Effect of UV irradiation on the expression of vaccinia virus gene products synthesized in a cell-free system coupling transcription and translation.

0022-538X/78/0026-0673$02.00/0

Copyright©1978 AmericanSociety for Microbiology Printedin U.S.A.

Effect of UV Irradiation

on

the Expression of Vaccinia Virus

Gene

Products

Synthesized in a Cell-Free System Coupling

Transcription

and

Translation

WALTER BOSSART, DONALD L. NUSS,* AND ENZO PAOLETTI

DivisionofLaboratories and Research, New York State Department of Health, Albany, New York 12201

Received for publication 7 December 1977

The effect of UV irradiation on the expression of the vaccinia virus genome

was investigated in a cell-free system coupling transcription with translation.

Exposure ofvaccinia virus to an increasing dose of irradiation resulted in differ-ential reduction in the syntheses ofvirus-specified polypeptides in the coupled

system,with sensitivity being proportional tothesize ofthe geneproduct.This

suggeststhateachtranslationallyfunctional mRNA species produced in vitro by vaccinia virus cores issynthesized from an individualpromotersite.

Vaccinia virus is alarge, DNA-containing vi- peptides bypolyacrylamide gelelectrophoresis.

ruswith a genomecomplexityofabout1.2x 108 Inthisreportwedescribe theeffects of increas-daltons(4, 6, 7,9). Synthesisof viralRNApro- ing UV irradiation of vaccinia virus upon the

ceedsreadilyunder invitro conditionsat alevel fmalexpressionofindividual viralgene products

which indicates that 40 to 50% of the entire in the

coupled

transcription-translation

system. genome is transcribed (19). This result suggests

that the RNA synthesizedunder in vitrocondi- MATERIALS AND METHODS

tions couldeasilycode for asmany as 150 to 200 UV

irradiation.

Purified vaccinia virus particles proteins in the size range of104 to

105

daltons. (strainWR), preparedasdescribedby Joklik (10), at

Although in vitro-synthesized vaccinia virus aconcentration of 45 optical density(OD260)units per

RNA has generallybeen described as a family ml in 10 mMTris(pH 7.5) wereexposedto incident

of molecules with an average sedimentation UV irradiation of 5 ergs/s per mm2

(supplied

by a

value ofbetween 8 and 12S (11, 26), the purifi- 253.7-nm-wavelengthsourcesituated30cmabove the

cation and partial characterization of a virion- sample). Exposures were conducted with continuous

associated speciesofhigh-molecular-weightvac- stirring at room temperature in a plastic cap7 mm in

cinia RNAhaverecently been described. 17,18). diameter and2.5mmindepth, with the thickness of

ciniaRNA haverecentlybeendescribed (17,18). the virus particle suspension measuring less than 1

The results derived from anumber ofdifferent mm.

experimental approaches are consistent witha Invitro RNA and protein synthesis. A cell-free

precursor-product relationship between the protein-synthesizing system was prepared from

Ni-high-molecular-weight RNA and the virion-re- black wheat germ by the method of Roberts and

leased 8 to 12SmRNA (16-19). Paterson(21). RNAsynthesiswasfollowed

by

deter-The technique of UV transcription mapping mining the incorporation of[3H]UTP (NewEngland

developed by Sauerbier andcolleagues (22, 23) Nuclear Corp.) into trichloroacetic acid-precipitable

originally with bacterial systems has recently material which was collected on Whatman GF/C

beensuccessfullyemployed to determineth glass-fiber filters (14). Conditions for in vitro RNA

been successfuly

employee

to

determine

the synthesis in the absence of the translation system were

order of tranScription of several animal virus asfollows.A

400-,ul

reaction mixture contained 50 mM genomes (1, 3, 8). We decided to apply this Tris-hydrochloride (pH 8.4); 10 mM MgCl2; 10 mM

techniquetothe vaccinia in vitrotranscription

dithiothreitol;

0.05%Nonidet

P-40;

2 mMeach

ATP,

system inanefforttodetectthe

transcription

of GTP, and CTP; 0.2 mM UTP; 40 ul of 0.5-mCi/ml

multiplegenes froma commonpromoter,oneof [3H]UTP; and 0.72 OD2we unit equivalent of virus.

thepossiblemodesoftranscription suggested by Incubation was at

37°C.

