Envelope proteins and replication of vesicular stomatitis virus: in vivo effects of RNA+ temperature-sensitive mutations on viral RNA synthesis.

JOURNAL OF VIROLOGY, Jan.1979,p.123-133

0022-538X/79/01-0123/11$02.00/0 Vol. 29, No. 1

Envelope

Proteins and Replication of Vesicular Stomatitis

Virus: In Vivo

Effects of

RNA'

Temperature-Sensitive

Mutations on Viral RNA Synthesis

CLAIRE MARTINET,* ARLETTE COMBARD, CHRISTIANE PRINTZ-ANE, AND PIERRE PRINTZ Institut deMicrobiologie, Universite de Paris-Sud, Centre d'Orsay, 91405 Orsay Cedex, France

Received for publication 19 May 1978

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to

complementation groups III and V were investigated for their in vivo RNA

synthesis. Thesucrose gradient patterns of the RNA species which theyproduced

at nonpermissive temperature

(39.2OC)

were systematically compared under

different experimental conditions: variationofinputmultiplicity andoftimeof infection, superinfection withTparticles, and temperatureshifts.Finally,amore

precise analysis of the various RNA species synthesized was carried out. It

appeared that thecharacteristics ofRNA synthesisspecifiedat

39.20C

bytsHI or

tsVmutantsdiffered from the normal RNA synthesis of vesicularstomatitisvirus

wildtype.Theircommon depression at

39.20C

invirion-like RNA (38S)

produc-tion-i.e.,so-calledgenomereplication-was tentatively paralleled with the

con-comitant ts eventswhich have been previously shown to affect the two viral envelope proteins. Anoverproduction of the RNA transcriptswasdescribed for

mutantsingroupIII andposed the question ofaregulationprocess todetermine

theamountofRNAtobe transcribed.

Inthe processof

elucidating

the

virus-speci-fied events in cells infected with vesicular

sto-matitis virus (VSV), much interest has been centered on temperature-sensitive (ts)mutants

of this virus. Uptonow, information about

mu-tantsclassifiedas

RNA' (able

tomakea

readily

detectable amount of

actinomycin

D-resistant RNA) was almost limited to

description

of the protein

synthesis they

induced at restrictive

temperature (21,

32).

Indeed,

onemaya

priori

assumethattheir RNA

syntheses

would be

fully

comparable

tothose of the wildtypeand there-fore that these groups are rather altered in a maturationstep late in theviral

cycle.

In

fact,

defects in

envelope

proteins

Mand G have been demonstrated forgroupsIII and

V,

respectively

(4, 9, 11,12,33).

Despite the sparseness of

published

results about the RNA

synthesis

induced

by

tsII and

t8Vmutants,atleastoneobservation

prompted

ustocharacterize inmore detail their intracel-lularRNAspecies:the

abnormally

highlevel of RNAsynthesis obtainedintsIII-infectedcellsat restrictive temperature, either in cumulative

measurement (20) orin a sucrose

gradient

pat-tern (29).

Therefore,

a

comparative

analysis

of

the RNA

syntheses

induced

by

tsIII and tsV

mutants was systematically

conducted,

and an

alterationintheirsyntheseswasdemonstrated.

MATERIALS AND METHODS

Cells and viruses. AU the analyses of RNA syntheses in vivowereperformedonHeLacell mono-layers grownasalready described (22) andonchicken embryo cultures whenalow level of residualcellular proteinsynthesiswasrequired.

VSV (Indiana serotype) strainsinvestigated were:

awild type which gives twiceasmanyplaquesat 30 as at39.20C,parentofthe mutants; tsmutantsbelonging

tocomplementationgroupm [ts023(III),

ts076(Il),

and

ts089(Ill)],

andts mutantsbelongingto

comple-mentation group V [tsO45(V), ts044(V), and

tsO110(V)]. Allmutantstocks, containing only

stan-dard Bparticles,werechecked forlow percentage of

revertants and low residualgrowthat39.2°C andby

complementationtestsasreported previously(22).

Defective interferingTparticleswerepreparedby

infecting chicken embryo monolayers with a third

high-multiplicity passage ofwild-type VSV. After a

low-speedcentrifugation toremove cells and debris,

the virions(Band Tparticles)werepelletedat33,000

rpmfor 1 hat40CinaSpinco45 rotor.Thepellets

weresuspended in asaline buffer (0.01 M Tris [pH

7.4], 1 M NaCl, 1 mM EDTA).Thesuspensionwas

layeredonto a 5 to40%sucrosegradientinthesame

buffer andcentrifuged (18,000rpmfor1.5hat4°Cin

a SpincoSW25 rotor). The band of Tparticleswas

removedbysuction, andtheyieldof Tparticlesinthe

preparationwas calculated in equivalent PFU from

optical densityat 260nm (7). The Tparticleswere

checked for homogeneity in size, corresponding to

shortTparticleswith19SRNA.

123

on November 10, 2019 by guest

http://jvi.asm.org/

124 MARTINET ET AL.

Sucrose gradient fractionation of uridine-la-beledproducts. HeLacells wereinfectedatthe mul-tiplicity specified for eachexperiment and treated with actinomycin D. After labeling with [3H]uridine (20

MCi/ml), cytoplasmic

extracts were prepared as

al-readydescribed (22). Fromthisstarting materialtwo

differentanalyses could be conducted.(i)RNAspecies

weresolubilized from thecytoplasmic extractby ad-dition ofsodiumdodecyl sulfateto afinal

concentra-tion of1%;theywerefractionatedby centrifugationin a15 to 30% sucrosegradientat21,000rpm for 15 hat

17°Cin aSpinco SW27.1rotor,asdescribedelsewhere (2).(ii) Thecytoplasmicextractwasalsofractionated

on a 15 to30%sucrose gradient inhighsalinebuffer

(0.01 MTris[pH 7.4],0.5 MNaCl,0.05 MMgCl2at 27,000 rpmfor3h at4°C in the Spinco SW27.1rotor.

Determination of the radioactivity in an aliquot of each fractioncollected enabledus tolocate the

poly-somes (positioned onboth sides of the 120S nucleo-capsidpeak) and the free mRNA present as ribonu-cleoprotein structuresmigratingjust above the ribo-somal optical density peak. Fractions were pooled (polysomeson theonehandand free mRNAon the other); the RNAwasphenolextracted(22), and

poly-adenylic acid-containing mRNA's were isolated

ac-cordingtotheiraffinityforoligodeoxythymidylicacid [oligo(dT)]-celluloseasdescribed below.The mRNA's

were analyzed on a 15 to 30% sucrose gradient in

sodiumdodecyl sulfateasusual.

Analyses of viral RNA. After separationon su-crose gradients, the various VSV RNA species were

analyzedin severalways.

