Copyright )1976 American Society for Microbiology Printed inU.S.A.
Synthesis of Turnip Yellow
Mosaic Virus Coat
Wheat Germ Cell-Free System
C. BENICOURT AND A. L. HAENNI*
Laboratoire de Biochimie duDeveloppement,Institut de BiologieMoleculairedu Centre National de la RechercheScientifique, Universite Paris VII, 75005 Paris, France
Received for publication19April 1976
Turnip YellowMosaic Virus RNA directs the synthesis in vitro ofits coat
defined, and the effect ofspermine on
specifically enhancingcoat protein
examined. Identity between thein vitro
protein and authenticcoat protein
of Turnip YellowMosaic Virus was
sulfate-polyacrylamide gel electrophoresis,
peptide mapping, and immunoprecipitation.
the use of a wheat
the translation of
plant viral RNAs have been used
and have directed the
(6, 7, 12, 15, 20, 23, 26-28, 30, 31).
Yellow Mosaic Virus (TYMV) RNA (2
x 106 daltons) is large enough to code for
molecular weights, and
proteins would be expected to be
the viral coat protein (20,000 daltons).
this RNA introduced into various in vitro
systems derived from
pro-caryotes or eupro-caryotes has never been reported
lead to the synthesis of viral coat protein (24,
this paper, we have used viral RNA in a
and present evidence for
synthesis of TYMVcoatprotein.
MATERIALS AND METHODS
Isolation of TYMV RNA and coat protein.
provided by S. Astier-Manifacier and P. Cornuet,
and the viruswas
purified accordingto Leberman
(17)by using polyethylene glycol and sodium
dex-transulfate.The RNAwasextractedfrom the virus
withwater-saturatedphenolasdescribedby Gierer and Schramm (10) andwasstoredat-80°C.TYMV coatproteinwasisolated from the virionby
precipi-tationof the RNAinthe presence of aceticacid, as indicated by Fraenkel-Conrat (8).
Chemicals. L-['4C]leucine(389mCi/mmol) and
L-[35S]methionine (260 to 500 Ci/mmol) were from
Amersham-Searle. TPCK-trypsin was from
Wor-thington Biochemicals Corp., T, RNase was from
Sankyo, and spermine tetrachloridewasfromSigma
Preparation of wheat germ extracts. Commercial wheat germwasagiftof B. Roberts and T. Hall. The extract was prepared essentially as described by
Daviesand Kaesberg (5), withafewmodifications
(T. Hall, personal communication). Two grams of
wheatgermwasground with broken sterilePasteur
pipettesfor1to2min; 8mlofextractionbuffer (100
mM imidazole, 90 mM potassium acetate, 2 mM
calcium acetate, 1 mM magnesium acetate, 1 mM
p8-mercaptoethanol,and 5 mM
broughttopH 7.3with aceticacid,wasadded, and
gentlegrindingwascontinuedfor30 s.The mixture
wascentrifugedfor 10 minat17,500rpm inaSpinco
ultracentrifuge (rotorno. 40). The clearyellow
su-pernatant appearing between the pellet and the
lipid layer was centrifuged again under the same
exten-sively againstasolutioncontaining 10 mMHEPES
acid), pH7.5, 90 mMpotassiumacetate, 1mM
mag-nesium acetate, and 1 mM dithioerythritol and
50mgofproteinsand 4.5 mgof nucleic acidsperml.
Invitroproteinsynthesis.The incubations for in vitro proteinsynthesiswereperformedfor1 h
(un-less otherwise stated)at30°C inconditions similar
tothose of DaviesandKaesberg (5)andcontained20
mMHEPES, pH7.5,2 mMATP,0.2mMGTP,8mM
phosphoenolpyruvate, 20,uMeach of19aminoacids,
the 20th amino acid being either [14C]leucine or
[35S]methionine, respectively,at 12.5 ,uMor0.5 to
,AM.Theconditionswereoptimized for Mg2+, K+, and TYMV RNA. A
15-,ulportion of wheat germ extract wasaddedtostartthe reaction(total incuba-tionvolume,50,ul). To controlthe amino acid incor-poration, samples were removed and spotted on Whatman 3MM diskspreviously soakedin10% tri-chloroacetic acid. The diskswereboiledfor10minin 5% trichloroacetic acid and washed successively withethanol,ethanol-ether, and ether. The radioac-tivity retained on the disks was determined with toluene-based scintillator.