Conditions used for the

the synthesis of a high-molecular-weight pre- syntheses of RNA and protein in the coupled

tran-sumptiveprecursor tovacciniamRNA (16-19). scription-translation system are described in detail in

Sincegood resolution of individual invitro-syn- Results. Protein synthesis was monitored in this

sys-thesized viral mRNA's

been,

has not

achieved,tem

bydeterminingtheincorporationof[

S]methio-wecoupledthesizedscriptionofthe

mRNAievmed

viral

nine

(New EnglandNuclearCorp.)

into polypeptides

we coupled thetranscriptionof theviralgenome asdescribed by Mans and Novelli (13). Globin and

to translation in a wheat germ protein-synthe- reovirus mRNA'swere obtained from C. Baglioni and

sizing system and resolved the resulting poly- A. J.Shatkin, respectively.

673

on November 10, 2019 by guest

http://jvi.asm.org/

674 NUSS, J. VIROL.

Polyacrylamide gel electrophoresis. Samples 800

containing [3S]methionine-labeledpolypeptidesfrom the coupledtranscription-translationsystemwere pre-pared foranalysis byaddinganequalvolume ofwater W

and 2 volumes of2xsample buffersothat the final

!

/

concentrations ofsample bufferingredientswere0.1 O 600 M dithiothreitol, 2% sodiumdodecylsulfate, 0.08M C

Tris-hydrochloride (pH 6.8), 10%glycerol, and 0.4% o bromophenol blue. Thesampleswereboiled for 2 min 0 and applied to 15% sodium dodecyl sulfate-polyac- 400 rylamideslabgelspreparedaccordingtoAndersonet

4L0_

al. (2) by the method of Laemmli (12).After

electro-phoresis, gels were fixed and stained with 0.025%

(wt/vol) Coomassie brilliant blue in 40% (wt/vol)

methanol-7.5%(wt/vol)aceticacid, usuallyovernight. , 200 Gelsweredestained in severalchangesof 40%(wt/vol) u

methanol-7.5% (wt/vol)acetic acid and then dried in / vacuoandautoradiographedonKodak BB54 medical E

X-ray film. Anappropriateexposure timewaschosen a inwhich the film gavealinear responsetotheamount

ofradioactivity. Autoradiographswere scanned with 0 60 120 180 240

aBeckman model R-112scanningdensitometer.The MINUTES

radioactivity incorporated into individual polypep- FIG. 1. RNA synthesis directed by vaccinia virus

tides wasestimatedbydeterminingtheareaunder the in a wheat germ cell-free translation system. The peaks in the autoradiograph tracings or measured reaction mixture contained

206

wheat

germ

extract,

directly by cutting out the polypeptide bands and 15 mM creatine phosphate, 11 U of creatine

phos-detesrining

prev(ously

theamountofradioactivityas phokinase per ml, 16 mM N-2-hydroxyethyl pipera-described(15).

zine-N'-2-ethanesulfuric

acid(pH

7.5),

10mM dithi-othreitol, 110mMpotassium acetate, 0.3mM sper-RESULTS midine, 0.01% Nonidet P-40, 0.01 mM

S-adenosyl-L-Syntheses of vaccinia virus RNA and methionine, 4 mMmagnesium acetate, 0.05 mM each

polypeptides in the wheat germ cell-free amino acids minus methionine, 1.7 mM ATP, 0.75

system. In considering thecoupling of vacinia mM GTP, 0.5 mM CTP,

200

mCi of

r3HJUTP

per

virus-directed RNAsynthesis totranslation, we 1tmolat a final concentration of 0.1mM;and1 OD20

first

dunit

ofpurified vaccinia virus per ml ofreaction

first

determined

the level andrateof RNA sy- mixture. Incubation was at

22.50C,

and

13H]UTP

thesis under standard conditions used for

protein

incorporation was determined as described in the

synthesis

inthewheat

germ

translationsystem. text.Symbols:

El,

r3HUTP

incorporation inthe ab-RNAsynthesisbegan slowlyandthenproceeded senceofvirus; 0,f3HJUTPincorporation directed by

linearly

under these conditions forover4h

(Fig.

addedvaccinia virus

particles.

1).Although the initialrateof RNA

synthesis

in

the translationsystem at22.5°C andpH7.6was since no delay was observed in the synthesis of

approximately 50% that observed under stan- proteins directed by exogenous vaccinia RNA

dard transcription conditions at

370C

and pH (Fig. 2). Rather, the lag probably reflects the

8.4

(data

not

shown),

the final level of RNA time required for the energy-dependent

extru-synthesiswasgreater in the translation system. sionof RNA from the viruscores(11).

