Gamma Ray-Induced Small Plaque Mutants of Western Equine Encephalitis Virus

JOURNAL OF VIROLOGY, Dec. 1973,p.1568-1578 Copyright0 1973 AmericanSocietyforMicrobiology

Vol.12,No.6 PrintedinU.S.A.

Gamma

Ray-Induced Small Plaque

Mutants of

Western

Equine Encephalitis Virus

B. SIMIZU, S. YAMAZAKI, K. SUZUKI, AND T. TERASIMA

Department of Virology and Rickettsiology, Central VirusDiagnostic Laboratory, Departmentof Chemistry, National Institute of Health,Shinagawa, Tokyo141, andDepartment ofPhysiologyandPathology,National

Institute of Radiological Sciences, Chiba 280, Japan

Received for publication 18July 1973

SmallplaquemutantsofWesternequine encephalitisviruswereobtained from thesurviving fractionsofwild-type virus whichwasirradiated withgammarays.

The frequency with which small plaque mutants appeared in the surviving fraction increased with the radiation dose. These mutants were not more resistant to radiation than wild-type virus.Thegrowthrate ofamutant, S127, waslower than that ofwild-type.Clonally purifiedmutantvirionspresentedtwo

peaks inavelocity sedimentation profile; peak1correspondedtothepeakofwild

typeandpeak 2 moved faster than peak 1. Virions of both peakswereinfectious andconsistently formed small plaques in chicken embryo cells. Virions reisolated fromeither peak and grown inchicken embryo cells also revealedtwopeaks in sedimentationanalysis.Inthe electron microscope examination peak2provedto

consist ofgiant form particles, eachofwhich containedmorethanonenucleoid

surrounded with a common envelope. Despite this remarkable morphological difference, densities of thewild-type and S127 mutant virions were similar in

cesium chloridegradients. The RNAsandproteinsof mutantvirionscould not be

distinguished from thoseofwild typeonthe basis of sizeorcharge.

Mutations can be induced in animal viruses

by treatment with chemical mutagens (7,

33),

However, induction of mutants from

single-strandedRNAvirusesby ionizing radiation has

been relatively limited thus far (33) although

radio-induced mutants of Rous sarcoma virus

have been reported recently

by

Golde (11). In

the course of isolation oftemperature-sensitive

mutants (28) we attempted to use gamma

radiation for induction of the mutants. As a

result, some small plaque mutants emerged

among the survivors of the radiation at

rela-tivelyhighrate,

although

temperature-sensitive

mutants were not detected inthe examination

of a few hundred plaques. The virions of the mutants sedimented faster than those of the

wild-type when analyzed in sucrose gradients,

and electron microscope examination showed

the morphological variations of the mutant

virions tobein sizeand shape.

Most investigations dealingwith plaque

mu-tants in RNA viruses have been concerned

primarily with theirbiological properties.

Evi-dence obtained by physico-chemical and

sero-logical studies indicate that the surface

struc-ture of some ofthese mutants may also differ. This isreflectedindifferences inadsorption and

elution pattern on some chromatographic col-umns(9, 16), in coatproteins testedin

serologi-cal studies (4, 35), and inamino acid composi-tion of mutant proteins (22). Studieson

mor-phological differencesinmutant virionsrelating

plaque type variations have been limitedthus

far.

In this investigation, we report the isolation

and preliminary characterization of small

plaque mutants ofWestern equineencephalitis

(WEE) virusobtainedfrom thesurviving

frac-tions aftergamma rayirradiation.Relationship

betweenmorphologicalvariations ofthemutant

virionsand smallplaque formation is discussed.

MATERIALS AND METHODS

Virus and cell culture. The McMillan strain of

WEE virus was used. Before this experiment, it was

purifiedby the plaque cloning technique.The proce-dures for the preparation of WEE virus stocks and chicken embryo (CE) monolayer cultures have been described (27, 28). Eagle minimal essential medium (MEM)supplementedwith 5% calf serum was used as the cell culturemedium.

Gammarayirradiation.Virus was suspendedin cell culture medium and put into glass ampoules. Those ampoules were exposed to gamma radiation

from acobalt-60source at adoserate of1.4 x 104 rads

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GAMMA RAY-INDUCEDWEE VIRUS MUTANT

per min. They were kept cold in ice water during irradiation to minimize heat inactivation and then storedat -80C untiluse.

