Transcription and Replication of Vesicular Stomatitis Virus: Effects of Temperature-Sensitive Mutations in Complementation Group IV

Transcription and Replication of Vesicular

Stomatitis Virus:

Effects of

Temperature-Sensitive

Mutations in

Complementation Group IV

ARLETTE COMBARD, CLAIRE MARTINET, CHRISTIANE PRINTZ ANE, ANNICK FRIEDMAN, AND PIERRE PRINTZ

Institut deMicrobiologie, Universite Paris-Sud, Centre d'Orsay, 91405 Orsay,France Received forpublication3 December 1973

Temperature-sensitive (ts) mutants ofvesicular stomatitisvirus belongingto

the RNA-complementationgroupIVwereinvestigatedunder various conditions tostudy both their RNA and protein syntheses. In infectedcells maintained at

39.2 C, viral RNA species were recovered only in the 13 to 15S region ofthe gradient in an amount depending on the ts mutant used. In the presence of

cycloheximide at 39.2C, the primary transcription wasdeficient, especially for

28SmRNA production. When mutant-infected cells wereshiftedto

nonpermis-sivetemperature, ashutofiof28S mRNAsynthesis occurredas ageneral feature.

On the contrary under thiscondition, thetwo mutants chosen, ts IV100 andts

IV111, behaved very differently in their 13 to 15S and 38S RNA production.

However, treatment with cycloheximide at the time of the transfer to 39.2 C resulted inasimilarrecoveryof 13to15SRNA in bothmutants,whereas the 28S remained very depressed. The viral proteins synthesized by cells infected with

thesametwomutantsalsoshowedadistinctpattern, especially regarding the N protein;acorrelation between 38S RNA andprotein N syntheseswastentatively

drawn. The whole set of data suggested that the lesion in group IV mutants

concerned aviralstructural proteinrequiredfortheprocessof in vivo transcrip-tion and which probably intervened inthereplication mechanism.

Progress hasrecently been madeinthestudy

ofprocessesinvolvedinthe productionof vesic-ular stomatitis virus (VSV), making use of mutants isolated invarious laboratories (9, 11,

30). Temperature-sensitive (ts) mutants,

una-bleto synthesize sizableamountsofviralRNA

at high temperature (RNA-), have been

ac-tively investigated as being the most useful

toolstodelineate the mechanisms ofboth viral

transcription and replication. New facts

emerged from our previous studies (16, 20) of

twomutants, tsIandtsIV100, representative of two RNA- complementation groups, I and IV

(F. Lafay, thesis, University of Paris, France, 1972) respectively. Firstly, transcription and

replication appear to be tightly linked, and we

suggest that a common subunit intervenes in vivo in transcription and replication. In addi-tion,results obtained withts IV100prompteda new hypothesis, in which the enzyme which functions in vitro as a transcriptase (2) would require another virus-coded polypeptide to

pro-duce faithful transcription in vivo. In this

paper, evidence obtained by studying various

group IV mutants and which further supports

thehypothesis ispresented.

MATERIALS AND METHODS

Cells. In all RNA analyses,HeLaconfluent

mono-layers were used under conditions already described (20). Forsomeproteinstudies,chickenembryo fibro-blasts (CEF)wereculturedasreported(9).

Virus.Temperature-sensitive(ts)mutantsofVSV (Indiana strain), herein investigated, belong exclu-sively to complementation group IV. Virus stocks containing only standard B particles wereobtained andwerechecked for low percentage of revertants,no

residual growth at 39.2C, and by complementation

test as described (20). The wild-type virus grew at 39.2C, aswellas at30C.

Chemicals. Actinomycin D, a generous gift from

MerckSharp andDohme, French branch,wasusedat a final concentration of 10 gg/ml. Cycloheximide, dilutedin medium to 100

ltg/ml,

andsodiumdodecyl sulfate (SDS) were purchased from Serva, Heidel-berg, Germany. RNase T,wasobtained from Sankyo (Tokyo, Japan) and pancreatic RNase A was obtained

from Nutritional Biochemicals Corp. (Cleveland, Ohio). All labeled compounds were from C.E.A.

