In Vitro Synthesis of Turnip Yellow Mosaic Virus Coat Protein in a Wheat Germ Cell-Free System

Copyright )1976 American Society for Microbiology Printed inU.S.A.

In Vitro

Synthesis of Turnip Yellow

Mosaic Virus Coat

Protein in

a

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 Yellow

Mosaic Virus RNA directs the synthesis in vitro ofits coat

protein in

a

wheat

germ

cell-free

extract.

Optimum

conditions

for

synthesis

have

been

defined, and the effect of

spermine on

specifically enhancing

coat protein

formation

has been

examined. Identity between the

in vitro

synthesized

coat

protein and authentic

coat protein

of Turnip Yellow

Mosaic Virus was

estab-lished by

analysis

on

sodium

dodecyl

sulfate-polyacrylamide gel electrophoresis,

peptide mapping, and immunoprecipitation.

Since the

initial reports

on

the use of a wheat

germ

extract

for

the translation of

mRNA's,

several

plant viral RNAs have been used

suc-cessfully

in

this system

and have directed the

synthesis

of

viral

proteins found

in

the virion

(6, 7, 12, 15, 20, 23, 26-28, 30, 31).

Tumip

Yellow Mosaic Virus (TYMV) RNA (2

x 106 daltons) is large enough to code for

sev-eral

proteins of

average

molecular weights, and

one

of these

proteins would be expected to be

the viral coat protein (20,000 daltons).

How-ever,

this RNA introduced into various in vitro

protein-synthesizing

systems derived from

pro-caryotes or eupro-caryotes has never been reported

to

lead to the synthesis of viral coat protein (24,

32).

In

this paper, we have used viral RNA in a

wheat

germ extract

and present evidence for

the

in

vitro

synthesis of TYMV

coatprotein.

MATERIALS AND METHODS

Isolation of TYMV RNA and coat protein.

TYMV-infectedChinesecabbageleaveswerekindly

provided by S. Astier-Manifacier and P. Cornuet,

and the viruswas

purified according

to 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

Chemical Co.

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

dithioerythritol),

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

conditionsafteradjustingto2 mMmagnesium

ace-tate. Theresultingsupernatantwasdialyzed

exten-sively againstasolutioncontaining 10 mMHEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid), pH7.5, 90 mMpotassiumacetate, 1mM

mag-nesium acetate, and 1 mM dithioerythritol and

storedat-80°Cinsmall

samples.

Itcontainedabout

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

1.7

,AM.

Theconditionswereoptimized for Mg2+, K+, and TYMV RNA. A

15-,ul

portion 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

gels,proteinsynthesiswasallowedtoproceed,after

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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-Al

samples 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

driedandautoradiographedonKodirexX-rayfilms.

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

370C

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).

['4C]leucine-

(notshown) or[35S]methionine-labeled

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

according

to Kerr and Martin (14). The first dimension consisted of

electrophoresisin apyridine-aceticacidbuffer

(pyri-dine-acetic acid-water, 25:1:474, pH 6.5) at 4,000 V

for30min.The second dimension consisted of

chro-matography inn-butanol-pyridine-acetic

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

polyacrylamidegels,asdescribedabove,was

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.

RESULTS

Optimal

conditions for

protein

synthesis.

The

optimum

Mg2+

and K+

concentrations for

protein synthesis determined with TYMV RNA

asmessenger are

3.3

and 140

mM,

respectively

(1).

The effect of

TYMV RNA concentration

on

protein

synthesis showed

that the level of

incor-poration

increased with

increasing

RNA

con-centrations

upto

80

to100

,ug/ml,

beyond which

protein synthesis

was

inhibited

(Fig. 1). This

might reflect interaction

of

Mg2+

with

the

mRNA, which would result in

a

decrease

of

free

Mg2+ in

the incubation

mixture.

Kinetic experiments indicated that in the

presence

of TYMV

RNA incorporation of

[I4C]leucine

into

proteins

was linear for about

60 min

at

300C (Fig. 2); there

was

virtually

no

incorporation in the absence of added mRNA

(see also Fig. 3). The products synthesized at

different times

were

analyzed by

SDS-poly-acrylamide gel

electrophoresis

and

autoradiog-raphy:

the

results showed that the

profile

of the

4

53

0

.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

pl)

wereremoved, spottedonWhatman 3MM

disks,

and

counted.

20,

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[image:2.508.280.428.418.611.2]

FiI

react

TYAI

vario

.nan

in vi

betv,

resu

prec

toyi at tk

SI

yses gels

ing 20%

(Fig

stror

mole

cons cal s Tr TYI cule

argii

dues thior two

mole yielc

bele(

to stained

peptides.

Four main radioactive

spots

reproducibly

appear, two

of which

corre-Z_

+ TYMV

RNA

spond to ninhydrin spots (spots 1

and 2) (Fig.

