Journal of General Virology (1996), 77, 587-592. Printedin Great Britain
587
S h o r t communication
Cooperative binding to nucleic acids by barley yellow mosaic bymovirus
coat protein and characterization of a nucleic acid-binding domain
C. Reichel,* C. M a a s , S. Schulze, J. Schell and H . - H . Steinbiss
Max-Planck-Institut fiir Ziichtungsforschung, Abteilung Genetisehe Grundlagen der Pflanzenziichtung,
Carl-von-Linnd Weg 10, D-50829 Krln, Germany
The capacity of several coat protein (CP) mutants of a
German isolate of barley yellow mosaic bymovirus
(BaYMV) to bind to nucleic acids was studied in vitro.
Recombinant CP, produced by overexpression in Escherichia eoli, was purified from inclusion bodies and
subsequently renatured. Binding to single-stranded (ss)
RNA and ssDNA oligonucleotides was found to be
cooperative and sequence non-specific. By deletion
mutagenesis, several truncated CP derivatives were
created and their nucleic acid-binding capacity was
investigated in order to define a protein domain
responsible for RNA- and DNA-binding. The nucleic
acid-binding domain consists of a core which was
located to an internal 23 amino acid peptide (aa
125-147) and an adjacent domain (aa 148-184) which
stimulates binding.
Barley yellow mosaic bymovirus (BaYMV) is one of the
causal agents of an important virus disease of winter
barley that occurs in several European and Asiatic
countries, and which can lead to severe yield reductions
(Huth & Lesemann, 1978). BaYMV is transmitted by the
soil-borne fungus Polymyxa graminis and is classified in
the genus Bymovirus of the Potyviridae, the largest family
of plant RNA-viruses (Murphy et al., 1995). Its bipartite,
plus-strand RNA genome codes for two large polyproteins, which are processed by virus-encoded proteases
(Kashiwazaki et al., 1989; Davidson et al., 1991).
RNA-binding has been demonstrated for the coat
protein (CP) of several viruses and the binding properties
have been intensively investigated. However, studies on
bymoviruses have so far been focussed on the sequence
analysis of the viral genomes and electron microscopy of
infected plants (Huth et al., 1984; Kashiwazaki et al.,
1990; Davidson et al., 1991; Peerenboom et al., 1992;
Schenk et al., 1993), and nothing is known about the
nucleic acid-binding characteristics of any bymoviral CP.
Therefore, we set out to characterize the nucleic acidbinding properties of the CP of a German isolate of
BaYMV. Since the amounts of CP that could be
extracted from BaYMV-infected barley tissue were low
and degradation of CP was observed upon storage,
recombinant CP was prepared from E. coll. The CP
coding region was placed downstream of the T7
promoter, adding a translation start site by PCR
(plasmid pT7CP; A. Davidson, unpublished). CP was
expressed in E. coli BL21(DE3) and, after induction with
1 mM-IPTG, inclusion bodies were purified according to
Schmidt et al. (1986). After solubilization in 6 N-urea
contaminating nucleic acids were removed by ionexchange chromatography (DEAE-Sephacel; Pharmacia) and CP was renatured by dialysis (50 mg-NaC1,
1 mM-EDTA, 50 mM-Tris-HC1, pH 8"5). Samples from
each purification step were subjected to SDS-PAGE and
the final CP preparation was judged to be pure (data not
shown). After renaturation a protein with higher mobility appeared, the intensity of which increased upon
storage. Immunodetection with BaYMV-specific polyclonal antibodies (Sanofi Sante Animale, France) suggested that this protein was a CP degradation product. In
SDS-PAGE the 32.3 kDa CP showed an apparent M r of
36000 (data not shown), similar to CP from infected
barley tissue. By sequencing the N terminus, the
recombinant protein was shown to be identical to the
authentic CP, since the N-terminal methionine added for
expression in E. coli, had been post-translationally
removed (data not shown).
UV-crosslinking was used to detect interactions between CP and R N A (Fig. 1). R N A transcripts were
prepared in vitro using T7 RNA polymerase, incorporating [~-3~P]CTP and 5-bromo-UTP (Sigma) to allow
efficient UV-crosstinking. CP and transcripts were
incubated for 15 rain at room temperature in 50 mMNaC1, 1 mM-EDTA, 33 mM-PIPES (pH 6-5) and then
crosslinked according to Citovsky et al. (1990). After
* Author for correspondence. Fax +49 221 5062213.
e-mail [email protected]
0001-3743 © 1996SGM
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588
C. Reichel and others
(a)
CP increase
t~
c.)
