including 80K and 90K polypeptides, although the strongest reaction of SCR 1
(although not anti-P5) with polypeptides larger than 23K
was with a 53K species (Fig. 4, lanes 5 and 6). The
doublet band in lane 6 that reacted weakly with anti-P5
but not SCR 1 may be some of the C-terminal part of P5
removed by the cleavage to form the 53K protein, but
further experiments are necessary to test this hypothesis.
When protein that had been extracted from leaves of
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:02:50
Readthrough in P L R V genome expression
potato or N. clevelandii plants infected with PLRV was
immunoblotted, infection-specific polypeptides were less
readily detected than in protoplast samples but those
detected were almost exclusively 23K and 90K species
(Fig. 5, lanes 1 to 3). In this experiment a sample from
PLRV-infected protoplasts contained a 90K polypeptide
that reacted with SCR 1 but the 80K species detected in
other experiments was absent (Fig. 5, lane 4). SCR 1 also
detected a small amount of a 53K polypeptide which
comigrated with the 53K species found in purified
preparations of PLRV. No reaction was detected when
protein from N. clevelandii tissue infected with BWYV
was immunoblotted with SCR 1 or anti-P5 (data not
shown).
Discussion
The results show that although most of the protein made
in infected cells that reacts with SCR 1 is found as a 23K
product, some is linked to the polypeptide encoded by the
next ORF downstream (ORF 6). No polypeptide corresponding to the primary translation product of ORF 6
(Mayo et al., 1989) was detected in infected protoplasts
or, in a more limited examination, in infected potato
tissue. It appears that this ORF is expressed only as a
result of suppression of the amber termination codon of
O R F 4 , the gene for the 23K coat protein (P3).
Presumably this suppression is mediated by a rare,
naturally occurring tRNA. This type of readthrough is
known to occur during the translation of several other
plant virus RNA species, for example suppression of the
amber termination codon of the tobacco mosaic virus
126K gene by tyrosine-accepting suppressor tRNA to
yield a 183K protein (Morch & Haenni, 1987) and during
translation of animal virus R N A in the synthesis of the
gag-pol precursor of murine leukaemia virus (Philipson
et al., 1978; Shinnick et al., 1981); in this case the amber
codon of the gag gene is read as glutamine (Yoshinaka et
al., 1985).
The predicted size for the PLRV readthrough protein
is therefore the sum of 23K, 53K (ORF 6) and the 4K
encoded by the sequence between ORF 4 and ORF 6, a
total of 80K. In most immunoblots of infected protoplasts, antisera to the 23K and 53K components reacted
with two polypeptides of 80K and 90K. In one protoplast
sample (Fig. 5, lane 4) and with several samples from
infected potato and N. clevelandii leaves (Fig. 5, lanes 1 to
3), only the 90K polypeptide was detected. Although the
predicted size of the readthrough protein is 80K, it is not
possible, with the available evidence, to determine
whether the 80K or the 90K polypeptide is the primary
translation product. Possibly the 80K polypeptide is the
primary product and is modified, for example by
2255
glycosylation (there are three potential glycosylation sites
in P5: QNY, YNY and K N K which are 64, 201 and 394
amino acid residues downstream respectively of the 23K
termination site), so that it migrates as if it were 90K
rather than 80K. Alternatively the 90K polypeptide may
be the primary product and may migrate unusually
slowly, the 80K polypeptide arising from it by
degradation.
The 53K polypeptide present in protein extracted
from purified particles of PLRV resembled the 80K
polypeptide in that it also reacted with SCR 1 and
anti-P5. It is probable that the 53K protein in virus
particles is derived by the loss of about 26K of the 80K
protein, presumably from its C-terminal end. When
partially purified particles were examined in one
experiment the proteins resembled those in unfractionated protoplasts but in the other experiment the result
suggested that some of the 80K/90K polypeptides had
been degraded to form polypeptides intermediate
between 80K and 53K but mainly 53K. This suggests
that the proteolysis occurs relatively rapidly after
infected cells are disrupted and that the 80K protein is
cleaved at a limited number of sites.