(i) Hybridization. Samples of[3H]RNAmeltedat

100°C for2minwerehybridized with unlabeled RNA extracted withphenol frompurifiedvirions.Annealing

was conducted in0.01 M Tris-0.5 M NaCl, pH 7.4

(final volume, 40 d), in the presence of 0.5

Pi

of diethylpyrocarbonate. Annealing was performed at

70°C for atleast 24 h. Then the amount of RNase-resistant radioactivity in each annealedsample was

determined aftera30 mindigestion with pancreatic

RNase A(10lg/ml,finalconcentration).

(ii)Melting. Samplesof[3H]RNAwereheated in 90%dimethyl sulfoxide for4min at60°C toseparate anypossiblecomplexedRNA strandsfromeach other

(31). ThenRNAswererecoveredby precipitationwith 2volumes ofethanol in the presenceof0.2 MNaCl

and separation on a 15 to 30% sucrose gradient as

describedabove.

(iii)Bindingto oligo(dT)-cellulose. Samples of [3H]RNAweremixedwitholigo(dT)-cellulose (0.2g) in0.01 MTris-hydrochloride (pH7.4)-0.5 MKCl.The reaction begansimplyas a batch kept for15 min at room temperature; then thesuspension was poured

intoasmallcolumn,andthefirst effluentwaspassed twice more overthe column. The column wasthen washed with about 4 ml of 0.01 M Tris-0.5 M KCl

while a slow flow rate was maintained throughout.

Adsorbed RNAwaselutedwithabout 1 ml of 0.01 M Tris. The measurement of theacid-precipitable

radio-activityofeach fraction madepossibletheselectionof

appropriate fractions, which werepooled and

even-tuallyanalyzedona15 to30%sucrosegradient.

(iv) Methylation. Infectedcells weremaintained

attheappropriate temperature (30 or 39.2°C) for 2 h

inEagleminimal essential medium withonly 10 ttM

methionine butsupplemented with 10jig of

actino-mycin D per ml and 2% fetal calf serum. At 2 h

postinfection (p.i.), cells were labeled with 10 yM

[methyl-3H]methionine (160 .tci/ml)inthe above me-dium without coldmethionine but in thepresence of 10mMsodiumformateand 10AMguanineand ade-nine. Inparallel, cultureswere labeled with

[3H]uri-dine (20,uCi/ml) inthesame mediumadjustedto 10

,M withmethionine.At4 hp.i.,cellswerescrapedoff the sides ofthe flasks and treated withproteinase K

(100lg/ml) for 1 h at 37°C, and RNA was twice extracted with phenol-chloroform and then alcohol

precipitated.

Analysis of viralproteins. Conditions for

infec-tion, labelingofviralproteins in chickenembryo cells, andtreatment of thecytoplasmicextractbefore elec-trophoresis,as well astheelectrophoretic procedure,

wereexactly thesame aspreviously described (2). Chemicals. Additionalcompounds,notmentioned

previously (2, 3, 22),weredimethylsulfoxide, fromE. MerckAG, Darmstadt, Germany;oligo(dT)-cellulose,

from Collaborative Research Inc., Waltham, Mass.;

anddiethylpyrocarbonate, from Hopkins & Williams, Chadwell-Heath, England.Actinomycin Dwas a

gen-erous gift from MerckSharp & Dohme, West Point,

Pa. Radioisotopes were obtained from the

Commuis-sariata l'EnergieAtomique, Saclay,France.

RESULTS

Results reported below refer to mutants

tsO23(III)andts045(V).It mustbepointedout

that within each of these complementation

groupsonlyonephenotype has been constantly

observed, whatever the mutant considered:

tsO23(III), tsO76(III), or tsO89(III) and

ts045(V), ts044(V),ortsO110(V).

Characteristics of RNA synthesis

in-duced at 39.2°C by mutants ofgroups m

and V. (i) Global pattern in sucrose

gra-dient. Auniformlabelingfrom 1 to 4 h p.i. with

[3H]uridinewasappliedtoequivalentbatches of

cells infected in duplicate with tsO23(III) or

tsO45(V)andmaintained at either 30 or 39.20C.

Cytoplasmic viral RNAs were analyzed by

su-crose velocity gradients. At 39.2°C both

tsO23(III)andtsO45(V) synthesized all the

char-acteristic RNA species normally seen at the

permissive temperature, butwithdifferent

pro-portions (Fig. 1). The levels of synthesis at

39.2°Cof the 13-15 and28S RNAs were approx-imately the same as those at 30°C. Some

in-crease at high temperature could even be

de-tected inthe 13-15S peak. Conversely, the

syn-thesis of the 38S RNA seemed to be depressed

at39.20C.Thesemodificationsarecharacteristic

oftsO(III) and tsO(V) mutants; in the case of

infection withwild-type VSV, the RNA profiles

wereonly slightlymodified with the increase of

temperature (data not shown).

(ii) Effect of input multiplicity on RNA

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

ENVELOPE PROTEINS AND REPLICATION OF VSV 125

multiplicity dependent,

and

they

became dis-2 tsOV 45

tinctly higher

than the

wild-type

ratio above the moderate

multiplicity

values that we

usually

used

(50

PFU per

cell). Looking

to the

corre-sponding

profiles,

we observed that the

over-production

here illustrated at

39.2°C

was

fully

1 It V1 due to excessive synthesis of the

so-called

mRNA's

(see,

e.g.,

Fig.

1),

especially

for the

tslI

mutant. In all cases the

38S

peak

was much more moderate at39.2 thanat

300C

andnever

accounted for the observed increase of the

39.2/30°C

ratio.

However,

the amount of 38S

to

, , , RNArose asthe

multiplicity

wasincreased.

_tsO IU 23 [1

(iii)

Effect of time on RNA

synthesis.

Since

X tsO IN 23 a long labelingperiod represents a steady-state

X. t

\condition,

shorter

pulses

were then

performed.

2 At different_uridine

times

of the infection cycle,

[3H]-wasaddedtothe

medium,

and the total

resulting

labeling

in each RNA

category

was

measuredastheareaof each

gradient peak.

This

alloweda

ready

comparison

of the

hourly

rates of

synthesis

of

38, 28,

and 13-15S at 30 and

1

39.2°C

by

eachof thetwo mutants

(Fig.

3).

The

curves obtained at

300C

for 13-15S

illustrated

the well-known

predominance

of the mRNA's

during

the first

stages

of the

normal

cycle

(24,

26).

The 38S RNA

emerged

more

slowly

and

culminated

rather

late,

just

before

being

released

10 20 30 40 as

part

of the virions. Because of their

similarity,

FRACTIONS results obtainedat39.2°Cwith either

ts023(Il)

FIG. 1. In vivo RNA synthesis directed by

ts023(III)

andts045(V).HeLacellswere

infected

for 2

45minatroomtemperaturewith eitherts023(III)or

ts045(V) (100PFUpercell). Thentheyreceived

com-plete Eagle essential medium with 10pgof

actino-mycinD perml,

[H]uridine

(20

ACi/mi)

from1 to4

hp.i. Cellswerewashed,scraped

off

the

flasks, pel-

/

leted, anddisruptedinreticulocytestandard

buffer.