2
1
3
4
NaCI conch (raM)
<
5
6
7
8
9
I0
l I 12
kDa
106.0
80-0
49.5
32.5
(b)
yeast RNA
kDa
106-0
80.0
1
2
3
RNA 1
4
5
:
6
7
ssDNA
8
9
dsDNA
10 11 12 13 14
15 16
/!i
i2:
;2
49-5
32.5
27-5
removal of uncrosslinked RNA by RNase A (0.5 mg/ml;
30 min at 37 °C), samples were analysed by SDS-PAGE.
Since initial experiments revealed that binding characteristics of CP to BaYMV RNA 1 and 2 were basically
identical (data not shown), the following experiments
exclusively present data for CP interactions with RNA 2.
Increasing amounts of CP ted to a proportional
increase in signal intensity. In addition, complexes of
high molecular mass became visible (Fig. 1 a). Formation
of similar high molecular mass complexes has been
previously described for the tobacco mosaic virus (TMV)
movement protein (MP) by Citovsky et al. (1990).
Presumably because of the interaction not only of
protein and RNA, but also between protein monomers,
longer RNA stretches were protected from RNAase
digestion. No protection from RNAase digestion was
observed in the absence of CP. The interaction of CP and
Fig. 1. (a) UV-crosslinking analysis of CP- RNA
interaction and effect o f NaC1. In each reaction
100 ng bromo-UTP/[~-:~2P]CTP-labelled BaYMV
R N A 2 was included. After UV-crosslinking and
RNase treatment samples were analysed by
SDS PAGE (12%). Lane 1, no CP; lanes 2-5,
increasing amounts of purified CP (0'7, 1.4, 7 and
14 gg, respectively); lanes 6 and 7, 14 gg CP; lane
6, 20gg yeast total RNA; lane 7, 3 5 g g B S A ;
lanes 8 12, 14 lag CP and increasing NaCI concentration (50, 100, 150, 200 and 250 mM-NaCt,
respectively). Arrows indicate complexes formed
by CP and crosslinked R N A transcript. (b)
Competition of interaction between CP and
RNA. In each reaction 100 ng bromo-UTP/[~3~PJCTP-labelled RNA 2 and 1-4 lag CP were
analysed in the presence of increasing amounts of
several different competitors. After UV-crosslinking and RNase treatment samples were analysed by S D S - P A G E (12%). Lanes 1-4, yeast
total RNA (0, 1, 5 and 10 lag, respectively); lanes
5 8, BaYMV RNA 1 (0, 1, 5 and 10gg,
respectively); lanes 9-12, ssDNA (heat-denatured
calf thymus DNA, approximately 1-4 kb fragments; 0, 1, 5 and 10 lag, respectively); lanes
13 16, dsDNA (calf thymus D N A ; 0, 1, 5 and
10lag, respectively). Arrow indicates complex
formed by full-length CP and crosslinked R N A
transcript.
RNA 2 was abolished by addition of excess of total yeast
RNA. Excess amounts of BSA did not lead to additional
complex formation (Fig. 1a).
To estimate the strength of the interaction between CP
and RNA, the NaC1 concentration in the reaction
mixtures was sequentially increased. The strong signal
gradually decreased with increasing NaCI concentration
and was completely abolished by 250 mM-NaC1. In some
lanes a weak signal of slightly higher mobility appeared,
presumably representing protection of RNA by the CP
degradation product (Fig. l a). The strength of interaction between RNA and CP was comparable to
reported values for the cauliflower mosaic virus (CaMV)
gene I product (stable up to 0.15-0-2 M; Citovsky el al.,
1991) and the MP of alfalfa mosaic virus (A1MV) (stable
up to 0.2 M; Schoumacher et al., 1992), while being
weaker than the values reported for the TMV and red
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Nucleic acid-binding to B a Y M V
(a)
CP
589
(b)
17 nt
2 3 4
. . ,q
37 nt
72 nt
5 6 7 8 9 101112
y
,*~ RNA
Fig. 2. (a) Cooperative interaction between CP and RNA. Twenty ng of a bromo-UTP/[c~-32P]CTP-labelled, 172 nt transcript (RNA
2 nt 3050-3222) was incubated with increasing amount of CP, UV-crosslinked and analysed by native PAGE (4%). Lane 1, no CP;
lanes 2 14, CP increase (10 ng, 15 ng, 35 ng, 70 ng, 100 ng, 140 ng, 350 ng, 700 ng, 1 lag, 1.4 lag, 3"5 lag, 7 lag and 14 lag, respectively);
lane 15, 20 lag BSA (no CP); lane 16, repetition of lane 14. The lower arrow (RNA) indicates the signal from unbound RNA; the upper
arrow indicates the complexes formed by CP and transcript. (b) Binding of CP to ssDNA oligonucleotides. In each sample, 5 ng of
radioactively labelled oligonucleotide (17, 37 and 72 nt) were incubated with increasing amounts o f CP. Reactions were analysed b y
native PAGE (10 %). Lanes 1, 5 and 9, no CP; lanes 2, 6 and 10, 0-7 lag CP; lanes 3, 7 and 11, 1.4 lag; lanes 4, 8 and 12, 7 lag. The arrow
indicates the complexes formed by CP and the oligonucleotides; asterisks indicate intermediate complexes entering the gel, which were
formed by CP and the 72 nt oligonucleotide.