SDS-polyacrylamide gel electrophoresis of
pro-teins. Toanalyze theinvitrosynthesizedproducts
on sodium dodecyl sulfate (SDS)-polyacrylamide
on November 10, 2019 by guest
SYNTHESIS OF TYMV COAT PROTEIN 197
which10mM EDTA and 2 U ofT1RNase per 5 ,ug of
TYMV RNA were added, and incubation followed
for 20 min at 30°C. The reaction was stopped by boiling the
50-Alsamples for 10minwith 25 ,ul of sample buffer (0.19 M Tris-hydrochloride, pH 6.8,
30%glycerol, 15%f3-mercaptoethanol, 6% SDS, and
0.03%bromophenolblue). A portion of the samples was layered over 0.1% SDS-15% acrylamide gels
prepared and run according to Laemmli (16). The
gelswerethenstainedwith a Coomassie blue solu-tion (2 g of Coomassie blue, 500 ml of methanol, 100 ml of acetic acid, 500 ml of water) for 20 min and
destained using amethanol-acetic acid-water
solu-tion (30:7.5:62.5, vol/vol/vol). Finally, the gels were
Forthe purpose of further characterization, the main radioactive band corresponding to protein syn-thesized in vitro and comigrating on
SDS-polyacryl-amidegels with authentic coat protein purified from
the virion was isolated. To this end, the gel was dried without previous staining and autoradio-graphed. The radioactive band presumably
corre-spondingtolabeledcoatprotein was eluted at
for24h in 0.1% SDS. TYMV coat protein wasthen
added as carrier, and SDS was eliminated by
re-peated precipitation with 20% cold trichloroacetic
acid andsolubilizationwith 0.2 N NaOH. The final
trichloroaceticacid pellet was washed with
ethanol-ether, followed by ether.
Tryptic peptide analysis. Performic acid
oxida-tionand tryptic digestion were carried out essentially
as described by Crawford and Gesteland (4).
proteins comigrating with authentic coat protein
and isolated as described above were dried after
removal of trichloroacetic acid. The pellets were
dissolvedinperformic acid to a final concentration
of 1 mg of protein per ml and incubated for 1 h at
0°C. The proteins werelyophilized and redissolved
to 1 mg/mlin 50 mM ammonium bicarbonate, pH
8.6. Theywereincubated for 4 h at 37°C with 1/100
(by weight) of TPCK (tolylsulfonyl phenylalanyl
chloromethyl ketone)-trypsinadded at time zero and
the same amountof enzyme added after 1 h. The
productswerethen lyophilizedandsuspended in 100
,ul of electrophoresis buffer.
The two-dimensional peptide analyses were
car-ried out on Whatman 3MM
accordingto Kerr and Martin (14). The first dimension consisted of
(pyri-dine-acetic acid-water, 25:1:474, pH 6.5) at 4,000 V
for30min.The second dimension consisted of
acid-wa-ter (90:60:18:72). After stainingwith a
ninhydrin-cadmiumacetate solution(2),the radioactive spots
were locatedbyautoradiography, using Kodirex X-rayfilms.
Immunodiffusion experiment. Immunodiffusion testswereperformedin 0.8%agaroseplates contain-ing 30mMveronal, pH 8.3, and 0.02% sodium azide.
The [35S]methionine-labeled material comigrating
with authentic coat protein and isolated from the
redis-solvedin150mMNaCl and loaded into the central well. Twoexternal wells wereloaded,respectively,
with anti-TYMV rabbitserumkindly provided byJ. M. Bove and normal rabbit serum. After
immuno-precipitation, the plates were washed for several
days in 10 mMTris-hydrochloride, pH 7.4, 150 mM NaCl. They weredried, stained for 20 min withan amido black solution (1 g of amido black, 0.42 M aceticacid, 0.42 M sodium acetate, 15%glycerol) and destained with methanol-acetic acid-water. The agarosepellicle wasautoradiographedon aKodirex X-ray film.
protein synthesis determined with TYMV RNA
The effect of
TYMV RNA concentrationon
that the level of
(Fig. 1). This
might reflect interactionof
mRNA, which would result ina
Mg2+ inthe incubation
Kinetic experiments indicated that in the
RNA incorporation of
proteinswas linear for about
300C (Fig. 2); therewas
incorporation in the absence of added mRNA
(see also Fig. 3). The products synthesized at
results showed that the
.00 200 300
TYMV RNA pg/mi
FIG. 1. Dependence on TYMV RNA
concentra-tion.The reactionswereperformedwithoptimal con-centrationsof Mg2+(3.3mM)and K+(140 mM)and variousTYMV RNA concentrations.Samples (2
wereremoved, spottedonWhatman 3MM
on November 10, 2019 by guest
toyi at tk
cons cal s Tr TYI cule
dues thior two
peptides.Four main radioactive
spond to ninhydrin spots (spots 1
and 2) (Fig.