(Saclay, France): [9Hluridine(20Ci/mmol), [3H

]leu-922

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VSV COMPLEMENTATION GROUP IV TS MUTANTS

cine (33Ci/mmol), [3H]tyrosine (49 Ci/mmol), and a mixture of [14C]amino acids (100 mCi/mmol).

Analysis of viral RNA. HeLa cells were infected (-10 PFU/cell, unless otherwise specified) and treated with actinomycin D as previously described (20). When cycloheximide was used, addition of radioactive nucleoside occurred 15 min later. After labelingw4h [3H luridine(20

gCi/ml

unless otherwise specified in figure legends), cells were harvested at the endofthe indicated incubation periods. From cyto-plasmic extracts in reticulocyte standard buffer, made1%with respect toSDS, solubilized RNAs were analyzed by centrifugation in the Spinco SW 27.1 rotor in 15 to 30%sucrose-SDS gradients. All methods and buffer compositions were previously described (20). After centrifugation (21,000 rpm at 17 C for 15 h), the gradients were fractionated and monitored as previously described (20).

Annealing conditions. Annealing of RNA species was achieved as follows: portions (1,500 3H-counts/ min) were mixed with virion RNA (4 to 10

Ag/ml)

extracted according to the method of Montagnier (17). Duplicate samples were made toafinal volume of 400 gliters and a final concentration of 2 x standard saline citrate (0.15 M NaCl, 0.015 M sodium citrate). They were boiled for 3 min, hybrid-ized 1 h at 70 C, and then divided into two equal portions. One of them was digested for 30min at 37 C by pancreatic RNase A (5Ag/ml) andT, (30 units/

ml). All samples were then acid-precipitated inthe presence of 0.08M sodium pyrophosphate and 100,g

ofyeast carrier RNA per ml. Materials trapped on membrane filters, type HA, were assayedfor radioac-tivity as usual. Controls simultaneously conducted lacked added virion RNA.

Labeling and analysis of viral proteins. CEFor

HeLa cells were first exposed for 90min at 37 C to

actinomycinD (1 ug/ml) and, afterviral adsorption (40 min at room temperature), were covered with diluted 1:10 (vol/vol) Eagle minimal essential

me-dium(Eurobio) containing 1%calfserum.Amixture of [3HIleucine and [3H]tyrosine was added at inter-vals after infection for 60-min pulses (20 MCi/ml).

Cellularextracts werepreparedbydisrupting the cells with aDounce homogenizer in awaysimilartothat used for RNA extraction(20). Proteinswereextracted

from cell fractions in precisely the same way as

previously described (21). After adding one-tenth volume of glacial acetic acid, the suspensions were

made0.5 M withurea and 1%with SDS, incubated for1hat37C,andfinally dialyzedatroom

tempera-turefor 16 h againstphosphate buffer (pH 7.2, 0.01

M) containing 0.1% SDS, 0.5 M urea, and 0.1% 2-mercaptoethanol. Viral proteins were analyzed by

polyacrylamide gel electrophoresis performed as

de-scribed(29),by using7.5%acrylamidegelat 5mA/gel for5h.Gels slicedinto 1-mmlengthswere

depolymer-izedin0.2 ml ofhydrogenperoxide (25%)at55Cfor4

h priortothe additionofBray scintillation fluid. For

theidentification ofviralproteins,complete reliance

was placed on the migration of intracellular [3H]proteins coelectrophoresed with authentic viral [4C] proteins extracted from purified virions (21).

Under these conditions it was regularly observed that intracellular [3HIprotein G migrates faster than the virion marker ["C ]protein G, as reported by Kang and Prevec (14). Radioactivity was measured in an IntertechniqueSL40 spectrometer assisted by a dou-ble-labeling computer program.

RESULTS

Viral RNA synthesis at 39.2 C. A first

approachto viral RNAsynthesis in

complemen-tation group IV has been done by using the prototype mutant tsIV100 (20). Parallel studies

of three mutants, ts IV62, ts IV111, and ts

IV100, now make it possible to generalize about some properties of group IV mutants. As

com-pared to wild type, the total amount of RNA

synthesized at 39.2 C is always less than 10%.