This is because RNA

synthesis

nornally

ends The abrupt cessation in

[3S]methionine

in-within30 to 40minwhen

transcription proceeds

corporation after 2 h of linear virus-directed

intheabsence ofan

energy-regenerating

system proteinsynthesisappears to be due to the

con-(18). sumption or inactivation of some factor(s) in the

[3S]methionine

incorporation into polypep- wheat gern extract itself. Of

all

the reaction tides under conditions whichsupport bothtran- ingredientstested,only the wheat

germ

extract

scriptionandtranslationin thewheat

germ

sys- permitted proteinsynthesistoresume. This

ef-temwas

dependent

upon theadditionof either fect cannot be attributedto RNase contamina-mRNA or vacciniavirus (Fig. 2).After addition tion of the system since addition of fresh RNA of virusparticlestothe reactionmixture,there failed to stimulate protein synthesis and

3H-la-was a 30-to45-minlag inthe incorporation of beled vacciniamRNAwasstable, as determined

[3S]methionine

into polypeptides,

followed

by by measuring acid-insoluble radioactivity, for

alinear increaseinproteinsynthesisfor approx- severalhours in thetranslation system (data not

imately

2 h, atwhich time

[wS]methionine

in- shown).

corporationendedabruptly.Thelag phasewas Inpreliminary experiments,acomparisonwas

not a characteristic ofthe translation system, made of the level of translation directed by

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.501.285.455.64.266.2]

8 efficient indirectingprotein synthesis than

vac-cinia RNA purified from the standard in vitro

/ transcription system. Thus, there is no

indica-tion thatgross quantities oftranslationally

in-O 6 active mRNA are

synthesized

and released

by

/ vaccinia virus cores in the coupled system. At

the same

time,

these measurements do not rule

outthepossibilitythat afraction of these RNA

/k

species

arenonfunctional.

[3S]methionineincorporation in the

virus-di-rected

coupled

system was

optimized

with

re-spect to each reaction ingredient (Table 1). As

previously determined for the translation of

ex-e 2- ogenousvaccinia RNA (25), the translation of

vaccinia virus RNA synthesized directly in the

coupled system was also dependent upon the

methyldonorS-adenosylmethioninebeing

pres-ent

during

RNA

synthesis.

Nonidet P-40 was

0 60 120 180 240 required for RNA synthesis to proceed; however,

[image:3.501.49.224.55.276.2]

ithadlittle effectonthe subsequenttranslation

MINUTES of the RNA over a concentration range of 0.01

FIG. 2. Protein synthesis in the wheat germ cell- to0.1%.RNAsynthesiswas alsodependentupon freetranslationsystemdirectedbyvacciniavirus or thepresence of

Mg2s

iwiththeoptimum

concen-exogenous vaccinia RNA. [uS]methionine incorpo-

tratio

fre

protein y

nthe

theo

couped

-ration intoprotein wasdetermined, asdescribed in

the text, under reaction conditions similar to those tem beig 2 mM above the total nucleoside

described in the legendtoFig. 1, except that unla- TABLE

1.

Optimalconditionsofproteinsynthesisin beled UTPwaspresentataconcentrationof0.5mM,

the

wheat

germ

cell-free

system coupling

and the reaction mixture contained45,uCi of

["S]7-

thenwheati

by

vcU-frus

particleit

methionine. Vaccinia virusRNAwasextractedfrom

transcrition

by vaccinia virusparticles

with

an in vitro transcription reaction by phenol

extrac-tion of the virus-free supernatant after removal of Reaction mixture constitutents Concn

virusparticles by centrifugation. The RNA wasfur-

Vaccinia virus"

1

OD2w

unit/mi

therpurified bygelfiltrationonSephadex G-50equil-

ATP.

1.70

mM

ibrated with0.01M sodiumacetate(pH5.0),0.05 M

GTP*

0.75

mM

NaCl, and 0.5% sodiumdodecyl sulfate, followed by

CTP

.. 050mM

threeethanolprecipitations (asthe sodium salt) to UTPc

0.10

mM remove traces of sodium dodecyl sulfate. Symbols:

Total nucleoside

triphos-O,

f'S]methionine

incorporationdirected by added

phatesc

. 3.05mM

vacciniavirusparticles; *,rS]methionineincorpo- Nonidet P

40.

