Download Mapping of the RNA-binding domain of the alfalfa mosaic virus

Journal of General Virology (1994), 75, 3199 3202.
3199
Printed in Great Britain
Mapping of the RNA-binding domain of the alfalfa mosaic virus
movement protein
Fabrice Schoumacher,t Christine Giovane, Michel Maira,:~ Anne Poirson,
Thrr~se Godefroy-Colburn* and Anne Berna
Institut de Biologie Moldculaire des Plantes, Universitd Louis Pasteur, 12 rue du G#n#ral Zimmer,
67084 Strasbourg Cedex, France
In-frame contiguous deletions were created in the
movement protein gene of alfalfa mosaic virus by sitedirected mutagenesis. The mutated movement proteins
were expressed in Escherichia coli, extracted and then
purified by denaturing gel electrophoresis and then
renatured. Their binding ability with RNA was assayed
by electrophoretic retardation and u.v.-crosslinking.
Results indicated that a domain included within amino
acids 36 to 81 was necessary for RNA binding.
The alfalfa mosaic virus (AMV) movement protein
encoded by RNA3 (P3,300 amino acids) has been shown
to bind single-stranded nucleic acids in vitro (Schoumacher et al., 1992a and b), as do several other viral
movement proteins. These are the 30K protein of tobacco
mosaic virus (TMV) (Citovsky et al., 1990), the gene I
product of cauliflower mosaic virus (CaMV; Citovsky et
al., 1991), the 35K protein of red clover necrotic mosaic
virus (RCNMV) (Osman et al., 1992), the ORF2 (open
reading frame 2) product of foxtail mosaic potexvirus
(Rouleau et al., 1993) and the putative 17K movement
protein of potato leafroll luteovirus (Tacke et al.,
1993). The nucleic acid binding domain(s) have been
localized in the movement proteins of TMV (Citovsky et
al., 1990, 1992)and RCNMV (Osman et al., 1992, 1993;
Giesman-Cookmeyer & Lommel, 1993). Deletion mapping identified two binding domains in the TMV
movement protein; one of them was active independently
of the rest of the molecule and is qualified as
'independent' but the other was not (Citovsky et al.,
1992). By the same approach, an independent RNA
binding domain was detected in the RCNMV movement
protein (Osman et al., 1993). In addition, alaninescanning mutations affecting several other regions of the
molecule decreased the affinity of the movement protein
for RNA and the cooperativity of binding; mutations in
one of these regions also affected the biological function
of the protein (Giesman-Cookmeyer & Lommel, 1993).
The nucleic acid binding activities of the TMV and
CaMV movement proteins are presumed to be relevant
to the movement function because, upon binding, the
nucleic acid molecule becomes elongated and 'shaped
into a form suitable for transport through plasmodesmatal channels' (Citovsky et aL, 1992). However,
binding of the RCNMV movement protein was shown
recently not to affect the overall length of RNA molecules
(Fujiwara et al., 1993).
There is no evidence that the mechanisms of transport
are identical for all plant viruses; indeed some evidence
to the contrary is emerging (Atabekov & Taliansky,
1990; Harrison et al., 1990). In particular, the movement
mechanisms of AMV and TMV may differ significantly
because AMV, unlike TMV, seems to require its coat
protein for cell-to-cell movement (van der Kuyl et al.,
1991) and also the AMV movement protein does not
have the same nucleic acid binding characteristics as the
TMV 30K protein possesses (Schoumacher et al., 1992a
and b). We undertook to locate the RNA binding
domain(s) of the P3 protein by deletion mapping in
order to compare its (their) structure with those of the
TMV and RCNMV movement proteins. For this
purpose, we created a set of eight contiguous in-frame
deletions in the P3 gene ORF and expressed the
corresponding proteins in Escherichia coli. The proteins
were purified and their RNA binding activity was tested.