NodegradationofRNAalready synthesizedcan

be detected, and the rate ofsynthesis appears

constant without amplification throughout the

cycle.Moreover, thepattern ofthe RNA

synthe-sized at 39.2 C shows no change at any time

after infection. It is characterized by the

pres-ence oflight RNAs at the top ofthe gradient

(less than lOS); in addition, among specific

VSV RNApeaks,only 13to15S RNAisreadily

identified (Fig. 1, full-circle curves).

Neverthe-less, in the same experimental conditions the

largest proportions of 13 to 15S RNA are

ob-served with ts IV111 and the smallest with ts

IV100. Therefore, a minimal and maximal

re-sidual viral RNA synthesis amongthe mutants

studied is provided by ts IV100 and ts IV111,

respectively. Consequently,further studieswere

carried outwith thesetwo mutants only.

Viral RNAsynthesisat39.2 C in

cyclohex-imide-treated cells. Results stated above

showed thatsomestep(s)intranscription,

espe-cially for thesynthesis of28S, is(are) affected

inthegroup IV mutants. Inordertodetermine

whether any alteration could be detectedfrom

theearly stage ofprimarytranscription defined

by Huang and Manders (13), RNAsynthesisat

39.2Cwasstudiedinthepresence of

cyclohexi-mide whichwasaddedjustafter infection(Fig.

1, open-circle curves). Whatever the mutant

used, the RNA pattern obtained under these

conditions differsfromthatobtainedat30C in

the presence of cycloheximide (Fig. 1, star

curve) which shows all the characteristics of

VSV primary transcription (13). The striking

facts werethe lack of28SRNA andthe similar-ity of the twoprofiles withorwithout

cyclohexi-mide. Despite the apparent importance ofthe

RNA synthesis by ts IV111 under these

condi-tions, this mutant behaves as RNA-, because here wecompare the RNAsynthesizedat39.2C

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4 C D39.2C, we observed a

depression

of all viral

A tomI" \RNA peaks with bothmutants (Fig. 2,

full-cir-28s Iss cle

curves).

The

single-stranded

28S

RNA,

com-$ +I* plementary to the virion RNA synthesized at

30C, was not recovered in the upward shift

a lL l conditions. The small amount of28S still

de-tected was

essentially

RNase-resistant. On the

other hand, the two profiles strikinglydiffered

4. ^\ *Iy \ A concerning 13 to 15Sand 38S RNAs. Actually,

after the

upward

shift, ts IV100 and ts IV111

produced

both RNAs

sedimenting

in the 13 to

15Sregion which,like truemRNAs,hybridized

x *. completely to virion genome (98%). However,

s44I when compared to the quantity of 13 to 15S

normallypresent at 30 C, the amount of 13 to

4. P / 15S was only decreased from 10 to 50% in the

case ofts

IV111,

whereas it was reduced less

than50% for ts

IV100.

c , $ . The38S

peak,

inour

upward

shift

conditions,

t.

m was never detected in the ts IV111 profile,

4 whereas it was always present in that of ts

IV100, although in various proportions (10 to

90% of its 30-Cyield).

I10 20 30 40 50 _______________________

FRACTIONS 2 A

tslIIItN

FIG. 1. Sucrosegradient separation ofRNAspecies

made by ts group IVmutants in the presence or ill|

absence of cycloheximide. Equivalent samples of

HeLa cellswereinfectedeitherbyts

IVI00,

tsIV62,or

E.I

\ / I\

ts

IVI11

atthesamemultiplicity ofinfection (- 20). 28S 18s

After 45 min of adsorption at room temperature,

/

actinomycinDwas addedto afinal concentrationof\

10

,g/ml.