Procedures for labeling virus. CE monolayers were infected with WEEvirusatamultiplicityof 10

plaque-forming units (PFU) per cell after a 60-min incubation with MEMcontaining1 ;tgofactinomycin Dperml. Viral RNAwaslabeled by incubating cells

from 1 hpostinoculationwithfreshMEM containing

3H-uridine (1 uCi/ml) for 12 h (28). Viral proteins werelabeled by incubating cellsfrom 3 h

postinocula-tion withfreshMEMcontaining 3H-tyrosine (5

;iCi/

ml)for 9h. The culturefluids containing thelabeled

viruseswereharvested and purifiedasdescribedlater.

Concentration and purification ofviruses. Cul-turefluids containing viruseswereclarified by centrif-ugationat3,000rpmfor15minandthenat10,000xg

for 15minat4C. Thesupernatantfluidwas

centrifu-gated to pellet virus in the no. 30 rotor (Spinco,

Beckman, L2-65B)at 74,000 x gfor120minat4 C.

Theviruspelletswereresuspendedinasmall volume

ofTNEbuffer(30) (0.05MTris,0.1MNaCl,0.001M ethylenediaminetetraacetate, pH 7.4) and pipetted vigorouslyto breakupaggregates.The virus suspen-sionwasthencentrifuged at10,000x gfor 15minto removeaggregates.Thesupernatantfluidwaslayered

over a preformed 0 to 40% (wt/wt) linear sucrose gradientprepared inTNEbuffer andcentrifuged in

the SW 27 rotor at 52,000 x g for 2 h (rate zonal

centrifugation). The visible band of virus near the

middleofthetubewasremoved anddialyzed

exten-sively againstTNEbufferand, ifnecessary,rebanded

inasecondsucrosegradientforfurtherpurification.

Extraction of RNA. RNA was extracted from

labeledvirusbyphenol-sodium dodecylsulfate(SDS)

at 60 C. Samples were diluted with phosphate

buf-feredsaline containing 0.15% SDS (final

concentra-tion).Anequalvolume ofwater-saturatedphenolwas

added and the mixturewasshakenfor3minat60 C.

Two phaseswereseparated by low-speed centrifuga-tion; the upper aqueous phase was removed and

reextractedonce more with freshphenol. RNAfrom

the finalaqueousphasewasprecipitatedtwicewith2

volumes ofice-cold ethanol. The RNAprecipitatewas

collected by centrifugation in the cold and was

resuspended in TNEbuffer solution with orwithout

1%SDS.

Polyacrylamide gel electrophoresis.

Polyacryl-amidegels, 10cminlength,werepolymerizedinglass

tubes 0.4 cm in diameter. The gelscontained 7.5%

(wt/vol) acrylamide, 0.2%N,N'-bismethylene acryl-amide, 0.05% ammonium persulphate, and 0.03%

N,N,N',N'-tetramethylenediamine, in 0.1 M

so-diumphosphate buffer, pH 7.2,with0.1% SDS (31).

Virus samples in phosphate buffer solution were

acidified with 0.1 volume ofglacial acetic acid and

then made 0.5Minureaand 1%inSDS. Afterheating

in a boiling water bath for 3 min, samples were dialyzed atroomtemperature overnight against0.01

M ofphosphate buffer solution with 0.5 M ofurea,

0.1% of 2-mercaptoethanol and 0.1% of SDS. After

dialysis Iwovolume of 60%sucroseandofbromophenol

bluewereadded, and200-,litersampleswerelayered

onto gels. Electrophoresis was carried out at room

temperature at 10 mA/column for 3 h. Gels were extruded from the tubes and frozenondry ice. Frozen gels were sliced into 2-mmsegments. Each slicewas dissolved in 0.5 ml of30%H202 at 80C for 2 hand mixed with5ml ofscintillation fluid (toluene, 1liter; TritonX 100,500ml;PPO,10g;POPOP,0.3g).The radioactivity was counted in a Beckman LS-200B liquidscintillation spectrometer.

Electron microscopy. Virus samples purified by sucrose gradients were mixed with 2% solution of uranyl acetate and incubated for 1 min at room temperature. Afterwashingoncewithsaline,samples werestained withan aqueous2% solution of sodium phosphotungstate at pH 7.0 for 30 s (T. Kitano, personal communication). The preparationswere ap-plied to carbon films on copper grids; they were examinedin aHitachiHU-11B electron microscopeat anoperatingvoltage of75 kVandat amagnification ofx40,000.

Hemagglutination (HA) test. The test was per-formed according to the procedure previously de-scribed (29) using goose erythrocytes. Hemagglutinin units (HAU) were expressed as reciprocals of the highest dilution of viruses.