4);

one

of

the

two

other

spots

(spot 3

or

4)

presum-ably

corresponds

to

the

NH2

terminal peptide,

the other

spot

corresponding

to

the incomplete

hydrolysis of

a

lysyl-glutamic

bond which

is

3_ / trypsinresistant (R. Peter,

Ph.D.

thesis, Univ.

of

Strasbourg, Strasbourg, France, 1972) and

located

near the

NH2

terminus: it

leads

to a

dodecapeptide

that

does

not react

with

ninhy-drin.

When,

instead

of

[35S]methionine,

[14C]leu-2-_

f5cine

is

used, the

in vitro

synthesized protein

comigrating

with TYMV

coatprotein

also gives

rise to

peptides

that

correspond

to

peptides

derived from authentic

coatprotein (not

shown

here).

_ /

Immunodiffusion

experiment. The TYMV

RNA-directed

protein

synthesized

in

vitro

and

TYMV RNA

comigrating

with

authentic

coat

protein

was

also characterized by immunoprecipitation.

The results

are

shown

in

Fig.

5.

With

anti-TYMV

rabbit

serum a

specific

precipitation

oc-1

20 30 40 so 60 75

curs with both authentic coat protein and

with

Minutes

the corresponding in vitro synthesized protein,

2. Kinetics ofin vitroproteinsynthesis. The as ascertained

by

the coincidence of

staining

G.

*T 7 and

autoradiography.

tion was

performed

inoptimized conditions for

fVRNA (where required), Mg2+, and K+. At From these results and from the tryptic

pep-)us times,

2-pl

samples were applied to What-

tide analysis,

we

conclude that the product

syn-3MM disks and counted.

thesized in vitro is the TYMV coat

protein.

Effect of

spermine

on

in

vitro

translation.

Spermine

has

been

reported

to

have

a

stimula-itro

synthesized polypeptides did

not vary tory effect on translation in the

wheat

germ

veen 2

and

75 min

(data

not

shown). These

cell-free

system

and

has

therefore been

used

for

ilts speak against the synthesis of

a

large

the

translation of several mRNA's

(19, 22,

30).

ursor protein

that

would undergo cleavage Sincepolyamines can replace Mg2+ and interact

ield

the

mature coat protein,

but

we cannot with RNA

molecules

(9, 29), we

checked the

uis

stage

rule

out

such

a

possibility.

optimal Mg2+ concentration for polymerization

)S-polyacrylamide

gel analysis. The anal-

in

the absence

orin the presence

of

spermine.

of the

autoradiographs

of

polyacrylamide

At

30

,uM,

spermine stimulates total

protein

show that

synthesis of

a

protein comigrat-

synthesis twofold and the optimal

Mg2+

concen-with TYMV

coat

protein

represents 15 to tration is

lowered

from

3.3 to1.5

mM

(Fig.

6).

of the

total

in

vitro

synthesized products

To

define whether

or not

the

stimulation

by

3).

Among the

many

weak

bands,

three spermine was

specific,

we

compared the

incor-nger ones,

corresponding

topolypeptides of poration

of

[35S]methionine

into

total protein

hcular

weights of

45,000,25,000,

and

13,000, and into coat protein in the presence or

absence

tantly appeared; their

possible physiologi- of spermine at various Mg2+

concentrations.

To

,ignificance will be discussed

later. this effect, the synthesized products were

ana-'yptic

peptide analysis. The sequence of lyzed by

SDS-polyacrylamide

gel

electrophore-4V coat protein is

known

(21).

This

mole- sis as

indicated

in

Materials

and

Methods.

In

contains 189 amino

acids,

of

which

10 are each

case,

the

radioactive

band

corresponding

nyl

plus lysyl

and four are methionyl resi- to TYMV coatprotein was cut out and its

radio-Since the

molecule carries an acetylme- activitywas compared to the total radioactivity

nyl residue

atits NH2 terminus, and since found forthat sample. The results are shown in

of

the other

methionyl groups within the Fig. 6. In the absence of spermine, the Mg2+

-cule

are

adjacent,

tryptic digestion should concentration yielding maximum synthesis of

I ten

ninhydrin-stained

and three 35S-la- coat protein also yields maximum

incorpora-d

peptides, of

which two should correspond tion ofmethionine into total protein, whereas

Is

4

a

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SYNTHESIS OF TYMV COAT PROTEIN 199

a

1 2 3 4

000

-ci>

000

000

--

d

000

-n

23.500

--->

1

1.700

-c0

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

pi

ofsamplebuffer 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

pl

ofawheatgermcell-freeincubation mixture performed intheabsence ofTYMV

RNA (10,000 cpm); (3) 20

pg

ofTYMV 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.

a

2

1

0 0

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

acetatemixture and(b)autoradiographed.

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

intocoatproteinovertherangeofMg2+

concen-trations tested. Whenspermineis added to the

incubationmixture,onedistinguishestwo

situ-M.W.

94

68

50

43

b

2 4

b

2

1 4

VOL.