The set of deletions of the P 3 0 R F shown in Fig. 1 was
obtained by site-directed mutagenesis (creation of unique
HindIII sites) and segment reassortment. Mutagenesis
was carried out on the plasmid designated pSel 1 .P3N
(Schoumacher et al., 1992b). The latter had been
constructed by placing the complete P 3 0 R F between
the SalI and B a m H I sites of a pAlter-1 phagemid
t Present address: Friedrich Miescher Institute, P.O. Box 2543,
CH-4002 Basel, Switzerland.
Present address: Laboratoire de Grnrtique Mol~culaire des
Eucaryotes, ~1, rue Humann, 67085 Strasbourg Cedex, France.
0001-2569 © 1994 SGM
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P3 wt
0
50
I
I
I
~ i
KM
100
150
200
250
1
I
I
I
I
KI
I
I
KL
I
KL
300
(a)
C
I
1
2
3
4
5
6
7
8
9
KL KI
MGKL
P3A[1 20]
~v
P3A[21 34]
~KL
P3A[36-811
~
P3,5182-112]
I
rI._L.
KI
tL
I
lq
P3A[113-156] I
P3,5[157-213] [
[L
]
Iq
[L
I
K~
P3A[215-2431 [
P3,51244-300] I
Fig. 1. Mutagenesis of the P 3 0 R F . The wild-type P 3 0 R F is
designated P3 wt and the deleted ORFs are designated P3A[x-y], where
x and y indicate respectively the first and the last amino acid of the
deleted portion, as numbered in the wild-type sequence (Ravelonandro
et al., 1984). The positions of the HindIII restriction sites that were
either originally present ( ~ ) or created by mutagenesis are represented
by vertical bars in the upper diagram (P3 wt), with the original
dipeptides indicated underneath and amino acid numbering given
above. In the deleted ORFs, the remaining portions of the wild-type
sequence are represented by rectangles above which the inserted amino
acids are indicated. The amino acids that border the deletions are
shown within the rectangles, with changes indicated in bold type.
(a)
I 2
(b)
3
4
5
6
7
8
9
1 2
3
4
5
6
7
8
9
Fig. 2. Analysis of the P3-related proteins purified from recombinant E.
coli. Proteins prepared from recombinant E. coli expressing the wild-
type or the mutant P3 genes (about 150 ng) were subjected to
SDS PAGE in a 10% gel (Laemmli, 1970). The gel was blotted to
nitrocellulose by contact, then stained with Coomassie Brilliant Blue
(a), and the nitrocellulose membrane was processed for detection of P3related proteins with an anti-P3 serum as described by Schoumacher et
al. (1992a) (b). The E. coli-expressed proteins were: full-length P3
(lane 1), P3A[1-20] (lane 2), P3A[21-34] (lane 3), P3A[36-81] (lane 4),
P3A[82 112] (lane 5), P3A[113-156] (lane 6), P3A[157-213] (lane 7),
P3A[215-243] (lane 8) and P3A[244~300] (lane 9). The arrowhead
points to the position of the full-length P3 protein.
(formerly pSelect; Promega Biotec) from which the
HindIII site had been eliminated, and by creating an
NcoI restriction site at the start codon in order to
(b)
Fig. 3. U.v.-crosslinking assay. The radioactive RNA (10 ng in 20 lal)
was incubated with 15 to 30 ng of the different proteins (lanes 1 to 9,
same as in Fig. 2) or without protein (lane C). The open microtubes
were then subjected to u.v.-light (3.6J) in the 'Stratalinker 1800'
apparatus (Stratagene). Unprotected RNA was digested for 30 min at
37 °C by 500 ng of ribonuclease A and the protein carrying residual
RNA was analysed as in Fig. 2, except that the gel was dried and
autoradiographed without staining. (a) shows the autoradiogram and
(b) the immunoblot.