0.01-1.1%

rationdirectedby exogenous vaccinia RNA;EL level

S-adenosyl-L-methionineb

.. 0.01mM

of

J`S]methionine

incorporationobserved in the ab- Creatine

phosphatec

.15.00 mM

senceof added virusorRNA. Creatine

phosphokinase'

11.00

U/mi

HEPES-KOH (pH7.6)cd 20.00mM

vaccinia RNA extracted from an mi vitro tran- Mg acetateb

5.00

mM

scription

reaction with the level of translation K

acetate"b

... 135.00mM

obtained with othermRNA

species,

e.g.,

globin

Amino acids minus

methio-and reovirusmRNA's.In

vitro-synthesized

vac- ninec 0.05 mM each

cinia mRNA formed functional initiation com- Spermidine" .. ... 0.03 mM

plexes efficiently in both the wheat germ and Dithiothreitol" ... 13.00 mM

thereticulocytecell-free system

(25).

Moreover, Wheatgerm

extract"b

.. 20.0OD20 units/ml

the level of amino acid

incorporation

obtained a The effect ofvarying theconcentrationof

individ-per microgram of input RNA suggested that ualreaction constituents on the level of

[3'S]methio-exogenous vaccinia RNA was

approximately

nineincorporationin thecoupledsystem

programmed

70% as efficientasglobinmRNAin stimulating byvaccinia virusparticleswas determined.

protein synthesis (data not shown). From cal- "Reagent which showed a

clear

concentration

op-culationsof the amountof RNA

synthesized

in timum.

thecoupled system, based onthelevel of [3H]- cReagentfor

which

noclearoptimumwas

observed,

UTPincoportionintRN

(Fi.itapparsbut

theconcentrationshownissaturatingforprotein

UTPincorporation into RNA (Fig. 1), it appears

synthesis.

that the RNAsynthesized directly in the cell- dHEPES, N-2-hydroxyethyl

piperazine-N'-2-free translation system is only slightly more ethanesulfonicacid.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.501.246.439.353.554.2]

676 BOSSART,

NUSS,

AND PAOLETTI J. VIROL.

triphosphate level in the reaction mixture. Ad- 1 2 3 4 5

dition of nucleosidetriphosphatesoramino acids to concentration levels in excessof that shown

in Table 1 resulted in no increase in protein

synthesis. The saltoptimumforprotein

synthe-sisin thecoupled systemwasestablished as135 G

-mM potassiumacetate. When thechloride salt

ofpotassium was used, however, the optimum

was muchlower,presumablydue tothe inhibi- N S

-toryeffect of thechloride ion

(23,

25).

Distinct N

-optima

were also observed for the amounts of -P 45

virusparticles andwheatgermextractaddedto

the reaction mixture.

Analysis of

polypeptides synthesized

in -p. 33. 4

the

coupled

system. 35S-labeled

polypeptides

M-synthesizedinthe

coupled

systemwere

analyzed

by

sodium

dodecyl

sulfate-polyacrylamide gel

blectrophoresisasshown inFig.3. Polypeptides

synthesized

in the wheatgermextract,directed - P

by RNApurifiedfrom the in vitro

transcription

- p. 20. 2

reaction,areshown in lane1.

Approximately

30 ___

to35

prominent

bandsare

observed,

ranging

in ' gjg 9 i - p 17. 6

molecular weight from 10,000 to 50,000. When

proteinsynthesiswasdirectedby RNA

resulting

fromtranscription byvirus particles

directly

in

the translation system, the same

polypeptides

bands were made, but a greater proportion of FIG. 3. Sodiumdodecylsulfate-polyacrylamidegel

the higher-molecular-weight polypeptides was analysis of 35S-labeled translation products

pro-the m . s s . * .

rgrammed

by

exogenousvaccinia RNAorbypurified observed with molecular

weights

in excess of virusparticles. Theautoradiograph shows the poly-65,000. Therelative synthesis ofindividual poly- peptidesresulting from translation ofexogenous

vac-peptideschanged

dramatically

when the system cinia RNA (lane 1) and the translation products

was programmed by the virus rather than by synthesizedin thecoupled system atincreasingvirus

exogenous RNA; e.g., notice the changein the concentrations: (lane 2)0.5OD26ounit/ml;(lane 3) 1 levels ofsynthesis ofp-17.5, p-20.2, p-22.6, and OD26o unit/ml;

(lane 4)

1.5OD260

units/ml;

and(lane

p-45. (cf. lane 1 with lanes 2 to 5). We also 5) 2OD260 units/ml The lettersontheleftdenote the

observed that the virus concentration affected locationsofprotein markers, which include the

vesic-the relative synthesis ofindividualproteins (cf. ular stomatitis virus glycoprotein (G), minor protein

lanes 2

through 5).