Severalsamples (seesymbols)weretreated i

with100lAgofcycloheximideper ml.Then allsamples

werelabeled with40

,Ci

of[3H]uridineper ml. Cells /

were harvested at 3 h p.i. Cytoplasmic RNAs were 0.5.

separated on 15 to 30% sucrose-SDS gradients, col- I lected into50fractions, andacid-precipitated by5% x

trichloroacetic acid. 0,RNA profile of infectedcells X I

at 39.2C; 0, RNA profile of infected cells treated I withcycloheximideat39.2 C;

4,

RNAprofile ofcells ts100 infected withtsIV111 and treated withcycloheximide

at30C. Peaksofradioactive RNAidentifiableat30C 0.5

in infections with ts IV62 and tsIV100, identicalto 0. thosesynthesized bytsIV111, are notshown.

with the RNA obtained at 30C in primary __

transcription whichaccounts foronly10%ofthe lb1To io 40

total RNAsynthesis (13, 28). FRACTIONS

Effect of anupward temperature shift on FIG. 2. Sucrosegradient analysis of RNA species

RNA synthesis. As was already mentioned synthesized bytsIVI00-andtsIVIII-infectedHeLa

with ts IV100 (20), a rapid change in RNA cells at 39.2 Cafterprior incubation at 30 C.Infected

synthesis follows a temperature shift to 39.2 C HeLa cells with tsIVI00ortsIVIII werelabeled in

after 3 hat 30C.Hence,suchexperimentswere the presence of actinomycin D with 20 MCiof

[3H]uri-simultaneously conducted withts IV100 andts

dine

perml 3to4hp.i.RNAswereanalyzedasinFig.

IV111. Immediately after_{IVil.Imediaely fterthe}theupward tempera-_{pwar temera-}a t i,_39.2Cts_atMutant-infected_{3 h p.i.; 0,} cells transferred from 30 C to_{ts mutant-infected cells at}

ture shift, RNAs werepulse labeledfor 1 hand 39.2C; *, infectedcellsmaintained at 30 C for 4 h.

compared with the corresponding control at As in Fig. 1,and for the same reason, only one profile

30C (Fig. 2, star curve). After transfer to isshown.

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VSV COMPLEMENTATION GROUP IV TS MUTANTS

The rate of RNAsynthesis declined after shift

to39.2 C as shownbythegradual decay of RNA

labeling, without specific alteration of any one

peakduring the second and third hour after the

shift (not shown). Finally, the effect of prior

incubation at 30 C disappeared, because 3 h

after a shiftthe profile became the same as if it

wasobtained from infected cells maintained at

39.2 C throughout the experimental time course

(Fig. 2, open-circle curves).

It canbe seen from the control curve in Fig.

2that even at 30C all the mutants in group IV

induce a remarkable RNA profile, because 38S

isgenerally the highest peak, and 28S is

especi-allybroad. Inthis important and heterogeneous

28S region, additional RNA categories appeared.

One ofthem, 23S RNA, was previously

men-tioned (20) but was not regularly observed

either at 30 C or at 39.2C depending on

un-known contingencies.

Combined effect of cycloheximide

treat-ment and upward temperature shift. In

up-ward temperature shift experiments, viral

pro-teins synthesized during the first 3 h at 30 C

may interfere withormask someconsequences

ofthe mutation taking place at 39.2 C.

There-fore, to interpret the former results,

interven-tion of these proteins must be estimated by

preventing further protein synthesis after the

shift. Thus, cycloheximide was added at the

time of the upward temperature shift (3 h

postinfection [p.i. ]) in cells infected

respec-tively by the ts IV100 and ts IV111 mutants

(Fig. 3), whereas a sample was maintained in

parallel at 30C as a control (Fig. 3, square

curve). The transferto39.2C ledto adecrease

ofboth classes ofmRNAs, whichwasespecially

drastic atthe level of28S.

We verified withwild-typevirus thatnoneof

these facts was the result either ofthe direct

action ofcycloheximideorof itscombined effect

with theupwardtemperatureshift. First,asdid

otherauthors(19,30),wefound thatadditionof

cycloheximide atany timeduringan infectious

cycle at 30C was conduciveto ashutoffof38S

RNA synthesis (visible on the control curve),

whereastranscriptionof28S and13 to15S RNA

was unaffected oreven enhanced as compared

with cells without the drug. Second, the

com-bined effect of a temperature change and

cy-cloheximide treatment was shown to

merely

promotean extraamplificationoftranscription.