20,

1976

I

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a

b

anti TYMAV

cow,-- *

prot e in

[image:5.508.70.463.74.276.2]

norma serum

FIG. 5. Immunodiffusiontest. The reactionwas

performed

asdescribed in Materials and Methods. The

wells wereloaded with200 pgofTYMVcoatproteinand 70,000cpm

of

[35S]methionine-labeled

product

comigratingwithauthentic coat protein onSDS-polyacrylamide gel and isolated from the gel (see Materials and Methods), or 30

pi

ofanti-TYMVrabbit serum (63mg/ml),or 30

PIu

of normal rabbit serum (58mg/ml).

(a)Stained with amido black; (b) autoradiographed.

m

0

E

C

.2

0

C

.2

EU

0

0.2

0

._

-W

4w

No Spermine 4_x0

E

0._ a U

-C

0 0.

a m 0

U 0 C C

.0

C~ 0 0. 0

1CL

Mg++[mM]

I 4

0 x

E

a

._0

-r w

EU

U

4._0

CL

Mg++[mM]

FIG. 6. Effect of spermineoncoatproteinsynthesis. Reactionswerecarriedout atoptimum K+ and TYMV RNA concentrations, varyingMg2+ concentrations, and in the absenceorpresenceof30

MM

spermine. The samples were treated as describedin Materialsand Methods,and25

pl

ofeach 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.

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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

Mg2+

conditions but has a

specific

stimulatory effect

on

coat protein synthesis at 2.5 mM

Mg2+.

DISCUSSION

Translation of TYMV RNA in a wheat germ

cell-free

system gives rise to coat protein

syn-thesis,

as

demonstrated by gel electrophoresis,

tryptic peptide analysis, and

immunoprecipita-tion.

The specific effect of spermine on TYMV coat

protein

synthesis cannot be explained entirely

on

the

basis of different

Mg2+

requirements for

the translation of various cistrons contained in

the genome. In another system (T4 mRNA and

an

Escherichia coli cell-free system) it has been

reported that the optimum

Mg2+

concentration

for a specific protein does not follow that for the

synthesis

of total protein (25). Further, it is

noteworthy that the optimum ionic

concentra-tions

for protein synthesis depend on the

mRNA

used (31), probably reflecting

differ-ences in

the structures of these

mRNA's.

Apart

from coat protein, three other main

polypeptides

are

synthesized with

TYMV RNA

(Fig.

4).

They appear not to be precursors or

fragments

of TYMV coat protein because their

ratios

remain constant throughout incubation

and

even

after protein synthesis has reached

the

plateau (not presented here).

Experiments are in progress to define the

nature

and possible biological activity of these

proteins. One of them could correspond to a

component

of the

replicating system of the

vi-rus.

The

Q,8

replicase is composed of one

phage-coded

subunit and three host

cell-coded

sub-units:

EF-Tu,

EF-Ts, and the ribosomal protein

Si

(3, 13). If

the

TYMV replicase

shows similar

properties, interesting experiments could be

de-signed

to

try to

elucidate

the amino acid

recep-tor

function

of the 3' terminal part of TYMV

and of other

plant viral

RNA genomes in

con-nection

with the observation that they form

complexes

with

the elongation factors and GTP

(11, 18).

It is known

(C. W. A.

Pleij,

Ph.D.

thesis,

State Univ. of

Leiden, Leiden, The

Nether-lands, 1973) that TYMV RNA when isolated

from the virion contains, besides

molecular

spe-cies

of

21to

23S

thought to represent the intact

genome,

large amounts (up to 50%) of shorter

RNA

molecules

(between 8 and 16S), which can

be

separated

from the 23S molecules after a

heat

treatment

and have

been

considered

to

be

degradation products of the 23S molecules.

Throughout

this

work,

unfractionated

TYMV

RNA was used for incubations, and

con-sequently the

experimental results

do

not

show

whether

the coat

protein

formed

is

a

translation

product of the intact RNA molecule.

Very

re-cently, experiments

by

Pleij

et

al.

(submitted

for

publication),

likewise

using

the wheat germ

translation system,

have

demonstrated

that the

isolated 23S RNA

molecules do

not

direct the

synthesis of the

coat

protein,

but that the

sedi-mentation constant

of

the molecules that

are

translated

into

coat

protein

is of the order of

8S

(250,000

daltons).

In

agreement

with the

results

reported here,

these

authors have also observed

that

unfractionated

TYMV

RNA leads

to

the

synthesis of

coat

protein.

It remains to

be

determined whether these 85

RNA

molecules

are

degradation

products

is-sued

from the

processing

of the viral

RNA,

or

whether

they represent

preferential replication

products

that

would

be

encapsidated

during

vir-ion

formation.

ACKNOWLEDGMENTS

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.

LITERATURE CITED

1. Benicourt, C.,and A. L. Haenni. 1975. Translation of TYMV RNA in awheatgerm cell-freesystem, p. 189-196. In A. L. Haenni and G. Beaud (ed.),In vitro transcription and translation of viralgenomes, vol. 47.INSERM, Paris.

2. Blackburn,S.1965.Thedetermination ofaminoacids by high-voltagepaperelectrophoresis,p. 1-45. In D. Glick(ed.), Methods ofbiochemical analysis,vol.13. IntersciencePublishers,NewYork.

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