The results also suggest that the readthrough protein
can be assembled into virus particles. One feature of the
amino acid sequence encoded by the nucleotide sequence
immediately downstream of the 23K termination codon
is a succession of proline residues alternating mainly
with serine or threonine residues (Mayo et al., 1989). This
feature is shared with other luteoviruses (Miller et al.,
1988; Veidt et al., 1988). Perhaps a function of this
unusual sequence is to separate the 23K domain of the
readthrough protein, which must assemble with other
23K polypeptides to form the capsid, from the 56K
domain which would thus protrude from the virus
particle. Indeed a small number of protrusions have been
detected on the surfaces of PLRV particles in favourably
stained preparations (Harrison, 1984). A similar readthrough protein composed of coat protein and part of
the downstream ORF has been detected in particles of
BYDV (P. M. Waterhouse, personal communication)
and Veidt et al. (1988) have demonstrated suppression in
vitro of the amber termination codon of the 23K protein
ORF for BWYV. Therefore this means of expression of
the ORF for P5 may be characteristic of luteoviruses.
A possible role of the P5 domain of the 80K protein is
suggested by the results of comparisons (M. A. Mayo &
C. A. Jolly, unpublished results) between the 23K
proteins of aphid-transmissible isolates of PLRV and an
isolate that is very poorly aphid-transmissible (strain V;
Massalski & Harrison, 1987). Although some monoclonal antibodies that react with particles of transmissible isolates failed to react with particles of
PLRV-V, the only amino acid changes between the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:02:50
2256
L Bahner and others
sequences of the 23K coat proteins were exchanges of
very similar amino acids (arginine to lysine and
isoleucine to valine). Thus it seems likely that at least one
determinant for aphid transmissibility is located in the
P5 domain. A 90K polypeptide that reacted with SCR-1
was detected in potato tissue infected with PLRV-V
(data not shown) and thus it is likely that the determinant
is altered rather than deleted in PLRV-V P5. The amino
acid sequences of the P5 proteins of PLRV, BWYV and
BYDV are appreciably similar in their N-terminal
halves but are largely (PLRV and BWYV) or completely
(BYDV and either PLRV or BWYV) distinct in their
C-terminal halves (Mayo et al., 1989). The similarities
among the N-terminal halves may be related to the
location of a determinant for aphid transmission in this
part of P5.
The results therefore suggest that P5 of PLRV is a part
of the virus particle that possibly contributes biological
properties to the virus. In evolutionary terms this
strategy has the advantage of separating distinct roles of
the virus particle so that changes in one do not cause
disadvantageous changes in another. Indeed, whereas
the sequences of the three known luteovirus 23K proteins
are strikingly similar (Mayo et al., 1989), the P5
polypeptides are less similar, particularly at their
C-terminal ends. However, even if correct for luteoviruses, this strategy is not shared by many other plant
viruses, the only other examples of readthrough of the
termination codon of the coat protein gene of a plant
virus being the furoviruses beet necrotic yellow vein
(Ziegler et al., 1985; Quillet et al., 1989) and soil-borne
wheat mosaic (Hsu & Brakke, 1985) viruses.
We thank Dr H. Barker for supplying PLRV-infected plant tissue
and Miss Anne Jolly for technical assistance.
References
HARRISON,B. D. (1984). Potato leafroU virus. CMI/AAB Descriptions of
Plant Viruses, no. 291.
Hsu, Y. H. & BRAKKE,M. K. (1985). Cell-free translation of soil-borne
wheat mosaic virus RNAs. Virology 143, 272-279.
KUBO, S., HARRISON,B. D., ROBINSON,D. J. & MAYO, M. A. (1975).
Tobacco rattle virus in tobacco mesophyll protoplasts: infection and
virus multiplication. Journal of General Virology 27, 293-304,
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, London 227,
680-685.
MASSALSKI,P. R. & HARRISON,B. D. (1987). Properties of monoclonal
antibodies to potato leafroll luteovirus and their use to distinguish
virus isolates differing in aphid transmissibility. Journal of General
Virology 68, 1813-1821.
MAYO, M. A. & BARKER,H. (1983). Effects of actinomycin D on the
infection of tobacco protoplasts by four viruses. Journal of General
Virology 64, 1775-1780.