clover necrotic mosaic virus (RCNMV) MPs (both stable
up to 0.6 M; Citovsky et al., 1990; Osman et al., 1992).
Interactions between CPs and RNA had been reported
to be stable in salt concentrations up to 0.8 M and 0"5 M
for brome mosaic virus (BMV) (Duggal & Hall, 1993)
and for turnip crinkle virus (TCV) (Wei & Morris, 1991),
respectively. However, for the latter to achieve complex
formation at salt concentrations higher than 0.18 M an
increase in CP concentration had been necessary, thus
indicating a comparable strength of interaction for the
TCV and BaYMV CPs.
Binding specificity between BaYMV CP and RNA 2
was investigated under the same assay conditions
described before using RNA (yeast total RNA, BaYMV
RNA 1) and DNA (heat-denatured and untreated calf
thymus DNA) competitors. Increasing amounts of these
nucleic acids were investigated for their effect on complex
formation by SDS-PAGE. Single- and double-stranded
(ss and ds) DNA and both RNA substrates led to a
strong signal reduction. In addition to the signal derived
from the interaction between transcripts and CP, a
strong signal with higher mobility was clearly visible,
again corresponding to a complex formed between
transcript and the CP degradation product, the relative
amount of which had increased upon storage (Fig. 1b).
The fact that yeast total RNA and both DNA
substrates competed nearly as well as one of the viral
RNAs supports the view that binding of the CP is
sequence non-specific. This seems to be unfavourable for
a process that has to be specific in packaging viral RNAs.
However, at least for some viruses particle assembly and
genome synthesis occur in separated dense structures,
called cytoplasmic viroplasm (Matthews, 1991). Therefore, all components for virus replication and assembly
are located in close vicinity, ensuring a specific encapsidation of the viral RNA by compartmentalization.
Cytoplasmic inclusions visualized by electron microscopy have been previously described for BaYMVinfected tissue (Huth et al., 1984; Schenk et al., 1993),
indicating a similar compartmentalization, a strategy
recently also discussed for TCV assembly (Skuzeski &
Morris, 1995).
To demonstrate cooperative RNA-binding of CP,
bound and unbound transcripts were separated by native
PAGE. An internal fragment of RNA 2 (nt 3050-3222)
was transcribed from plasmid pSY3050-3439, again
incorporating [~-32P]CTP and 5-bromo-UTP, and was
then UV-crosslinked with increasing amounts of CP.