Thensodiumdodecylsulfatewasadded

(1%[wt/volJ

z /

finalconcentration)tosolubilize

cytoplasmic RNAs,

whichwerethen analyzed byvelocity centrifugation ina15to30%sucrosegradientwith sodiumdodecyl

sulfate at21,000rpmfor15h in theSpincoSW27.1

_-rotor at 17°C. Arrowson thefigure show thepeak

positionsofthe 18and28S ribosomal markers

from

HeLa cells. Symbols:0, 30°C;

0,

39.20C. d

20 100 200 40000O iO

synthesis.

The influence of the

multiplicity

of MULTIPLICITY OF INECTIO0

infectionon anextended

labeling

asabove

(1

to FIG. 2. Effect of

multiplicity

of infection on total 4 h

p.i.)

was

investigated

for both groups of RNA synthesis by mutants

from

groups IIIand V. mutants.

By

summing the total

radioactivity

For eachmultiplicity of infectionused,the amount

of

within each of the three main

peaks

in the viral RNA was calculated at either 30or

39.2°C

by

gradient

profiles,

we could evaluate theratioof summing the total radioactivity of the three main the RNA induced at 39.20Cto that induced at peaks observedin a sucrose gradient profile as in

300C and follow its variation versusthe multi-

Fig.

1. The evaluated ratio of counts per minute at

plctfinfection (Fig. 2). The ratio for our

39.20C

to countsperminuteat

300

Cwasthenplotted

plicity

ofsinfein

independe

ofor

mul versus the multiplicity of infection.

Symbols:

0,

wild-type

strain,

being

independent

of the mul-

ts023(III);

0,

ts045(V).

The broken line corresponds

tiplicityover a widerange, was taken as aref- to the value observed for a wild-typestrain close to erence

(equal

to

=0.6).

Strikingly,

for both

group

the strain

from

which themutants wereisolated and

IIIandgroup Vmutantsthe ratioswere

strongly

whichwas not

completely

heat-resistant.

VOL. 29, 1979

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.501.74.215.67.370.2] [image:3.501.255.449.377.541.2]

J. VIROL.

126 MARTINET ET AL.

10

'C~~~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~~~~~.

5

,,,,~~~~~~~I,i,L,

[image:4.501.59.248.57.306.2]

1 2 3 4 1 2 3 4 1 2 3 4 HOURS p.i

FIG. 3. Effect oftimeonthesynthesis ofeachRNA

species. HeLa cells were infected with either

ts023(III)orts045(V) (multiplicityof infection = 100

PFUper cell).After adsorptionatroomtemperature,

the cells received complete Eagle medium supple-mented with actinomycin D. For each mutant, two seriesof monolayers were used: one was incubated

at 30°C, and the other was incubated at 39.20C. [3H]uridine(20jiCi/ml)wasaddedatdifferenttimes ofinfection, and cytoplasmic extract wasprepared

after 1 h oflabeling. RNAs were solubilized and

layeredontolinear 15to30%osucrosegradients. The

totalradioactivitywascalculatedforeachpeak

ob-served aftergradientfractionationandwasplotted

atthetimevaluecorrespondingtothemeantimeof

the uridinepulse considered. Symbols: 0, 30°C, 0,

39.20C.

or tsO45(V) may be now discussed together.

During the first hours (up to2 to3 hp.i.), the

synthesis of all viral RNAs was impaired at 39.2°Cwithoutaselective inhibitionofanypeak and thus led to a qualitatively normal RNA

profile. Later in the cycle, this normal

equilib-rium between the three main RNA peaks

be-came

progressively

alteredinthefollowingway.

First,noincreasedratecould beobserved in 38S

formation: this RNAappearedmerelyata

con-stant restrictedrate. Onthecontrary, mRNA's weresynthesizedwithincreasing speedand

be-came several times more important than they

wereatpermissivetemperature.Onemaynotice

the different slopes of thecurvesof the 13-15S

RNA, which showed again that mutant

tsO23(III) synthesized larger amounts of this

RNA than didmutantts045(V).

Question of a replicative defect in tsHl

and tsV mutants. The

observations

reported

above have

demonstrated

thedeficientamount

of38S RNA

produced

at 39.2°C

by

either

tsIII

or tsV mutants. The

possibility

of an actual

defective step in the

replication

process of the viralgenome

will

nowbe

examined.

(i)

Superinfection

withT

particles.

Inter-ferencewith the

production

of viral

standard

B

particles

ofVSVbyits T

particles

is

thought

to

occur via a

competition

atthelevel ofthe

rep-lication ofthe twotypes ofgenomes (7, 16, 18).

This is

characterized

inviralRNA

synthesis

by

theappearance inthe

gradient

profile

ofa

pre-dominant

19S

peak,

yielding

new

T-particle

ge-nomes (26). Such a deviation is shown in the

control

(wild

typeormutantsat

permissive

tem-perature)

panel

ofFig.4.Inthis

experiment

the

superinfection

with T

particles

wasdone as

in-dicated

by

Stampfer

etal.

(26):

T

particles

were added2.5 haftertheB

particles

at an

equivalent

multiplicity

to that of the B

particles

already

adsorbed.

Cells

were

exposed

to

[3H]uridine

from 3 to6hp.i.Wedidnotobserve

(Fig.

4) any

change at

all

in the 39.2°C RNA

profiles

of tsO23(III) ortsO45(V),and no inhibition ofthe

exaggerated

13-15SRNA

synthesis

or

accumu-lation of T genomes could be detected, even when the resolution of the

gradient

was

im-proved

byextendingthe time of

centrifugation

(bottom

panels).

Therefore, at

nonpermissive

temperature the

replication

ofT

particles

was

prevented

in

tsO23(III)-

or

tsO45(V)-infected

cells.

Thisresult arguesforanalteration of the replicative process of these mutants. The two

following experiments

were

performed

to find

outwhich

possible

stepcouldbe

impaired.