facilitate subsequent insertion of the ORF in the
expression vector pET-3d (Studier et al., 1990). After
eliminating conservatively the natural HindIII restriction
site of the P3 gene (encoding a KL dipeptide at positions
112 to 113 of the amino acid sequence), several other
hexanucteotides, encoding identical or similar dipeptides
(KL, KI or KM at nucleotide positions 35 to 36, 81 to 82,
156 to 157, 213 to 214 and 242 to 243) were substituted
to create unique HindIII sites. In addition, a mutant
(designated 'A') was created by inserting a HindIII site
immediately upstream of the twenty-first codon. The
original pSel 1 .P3N plasmid and the mutagenized plasmids derived from it were digested by HindlII (in the P3
gene ORF) and by E c o R V (at a unique site located about
580 nucleotides upstream of the NcoI site). The internal
deletions were created by reassembling the small E c o R V HindIII fragment and the large H i n d l I I - E c o R V fragment from two mutants carrying the HindIII site at
successive locations. To obtain the N-terminally deleted
gene, a linker carrying an NcoI site with the ATG
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in phase with the rest of the sequence was inserted upstream of the HindIII site of mutant A. To obtain the
C-terminally-deleted gene, a TGA triplet followed by
a B a m H I site was inserted after the 243rd codon. The
sequence of 150 to 200 nucleotides bordering the
mutations was confirmed with the dideoxynucleotide
'T7' chain termination system (Pharmacia), primed by
the next mutagenic oligonucleotide downstream of the
deletion created, or by a universal primer hybridizing to
the phage T7 promoter adjacent to the polylinker of
pAlter-1. Cloning techniques used in these and subsequent operations were as described by Sambrook et al.
(1989).
The coding sequences to be expressed, obtained as
N c o I - B a m H I fragments from the pSell .P3N-derived
plasmids, were inserted between the NcoI and the B a m H I
sites of the expression vector pET3d. The recombinant
plasmids were cloned into E. coli strain BL21(DE3) and
the proteins were extracted from inclusion bodies of the
IPTG-induced bacteria with a urea-containing buffer
(Schoumacher et al., 1992b). They were purified by
SDS-PAGE in a 10 % gel (Laemmli, 1970), eluted from
the gel according to Hager & Burgess (1980), renatured
by dialysis against 50 mu-Tris-HC1, pH 7.5, containing
10 mM-2-mercaptoethanol and 1% Tween-20 and stored
at - 2 0 °C after addition of glycerol (50% v/v final
concentration). The purified mutant proteins were
analysed in parallel with the full-length protein
(Schoumacher et al., 1992b) by denaturing electrophoresis (Fig. 2a) and immunoblotting with an anti-P3
serum (Fig. 2b). A~ 0 scanning of the gel shown in Fig.
2(a) indicated that all these proteins were at least 90 %
pure. All the mutants reacted with the anti-P3 serum in
the presence of 1% Tween-20, although the efficiency of
detection was variable.
The renatured deleted proteins were tested for their
ability to bind radioactively labelled RNA, using u.v.crosslinking and electrophoretic retardation assays adapted from Citovsky et al. (1990). The incubations were
done for 1 h at 27 °C in 40 mM-HEPES-NaOH, pH 7.0
(containing 12.5 mM-Tris-HC1, 2'5 mM-2-mercaptoethanol, 0.25 % Tween-20 and 25 % glycerol originating from
the protein sample), in polypropylene microtubes which
had been treated with dimethyldichlorosilane (2%
solution in trichlorethane, obtained from LKB) and
rinsed with deionized water. The RNA (141 nucleotides,
collinear to the 5' end of AMV RNA3) was synthesized
by the phage T7 RNA polymerase from an HaeIII
fragment of the transcription plasmid 3BS(12.0) (Dore
et al., 1990), in the presence of [~-a2P]UTP
(0"05 Ci/pmol).
The u.v.-crosslinking assay showed that all the deleted
proteins (Fig. 3 lanes 2 to 9), except P3A[36-81] (Fig. 3
lane 4), could bind RNA as did full-length P3 (Fig. 3 lane
C
1
2
3
4
5
6
7
8
9
Fig. 4. Electrophoretic retardation of R N A by the P3-related proteins.