This

phenomenon appeared

(NS),

nucleocapsidprotein

(N),

andmembrane matrix

lanes2through5).Thispprotein (M) with respective molecular weights of

to be dependent upon the actual virusparticle 66,000, 52,000, 47,000, and 30,000. MG denotes the concentration andnotupon therelative amounts positionofmyoglobin (molecularweight,

17,000).

The of viral RNA made in the system. A similar numberson therightindicatethecalculated molec-effect onthesynthesisofindividual

polypeptides

ularweights ofseveralpolypeptidebandsofinterest. was notobserved when theamountofexogenous

vaccinia mRNA was variedover aconcentration upon theexpressionofviral geneproducts

range similar to thelevelofRNA

synthesized

in in the coupledsystem.High-molecular-weight

the coupledsystem

(data

not

shown).

Also,

the RNAsaresynthesizedinvitro by vacciniavirus,

gel profiles ofpolypeptides synthesized in the and these RNAs have a number of properties

coupledsystem did notchangewith timeduring consistent with their being precursors to 8 to

thereaction, i.e.,asmRNAsynthesisincreased, 12S mRNA (17, 18).Thisfinding stimulatedus

for reactionscontainingagivenconcentration of to investigate the mechanism of vaccinia virus virusparticles.Thus, it appears that the relative transcriptionbyusing thecoupled

transcription-synthesisand/orrelative translation of individ- translation system as a means ofresolvingand ual mRNA's is affected by the virus particle quantitating the viral geneproducts after expo-concentration rather than themRNAconcentra- sure of the virus to UV irradiation. Conse-tion. Whether this effect has any relation to a quently, we first determined the effect of UV control mechanism

normall_

operating in the irradiation on the infectivity ofthe virus (Fig. infectedcellsisnotpresentlyknown. 4A).Infectivity decreasedat anexponentialrate, Effect of UVirradiationofvacciniavirus witha

37%

survival doseof60

ergs/mm2.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.501.275.465.70.318.2]

larly, RNA synthesis, as measured either in the resolved into three polypeptide bands having

standardinvitrotranscriptionreaction or in the sensitivities similartothose of theirimmediate

coupled system, decreasedwith single-hit kinet- neighbors; i.e.,theyfailedtoshow theresistance

icsafterexposureto UVirradiation, as did pro- exhibited by the unresolved band. The nature

teinsynthesisinthe coupled system directed by and significance of thispuzzling phenomenaare

virus particles exposed to increasing doses of

irradiation (Fig. 4B). Similar results were ob- '0 A 0 B

tainedwhether or not exposure of virus particles 8c

to UVirradiationwas preceded by sonic oscil- , 60

lation to disrupt aggregates. z E

When the viral polypeptide products which 4 0 40

were synthesized in the coupled system pro- w \

grammed byirradiated virus particles were an-

-alyzed by polyacrylamide gelelectrophoresis, it

was observed that the individual

polypeptides

20

were differentially inhibited in their synthesis 0 20 60 0 100 200 300

(Fig.5). Thisexperimentwasrepeated a total of UV DOSE(sec)

five times, and in each case visual inspection FIG. 4. Effect ofUV irradiationon vacciniavirus

suggested that the sensitivity of the individual infectivity andon the rates ofRNA

synthesis

and

proteinswas directly related to their molecular protein synthesis in thecoupled system directedby

weights. On severaloccasions, a diffuse band of UV-treated virusparticles. (A)Effect of UV

irradia-approximately

55,000 molecular

weight

ap- tionon

infectivity. Purified

virus

particles

were

ex-peapproxitel

55,000b

moleula

weight

ap

-V

posedto

UVirradiation,

asdescribed in the text, and

peared to beconsiderably more resistantto UV the infectivity was subsequently determined on

L-cell

inactivationthan did other polypeptide bands in monolayers. (B) Reduction in RNA synthesis

(0)

and

thatmolecular weight range. When gel electro- protein synthesis (0) programed by virus particles

phoresis conditions were altered to give maxi- which had been exposed to UV irradiation under

mum resolution in this region, the band was identical conditions.

1

2

3

4

5

6

7

8

9

G-

-p. 66

-p.

55

N-

-p.

45

J~I4~.

-p.

37.5

M

l£tZ

-p.

29.2

-p. 26

-p. 22.6

-p.

20.2

-p.