(ii) Melting of28S RNA material. A first

explanation

oftherestrictedappearance ofthe

genome-like

RNA among the RNAsynthesized

by tsO(III) or tsV mutants was a piling up of

most of the 38S RNA. This might occur by

prevention

of itsleaving thepool of

replicating

andtranscribing complexes present in the 28S

regionofa

gradient

(25, 26). Therefore, after a

labelingfrom 2 to 5 hp.i.,cytoplasmic extracts

from

cells

infected with either tsO23(III) or

tsO45(V)werefractionatedinsucrosegradients

asusual. The fractions sedimenting inthe 28S

regionwere

pooled

and

separated

into two

ali-quots,oneof which was melted in 90%dimethyl sulfoxide before being ethanol

precipitated,

as

was done directly for the other. Both

precipi-tatedRNAswereanalyzedin 15 to30%sucrose gradients (Fig. 5). As a control, the 28SRNAs

synthesized

at 30°C by each mutant showed

aftermelting the sameproportionofradioactive

38S RNA as is

normally

presentinsuch

mate-rial. In case of infection at 39.2°C by either

mutant, the melting procedure released

on November 10, 2019 by guest

http://jvi.asm.org/

ENVELOPE PROTEINS AND REPLICATION OF VSV

FRACTIONS

FIG. 4. SucrosegradientpatternsoftheRNA synthesizedintsmutant-infectedcellssuperinfectedwith

VSV Tparticles. HeLa cellswere infectedwith eithertsO23(III), tsO45(V), orwild-type(WT) Bparticles (multiplicity of infection=50PFUpercell)andincubatedat39.2°C. After2.5hp.i.themediumwasdiscarded

andthecells weresuperinfected with defective interfering Tparticlesatanequivalentmultiplicitytothat of

theBparticles; half ofthecultureswereparallely mock infected with saline buffer. Thirty minutes later, the

inoculumwassubstitutedwithmediumcontainingactinomycin D.At 3h afterthefirst infection,[3HJuridine wasadded, andcellswereharvestedat6hp.i.CytoplasmicextractswereprocessedasforFig. 1. In thecase

ofthethree bottompanels,the RNAswereanalyzedin thesame sucrosegradientbutat33,000rpmfor17 h

inaSpincoSW41rotor at17°C. Symbols:0,single infection; *,superinfectionwithTparticles.

8

4

03

2

[image:5.501.105.388.65.306.2]

10 20 30FRACTIONS10 20 30

FIG. 5. Analysisofproducts obtained bymelting

28S RNAs. Viral RNAs induced by tsO23(III) or

tsO45(M

and labeled with[3H]uridine from2to 5 h

wereanalyzedasindicated inFig.1.After localiza-tionofthe28Speak byprecipitatingaliquots along thegradientwithtrichloroaceticacid, thefractions

tially light RNAs (i.e., <38S) andnot-ascould

beexpected-an increasedamountof 38S RNA.

The significance of this result could be

rein-forced when the RNA complexes recovered in

the 28S region were shown not to be artifacts

arising from a random association during our

wholeexperimentalprocedure. Indeed, addition

of radioactive 38S RNA extracted from VSV

purified virions to cold VSV-infected cellsjust

atthe timewhentheywerescraped into

reticu-locyte standard buffer before beingsubmittedto

theusualextraction-fractionationprocedure did

notlead to the appearance ofradioactivity in

ofthisarea werepooledandseparatedinduplicate:

one wasdirectlyprecipitated byadditionof2volumes

of ethanol, whereas the otherfirstreceived

dimeth-ylsulfoxidetoafinalconcentrationof90% andwas

melted at 60°C for4 min. Both aliquots were

sus-pended in reticulocyte standard buffer-1% sodium

dodecyl sulfateandanalyzedon sucrosegradientsas usual. Cold ribosomalmarkerswereaddedtoeach

gradient. Symbols: 0,original28SRNA; 0,melted 28SRNA.

lSs

Li1

ts0 IN 23

1

tsOV45

31C4c

3- i i

~

~~~39.2'C

4 4

I

CA

Ie

127

VOL. 29,1979

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.501.55.244.407.593.2]

128 MARTINET ET AL.

the28S region ofa 15to30%sucrose gradient.

From this experiment, the hypothesis ofsome

hidden38S RNA within the 28Scomplexes has

toberuledout.

(iii) Polarity of RNAs synthesized at

39.2°C by tsO23(III) and ts045(V). If the

replicative defect demonstrated above forboth

group III and group V mutants consisted in a

deficient 38Sgenome completion, accumulation

ofnegative strands could be expected in other

regions of thegradient. Therefore, the polarity

of the molecules present in each radioactive

peak detectedat39.2°Cwasdetermined by

an-nealing them with unlabeled virion genome,

after melting of the radioactive material. The

comparison (Table 1) of the negative-strand

yields thus obtained with the normalones (i.e.,

in the wild type at 30 or 39.2°C or in each

mutant at 30°C) didnot reveal anyspecial

ac-cumulationat39.2°C ofnegative strands in each

RNAcategoryofthe15 to30%sucrosegradient,

whatever the mutant used. In fact, more 38S

RNA could be annealed toviral genome than

normally when it had been synthesized at

39.2°C. But this was notreally significant,

be-cause-despite the special care taken to avoid

contamination with short nascent RNA

strands-the 38S RNA analyzed presented

about 11%self-annealing.

Effects of temperature shifts. The use of

temperature shifts demonstrated successfully

that the G and M proteins behave abnormally

at 39.2°C during the maturation of tsO23(III)

and ts045(V), respectively (11, 12). Therefore,

similar temperature shifts were imposed on

tsO45(V)- or tsO23(III)-infected cells, and the

viral RNA syntheses were then analyzed. For

bothmutants,wecompared (Fig. 6) the profiles

oftheRNAs labeled from 5 to 6.5h p.i. under

threedifferent conditions: (i)labelingat30°C in

cellsmaintainedatthistemperaturethroughout,

(ii) labeling at 39.2°C in cells which had been

transferred from 30to 39.2°C at 2.5h p.i., and

(iii) labelingat30°C incellswhich hadjustbeen

incubatedbackat30°C afterafirstshift from 30

to39.20C. Attention hastobepaidtothe

differ-ence of the scale used for each mutant.

Com-paredtonormal 300Cprofiles (whichwerevery

similar for both mutants), the simple shiftfrom

30 to 39.20C induced a marked change in the

RNAprofiles of bothmutants,wherethe

alter-ations already described were particularly

sig-nificant: an evident disequilibrium took place

which favored the transcribed RNAsatthe

ex-pense of 38S RNA accumulation (especially

weak). In thecaseofts023(III), the 13-15Speak

couldamount toasmuchas20 times itsnormal

yield, even by 1 hafter the temperature shift.

TABLE 1. Annealingof intracellular viral RNAs with virion RNA'

RNaseresistance(%)b

Virus Type of labeled Afterannealing

RNA

Beoane-

with

genome

Self-annealing

ing RNA

Wildtype at 30 or39.2°Cormutants at 13-15S 7+10 99±8 9±6

300C 28S 12±8 88±10 26±5

38S 2±5 26±7 30±4

ts023(III)at39.20C 13-15S 2±3 100±5 8+ 5

28S 13±5 100±9 21±7

38Sc 11±7 40±2 11±9

tsO45(V)at39.2°C 13-15S 9±2 89±8 10 ± 14

28S 15±3 78±12 22±5

38Sc 7±5 35±7 16±6

Afterextraction and isolationof3H-labeledviral RNAs asdescribedin Fig. 1,materialfrom each peak (at

least1,500cpm) wasmixedwithunlabeledRNAextractedfromVSVvirions.Annealing wasallowedat70°C for atleast24h, andtheproportionofradioactivityrenderedresistant to a30-mindigestionwithpancreaticRNase wasestimatedbyacid precipitation.Theexcess of genome RNA was checked byhybridizationofincreasing levels ofthis one withtotal labeledinfected-cellRNA. The valueobtainedfortsO23(III)-infectedcells at 39.2°C

beingthehighest, it was chosen as abasisto calculate thequantity of genome RNA to be added in each

hybridization sample(=3,ug).

bMean values of several

experiments.

cThe 38Speakwasalwayscontaminatedwith short nascent RNAs

essentially

ofpositivepolarity. In the caseofthepoorly defined38S RNApeak observedduringanalysisof the RNAsynthesizedat 39.2°C by either mutant,thiscontaminationwasespecially inconvenientforsubsequent characterization.Therefore, the fractions

corresponding to the 38S zone in afirst gradientwerepooled, meltedin 90% dimethylsulfoxide, and then

fractionatedonasecond15to30% sucrosegradient.The onlymolecules migratingagain as 38Smaterialwere

annealed.

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.501.66.452.382.531.2]

ENVELOPE PROTEINS AND REPLICATION OF VSV 129

,31c 39o2c