The radioactive R N A (0.5 ng in 20 gl) was incubated without protein
(lane C) or with 25 to 30 ng of the different proteins (lanes 1 to 9, as in
Fig. 2). The samples were electrophoresed for 1 h at 13 V / c m in a 6 %
polyacrylamide gel (acrylamide:bisacrylamide 36:1) made in 20 mMTris acetic acid, 0.5 mN-EDTA, pH 7.5. ~fhe gel was dried and
autoradiographed. The positions of free and completely retarded R N A
are indicated respectively by the closed triangle and the arrowhead.
1). This was confirmed by the electrophoretic retardation
assay (Fig. 4). Indeed all the mutant proteins except
P3A[36-81] (Fig. 4 lane 4) decreased the mobility of
RNA to some extent. Those which did bind RNA could
be placed in two groups according to the degree of
retardation observed. The first group comprising
P3A[1-20] (Fig. 4 lane 2), P3A[21-34] (lane 3),
P3A[l13-156] (lane 6), P3A[215-243] (lane 8) and
P3A[244-300] (lane 9) seemed to bind RNA as cooperatively as did full-length P3 (lane 1) because the only
detectable species were the free and the fully retarded
RNA. However, complete retardation of the bound
RNA may have been due in some cases to aggregation of
the protein. Aggregation, or incorrect conformation of
the protein, could also cause the low efficiency of binding
observed in lane 9 of Fig. 4. Indeed, P3A[244-300] had the
characteristics of thermodynamically unstable proteins
(Schein, 1990): poor solubility (of the order of 5 to
10 ~tg/ml after centrifugation for 20 min at 15000 g) and
tendency to adsorb to polypropylene in spite of the
presence of detergent. The lack of RNA binding activity
of P3A[36-81] could also have been caused by incorrect
renaturation. This seems unlikely however, because the
latter protein did not aggregate or adsorb to plastic. An
additional argument against improper renaturation is
that this mutant had the same antibody reactivity as fulllength P3 (Fig. 2). The second group of mutants,
P3A[82-112] (Fig. 4, lane 5) and P3A[157-213] (Fig. 4
lane 7) bound the RNA with sufficient affinity to decrease
its mobility but a significant proportion of the complexes
(66% in the case of P3A[82-112] and 34% in the case of
P3A[157 213], evaluated by A600 measurement) were
incompletely retarded, indicating a low degree of
cooperativity or a low binding constant. A similar
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behaviour has been observed with some alanine-scanning
mutants of the RCNMV movement protein (GiesmanCookmeyer & Lommel, 1993).
In summary the region spanning amino acids 36 to 81
of P3 appears necessary for RNA binding. For reasons
explained above, we think it is unlikely that this region
enables binding by an indirect effect on the conformation
of the polypeptides as does the 65 to 86 amino acid
region of TMV 30K (Citovsky et al., 1992). Thus the
amino acids 36 to 81 region of P3 should interact directly
with the RNA. Indeed, it includes the major portion of
a positively charged domain: (69) K+E K+K+SILNR +MLPK+IGQR+MYVH+H+H + (90). Moreover, amino
acids 69 to 72 have a higher surface probability than any
other region of P3 according to Emini et al. (1985). The
positive charge and high probability of surface exposure
are shared by the independent RNA binding domains of
the TMV and RCNMV movement proteins (Citovsky
et al., 1992; Osman et al., 1993), but this is the only
similarity. Other experiments now in progress will
determine the relevance of the RNA binding domain of
P3 to its biological activity.
We thank Drs W. F. Studier (Brookhaven National Laboratory,
Upton N.Y., U.S.A.) for the pET vector and E. coli strain BL21(DE3),
L. Pinck and J.-M. Dore (IBMP, Strasbourg, France) for plasmid
3BS(12.0) and C. Stussi-Garaud (IBMP) for critical reading of the
manuscript. This work was supported by the Commission of the
European Communities in the context of a BRIDGE contract no.
BIOT-CT90-0156 and F.S. was supported by a fellowship from the
Minist~re de la Recherche et de la Technologic.
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