Now comparisonofthetwoprofilesobtained

for each mutant after an upward temperature

shift in the presence or absence of

cyclohexi-midepointedoutthe roleofnewly synthesized

proteins at 39.2 C. Although both types of

profiles have already been reported, but from

nonsimultaneous analyses, we preferred to

com-pare new curves resulting from another

experi-ment. De novo protein synthesis at 39.2 C

resulted for both mutants in a dramatic

reduc-tion of the amount of 13 to 15S and almost a

disappearance of 28S RNA (Fig. 4). Moreover,

38S RNA appeared when cycloheximide was

omitted, but only with ts IV100.

Protein synthesis by mutants ts IVIOO and

ts IVlll. The synthesis of viral proteins has

been investigatedinconditions similar tothose

used forthe RNAsynthesis. In our hands, CEF

supporting a much more active and rapid

pro-teinsynthesis than HeLa cells, without

exhibit-ing consistent differences, were preferentially

used.

Figure 5showsthe electrophoretic profiles of

the viral proteins extracted from CEF infected with the mutants ts IV100 and ts IV111 at 30 C,

39.2C, and inupward temperature shift

condi-tions. All five viral proteins were present in

normal amounts in cells infected at 30 C with

thetwomutantsand werelabeledfrom2to 3h

p.i. At nonpermissive temperature and during

the same labelingperiod, the level ofsynthesis

was markedly reduced, espeically with the

mu-28s ls

x~~~~~~~~

FRACYONS

FIG. 3. Sucrose gradient pattern of RNA synthe-sized by ts IV100 and ts IV111 after addition of cycloheximide in upwardshift experiments.Infected

andactinomycin D-treated HeLa cellswereincubated

3hat 30C.At3hp.i., cycloheximidewasadded(100 ug/ml). Fifteenminuteslater,cellswerelabeled with

20AtCiof[8H]uridineper mlfor1handanalyzedasin

Fig.1.0,tsIVI I I-Infectedcellstransferred from30C

to 39.2Cat 3hp.i. timeofadditionoftheantibiotic; 0,tsIV100-infectedcells treatedastsIV111-infected

cells; 0, control infected cells maintained at 30C after additionof thedrug.

VOL.13,1974 925

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T.

FIG. 4. Sucrose gradient centrifugation of viral

RNA madebytsIV100-ortsIV111-infected cells,in

thepresence orabsenceofcycloheximide inupward

temperatureshift experiments. After infectionwith ts

IV100 ortsIVi11, actinomycinD-treated cells were incubated 3 h at 30C. At 3 h p.i., cells were transferred to 39.2C, and one of the duplicates

received cycloheximide (100 Ag/ml). At 3.15 h, all

cellswerelabeled with20uCiof[3Hjuridinepermlfor

1h.0,Infectedcells withcycloheximideadded at the

timeoftheshift;0,infectedcells without

cyclohexi-mide.

tant tsIV100;inthe lastcase,onlytheproteins N and NS emerged from the background. At

39.2C, thetsIV111-infectedcellsgavea

better-defined profile, but the N protein peak

ap-peared low as compared with the other ones.

With bothmutants, theLproteinwasvirtually

absent. The pattern of protein synthesis

ob-tainedwiththetwomutantsin theupwardshift condition are shown in panel B. The main

difference between thetwomutantswas inthe

behavior of the N protein. During the 1-h pulse

at39.2 C whichfollowed the 2-hpulse at30C, the N protein peak decayed drastically incells

infected withts IV111, but wasnot affected in

thecaseofthe mutanttsIV100.Thisresult has

been regularly observed in repeated experi-ments.Thedrop of theNproteinwasduetoan

absence of synthesis rather than to a rapid

destruction ofnewlysynthesized polypeptides, because in a chase experiment of labeled

pro-teins at 30C no drastic effect wasobserved at

39.2C atthelevelof anyviralprotein

(unpub-lished data). The L protein synthesis was

re-duced in a similarway forboth mutants.

A peculiar effect of the upward shift was

observed at the level of the G protein; its

synthesis was markedly increased in cells

in-fected with ts IV100 and ts IV111. This effect

wasnottransitoryand continuedtobeobserved

during successive 1-h pulses.