~~~~~~~~x

31oc 3i92c

oO~ ~ ~5E~S ~~~~2

01 2 3 4 5 6h a.

*dsorption

10 20 30 FRACTIONS 10 20 30

FIG. 6. RNA species synthesized bytsO23(III)- and ts045(V)-infected cells underdifferent temperature

conditions.After infection with ts023(III)orts045(V),actinomycin D-treatedcells wereincubatedat30°C.At

2.5andthenat5hp.i., the cellswereshiftedupto39.2°Cand then downto30°Casindicatedin theleftpanel.

Allsampleswerelabeled with[3H]uridine(20

ACi/ml)

from5to6.5hp.i.Cytoplasmicextractswereprepared

and analyzed as in Fig. 1. Symbols: 0, permissive temperature; 0, upward shift conditions; A, doubk

temperatureshift.

By extending the timne centrifugation of the

gra-dient, the various RNA species inthe 13-15S

peak could be resolvedas three distinct peaks

(i.e., 17, 15, and llS), which had the same in-crease (Fig. 4, bottom panels). Except for this

difference inmagnitudeofRNAproductionafter

the shift, the profiles for both mutants were

rather similar. On thecontrary,thetwo mutants completelydiffered in thecaseofadouble tem-perature shift. In ts023(III)-infected cells,

al-teredRNAsynthesis persisteddespitethereturn

to permissive temperature. Conversely, when

shifted backto

300C,

the

tsO45(V)-infected

cells

ledtoarapid change towardsa newunbalance

between the three peaks of the RNA profile,

yieldingatremendous amount of38S RNA at theexpenseof the 13-15S RNA. An actual burst in 38S RNA led to the normal level of 38S

accumulation within 1.5 h.

Overproductionof RNAby tsmmutants

at39.20C. The temperatureshiftup justshowed

moredramaticallythe excessivesynthesisof 28

and 13-15S RNAs by tsO23(III) at 39.2°C, as

described aboveonseveral occasions. The

ques-tion arises whether this really concerned the

transcription process,which would be exagger-ated owingto the defect ofgroup m mutants.

First, the possibilitywasruled outthat the in-crease of radioactivity in the 28 and 13-15S

species made at 39.20C was simply due to an increase of the specific radioactivity, as that

could besuspectedwithregardtotheabnornal

effect ofgroupIIImutantsonthepermeability

of chickenembryocellstouridine(5)at39.20C.

Infact, this phenomenonwasnotobserved on

HeLa cells (5; personal observation);

further-more,byextendingthespintime(asforFig. 4),

the viral RNAs in 13-15S peak could be

sepa-rated from the 188 ribosomal RNA, and the

ratio ofcountsperminutetoopticaldensitywas

found to be identical for viral RNA peaks at both30and39.2°C (datanotshown).Therefore, at39.2°C themutant tM023(I) actuallyinduced thesynthesis ofanexcessnumber of13-15S,as

of28S, molecules. They were then examinated

fortheirputativemessengercharacter.

(i) Analyses of28and 13-15S RNAs.One

ofthe characteristics ofVSV mRNA's is their complementaritytothe viriongenome.InTable 1,onecan seethatthe positive polarity of28and

13-158 RNAs induced at 39.20C by ts023(II)

wasconsistent with this criterion. Asasecond

characteristic, the presence of polyadenylated

tails was checked by chromatography on

oligo(dT)-cellulose, andtheamountof polyad-enylic acid-containing RNAs in the 28 and 13-15S material recoveredat39.20C (from 1to

3hp.i.) wascomparedwith thenormalamount

observed at 300C. Virtually no difference in

thesetwoproportionswasrevealed(Fig. 7). One

may conclude from these tworesults that the

overproduction of 28 and 13-15S RNA by ts023(III)at39.20Cwasrelated withtheprocess oftranscription.

(ii) Translation.Toseewhetheranexcessof

proteinsynthesistookplaceconcomitantly,cells

were infected with ts023(llI) under the same

conditionsasinFig. 7,and viralprotein synthesis

was followed by incorporation of [3H]leucine

(Fig. 8). Approximatelythesameamountofeach

viralpolypeptide (exceptforthe Lprotein,but

this is a general feature of all mutants) was

translatedat30 and

39.20C,

despitethe double

amountof available mRNA observedat

restric-31°c O

avL__ I I .,.^ F

VOL. 29, 1979

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.501.50.444.64.212.2]

130 MARTINET ET AL.

E_0.

39.2°

C

20

10

10 20 30

FRACTIONS

FIG. 7. Sucrosegradientanalysis

ofpolyadenyla-tedRNAsynthesizedbyts023(III)at30and39.2°C.

Total cytoplasmic RNAs from cells infected with

tsO23(III)at30 or39.20Cwereeachdivided intotwo

aliquots. One aliquotwas directlyanalyzed by

su-crose gradient centrifugation; the other one was

loadedontoanoligo(dT)-cellulosecolumntoadsorb the RNAcontainingpolyadenylatedtracks.After elu-tion, these RNAswereanalyzedincomparisonwith their counterparts. Symbols: 0,totalRNA; 0, poly-adenylicacid-containingRNA.

tive temperature in this

experiment.