3_ / trypsinresistant (R. Peter,
Strasbourg, Strasbourg, France, 1972) and
used, thein vitro
derived from authenticcoatprotein (not
experiment. The TYMV
also characterized by immunoprecipitation.
oc-120 30 40 so 60 75
curs with both authentic coat protein andwith
the corresponding in vitro synthesized protein,
2. Kinetics ofin vitroproteinsynthesis. The as ascertained
bythe coincidence of
G.*T 7 and
performedinoptimized conditions for
fVRNA (where required), Mg2+, and K+. At From these results and from the tryptic
2-plsamples were applied to What-
conclude that the product
syn-3MM disks and counted.
thesized in vitro is the TYMV coat
synthesized polypeptides didnot vary tory effect on translation in the
ilts speak against the synthesis ofa
translation of several mRNA's(19, 22,
thatwould undergo cleavage Sincepolyamines can replace Mg2+ and interact
themature coat protein,
butwe cannot with RNA
molecules(9, 29), we
possibility.optimal Mg2+ concentration for polymerization
gel analysis. The anal-in
the absenceorin the presence
spermine stimulates total
synthesis twofold and the optimal
proteinrepresents 15 to tration is
define whetheror not
bands,three spermine was
correspondingtopolypeptides of poration
and13,000, and into coat protein in the presence or
tantly appeared; theirpossible physiologi- of spermine at various Mg2+
,ignificance will be discussedlater. this effect, the synthesized products were
ana-'ypticpeptide analysis. The sequence of lyzed by
electrophore-4V coat protein is
Thismole- sis as
contains 189 amino
which10 are each
plus lysyland four are methionyl resi- to TYMV coatprotein was cut out and its
radio-Since themolecule carries an acetylme- activitywas compared to the total radioactivity
nyl residueatits NH2 terminus, and since found forthat sample. The results are shown in
the othermethionyl groups within the Fig. 6. In the absence of spermine, the Mg2+
adjacent,tryptic digestion should concentration yielding maximum synthesis of
ninhydrin-stainedand three 35S-la- coat protein also yields maximum
peptides, ofwhich two should correspond tion ofmethionine into total protein, whereas
on November 10, 2019 by guest
SYNTHESIS OF TYMV COAT PROTEIN 199
1 2 3 4
FIG. 3. Analysis by SDS-polyacrylamide gel electrophoresis of in vitro synthesized products. Protein
synthesiswasperformedatoptimal concentrations of Mg2+ and K+ using[35S]methionineaslabeled amino
acid.Eachslotwasloadedwith30 lofsample containing10
piofsamplebuffer and, from lefttoright:(1) 20
pg eachof the following protein markers: phosphorylaseA(molwt, 94,000),bovineserumalbumin (molwt,
68,000), ovalbumin (mol wt, 43,000), reduced gamma globulin (mol wt, 50,000 and 23,000), and cytochrome c (mol wt, 11,700); (2) 20
plofawheatgermcell-freeincubation mixture performed intheabsence ofTYMV
RNA (10,000 cpm); (3) 20
pgofTYMV coat protein (mol wt, 20,000); (4) 20 piof a wheatgermcell-free
incubation mixture carried out in the presence of TYMV RNA at0.1mg/ml(825,000cpm). Aftermigration,
the gelwas (a)stainedwithaCoomassiebluesolution,dried, and (b)autoradiographed.