Chemicals. Actinomycin D was provided by

Merck, Sharp & Dohme, West Point, Pa. 'H-uridine (20 Ci/mmol) and 3H-tyrosine (36 Ci/mmol) were obtained from theRadiochemical Centre, Amersham, England. Ribonuclease-free sucrose was purchased fromNakaraiChemicals Ltd., Japan. Acrylamide and N,N'-bis methylene acrylamide were bought from the Wako PureChemical Ind. Ltd., Japan.

RESULTS

Effect of gamma ray irradiation on WEE

virus. The lethal and mutagenic effects of

gamma rays on WEE virus were examined.

When

avirussuspensionincell culture medium

wasexposed togammarays, therewas alinear

relation between the dose of radiation andvirus

inactivationasseen inTable 1 (also Fig. 2). In

assaying survivors, we sometimes noted small

plaquesamong plaquesofwild-type virus(Fig.

1).Thefrequency ofsmallplaqueformers inthe

survivingfraction wasincreased with increasing

radiation dose (Table 1). Small plaques were

isolated and clonally purified by three

succes-sive passages from plaque to plaque. These

small plaque formers were found to be true

mutants because the characters described

be-low, especiallyin plaque size, weremaintained

TABLE 1. Effect of gamma ray irradiationon WEE virus

Gamma ray Survival (PFU) Frequencyofsmall (rad) plaquesinsurvivors

0 107.8 3/2042 (0.14%)

500 106.1 18/1782(1.01%)

1,000 104.4 24/1384(1.73%) 1,500 102.4 26/ 788(3.29%)

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SIMIZU ET AL.

afterseveral passages in CE cells and BHK-21

cellsoradultmousebrains.

Inordertodetermine whether thesemutants

were induced or selected by radiation, the

wild type and three mutants were

exposed

to

gamma rayirradiation and the survivingcurves

obtained were compared. Figure 2 indicates

that the wildtype and the mutants showed a

similar dose-response curve, suggesting that

these mutants were not obtained

by

selection

forrelativeresistance tothe radiation.

Further-more,the factthatfrequency of mutants among survivors wasincreased with the radiation dose

favors the hypothesis that the mutants were

induced by radiation.

Biological properties of small plaque

mutants. Six

small-plaque

mutants obtained

from the irradiated virus and onespontaneous

mutant werecompared with wildtypein

regard

tothe

biological

markers which had been

inves-tigated on WEE virus in our

laboratory,

i.e.,

temperature sensitivity, heat

stability,

plaqu-ing efficiency under dextran sulfate agar, and

virulence for mice (26-28,

32).

As summarized

in Table 2, the results obtained with these

mutants vary from one mutant to another but

marked differences in characteristics of wild

typeandthesemutants are not seen.

In a one-step growth experiment, growth patterns of the wild type and a small plaque mutant, S127, were compared in CE monolayer

cells (Fig. 3). Mutant virus grew more slowly

than wildtypeand the yieldof the mutant was

lower thanthat of wild type for the test period.

Even at 24 h after inoculation, the yield of the mutant didnotreachthelevel of the wild type.

The total (released and cell associated) virus

yieldofthemutant was also lower than that of

wildtype. The limited growth in CE cells is a prominentcharacteristic of this mutant.

Sedimentation behaviors of S127 mutant.

When the partially purified S127 mutant virus

was examined by rate zonal centrifugation, we

noted that it showed two visible bands in sucrose gradients. Therefore, mutant viruses

were labeled with 3H-uridine and analyzed by

rate-zonal centrifugationto seethedistribution

oftheirradioactivity, infectivity, and HA

activ-ity. Asshown in Fig. 4, tworadioactivepeaks of

infectivity and HA activity are seen in the

velocity sedimentation profile of the mutant

S127. Toseparatethesetwo

peaks,

fraction 14

ofpeak 1and fractions6 to 9ofpeak2inFig. 4

were collected and resedimented by rate zonal

centrifugation. As seen in

Fig.

5, two peaks

sedimented separately corresponding to each

I

FIG. 1. Plaquesproduced with the wild type (left) and a smallplaquemutant, S092(right) in CE cells.

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GAMMA RAY-INDUCEDWEEVIRUSMUTANT

e 0.01 0

C-\-0.001

-I

0 250 500 750 1000

[image:4.497.51.244.59.350.2]

GAMMA-RAY(KILORADS)

FIG. 2. Survivingfractionsof wild type and small plaque mutants irradiated with gamma rays.

Sym-bols:0,wild-type;0, S113 mutant; *, S118 mutant;

[image:4.497.257.449.238.533.2]

0,S127mutant.