Thisseems

to be correlated with the distribution of the mRNAmoleculeswithinthe

cytoplasm. Indeed,

two

pools

of viral mRNA have been demon-strated: (i)

polysomal

and

(ii)

free mRNA in

ribonucleoprotein structures

(6).

Therefore,

we

prepared

bothfractions from

tsO23(III)-infected

cells as described in Materials and Methods.

From the RNAs extractedfrom both fractions,

mRNA'swere

separated according

totheir

bind-ingto

oligo(dT)-cellulose.

Results(Table 2)

ob-tained at

300C

showed the normal distribution

of RNA

transcripts:

about 20%weretrappedas

polysomes, whereas most ofthe molecules

re-mained free in the

cytoplasm,

inaccordance with

Huang et al. (6). This proportion wasnot

pre-served in thecaseof infection withtsO23(III)at

39.2°C:no moremRNAwasfoundaspolysomal

RNA,but theamount of free mRNAwastwice

that observed at

300C.

As

methylation

of viral

mRNAseems tobeanimportantfactor for their

subsequent translation, at least in vitro (1, 28),

we checked the extent of methylation of the

mRNAsynthesizedat39.2°C bytsO23(III) (Fig.

9). Whereas in this experiment the mRNA's

10 20 30

FRACTIONS

40 50

30

20

100

x

0,

30

20

[image:8.501.67.251.50.338.2]

10

FIG. 8. Synthesis of virus-specified proteins in cells infected with ts023(III). Monolayers infected with tsO23(III) werecovered with 1/10(vol/vol) di-luted minimal essential medium and incubated at

either 30 or 39.2°C. [3H]leucine (10 uCi/ml) was

added 1.5 h later. Cells were harvested at3 hp.i. Proteinswereextractedfrom the cytoplasmiccontent

of disrupted cellsbyboilingwith10%oacetic acid-0.5 Murea-1% sodiumdodecylsulfate-0.1% 2-mercapto-ethanol. Extracted3H-proteinsweresubjectedto elec-trophoresis in 7.5% neutral sodium dodecyl sulfate-acrylamide gels along with "C-proteins

ex-tracted from purified virions as markers (arrows). The insertshowsthe RNAsyntheses under thesame

conditions at30°C(-- -)or39.20C ( ).

TABLE 2. Locationof mRNA in

tsO23(III)-infected

cellsa

cpm

Location Temp

Oligo(dT)-(OC) Total cellulose bound

Polysomes 30 19,600 1,400

39.2 12,000 1,300

Free ribonu- 30 74,000 6,900

cleoprotein

39.2 153,000 13,500

aCytoplasmic extracts from tsO23(III)-infected

cellsat 30and39.2°Cwerefractionatedon 15to30%

sucrosegradients inhigh saline buffer(27,000rpm,3

h, 4°C intheSpinco SW27 rotor). Fractions lighter than the 80S ribosomal peak and fractions of the

polysomalarea werepooled separately,and their RNA wasextractedaccordingtothephenol method.Their content in mRNAwas estimated by the amount of RNA boundtooligo(dT)-celluloseasinFig.8.

RNA NS 39'2C

RbA~~~~~~~~NS

'-I

Lc

30'C

tI

! *as ,', , X

oXWoeo o.owO,o, o

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:8.501.262.458.123.305.2]

ENVELOPE PROTEINS AND REPLICATION OF VSV 131

N*0 a

FRACTIONS

FIG. 9. Velocity sedimentation analysis of

meth-ylated viral RNA fromts023(III)-infectedcells. Four

batchesofHeLa ceUswereinfectedwith

ts023aIII)

(=50PFUpercell);twowereincubatedat30°C,and

the otherswereincubatedat39.20Cfor2h inEagle minimalessential medium withonly 10C2mM methi-onine, 2% fetal calfserum,andactinomycinD.Labels

([3H]uridine or[methyl-3H]methionine) were

intro-ducedat2hp.i. inthesamemediumsupplemented with 10mM sodiumformateand 10 l guanineand

adenine. At4hp.i.theRNAsweredeproteinizedwith

proteinase K and then twice extracted with

phenol-chloroform. Theywereanalyzedon15to30%

sucrose gradients as usual. Symbols: 0, 30°C; 0,

39.20C.

made at 39.20C were at least six times more numerousthan thosemade at

300C,

theyieldof themethylgroupstheybore didnotexceedthat

observed at 300C. Moreover, the methylated

mRNA ratio at 39.2 to 300C wds only 0.5,but

this reflected only a difference in methionine

metabolism between thetwotemperatures,asit

could be seen on the figure at the levelof the

tRNA peaks and as we observed for the wild-typeVSV(datanotshown).

DISCUSSION

ts mutants of groups III and V induce at restrictive temperature a large amount of viral RNA, which determines their RNA' phenotype (10). After sucrose gradient analysis, however,

an abnormal profile is observed for the RNA synthesized at39.2°C: for both groups the rela-tive proportion between the three main RNA species, i.e., 38, 28, and 13-15S RNA, ischanged whencompared with what is seen at

30°C.

The patternobtainedresembles thatdescribed under othercircumstances with mutants or even with the wild type (2, 17, 30) andshows also a dimi-nution ofthe yield of thevirion-like RNA, coun-terbalanced by enhancement of the RNA

tran-scripts.

Such a result has

generally

been ex-plained by the existence of anequilibrium be-tweengenomereplication and transcription (17, 22), which arethought to share the same tem-plate and the same enzyme(s). In the case of tsIIIand tsVmutants, high temperature appar-ently promotesupsetting of the normal equilib-rium. The quantity of the 38S RNA, which is the final replication product, is reduced, more especially as the absence of budding out at 39.20C would have lead to its expected accu-mulation. Further

analyses

here reported dem-onstrate that the paucity of the 388 RNA ob-served ina

gradient profile

cannotbe related to

a

trapping

of this RNA species among other viralproducts. Therefore, the synthesis of new genome RNA looks tobe

really

affected when infection with tsIIIortsVmutantsis carried out

at39.20C. The

inability

of

superinfecting

T par-ticles to

replicate

at 39.2°C when mixed with

tsHI ortsVmutantssustains the idea that

rep-lication of these mutants isdeficient at restric-tive temperature. Kinetic curves enable us to

explain

more

precisely

what occurs at 39.20C: there isnot an arrestof the

synthesis

ofthe38S RNA

but,

ratheranabolishment of the normal amplification of the

replication

process

by

limi-tation of the number of

synthesized

molecules. The rate of

synthesis

seems to berestrictedto

itsinitial valueas

if,

just

after the entry of the

parental

virions,

the

replication

round

began

nornally

at

39.20C

within thecell.An

interpre-tation of these observations is to suppose two

statesof the defective

polypeptide

ingroup III

andgroupV mutants

depending

onthe

temper-ature at whichtheyhave beensynthesized: the

alteration of the new

polypeptides synthesized

at

39.20C

would lead to a restriction of the

synthesis of new VSV genomes, whereas the

parental proteins

brought

by

virions grownat

300C

would operate

normally

in this process. Thislatter

assumption

is favored

by

two

obser-vations: for both tsIII and tsV mutants the

VOL. 29, 1979

on November 10, 2019 by guest

http://jvi.asm.org/

[image:9.501.50.236.55.404.2]

132 MARTINET ET AL.

amount of38S RNA at39.2°Cisdependenton

inputmultiplicity,and in the caseofgroup V the

parental G protein can be reused in rescue of

UV-irradiated virions (4).