Increase of CP led to strong retardation of the
transcripts, forming complexes unable to enter the gel
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590
C. Reichel and others
(a)
(b)
kOa
kOa
106.0
80.0
49.5
106.0
80.0
49.5
32.5
27.5
32.5
27.5
18.5
18.5
(c) Lane
Binding
0, 12 CP
e
1
CPdel1
e
D
(aa 1 298)
+
(aa 2(~298)
+
i
i
i
2
CPdel2
(aa 4 ~ 2 9 8 )
+
3
CPdel3
(aa 47-298)
+
4
CPdel4
5
CPdel5
6
CPdel6
7
CPdel7
8
CPdel8
9
CPdel9
10
CPdell0
11
CPdell 1
|
i
i
,
,
o
i
¢
,
i
m
m
o
,
u
i
i
l
m
i
i
o
*
o
o
m
,
(aa 6 3 ~ 9 8 )
+
(aa 1 184)
+
(aa 1 184A26-58)
+
(aa 1 184 A59-91)
+
(aa 1-184 A92 124)
+
(aa 1 184A125-157)
(aa 1 147)
i
a
i
o
i
+
(aa 1-120)
Fig. 3. Western and Northwestern analysis of CP deletions. (a) Equivalent amounts of mutant CPs were analysed immunologically
using a BaYMV CP-specific polyclonal antibody. In all lanes, proteins of the expected size were immunodetected. Lanes 0 and 12
contain wild-type CP. In some lanes, faint bands with higher mobility appeared, owing to interaction of the antibody with degradation
products of the CP (lanes 7 10). (b) CP and CP deletions were subjected to Northwestern analysis. Approximately0.5 lagof each protein
was analysed. Proteins bound to nitrocellulose filters were incubated with about 10 lag of radiolabelled BaYMV RNA 2. Lanes 0 and
12 contain wild-type CP. With the exception of CPdel9 and CPdelll (lanes 9 and 11) all constructs bound RNA. In lane 10, an
additional signal from an E. coli protein of about 45 kDa is visible. To clearly demonstrate that CPdel9 and CPdell 1 do not bind to
RNA, a double amount of protein (1 lag) was used for each, together with 0.375 lag protein of the constructs CPdel5 and 7 to allow
longer exposure times (right-hand panel). (c) Coding regions of the analysed proteins. Binding properties are summarized and the
resulting regions responsible for nucleic acid-binding are depicted below.
m a t r i x . F o r m a t i o n o f these c o m p l e x e s b y R N A - b i n d i n g
to C P aggregates as p r e v i o u s l y d e m o n s t r a t e d for the
C a M V M P ( T h o m a s & M a u l e , 1995) can be excluded,
since insoluble m a t e r i a l in the C P p r e p a r a t i o n h a d been
r e m o v e d b y c e n t r i f u g a t i o n at 2 0 0 0 0 g . S u b s t i t u t i o n o f
C P b y excess a m o u n t s o f B S A d i d n o t l e a d to c o m p l e x
f o r m a t i o n (Fig. 2a).
L a c k o f a n y i n t e r m e d i a t e complexes entering the gel
a n d an ' a l l - o r - n o t h i n g ' b i n d i n g b e h a v i o u r indicates a
very s t r o n g c o o p e r a t i v i t y (Fig. 2a). C o o p e r a t i v i t y for the
B a Y M V C P seems to be even s t r o n g e r t h a n for the T C V
a n d B M V CPs, where the f o r m a t i o n o f i n t e r m e d i a t e
c o m p l e x e s entering the gel was o b s e r v e d to v a r y i n g
degrees (Wei & M o r r i s , 1991; D u g g a l & Hall, 1993), as
well as for the T M V a n d R C N M V M P s , where b o u n d
a n d u n b o u n d R N A were s i m u l t a n e o u s l y o b s e r v e d over a
b r o a d range o f p r o t e i n c o n c e n t r a t i o n s ( C i t o v s k y et al.,
1990; O s m a n et al., 1992). Still, it c a n n o t be excluded
t h a t these differences were due to differences in the
e x p e r i m e n t a l p r o c e d u r e s . Nevertheless, the s t r o n g coo p e r a t i v i t y m a y c o m p l e m e n t the r a t h e r w e a k i n t e r a c t i o n
o f B a Y M V C P a n d R N A , a n d t o g e t h e r with the
p r o p o s e d virus a s s e m b l y in a subcellular structure m a y
lead to a sufficiently specific e n c a p s i d a t i o n o f viral R N A s
in vivo. T h e d a t a p r e s e n t e d in this s t u d y f a v o u r a
sequence non-specific i n t e r a c t i o n between C P a n d R N A .
I n v e s t i g a t i o n o f R N A - b i n d i n g with small s u b - f r a g m e n t s
o f the viral g e n o m e might, however, elucidate a nuc l e a t i o n s t a r t site specifically b o u n d by the CP. H o w e v e r ,
non-specific, c o o p e r a t i v e nucleic a c i d - b i n d i n g has o n l y
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Nucleic acid-binding to B a Y M V
recently been demonstrated for TCV CP, making
nucleation at a unique site in addition unlikely for this
virus (Skuzeski & Morris, 1995).