The transcription process in cells infected

withmutant ts IV100 or ts IV111 wasincreased

when upward shiftwas combined with a

treat-ment ofcycloheximide (Fig. 4). Toobservethe

effecton protein synthesis, HeLa cells or CEF

both infected with ts IV111 havebeen treated

bycycloheximide (100,ug/ml)atthetime ofthe

upward shiftto 39.2 C and extensively washed

before the 1-h amino acid pulse. Compared with

Fig. 5, Fig. 6 shows that the same profile of

proteins is obtained, but the proteinpeaks are

higher; this may result from the presence of

moremRNAinthat condition.Parenthetically,

the electrophoretic profiles were qualitatively

similar with proteins extracted from CEF or

HeLa cells.

DISCUSSION

Analysis of viral RNAs made at 39.2C after

infection with any one ofthegroupIVmutants

demonstrates that both the expected

transcrip-tionandreplication cannottake place.

Discuss-ing our previous data (20), we proposed that

transcriptionwasthestepdirectlyconcerned by

the mutation. In the present paper, shorter

labeling pulses and upward temperature shift

experimentsconductedtoamplify theeffects of

the mutation confirm that transcription is

un-doubtedly affected. Thisappearsfromthe early

phase of primary transcription during whichare

observed thealterationsinRNA synthesis

char-acteristic ofgroup IV mutants, i.e., lack of28S

and a lower degree of 13 to 15S transcription

(Fig. 1). In a recent study, Unger and

Reich-mann(28) reported the deficient RNAsynthesis

of one mutant of group IV at nonpermissive

temperature. They argued about the special

alteration of the long mRNA, but there is no

real discrepancybetween theirobservationand

ourown, because in conditions similar to theirs (5 h at 39.2 C) we actually found labeling in the 28S region, although it was not recovered as one

conspicuous peak (20). In addition, when they

examined the primary transcription of their

group IV mutant, theynoticed thesame quanti-tative change atnonpermissive temperature as we related. Because, bydefinition, among viral

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VSVCOMPLEMENTATION GROUP IV TS MUTANTS

N

' I --

4Atd

4;=;RS-/

V\f\

^A

l

N

A

88

L G (P5 64(5

44

4

4

1 20 30 40 50 ¶0

io

30 4' 50

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FRACTIONS

FIG. 5. Electropherograms of virus-specific proteins extracted from CEF infected at a multiplicity of- 50

with ts IV100 or tsIViii underpermissive, nonpermissive, or upward shift conditions. After an adsorption period of 40 min at room temperature, three plates (60 mm in diameter) for each mutant were covered with 2 ml of1:10(vol/vol)-diluted minimal essential medium and incubated at 30 C (two plates) or 39.2 (one plate). Two hourslater, [3H]leucineand [3H]tyrosine (10gCi/mleach) wereadded to one culture at 30 C and to the other at 39.2C; cells were harvested after 1 h of labeling (2 to 3 hp.i.). The remaining plate at 30 C was brought to 39.2Cat2.30hp.i. and labeled from 3 to 4 h p.i. Proteins were extracted from cytoplasmic contents ofcells, disruptedwithaDouncehomogenizer, with 10% acetic acid, 0.5 M urea, 1% SDS, and 0.1%2-mercaptoethanol. Extracted [3Hlproteins were subjected to electrophoresis in 7.5% neutral SDS acrylamide gels along with marker[14C]proteinsextracted from purified virions. Vertical arrows show the peak positions of the four marker

[14C]proteins. Left hand side, ts IV100; right hand side, ts IV111. Panels A: 0, permissive temperature; 0

nonpermissivetemperature. Panels B: upward shift conditions.

proteins only those associated with the virion

are involved in primary transcription (13), it

follows that one of these structural proteins,

essential particularlyfor 28S RNAsynthesis, is

alteredin group IVmutants.Moreover, in vitro

transcriptase activity at 39.2C has been

dem-onstrated with this group ofmutants (20, 26),

whereas it failed for group I mutants; in the

lattergroupthe lesionseems tobemorelikelyin

the enzyme thanin the template (P. Printz et

al., in R. D. Barry and B. W. J. Mahy, (ed.),

Negative strandviruses,inpress; R. R.Wagner,

personal communication). Therefore, besides

eventual host factors (19), we mustemphasize

that another viral structural protein, which for

convenience will betermedprotein IV,is neces-sary to obtain a faithful in vivo transcription.