TABLE 2. Biological propertiesof wild-type and small plaquemutantsof WEE virus

EOPa EOP5 Heatingc

Virus at42/ under at 50 C, LD,O/PFUd

37C DS-agar 30min

(%)

(%)

(%)

Wild-type 69 100 3.0 6.7/7.9(2)

S025 29 109 3.1 6.5/7.6(4)

S092 32 92 2.2 6.9/8.9(3)

S113 19 120 6.8 7.1/8.0(3)

S118 39 114 2.7 6.9/8.0(3)

S127 66 93 0.8 6.3/7.7(3)

S132 32 38 10.7 7.1/7.9(2.5)

Spontaneous 90 Not done 4.4 6.8/8.6(4)

aEfficiencyofplaquingat37/42C (28).

'Efficiency of plaquing under agar containing dextran

sulfate (32).

cSurvivingfraction afterheatingat50C for 30 min.

dLD,.inadultmice/PFUinCE cells. Parentheses indicate themeansurvival time(days)of 10miceinoculatedwith 100

LD,, ofeachvirus.

peakfractionshown in thebottom

panel

of

Fig.

4;peak2movesfaster, andisbroader than

peak

1 inboth radioactivity andinfectivity.

Virions in both peaks in Fig. 5 formed small

plaques in CE cells and their plaques were

reducedbyantiserum raised ina rabbit

immu-nized with wild type virus to the same degree

asthose of wildtype (data not shown).

In thenext experiment, density of virions was

examined by equilibrium centrifugation in a

cesium chloride gradient. Mutant virions from fractions 6 to 14 and wild type virions from

fractions 14 and 15 in Fig. 4 were collected and

dialyzed to remove sucrose and then mixed in

the solution of cesium chloride(startingspecific

gravity 1.243g/ml). As seen in Fig. 6, densities of wild type and mutant virions were 1.234

g/cm3

and 1.226

g/cm3,

respectively. In a 20 to

60% linear sucrose density gradient, both

vi-ruses showed 1.19 g/cm3 in density, although

the peak of mutant was broader than that of

10

9

-.

J

'" 8

Q-co

7

7.:

6

5

O 1 2 4 6 8 10 " 24

HOURS AFTER INOCULATION

FIG. 3. One-step growth ofthewild-typeand S127 mutant of WEE virus in CE cells. CE cells were

inoculated with the virusat aninput multiplicity of

10PFUpercell.Aftera60-min adsorption period,the

inoculated cultures were washed twice with MEM

and then incubated at 37C; duplicate plates were

sampledatselected intervals.A partofculturefluid

wasstoredafter clarifying by low-speed centrifugation

for released virus assay.Infected cellswere collected

into culturefluidwitha rubberpolicemanand then

frozen and thawed three cycles toobtain totalvirus.

Symbols: 0, wild-type released virus; 0, wild-type

totalvirus;*,mutantreleasedvirus;0,mutanttotal

virus.

i

0

o ,

I I

W

I I I

II

I I I

II II

I I I I I I

II II

I I

-P

"W,"

I I I I I IAl

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SIMIZU ETAL.

7.;.