The implication of proteinsalteredin

comple-mentation groups III and V in the process of VSVreplication is apuzzling question. Indeed,

it hasbeen demonstratedthatproteins Gand M

are not functional at39.20C in mutants of groups

V and III, respectively (9, 11, 12). Our results

seem toindicatethatthetwoenvelope proteins

of VSV may also have a role in the

intracyto-plasmic synthesis of viral RNA. Such a

state-ment may appear surprising; however, there is

a good agreement between what is observed

about the synthesisof 38S RNA after different

temperature shifts and thereversibility (orlack

ofit) of thealterationinduced byhigh

temper-ature in polypeptide M or G for tsIII or tsV

mutants.Lafay and co-worker (11, 13) have

dem-onstrated that formutants of group III the M

protein does not recover a functional state at

permissive temperature when it has been

syn-thesizedat39.20C,whereas under the same

con-ditions theGprotein ofgroup Vmutantsrapidly

reversestowards normal behavior (12). In

par-allel, we observed that the 38S RNA did not

recovernormalsynthesis (atleast notfor1.5h)

when tsIII-infected cells were transferredfrom

39.2 to 300C; on the contrary, in ts-V-infected

cells the return to300C

immediately

(in another

experiment,notshownhere,we sawthat 30 min wasenough) producestherecuperationof a

nor-mal level of38S RNA synthesis. Thus,we can

draw a

parallel

between the replication of38S

RNA and the behavior of M and G proteins.

Little is known about the mechanism of new

genome synthesis, but it wasshown to require

thenucleocapsid. Ourownprevious studies

deal-ing with mutants in groups I, II, and IV (2, 3,

22), tentatively correlated with the three

pro-teins ofthecapsid, haveshown theirimplication

in the replicative process. With the present

knowledge, thereis no reason toassignadirect

roletothe Mand Gproteinsin the same process,

but one may consider that these two envelope

proteins have indirect intervention. Perhaps,

owingtotheirspecificabilitytobe inserted into the cellularmembranes, they would determine some special sites in which the nucleocapsid apparatus would be devoted more to the

pro-duction of genomic RNA than to that of

mRNA's. Thelong cytoplasmic lifetime of the G

protein and its specific insertion in the

mem-brane compartment of the rough endoplasmic

reticulum(8, 9, 23) as soon as it is translated are

factsaprioriconsistent with such an hypothesis.

The second point to be discussed now also

implies that the M protein might have a role

other than the simple construction of the viral

envelope. We have alreadymentioned that re-striction of 38S RNA synthesis favors

produc-tion of the mRNA because of the upsetting of

theequilibriumbetween thereplicatingand the

transcribing nucleocapsids. This phenomenon

may well account for the increase of the 13-15S

peak observed for mutants of group V at39.20C,

butnotfor that for mutants ingroup III because

of thestriking increase of thequantityof 28 and

13-15S RNA species (up to 20times the 300C

yield) observed as soon as tsIII-infected cells are incubated at 39.2°C. However, this increase does not take place when primary transcription is

studied at39.2°C (personal observation).In this

case, onlyparental proteins synthesizedat

per-missivetemperature areimplicated, and,as

dis-cussedabove forreplication of38S RNA, they

might have preserved their expected function.

Theexcessivesynthesisof 28 and 13-15S RNAs

otherwiseobserved at 39.20Cconcerns actually

the process oftranscription, since these RNAs

appear to becomplementarytothe viral genome

and possess polyadenylic acid-terminal

se-quences.However,theycannotbe characterized

as polysomal RNAs: only a fraction of these

RNAs is recovered aspolysomes, leading

never-thelesstothe normalquantityof mRNA bound to ribosomes and to the same quantity of

trans-lated viral polypeptides as those during the

course of an infection at 300C. Whether this

impliesthat the translational machinery of

eu-caryoticcells isfullysaturatedwhen functioning

(assuggested by Laskeyetal.[14]) orthat viral or cellular regulation controls the quantity of

protein synthesiscannotbe presently resolved.

It must be noticed that such regulation could

occur via the degree of methylation of the

mRNA's,whichappeared defectivefor most of

them.Anyway, it is clear thatafterthealteration

of the M protein an exaggeration of the VSV

transcriptionrateappears. This in turn suggests

thatthepotential transcription capacitypresent

inVSV-infectedcells is notfullyexpressed

dur-ingthe courseofanormalcycleand that the M

proteinisimplicatedinsomeway in this

repres-sion. Inrelation to this statement, previous

re-sults may be considered. Inhibition of the in

vitrotranscriptionactivity by envelope proteins

has been shown in avirus related to the

rhab-dovirus class (15). Uncoatednucleocapsid cores

from VSV appear to be more active in the in

vitro polymerase reaction than are complete

VSV virions (27), and Perrault and Kingsbury

specifiedthat aninhibitor of this reaction, which

is not theG protein, is present in the envelope

of VSV (19).These data support our conclusion,

althoughitmightbeconceivable thatregulation

of the transcription activity by the M protein

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

ENVELOPE PROTEINS AND REPLICATION OF VSV 133 would not benecessarily observed in thevirion

butwould rather beanintracytoplasmic process. This putative role for the M protein as well as the reason for the lack of translation of the excess of mRNA made at 39.2°C by tsIII mu-tants,iscurrently under investigation.

ACKNOWLEDGMENTS

This work was partially supported by grants from the Centre National dela RechercheScientifique (L.A.136) and theCommissariat a l'Energie Atomique.

We thank C. LaBonnardiere and M. Caboche, Station de Virologie et d'Immunologie, Institut National de la Recherche Agronomique 78650 Thiverval Grignon, France, for helping us in the preparation of themethylation assays. We also thank Annick Friedmann forher excellent technical assistance.

LITERATURE CITED

1. Both,G.W., A. K.Banerjee,and A. J. Shatkin.1975. Methylation-dependenttranslation of viral messenger RNAs in vitro. Proc. Natl. Acad. Sci. U.S.A. 72: 1189-1193.

2. Combard, A.,C.Martinet,C. PrintzAne,A. Fried-man, and P. Printz.1974.Transcriptionand replica-tion of vesicular stomatitus virus: effects of tempera-ture-sensitive mutations incomplementation groupIV. J.Virol. 13:922-930.