MAYO, M. A., ROBINSON,D. J., JOLLY, C. A. & HYMAN, L. (1989).
Nucteotide sequence of potato leafroll luteovirus RNA. Journal of
General Virology 70, 1037-1051.
MILLER, W. A., WATERHOUSE,P. M. & GERLACH, W. L. (1988).
Sequence and organization of barley yellow dwarf virus genomic
RNA. Nucleic Acids Research 16, 6097~6111.
MORCrI, M. D. & HAENNI, A. L. (1987). Organization of plant virus
genomes that comprise a single RNA molecule. In The Molecular
Biology of the Positive Strand RNA Viruses, pp. 153-175. Edited by
D. J. Rowlands, M. A. Mayo & B. W. J. Mahy. London: Academic
Press.
MURANI, m. F., WATERHOUSE,P. M., RAscrm~, J. H. & ROBINSON,
D. J. (1985). Carrot red leaf and carrot mottle viruses: observations
on the composition of the particles in single and mixed infections.
Journal of General Virology 66, 1575-1579.
NILSSON, B., ABRAHAMSEN,L. & UHLEN, M. (1985). Immobilization
and purification of enzymes with staphylococcal protein A gene
fusion vectors. EMBO Journal 4, 1075-1080.
PHILIPSON, L., ANDERSSON, P., OLSHEVSKY, U., WEINBERG, R.,
BALTIMORE,D. & GESTELAND,R. (1978). Translation of MuLV and
MSV RNAs in nuclease-treated reticulocyte extracts: enhancement
of the gag-pol polypeptide with a yeast suppressor tRNA. Cell 13,
189-199.
QUILLET,L., GUILLEY,H., JONARD,G. & RICHARI~S,K. (1989). In vitro
synthesis of biologically active beet necrotic yellow vein virus RNA.
Virology 172, 293-301.
RANDALL, R. E. & YOUNG, D. F. (1988). Comparison between
parainfluenza virus type 2 and simian virus 5 : monoclonal antibodies
reveal major antigenic differences. Journal of General Virology 69,
2051-2060.
ROCrlOW, W. F. & DUFFUS, J. E. (1981). Luteoviruses and yellows
diseases. In Handbook of Plant Virus Infections and Comparative
Diagnosis, pp. 147-170. Edited by E. K urstak. Amsterdam: Elsevier/
North-Holland.
SmNNICK, T. M., LERNER,R. A. & SUTCLIFFE,J. G. (1981). Nucleofide
sequence of Moloney murine leukaemia virus. Nature, London 29#,
543-548.
TAMADA, T., HARRISON, B. D. & ROBERTS, I. M. (1984). Variation
among British isolates of potato leafroll virus. Annals of Applied
Biology 104, 107-116.
VANDER WILK, F., HUISMAN,M. J., CORNELISSEN,B. J. C., HUTTINGA,
H. • GOLDBACH,R. (1989). N ucleotide sequence and organization of
potato leafroll virus genomic RNA. FEBS Letters 245, 51-56.
VEIDT, I., LOT, H., LEISER, M., SACHEIDECKER,D., GUILLEY, H.,
RICHARDS,K. E. & JONARD,G. (1988). Nucleotide sequence of beet
western yellows virus RNA. Nucleic Acids Research 16, 9917-9932.
WATERHOUSE,P. M. (1981). Purification, properties and relationships of
carrot red leaf virus, and its interaction with carrot mottle virus. Ph.D.
Thesis, University of Dundee.
YOSHINAKA,Y., KATOH, I., COPELAND,T. n. & OROSZLAN,S. (1985).
Murine leukemia virus protease is encoded by the gag-polgene and is
synthesized through suppression of an amber termination codon.
Proceedings of the National Academy of Sciences, U.S.A. 82,
1618-1622.
ZIEGLER, V., RICHARDS,K., GUILLEY, H., JONARD, G. &; PUTZ, C.
(1985). Cell-free translation of beet necrotic yellow vein virus:
readthrough of the coat protein cistron. Journal of General Virology
66, 2079-2087.
(Received 22 February 1990; Accepted 11 June 1990)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:02:50