If\

~~~~~~~~~9

CLL

T T

Cfl

C5-,

6

250

2.5

150~

50

PEAK

2-

-.-PEAK

I

S

127

9

C-,)5.o0

A C

1

10

23

30

1-C,,~~~~ ~ ~ ~ ~ ~~~~~~~~C

2s5

- 6

16

0

120

1 1~~~I0 21 30

[image:5.497.108.388.53.539.2]

FRACTION

NUMBER

TOP

FIG. 4. Sucrosegradient analysis ofthewild-typeandS127mutantvirionssynthesizedin CE cells. Twenty

plates ofCE cellswereinfectedwith eitherwild-typeorS127mutantin thepresenceof1.5,ugof actinomycinD perml and 1 UCi of 3H-uridine per ml.At 12h afterinoculation, the culturefluids wereharvested and the labeled virionswereconcentrated. Concentrated virionswerelayeredover apreformed linear0 to40%osucrose gradientand then centrifuged in theSW27rotor at52,000 x gfor2h. Fractions(1 ml) were collectedand sampleswereassayed foracid-precipitable radioactivity(0), infectivity(A),and HAactivity(0).

wild type (data not shown). These data taken to confirm these observations, the virions of

together reveal that the wild type and mutant wildtype andS127 mutant were examined in an

virions are similar in density, but different in electron microscope (Fig. 7). The wild type

shape orsize. virions showed quiteuniform enveloped

spheri-Electron microscope observation. In order cal particles about 60 nm in diameters, as

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GAMMARAY-INDUCED WEE VIRUSMUTANT

4

CD

10

CDJ

- 2-c 2 c-3

PEAK 1

PEAK2

8

7

7LL.

c, O

C0~

6 °=

5

10

D

5 =

8

7Lh.

7fi 60~

5

10 =

5 =

1

1

0

20

30

FRACTION

NUMBER

TOP

FIG. 5. Separation oftwopeaksof S127mutantinsucrosegradients. Peak1from fraction14andpeak2from fractions 6to 9inFig.4werecollected and resedimented byrate zonalcentrifugation. Fractions (I ml) were collected and samples wereassayed foracid-precipitable radioactivity (0), infectivity (A), and HA activity (0).

reported by Morgan et al. (21) and Grimley et

al. (12), whereas the mutant virions

evidently

showedtwosizetypes,i.e., the

regular

spherical

particle (peak 1) and the giant particle(peak 2),

which consistedof two tofournucleoids. These giant form particles already appeared in the

concentrated viruspreparation before

purifica-tion insucrose(Fig.7B).Therefore,theyare not

likely to be an artefact due to exposure to

sucrose duringpurification.

Viral components of S127 mutant. The

mutantviruswasfurther examined for its RNA

and protein content to compare with those of

wild type virus. Viral RNAwhich was extracted

from the purified mutant virions was

sedi-mentedinalinear15to30% sucrosegradientat

the same rate as the 41SRNA extracted from wild type (Fig. 8). Viral proteins extracted from

similarlypurified mutant virions were analyzed

by polyacrylamidegelelectrophoresis.Asshown

in Fig. 9, viral core and envelope polypeptides migrated to positions identical with those of wild type. The molecularweight ofeach

poly-peptide was 6.0 x 104 and 3.8 x 10',

respec-tively, as estimated by electrophoresis with

standard marker proteins. These patterns of

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6

0.l

|

|

l

l135

0 * 1

250

10~~~~~

CL

L

2b

0 0

@0

2

100

80

Wild

a

_a'~~~~~

_60~

-40

0. - 20

-J

- 0

i

C,

,1.35

00

C-,

*

-41.30

00

5

10

c-10

0

*

P*0C

4

1.1 5

20

S127

Ijis

aa

~~~~~~~10=

1

5

10

15

20T

P

FRACTIONS

FIG. 6. BuoyantdensitiesofpurifiedWEEvirions in cesium chloride

density

gradients.

A 1.5-mlamount

of

3H-uridine-labeledvirionspurifiedtwiceinsucrosegradientswasmixedwith3.5ml

of

TNEsolution

containing

1.6 g of cesium chloride. Centrifugation wasperformed in SW50L rotor at 100,000 x g

for

46h at 4 C. Fifteen-drop fractionswerecollectedfromthebottomofthetube,andsampleswereassayed

for

acid-precipita-bleradioactivity (filledcircles), and HAactivity (opencircles).Measurementofrefractiveindexwasmadeon

allfractions, and densitiesat25Cwerecalculatedfromanempiricallydetermined relationbetweenrefractive

index anddensity.

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GAMMARAY-INDUCEDWEE VIRUS MUTANT

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.inw _ - - * w.

FIG. 7. Electronmicrographs ofWEEvirions. x100,000.A,wild-type; B,concentrated crude S127 mutant;

C, peak 1(Fig. 5) particles ofS127 mutant; D,peak2(Fig. 5)particles ofS127 mutant.

RNA andproteinsaresimilartothosedescribed

forgroup Aarbovirusesby Sreevalsanetal.(29)

and Acheson and Tamm

(2).

Geneticstability of small

plaque

mutants.

To determine whether these altered properties

of small plaque mutants resulted from a true

genotypic alteration and, if so, whether this

property was stable, the following experiments

weredone.Viruses labeled with3H-uridinewere

sedimentedin sucrose

gradients

under thesame

conditions stated

above,

and 30 fractions

col-lectedfrom eachtubewereassayed for

radioac-tivity and HA activity. As a result, wild type

and its large

plaque

mutant, L49

(unpublished

data),

showed a

single peak,

whereas S118

mutant aswell as S127 mutant showed 2peaks.

A spontaneous small plaque mutant derived

from wild type alsoshowed2peaks. However,a revertantpresentedasingle peak corresponding

tothat of wild type. The revertantwasobtained

from a wild type sized plaque that rarely

appearedafterseveralpassages ofS127mutant

inCE cells.