To check whether binding could also occur with
ssDNA, randomly selected oligonucleotides of different
lengths (17, 37 and 72 nt) were radioactively labelled
using [~,-3"P]ATP and T4 polynucleotide kinase. UVcrosslinking was not necessary. With increasing amounts
o f CP, all three oligonucleotides formed complexes, some
of which entered the gel. In parallel to RNA-binding, the
binding of CP to ssDNA was also cooperative (Fig. 2b).
Our investigations demonstrated that CP not only
interacted with its natural substrate (RNA), but also
with ssDNA. DNA-binding by RNA-binding proteins
has been previously reported for the TMV and R C N M V
MPs which naturally bind to R N A (Citovsky et al.,
1990; Osman et al., 1992).
For the determination of a CP domain responsible for
nucleic acid-binding, several deletions were created by
site-directed and 'loop-out'-mutagenesis and subsequent
overexpression o f the different clones in E. coli. Equivalent amounts o f the proteins, as judged by Coomassie
stained gels (data not shown) were first analysed
immunologicaUy with a CP-specific polyclonal antibody.
Cross-reactions to E. coli proteins were not visible (Fig.
3a).
The nucleic acid-binding capacity of the CP deletions
was determined by Northwestern analysis of equivalent
amounts of protein, which were separated by S D S PAGE. Proteins were electroblotted to nitrocellulose and
renatured on the membrane by incubation in binding
buffer (50 mM-NaC1, 1 mM-EDTA, 0-02 % BSA, 0-02 %
Ficoll, 0.02 % PVP, 400 rag/1 yeast total RNA, 20 mMMES, p H 6"5) for 90 rain, changing the buffer twice.
After incubation for 1 h with radioactively labelled R N A
2, filters were washed for 15 min with binding buffer to
remove non-specifically bound radioactivity. None of the
N-terminal deletions (Fig. 3b; C P d e l L 4 ) abolished
RNA-binding, but a C-terminal deletion down to aa 120
(CPdell 1) totally inhibited complex formation. The Cterminal deletion of only a smaller portion generated a
protein that was 27 aa larger (CPdell0) and showed
weak RNA-binding activity. Binding activity was completely abolished for the internal deletion derivative
CPdel9. The weak signal of a protein of about 45 k D a
visible in lane 10 presumably corresponds to an E. coli
nucleic acid-binding protein. Lack of immunodetection
of a protein of that size by CP-specific antibodies
supported this view (Fig. 3 a).
Since the binding-deficient clone CPdel9 was flanked
by the strong binding CPdel8 (Fig. 3 b, left-hand panel),
in order to clearly demonstrate the lack of binding
capacity of the proteins CPdel9 and C P d e l l l Northwestern analysis was repeated with different amounts of
CP
591
protein (Fig. 3b, right-hand panel). The amount of
protein used in the binding reaction was doubled for
CPdel9 and 11, while less protein was used for CPdet5
and CPdel7. Lack of RNA-binding activity for the Cterminal truncated CPdell 1 (aa 1-120) and CPdel9 (aa
1-184 A 125-157) was thereby confirmed.
These data indicate that the BaYMV CP contains a
core
binding
domain
of
23
aa
(125-147;
L E S E L K A W T D A V R T S L G I T T D E A ) necessary for nucleic acid-binding, while an adjacent portion (aa
148-184) seems to be necessary to restore maximal
binding (Fig. 3 b, c).
Data on size and sequence of nucleic acid-binding
domains of proteins from plant RNA-viruses are limited.
Subjects of investigation have been CPs of rather
distantly related or unrelated viruses. A detailed investigation of a nucleic acid-binding domain has been done
only for AIMV CP. N-terminal peptides o f 25 and 38 aa
were shown to efficiently bind R N A (Baer et al., 1994).
Sequence comparison of this domain to aa 125-184 of
the BaYMV CP showed no significant identity (data not
shown). To further elucidate the role of this domain in
nucleic acid-binding, studies analogous to the ones
performed with T M V M P fusion proteins (Citovsky et
al., 1992) or with oligopeptides of the A1MV CP (Baer et
al., 1994) might be helpful in future.
We appreciatethe gift ofplasmid pT7CP by Dr A. Davidson. Special
thanks to Dr F. Heyraud and Dr E. Tacke for critical reading of the
manuscript and to E. Luley for protein sequencing.We wish to thank
M. Kalda for photographic work. Parts of this work was supported by
the Volkswagen-Stiftungand the Deutsche Forschungsgemeinschaft
(DFG; Ste 532/3).
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(Received 13 November 1995; Accepted 11 December 1995)
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