17.6

FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel analysis of translation products synthesized in the coupledsystemprogrammed byvirusparticles exposedtoincreasing doses ofUV irradiation. As inFig. 3, the

lettersontheleftofthisautoradiograph show the positions of the marker polypeptides, and the numbers on therightdenotethecalculated molecularweights ofseveralprominent polypeptidebands. Lane 1,Translation productsprogrammedbyvirusparticles which receivednoUV irradiation.Lanes 2 to9,Translationproducts

programmedby virusparticlesexposedtothefollowing UV doses:40, 100, 140, 200, 300, 400, 600,and1,000s. Eachwellreceivedidenticalamountsofthecoupled cell-freeextract.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.501.240.437.127.236.2] [image:5.501.94.385.348.585.2]

678 BOSSART, NUSS, AND PAOLETTI J. VIROL.

unclear since thedifferences in the relative

sen-sitivities of all other polypeptide bands remained

the same under the varying conditions of gel

0010/

analysis. In addition, it should be mentioned thatexposure of virus to UV irradiation resulted neither in the appearance of new polypeptides ' synthesized in the coupled system, nor in any 0.008 evidence of increasedearlyquitting (Fig. 5). w

Quantitation of the major discretepolypeptide

!Z*

bands over a molecular weight range revealed

thatthe reduction insynthesisofthesepolypep- o 0006

tides after exposuretoUVirradiation exhibited

!Z/

single-hitkinetics (Fig. 6) forseveral represent- > .

ative polypeptides. In addition, whentherates

of inactivation for the synthesis of 17polypep- z-tideswereplottedversusthemolecularweights >

of these polypeptides, a linear relationship re- /

sulted (Fig. 7). This relationship suggests that

0.002

-the mRNA's coding for the individual viral translationproducts synthesizedin thecoupled systemaretranscribed fromindividualpromoter

sites; i.e., they appear not to be derived from O I

polycistronicmRNA precursormolecules which 0 2 4 6 8

are the result oftranscription ofseveral genes MOLECULAR WEIGHT x 104

fromasinglepromoter site

~~FIG.

7.

Relationship

between the rates of

inacti-froma

singlepromotersite.vation

and themolecularweightsoftranslation

prod-uctssynthesizedin thecoupled systemprogrammed

100i by UV-treatedvirusparticles. From survivalcurves

suchasshown inFig. 6,theinactivationratesfor17

80 \\sR\^ polypeptide bandsweredetermined andareplotted

against the calculated molecular weights of the

re-- \ \\ \\ spectivepolypeptides.

60\ \ X \DISCUSSION

O -\ \ \ \ 8\ The in vitro

synthesis

of RNA

by

vaccinia

z virusparticles and the translation of the

result-< 40 ingtranscription products were successfully

cou-pled

inthewheatgermcell-free

protein-synthe-Wr \ \ \ \ sizing system. Since evidence forthe synthesis

of a

high-molecular-weight

virion-associated

RNA species which may be the precursors to vaccinia virus8 to

12S

mRNAhas

recently

been reported

(17,

18),we decided to use this coupled system as a means ofanalyzing the effect of UV

20\ irradiation on the synthesis of the viral gene

products. Although coupling oftranscription to

0

\\ translation provides only an indirect method of analyzing and quantitating the survival of the viral genetranscripts after exposure of virus to 0) 200 400 UV irradiation, it is the only method immedi-_{ately available for this task, since resolution of}

UV DOSE (sec) individual viral mRNA's has not been

satisfac-FIG. 6. Survivalcurves for severalrepresentative

torily

achieved. Inactivation of virus infectivity

virus-programed polypeptides. Thisfigure shows the 4A),a nsctinan of trslation effect ofincreased UVirradiation of virus particles

(Fig.

4A),transcription, and resultingtranslation

on thesyntheses in the coupled system of five poly- in thecoupled system (Fig. 4B) did follow

single-peptides of different molecular weights. The calcu- hit kinetics after exposure ofthe virus to UV latedmolecularweights of the five polypeptides are irradiation.Analysisof thepolypeptides synthe-17,600

(LJ),

22,600 (A),

29,000

(0),

45,000

(0), and sized atthe direction of virus particles exposed

80,000 (-). toincreasing doses of irradiation showed a

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.501.73.267.333.580.2]

ferential inhibition of synthesis of individual the translation products to be identical to the

polypeptides (Fig. 5 and 6). Finally, a linear translation productsof 8 to 12S in vitro mRNA

relationship between the rates ofUV inactiva- (manuscript inpreparation). Additional

experi-tion and the molecular weights of individual ments in progress suggest that the sequences

polypeptidessynthesizedin thecoupled system translated arelocated at the 5' ends oftheselong

wasobserved (Fig. 7). We obtained no evidence transcripts. Third, a detailed comparison of the

whichwouldsuggestthe transcription of multi- polypeptides synthesized in thecoupled system