The ts event renders it rapidly inactive at

nonpermissive temperature.This is observed for

protein IV of the incoming virions in primary transcription (Fig. 1), as well as for the new

molecules made at 30 C and shifted to 39.2 C

when additional protein synthesis was

pre-vented by cycloheximide (Fig. 3). However,

whencells infected either by tsIV100or tsIVIli

werekept 3 h at thenonpermissive temperature

and then shifted downward to the permissive

one inthe presenceofcycloheximide,

transcrip-tion was completely recovered (not shown).

Therefore, the ts event does not seem to be

irreversible, and protein IV is not degraded at

high temperature.

Upward temperature shift experiments, with

and without cycloheximide addition, posed the

problem of the effect of the mutation in the

group IV mutants at the level of replication.

First, there is apersistant synthesis of 38S with

tsIV100 after anupwardshiftwithout

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(NS .

: ~ ~

- 1b 2b 3'0 FRACTIONS

FIG. 6. Electropherograms of virus-specific pro-teins extracted from CEF or HeLa cells infected at a

multiplicity of '-.50with ts IVIII in an upward shift

experiment combined with a cycloheximide

treat-ment. Afteradsorption, the plates (60 mm in

diame-ter) were covered wvith1:10 (vol/vol) dilutedminimal essential medium andincubated at 30 C for 2 h (CEF)

or 3 h (HeLa cells). After that period at 30 C, the

medium was drained, and new medium heated at

39.2 C and containingcycloheximide (1001lg/ml) was added. The plates, kept at 39.2 C for 50 min, were then washed extensively and covered with 2 ml of

medium devoid of cycloheximide, but heated at

39.2 C. Cells, maintained at 39.2 C, were pulse

la-beled with [3Hjleucine and [3H]tyrosine (10y.Ci/ml each) from 3 to 4 h p.i. (CEF) or from 4 to 5 h p.i.

(HeLa cells). Cytoplasmic extracts were treated as

described in Fig. 5. Vertical arrows show the peak

position7s of the four marker ['4C]proteins. *, CE fibroblasts; *, HeLa cells.

imide. Second, neither 38S nor any RNA species noncomplementary to virion genome are de-tected with ts WIVllunder the same conditions. Recent reports (19, 30; P. Printz et al., in press) gave evidence ofan equilibrium between tran-scription and replication and of a shift of this equilibrium towards transcription in the ab-sence of replication by devotion of the whole RNA polymerase activities to mRNA produc-tion. After an upward temperature shift, only the addition ofcycloheximide allowed this in-creased transcriptase activity in both mutants (Fig. 4). Accordingly, even with tsIVi11 in the absence of cycloheximide, transcription was still restricted as if equilibrium was preserved, arguing for a potentially present replicase func-tion although unable to finally lead to 38S or possible precursors (15). Whereas the recovera-ble transcriptase activity is nearly identical for both mutants in the presence of cycloheximide (Fig. 3), in upward shift experiments without the drug (Fig. 2) the balance between

replica-tion and transcription is notthesame. Lack of

28Sandaloweramountof 13 to15S RNA being

a common effect oftheir mutations, weascribe theapparentdiscrepancybetween the behavior ofthe two mutants to some subtle differences inprotein IV, relevanttothe site of the

muta-tion.

Study of translationwasundertakento

possi-bly reveal other differencesatanylevelevenin

transcription (undetected by RNA gradient

analyses). Analysis of protein synthesis at

39.2C merely reflects the respective deficiency

of RNA synthesis in ts IV100- or ts

IV111-infected cells (Fig. 5). Thisis in agreementwith

Wunner andPringle's results (31). Thus, as in

RNA study, upward temperature shift was

performed to sensitize the detection of the

mutational effects. In such experiments, three

particularities appear intheelectropherograms

(Fig. 5 and 6). The behavior of G protein, as

discussed elsewhere (P. Printz et al., in press),

is notgroupIV-specific and could be duetoits

particular biosynthesis (10). The reduced

syn-thesis of Lprotein, in the sameproportion for

both mutants, may well be ascribed to their

common deficiency of28S mRNA (25; D.