Two clones were isolated from peak 1

(frac-15"I5

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l l killing effect of radiation which is presumably

due to irreparable chain breaks.Therefore, the

observed dose-dependent occurrence of

small

plaque mutants in the wild typeWEE

viruses

3 mightbe explained by radiation-induced

muta-tion. However, we should note that the wild type used here is relatively mutable in its

ild

plaque

type

character. After being extensively

purified by plaquing, the wild type virus still

- S 121 contained

spontaneous

small

plaque

mutants at

2- the rate of

0.1%

(Table

1).This is not a

special

l-

lcase

inplaque-type mutationsofanimalviruses

28S 18S (7, 20, 24, 33). In view of such highmutability, it still remains a possibility that the mutants

wereselectedbythe relative resistance to

radia-tion, although a significant difference in

sensi-tivity to radiation

between

wild type and

mu-tants was not demonstrated. A

significant

difference in sensitivity to gamma radiation

might

not be

readily

detectable when the

ma-1

10O

20O

30

jority

of the infectious

particles

are

"mono-FRACTION NUMBER TOP ploid" in a mutant

population.

We would

ex-FIG. 8. Sedimentation analysis of WEE viral

RNA.

pectthat

preexisting

mutants

could be selected

RNA was extractedfrompurified 3H-uridine labeled

virions and was sedimented in a 15 to 30% (w/w) Wild

sucrose densitygradient. Centrifugation wasfor12h at 24,000 rpm in theSW27rotorand 1-ml fractions were collected. Arrows indicate the positions of co- 2_

sedimented 28S RNA and 18S RNA markers

ex-tractedfrom CE cells.

tion 12) and peak2 (fraction4) in Fig. 5.They

were grown once in CE cells for labeling virus

with 3H-uridine, and then applied to sucrose

gradients.

These two clones

presented

two2 peaks again, indicating that the regular spheri- c

cal particle of peak 1 is not a revertant but a SS127

mutant which is stable in theability to

repro-duceparticles oftwo types.

DISCUSSION

Since ionizingradiation causes breaks in the

polynucleotide chains, arboviruses containing 2

single-stranded RNA polynucleotides (3) are

highly

sensitive

to radiation compared with

viruses

which

contain

double-stranded

polynu-cleotides

(10).

Inactivationof theWEEvirusby I0 ,5 2'0 25 3'0 35 4 45

gamma ray followed a one-hit curve, and the E FRACTION NUMBER

37% survivaldoseofthis virusdetermined from FIG. 9. SDS-polyacrylamide gel electrophoresis of

Fig. 2 was 150krads.This value is inagreement proteins extractedfrompurified WEE virions. Wild-with those reported for single-stranded RNA type and S127 mutant viruses were labeled with

viruses (10, 12). 3H-tyrosine. One hour before infection CE cells were

Ionizing radiation yields highly reactive and incubated with1.5pgofactinomycin D perml. Three

short-lived radicals inthe medium surrounding hours after infection the medium was removed, and

orwithinviruses(10, 14) and ultimately leads to MEMwith 5

'Ci

of

3H-tyrosine

permlwasadded. At

alterationsofviralgenometroughthprod 12 h, culture fluids were harvested and virions were

alterations ofvlralgenomethroughthe produc- purifiedasdescribedinthetext. Thevirions

dis-tion ofreparable chain breaks orexcitations in rupted in 1% SDS, 0.5% ureaand0.1%

2-mercapto-DNA or RNA molecules. Accordingly, viruses ethanol andelectrophoresedina7.5%gelat10mAfor could receive a mutagenic effect other than a 3h.

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GAMMARAY-INDUCED WEE VIRUS MUTANT

by

gamma raysif each genome within a

"multi-ploid" particle is intrinsically infectious and

the multiploid particles are significantly more

resistant to irradiation. The fact that a spon-taneous mutant closely resembled the induced mutants in biological and physical characters mayalsosuggest this possibility.

We demonstrated two types of

morphologi-cally different particles in mutant virions by

sedimentation analysis and electron

micro-graphs. Both were infectious and formedsmall

plaques inCE cells, and contained the complete

RNAs andproteins

comparable

tothose of wild

type.Therefore, thesemutants arequite

differ-ent from the incomplete or defective particles

recently reported forSindbis virus (23, 25)

and

for other animal viruses (17). Despite this

morphological difference the densityof

mutants-wassimilartothatofwildtype.Variantstrains

of some animal viruses are known to have

different densitiesin cesiumchloridefrom those

of their parental strains (6, 19).

As shown in electron micrographs, our

mu-tants basically consisted of aroughly spherical

orsomewhat polygonal nucleoid havinga

well-defined envelope with fine projections.