plegenes from a commonpromoter site. While programmed by virus particle with the

virus-this work was in progress, similar resultswere specific polypeptides synthesized in infected

reported byPelham (20) for a coupled system, cells has not yet been completed. However, a using amessenger-dependent reticulocyte cell- comparisonofthe cell-freetranslation products

freetranslationsystem. ofvaccinia invitro-synthesizedmRNA with the

There are,however,several limitationsinher- cell-freetranslationproducts of vaccinia mRNA

entinthesesystems.First, there is currentlyno isolatedfrompolyribosomesofinfected cells has

way ofdetermining the actual relationship be- revealed that many of the polypeptides have

tweenthe relative sizes of the polypeptides and similar migration rates on polyacrylamide gels

the corresponding mRNA molecules coding for (J. Cooper and B. Moss, personal

communica-thosepolypeptides; i.e., just because polypeptide tion). Furthermore, since evidence for a

high-A is twice the molecularweight of polypeptide molecular-weight presumptiveprecursor mRNA

B, itdoesnotnecessarily follow that themRNA has so far been obtained only under in vitro

coding for

polypeptide

Ais twice the sizeofthe transcription conditions (16, 17-19), wefeel

jus-mRNA coded for

polypeptide

B. This uncer- tifiedinusingthe system in its present state of

tainty most

probably

affects the results

plotted

characterizationtoinvestigate possible modes of

in Fig. 7. Second, we have little information transcriptionas itproceeds in the purified viral

concerning therelationship between the relative particles.

abundanceofindividual mRNA'ssynthesized by Additional studiesarerequired both to

char-the virus

particle

and the relative

synthesis

of acterizethecoupledsystem, which should prove

polypeptides in the coupled system as deter- a usefultoolin elucidating controlmechanisms

mined byquantitation after

polyacrylamide gel

operativeininfectedcells, andtodetermine the

electrophoresis.

Boone and Moss (5) have re- functional significance of the virus-associated

ported thatasmallportionof thecomplexityof high-molecular-weightRNA.

in vitro vacciniamRNAispresentat a concen-

ACKNOWLEDGMENTS

tration of about 150-fold in excess of other

se-quences (J. Virol., in press). This may explain_{quences(JVirol.inpress).This}

_{mayeFoundation}

Walter Bossart is a recipient of a Swiss National Science_{Postdoctoral Fellowship. This work was}

sup-why only 30 to 40 polypeptides, ratherthan a ported by Public Health Service grant 1RO1 GM23853-01

possible 200 to 300 vaccinia polypeptides, are from theNational Institute of General MedicalSciences.

detectedin cell-freetranslation systems.

Alter-nately,the

polypeptides synthesized

inthecou- LITERATURE

CITED

pledsystem may representthe translation

prod-

1. Abraham, G., and A. K. Banerjee. 1976. Sequential

uctsofonlyafraction of the bulk oftranscripts transcriptionof thegenes of vesicularstomatitisvirus.

normallysynthesized b the, .r il

a...

Proc. Natl. Acad.Sci.U.S.A. 73:1504-1508.

normally

synthesized

by

the virus particles,

a 2.

Anderson,

C. W., P. R. Baum, and R. F. Gestland.

portion of which may be translationally non- 1973. Processingofadenovirus 2-induced proteins.J.

functional.Inthis

regard,

a

comparison

ofiniti- Virol.12:241-252.

ation

complex

formation and thelevel of amino 3. Ball,LA., and C. N. White. 1976. Order of transcription

acid incorporation stimulated by vaccinia ofgenes of vesicular stomatitis virus. Proc. Natl.Acad.

Sci.U.S.A.73:442-446.

mRNAwith that observed for other functional 4. Berns, K. I., and C.Silverman. 1970. Natural occur-mRNAspeciesdoessuggestthat the RNAsyn- rence of cross-linked vaccinia virus deoxyribonucleic

thesized andreleased

by

vaccinia viruscoresis acid. J. Virol. 5:299-304.

contaminated with nonfunctional 5. Boone, R.F.,andB.Moss.1978.Sequence complexity

not grossly contammatea wltn nontunctlonal and relative abundance of vaccinia virus mRNA's syn-mRNA (25; unpublished data). However, we thesized in vivo and in vitro. J. Virol.26:554-569.

cannotruleoutthepossibilitythat thepolypep- 6. Gangemi, J. D., and D. G.Sharp.1976.Use ofa

restric-tides expressed in the coupled system are the tionendonucleaseinanalyzingthegenomesfromtwo products_produtsof_{ofsequnces oundvery ear he 5'end}sequencesfoundverynearthe 5' end

_Geshelin,

different strains of vaccinia virus. J. Virol. 20:319-323._{P.,and K.}

_I.