Bal-timoreetal.,in R. D. Barryand B. W. J.Mahy,

(ed.), Negative strand viruses, in press). If L

protein is responsible for the transcriptase

ac-tivity detected in vitro (7), absence of new

moleculesofL transcriptase,alongwithfailure

ofreplicationat39.2 C withoutpriorincubation

at 30C, would explain that no amplification

occurs in RNA synthesis with group IV mu-tants. Thus, we conciliate two explanations

suggested by Unger and Reichmann, namely,

lack of replication and block in additional

synthesis ofproteins required for transcription

(28). Finally, the major difference in protein

synthesisbetweenthetwo mutantsinvestigated

concerns the synthesis of N protein; they also

differintheir 38Sproduction (Fig. 2).Asanew

protein synthesis is required for 38SRNA

for-mation(19, 30; Fig. 3),acausalrelationshipcan

be drawn from these two observations. If the recovery ofintracellularvirion RNAwas

pack-aged primarily with Nprotein, unstabilization

of38S RNA might result from the lackofthis

protein. This would partially explain protein

requirement forreplication, becauseother viral

proteins also appear to be involved inthe 38S

RNAsynthesis (manuscript inpreparation).

Anotherexplanation forthe different

behav-ior, with regard to the replication process,

between the two mutants in the same

com-plementationgroupcould be given if protein IV,

differently affected by the various ts point

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[image:7.494.65.254.55.229.2]

VOL.13, 1974 VSVCOMPLEMENTATION

mutations, is involved in severalfunctions. Our

results make it conceivable that protein IV is

concerned with some aspects of signal

recogni-tion and, thus, could intervene both in tran-scription andreplication. Effectively, transcrip-tion is supposedto set on the putative start or stop signals (18, 23) along the template. The presence ofintracellular38S RNA complemen-tary to virion RNA (D. Baltimore et al., in press; S.M. Perlman and A.S. Huang,

submit-ted for publication) suggested that replication

might also function by opening one initiation

site, with all others remaining masked.

In-teracting with either the template, the tran-scriptase, the putative replicase, or with any

complex between them, protein IV should be

devotedtosignals switch-on and -off orsimply

to their recognition, giving quantitative and

qualitativeefficiency to transcription or

replica-tion orboth. This hypothesis wouldaccount for

the observation thatingroup IV mutants RNA

synthesis in vivo at 39.2C occurs as if

tran-scriptasealonewas atwork, becauseas inthe in

vitro reaction (3) (i) input genomes of group IV

mutants arecompletely transcribed (8),and (ii)

thesemutantsgivemostly RNA speciesofshort

sizes (4, 12, 13, 20, 30). Inaddition,we

demon-stratedthat transcriptionof28S RNA and13 to

15S RNA may occur independently, which is

consistent with some sort of sequential

tran-scriptionofthe VSV RNA (22).

Protein IV cannot be identified by merely

studying in vivo protein synthesis, because,

besides the transcriptase L protein andgenome

RNA, the complete VSV transcription

appara-tuscomprises in vitro(5,6, 27), aswellas in vivo

(1), protein N,

NS,

and eventual minor

pro-teins; protein IV could be any one ofthem. Its

proposedfunctions maybe modulated bysome

chemical transformation (24).

ACKNOWLEDGMENTS

ThisresearchwassupportedbytheCentre National de la Recherche Scientifiquethroughthe L.A. 136, theFondation pour laRechercheMedicaleFrancaise, and the Commissariat al'EngergieAtomique.Saclay, France.

We thankA.Berkaloffformanyhelpfuldiscussions. For twoofus,C. Martinet and C. PrintzAne,thisworkispartofa

thesis(Doctoratd'Etat).

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