3. Combard, A., C. Printz-Ane, C. Martinet, and P. Printz.1977.Temperature-sensitive defect of vesicular stomatitis virus incomplementationgroupII. J. Virol. 21:913-923.

4. Deutsch,V. 1976. Parental Gprotein reincorporation by aVesicular StomatitisVirustemperature-sensitive mu-tant ofcomplementation group V at nonpermissive temperature.Virology69:607-616.

5. Genty, N. 1975. Analysis of uridine incorporation in chickenembryo cells infected byvesicular stomatitis virus and its temperature-sensitive mutants: uridine transport. J.Virol.15:8-15.

6. Huang,A.S.,D.Baltimore,and M.Stampfer.1970. Ribonucleic acidspeciesof Vesicular Stomatitis Virus. III. Multiple complementary messenger RNA mole-cules.Virology42:946-957.

7.Huang, A. S.,and E. K. Manders. 1972. Ribonucleic acidsynthesis of vesicular stomatitis virus. IV. Tran-scriptionbystandard virus in the presence of defective interfering particles.J. Virol. 9:909-916.

8. Knipe,D.M.,H. F.Lodish,and D. Baltimore. 1977. Localization oftwocellular forms of the vesicular sto-matitis viralglycoprotein.J.Virol. 21:1121-1127. 9. Knipe,D.M.,H. F.Lodish,and D. Baltimore. 1977.

Analysis of the defects oftemperature-sensitive mu-tantsofvesicular stomatitis virus: intracellular degra-dation ofspecificviralproteins.J. Virol.21:1140-1148. 10. Lafay, F. 1969.Etudedesmutantsthermosensibles du virus de la Stomatite Vesiculaire: classification de quelquesmutantsd'apris descritkres de fonctionne-ment.C.R.Acad. Sci.268:2365-2388.

11.Lafay,F. 1971. Etude des fonctions du virus de la Sto-matiteVesiculairealtereesparunemutation thermo-sensible: mise en6vidence de la proteine structurale affecteepar lamutation ts 23.J. Gen. Virol.13:449-453. 12.Lafay,F.1974.Envelope proteinsofvesicularstomatitis virus: effectoftemperature-sensitivemutations in com-plementationgroupsIIIand V. J.Virol.14:1220-1228. 13.Lafay, F.,and A.Berkaloff. 1969.Etudedesmutants

thermosensibles du VSV:mutantsde maturation. C.R. Acad.Sci.269:1031-1035.

14. Laskey,R.A.,A. D.Mills,J. B.Gurdon,andG. A. Partington.1977.Proteinsynthesisinoocytesof

Xen-opuslaevis isnotregulated by the supply of messenger RNA. Cell 11:345-351.

15.Marx, P. A., A. Portner, and D. W. Kingsbury. 1974. Sendai virion transcriptase complex: polypeptide com-position and inhibition by virion envelope proteins. J. Virol. 13:107-112.

16.Palma, E. L., S. M. Perlman, and A. S. Huang. 1974. Ribonucleic acid synthesis of Vesicular Stomatitis Vi-rus. VI. Correlation of defective particle RNA synthesis with standard RNA replication. J. Mol. Biol. 86: 127-136.

17.Perlnan, S. M., and A. S. Huang. 1973. RNA synthesis ofvesicular stomatitis virus. V. Interactions between transcription and replication.J.Virol.12:1395-1400. 18. Perrault, J., and J. J. Holland. 1972. Absence of

tran-scriptase activity oftranscription-inhibiting ability in defective particles of Vesicular Stomatitis Virus. Virol-ogy50:159-170.

19. Perrault, J., and D. T. Kingsbury. 1974. Inhibitor of Vesicular Stomatitis Virus transcriptase in purified vir-ions. Nature (London)248:45-47.

20. Pringle, C. R., and L. B.Duncan. 1971. Preliminary physiologicalcharacterization of temperature-sensitive mutants ofvesicular stomatitis virus. J. Virol. 8:56-61. 21. Printz, P., and R. R. Wagner. 1971. Temperature-sen-sitive mutants of vesicular stomatitis virus: synthesis of virus-specific proteins. J. Virol. 7:651-662.

22. Printz-Ane, C., A. Combard, and C. Martinet. 1972. Study of the transcription of the replication of vesicular stomatitis virus by using temperature-sensitive mu-tants.J. Virol. 10:889-895.

23. Rothman,J.E., and H. F. Lodish. 1977. Synchronized transmembrane insertionandglycosylation of a nascent membrane protein. Nature (London) 269:775-780. 24. Schincariol,A.L.,and A. F.Howatson. 1970.

Repli-cation of Vesicular Stomatitis Virus. I. Viral specific RNA andnucleoprotein in infected Lcells. Virology 42: 732-74"

25. Schincariol,A.L., and A. F. Howatson. 1972. Repli-cation of VesicularStomatitisVirus. II.Separationand characterization ofvirus-specific RNA species. Virology 49:766-783.

26. Stampfer, M.,D.Baltimore,and A. S.Huang.1969. Ribonucleic acid synthesis of vesicular stomatitis virus. I.Speciesofribonucleic acid found inChinesehamster ovary cells infected withplaque-forminganddefective particles. J. Virol.4:154-161.

27. Szilagyi,J.F.,and L.Uryvayev. 1973.Isolation of an infectiousribonucleoproteinfrom vesicular stomatitis virus containing an active RNAtranscriptase. J. Virol. 11:279-286.

28. Toneguzzo, F.,and H. P.Ghosh.1976.Characterization andtranslation ofmethylated andunnethylated vesic-ular stomatitis virus mRNA synthesized in vitro by ribonucleoproteinparticles from vesicular stomatitis vi-rusinfected L cells. J. Virol.17:477491.

29. Unger,J.T.,and M. E. Reichmann. 1973.RNA syn-thesis in temperature-sensitive mutants of vesicular stomatitis virus. J. Virol. 12:570-578.

30. Wertz,G.W.,and M.Levine.1973.RNAsynthesis by vesicularstomatitis virus and asmallplaque mutant: effects ofcycloheximide.J.Virol. 12:253-264. 31. Wild,T. F.1971.Replicationof vesicular stomatitis virus:

characterization of the virus-induced RNA. J. Gen. Virol. 13:295-310.

32. Wunner,W.H.,andC. R.Pringle.1972.Protein syn-thesis inBHK21cells infected with vesicular stomatitis virus. I.tsmutantsof the Indianaserotype.Virology 48:104-111.

33. Zavada, J.1972.Pseudotypeofvesicularstomatitisvirus with the coatof murineleukaemia andof avian myelo-blastosis viruses. J.Gen.Virol. 15:183-191.

VOL. 29, 1979

on November 10, 2019 by guest

http://jvi.asm.org/