How-ever, the virions collected from peak 2 in a sucrose gradient (Fig. 5) contained morethan

onenucleoid surrounded byacommonenvelope

(giantform). Itisunlikely that thesegiant form

particles are aggregates made up during the

process of purification, since an attempt to

break up these particles by sonication failed

and, in addition, these particles were found

even inthe crude viral material.

Envelopment of nucleocapsids to form

ma-turevirions occurseitheratthe cellsurface (1)

or along cytoplasmic membranes (12), and

ac-cumulation of nucleocapsids around

membra-nous vacuoles is characteristic in group A

ar-bovirus morphogenesis (5, 8, 12, 21). Judging

from our electron micrographs, we concluded

thatafewnucleoidswere

presumably

enveloped

by a common membrane at the final step of maturation. Similar giant forms have been

noted

by

Klimenko et al. (18) for Venezuelan

equine

encephalitis

virus,

Higashi

etal. (15) for

Chikungunya

virus, and Tan

(34)

for Semliki

Forest virus in arboviruses, although these

au-thors did not report physical properties, and

plaque morphologyofthe viruses. The

produc-tion of the two type particles of S127 mutant

wasconstantly observed at the same rate after

recloning of the mutant in CE cells or BHK

cells. Furthermore, the particles which resem-bled those of wild type were not revertants of

the mutants, because

they

formed small

plaques in CE cells. Therefore, we concluded

that the production oftwo

particle

typeswas a

function

of themutantgenomerather than that

ofthe host. The mechanism of the production of

both types of particles is not known at the

present time.

Apreliminary experiment showed that

infec-tivity of S127 mutant was about seven times

lower than that of wildtype whencomparedon

the basis of RNA content. This result shows

that most of the lower PFU to RNA ratio is

ascribed tothe multiploidnature ofthe

parti-cles. Aparticle with a few pieces ofviral RNA

can only makeone plaque. Furthermore, these

particles may have some disadvantages in the

replicating process.Thus limitedgrowth of the

virusmay result in developingasmall plaque.

ACKNOWLEDGMENTS

We aremostgratefultoT. Kitano,M.Kobayashi, and K. Hashimotointhislaboratory for theirtechnical advices. We are also indebted toMasako Wagatsuma for her excellent technical assistance.

A part ofthis investigationwas done in Department of Microbiology SchoolofMedicine ChibaUniversitywhile the authors(B. Simizu and S. Yamazaki) stayed there.

LITERATURE CITED

1. Acheson, N. H., and I. Tamm. 1967. Replication of Semliki Forest virus: an electron microscopic study. Virology 32:128-143.

2. Acheson, N. H., and I. Tamm.1970.Structuralproteins ofSemliki Forest virus and itsnucleocapsid. Virology 41:321-329.

3. Arif, B. M., and P. Faulkner.1972.Genome ofSindbis virus.J. Virol. 9:102-109.

4. Barron, A. L., and D. T. Karzon. 1965. Studies of mutantsof ECHO6 virus. 1. Biologic and serologic characteristics. Amer. J. Epidemiol.81:323-331. 5. Brown, D. T., M. R. F. Waite, and E. R. Pfefferkorn.

1972.Morphology andmorphogenesis ofSindbis virus as seen with freeze-etching techniques. J. Virol. 10:524-536.

6. Crawford, L.V. 1960. AstudyoftheRous sarcomavirus by density gradient centrifugation. Virology 12:143-153.

7. Fenner, F., and J. F. Sambrook. 1964.The genetics of animal viruses. Annu. Rev. Microbiol. 18:199-226. 8. Friedman, R. M., J. G. Levin, P. M. Grimley, and I.K.

Berezesky. 1972. Membrane-associated replication compex inarbovirus infection. J. Virol. 10:504-515.

9. Germanov, A. B., M. I. Sokolov, and N. A. Parasiuk.

1972.TheapplicationofDEAE-cellulose column chro-matography to the selection of S+ revertants and determination of reversionfrequencyin populationof S- mutants of fowl plague virus. J. Gen. Virol. 17:49-54.

10. Ginoza, W. 1970. The action ofradiation onviruses,p. 174-194.In H.Dertinger andH.Jung(ed.),Molecular radiationbiology.Springer-Verlag,New York. 11. Golde,A. 1970. Radio-induced mutants ofthe

Schmidt-Ruppin strain of Rous sarcoma virus. Virology 40:1022-1029.

12. Grimley, P.M.,J.G.Levin,I. K. Berezesky, andR.M. Friedman.1972.Specificmembranousstructures

asso-ciated withthereplicationof group Aarboviruses. J. Virol.10:492-503.

13. Gruber,J. 1971.ImmunogenicityofpurifiedVenezuelan equineencephalitisvirusinactivatedby ionizing radia-tion.Infect.Immunity3:574-579.