_{Berns. 1974. Characterization}

of several long transcriptional units, whereas and localization of the naturallyoccurringcross-linksin

other sequences that are

normally

expressed

vacciniavirusDNA.J.Mol. Biol.88:785-796.

only after processing of these long transcripts 8. Goldberg, S., J. Weber, and J. E. Darnell. 1977.The

aresilent inoursystem. In this respect,wehave

definition

ofa

largeL

iral

taping

bpy

effects

of

ultraviolet

recently translated

purified

virion-associated

irradiation.

Cell 10:617-621.

high-molecular-weightvaccinia RNAandfound 9. Grady,L.J.,and E.Paoletti. 1977.Molecular

on November 10, 2019 by guest

http://jvi.asm.org/

ity of vaccinia DNA and the presence ofreiterated atedRNA of vaccinia: apossible precursor to8-12S sequencesinthe genome.Virology79:337-341. mRNA.J.Biol.Chem.252:872-877.

10. Joklik,W. K. 1962. Thepreparationandcharacteristics 19. Paoletti, E., and L. J. Grady. 1977. Transcriptional of highly purified radioactively labelled poxvirus. complexity of vaccinia virus in vivo and in vitro. J. Biochim.Biophys. Acta61:290-301. Virol.23:608-615.

11. Kates, J.,and J.Beeson.1970.Ribonucleicacidsynthe- 20. Pelham,H. R.B.1977. Use ofcoupledtranscription and sisinvacciniavirus. I.The mechanism ofsynthesisand translation to study mRNA production by vaccinia release of RNA invaccinia cores. J.Mol.Biol.50:1-18. cores.Nature(London)269:532-534.

12. Laemmli, J. K. 1970. Cleavage of structural proteins 21. Roberts, B. E., and B. M. Paterson. 1973. Efficient duringthe assemblyof theheadofbacteriophageT4. translation of tobacco mosaic virus RNA and rabbit Nature(London)227:680-685. globin 9S RNAinacell-free system fromcommercial 13. Mans, J.R., and G. D. Novelli. 1961.Measurementof wheatgerm.Proc. Natl. Acad.Sci.U.S.A.70:2330-2334. theincorporationofradioactiveaminoacids into pro- 22. Sauerbier, W., and A. R. Brauetigam. 1970. A simple tein by a filter-paper disc method. Arch. Biochem. method of isolating RNA frombacteria. Biochim.

Bio-Biophys.94:48-53. phys.Acta 199:36-40.

14. Nuss, D.L., andE. J.Herbst. 1975. Stimulationof in 23. Sauerbier, W., R. L. Millette, and P. B. Hackett.1970. vitrotranscription ofT,DNAby the polyamine sper- Theeffects ofultraviolet irradiation on the transcrip-midine.Arch.Biochem.Biophys.169:513-521. tionofT4DNA.Biochim. Biophys.Acta209:368-386. 15. Nuss, D.L., and G. Koch.1976.Variationinthe relative 23. Weber,L. A., E. D. Hickey, P. A.Maroney, and C. synthesisofimmunoglobulinGandnon-immunoglobu- Baglioni. 1977. Inhibition of protein synthesis byCl-. linG proteinsincultured MPC-11cells withchangesin J. Biol. Chem.252:4007-4010.

the overall rate of polypeptide chain initiation and 25. Weber, L. A., E. D. Hickey, D. L. Nuss, and C. Bag-elongation.J. Mol.Biol. 102:601-612. lioni.1977.5'-Terminal7-methylguanosineandmRNA 16. Nuss,D.L., andE.Paoletti.1977.Methylgroupanalysis function: influence of potassiumconcentration on trans-ofvirion-associated high-molecular-weight RNA syn- lation in vitro. Proc. Natl. Acad. Sci. U.S.A. thesized in vitro by purified vaccinia virus. J.Virol. 74:3254-3258.

23:110-116. 26. Wei, C. M., and B.Moss. 1974. Methylation of newly 17. Paoletti,E. 1977. In vitrosynthesisofahighmolecular synthesized viral messenger RNA by an enzyme in weight virion-associated RNA by vaccinia. J. Biol. vaccinia virus. Proc. Natl. Acad. Sci. U.S.A.

Chem.252:866-871. 71:3014-3018.

18. Paoletti, E. 1977. High molecular weight

on November 10, 2019 by guest

http://jvi.asm.org/