FIG. 4. Fingerprint analysis ofTYMV coatprotein added to in vitro synthesized material comigrating withcoatproteinanddigested by trypsin.Electrophoresisandchromatographywereperformedasdescribed
inMaterials and Methods.Asample (200 p)containing700pgofTYMVcoatproteinand110,000cpmof [35S]methionine-labeled material comigrating with coat protein was spotted on Whatman 3MM after
performicacidtreatmentandtrypsinhydrolysis. Thepeptides were(a)stained withaninhydrin-cadmium
in the presence of spermine the optimal Mg2+
concentrations for coat protein and for total
protein synthesis are,respectively, 2.5 and 1.3
mM. Moreover, in the absence of spermine
about 15% of the methionine is incorporated
concen-trations tested. Whenspermineis added to the
on November 10, 2019 by guest
prot e in
FIG. 5. Immunodiffusiontest. The reactionwas
performedasdescribed in Materials and Methods. The
wells wereloaded with200 pgofTYMVcoatproteinand 70,000cpm
comigratingwithauthentic coat protein onSDS-polyacrylamide gel and isolated from the gel (see Materials and Methods), or 30
piofanti-TYMVrabbit serum (63mg/ml),or 30
PIuof normal rabbit serum (58mg/ml).
(a)Stained with amido black; (b) autoradiographed.
No Spermine 4x0
0._ a U
a m 0
U 0 C C
C~ 0 0. 0
FIG. 6. Effect of spermineoncoatproteinsynthesis. Reactionswerecarriedout atoptimum K+ and TYMV RNA concentrations, varyingMg2+ concentrations, and in the absenceorpresenceof30
MMspermine. The samples were treated as describedin Materialsand Methods,and25
plofeach was layeredon an SDS-polyacrylamide gel. After electrophoresis, thegel wasstained, dried,andautoradiographed to detectcoat
protein.It wasthen sliced, and the sliceswereincubatedat roomtemperaturefor24hinthe presenceof1 mlof
Soluene350(Packard)and10mlofTriton-Fluorpriortocounting. Theamountofradioactivityinthe slices containingthe in vitrosynthesizedcoatproteinwascomparedtothe totalradioactivity.
on November 10, 2019 by guest
SYNTHESIS OF TYMV COAT PROTEIN 201
ations: at 1.3 mM Mg2+ 15%
of the methionine
gives rise to coat protein, whereas at 2.5 mM
more than 30% of the methionine is
incorpo-rated into coat protein. This could mean that
spermine has an overall stimulatory effect on
total protein synthesis in optimized
conditions but has a
coat protein synthesis at 2.5 mM
Translation of TYMV RNA in a wheat germ
system gives rise to coat protein
demonstrated by gel electrophoresis,
tryptic peptide analysis, and
The specific effect of spermine on TYMV coat
synthesis cannot be explained entirely
basis of different
the translation of various cistrons contained in
the genome. In another system (T4 mRNA and
Escherichia coli cell-free system) it has been
reported that the optimum
for a specific protein does not follow that for the
of total protein (25). Further, it is
noteworthy that the optimum ionic
for protein synthesis depend on the
used (31), probably reflecting
the structures of these
from coat protein, three other main
They appear not to be precursors or
of TYMV coat protein because their
remain constant throughout incubation
after protein synthesis has reached
plateau (not presented here).
Experiments are in progress to define the
and possible biological activity of these
proteins. One of them could correspond to a
replicating system of the
replicase is composed of one
subunit and three host
EF-Ts, and the ribosomal protein
(3, 13). If
properties, interesting experiments could be
the amino acidrecep-tor
of the 3' terminal part of TYMV
and of other
RNA genomes in
with the observation that they form
the elongation factors and GTP
It is known
(C. W. A.
State Univ. of
Leiden, Leiden, The
Nether-lands, 1973) that TYMV RNA when isolated
from the virion contains, besides
thought to represent the intact
large amounts (up to 50%) of shorter
(between 8 and 16S), which can
from the 23S molecules after a
degradation products of the 23S molecules.
RNA was used for incubations, and
product of the intact RNA molecule.
the wheat germ
isolated 23S RNA
synthesis of thecoat
but that the
the molecules thatare
is of the order of
authors have also observed
It remains to
determined whether these 85
of the viral
We are indebtedtoF. Chapeville, inwhoselaboratory this workwascarriedout,for hisenthusiasticinterestand support, andtoL. Bosch forallowingustorefertowork performedinhislaboratory(C.W. A.Pleijetal.) priortoits publication.WeareverygratefultoB.E.Robertsand R. C. Mulligan, whoinformedusofexperimentstheyhad per-formed withTYMVRNAasmRNA, andtoA. Delfourfor hisadvice concerning thepeptideanalyses. WethankM. Garafoli forpurifying theTYMV, andG. Beaud;A. Pro-chiantz, and S. Teixeira for useful discussions.
This workwassupported bygrantsfrom NATO(no. 769) and from theATPDiff6renciation Cellulaire (no. 1.394),
Centre National de la Recherche Scientifique.
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