14. Heine, J. W., and G. J. Galasso. 1968. Effects of

X-1577 VOL.12,1973

on November 10, 2019 by guest

http://jvi.asm.org/

SIMIZUET AL.

irradiated aqueous solutions on vesicular stomatitis

virus. J.Virol.2:1147-1153.

15. Higashi,N., A. Matsumoto, K.Tabata, andY.

Nagato-mo.1967.Electronmicroscopestudy of development of

Chikungunya virus in green monkey kidney stable

(VERO) cells. Virology 33:55-69.

16. Hodes, H. L., H. D. Zepp, and E. Ainbender. 1960. A physical property as a virus marker. Difference in avidity of cellulose resinforvirulent(Mahoney) and attenuated (LSc, 2ab) strain of type 1 poliovirus. Virology11:306-308.

17. Huang, A. S., and D. Baltimore. 1970. Defectiveviral particles andviraldiseaseprocesses.Nature(London)

226:325-327.

18. Klimenko, S. M., F.I. Yershov, V. P. Gofman, A. P.

Nabatnikov, and V. M. Zhdanov.1965.Architecture of Venezuelan equine encephalomyelitis virus. Virology 27:125-128.

19. Martin, S. A., and H. J.Zweerink. 1972. Isolationand characterization of two types of bluetongue virus particles. Virology 50:495-506.

20. Marshall, L. D., R. P. Scivani, and W. C. Reeves. 1962. Variation in the size of plaques produced in tissue cultureby strainofwestern equine encephalitisvirus. Amer. J. Hyg.76:216-224.

21. Morgan, C., C. Howe, andH.M. Rose. 1961.Structure anddevelopmentofvirusesasobserved inthe electron microscopeV. Western equine encephalomyelitis virus. J.Exp. Med.113:219-234.

22. Moscarello, M. A., andM. E.Kaighn.1964.Theamino acid composition of three plaque-type mutants of encephalomyocarditis virus. Biochim. Biophys. Acta 90:161-163.

23. Mussgay, M.,and R. Rott. 1964.Studiesonthestructure

ofahemagglutinating component ofa groupA arbo

virus(Sindbis). Virology23:573-581.

24. Quersin-Thiry,L. 1961. Nutritiverequirementsofasmall

plaque mutant ofwestern equine encephalitis virus. Brit.J.Exp.Pathol. 42:511-522.

25. Schlesinger, S., M. Schlesinger, and B. W. Burge.1972. DefectivevirusparticlesfromSindbisvirus.Virology 48:615-617.

26. Simizu, B., and N. Takayama. 1969. Isolation oftwo

plaque mutantsofwestern equine encephalitis virus differinginvirulencefor mice. J.Virol.4:799-800. 27. Simizu, B., and N. Takayama. 1971. Relationship

be-tweenneurovirulence andtemperaturesensitivity ofan

attenuated western equine encephalitis virus. Arch. GesamteVirusforsch.35:242-250.

28. Simizu, B., and N. Takayama. 1972. Virulence of a

temperature-sensitive mutant ofwestern equine

en-cephalitis virus. Arch. Gesamte Virusforsch. 38:328-337.

29. Sreevalsan, T., and P. T. Allen. 1968. Replication of

western equine encephalomyelitis virus.II.

Cytoplas-micstructure involved in thesynthesis and

develop-mentofthevirions.J. Virol.2:1038-1046.

30. Strauss, J. H., Jr.,B. W.Burge,and J. E. Darnell. 1969. Sindbis virus infection of chick and hamster cells: synthesis of virus-specific proteins. Virology 37:367-376.

31. Summers, D. F., J. V. Maizel, Jr., J. E. Damell,Jr. 1965. Evidence forvirus-specificnoncapsid proteinsin poli-ovirus-infected HeLa cells. Proc. Nat. Acad. Sci. U.S.A.54:505-513.

32. Takayama, N. 1972. Further characterizationofan

at-tenuatedwesternequineencephalitis virus: searchfor in vitro markers. Arch. Gesamte Virusforsch. 36:363-371.

33. Takemoto, K. K.1966.Plaquemutantsof animal viruses. Progr.Med.Virol.8:314-348.

34. Tan, K. B. 1970. Electron microscopy of cells infected with Semliki Forest virus temperature-sensitive

mu-tants: correlation ofultrastructural and physiological observation. J. Virol.5:632-638.

35. Thiry,L. 1964. Someproperties of chemically induced small-plaque mutantsofNewcastledisease virus. Vi-rology24:146-154.

1578 J.VIROL.

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