Envelope Proteins of Vesicular Stomatitis Virus: Effect of Temperature-Sensitive Mutations in Complementation Groups III and V

JouRNAL OFVIROLOGY, Nov. 1974,p.1220-1228 Copyrighti 1974 American Society forMicrobiology

Vol.14,No. 5 PrintedinU.SA.

Envelope Proteins of Vesicular

Stomatitis

Virus: Effect of

Temperature-Sensitive

Mutations in

Complementation Groups

III and

V

FLORENCE LAFAY

Laboratoire de Biologie Experim'entale, Bat. 400; Institut deMicrobiologie, Bat. 409, UniversiteParis-Sud Centre d'Orsay, 91405 Orsay, France

Received forpublication 8 July 1974

All fivemajor viral proteins weresynthesized inchickenembryocellsinfected

with vesicular stomatitis virustemperature-sensitive (ts)mutantsof

complemen-tation groups III and V and maintained at thenonpermissive temperature. The

distribution of theseproteins among cytoplasmic cellular fractions separated on

discontinuous sucrose gradients was identical for wild-type and tsllI-infected

cells. Strikingly different patternswere observed for the Gprotein in gradients

fromcells infectedbytsV mutants; verylittle,if any, Gproteinwasfound inthe

lightest fraction. Pulse and chase experiments with wild-type, virus-infected cells showed that protein G moves from the heaviest to thelightest fraction before being incorporated into the virion. After shift down tothe permissive

tempera-ture (30 C), G protein synthesized at 39.6C in tsV-infected cells became

associated with the lightestcellular fraction and later with the released virions.

In contrast, M protein, synthesized at 39.6C in tsIII-infected cells, was not

incorporated into the virions after shift down. These datastronglysuggest,first,

that M protein isencodedbythe vesicular stomatitisgenem, andsecond, that

incorporation ofG proteininthe lightest cellular fraction isanecessary step of

vesicular stomatitis maturation. This step isimpairedby tsV mutations.

The vesicular stomatitis virion contains at

least fiveproteinsdesignatedaccording to

Wag-ner et al. (23): L, G, N, NS, and M (12, 24).

These proteins constitute the major proteins

found in cytoplasmic extracts ofinfected cells

(13), thoughadditional viralminorcomponents

maybe present (1, 19, 20, 25).

These extracts can be fractionated by

high-speed centrifugation into soluble andinsoluble

components.NS and half of the Narerecovered

in the soluble fraction, and the majorityofM

and G and halfof the N are recovered in the

insoluble one (25). Association ofviralproteins

with cellular insoluble components has been

investigated by several methods (4-6,8).

Wag-ner et al. (22) observed an unequal distribution

ofthe viral proteins among four cellular

frac-tions which can beseparated on discontinuous

sucrose gradients. The lightest one contains

mainly protein G, whereas protein N is the

major viral component of the third fraction.

Inthispaper we show that

temperature-sensi-tive (ts) mutations classified in

complementa-tion group V (10, 11) specifically affect the

association of the G protein with the lightest

cellularfraction atthenonpermissive

tempera-ture.

(This work is part of a thesis [Doctorat

d'Etat]).

MATERIALS AND METHODS

Virus,cells,and media.Wild-typets+strainand the following spontaneous ts mutants of vesicular stomatitisvirus, serotype Indiana,describedby Lafay (15-17) and Flamand (10), have been used: tsV 44,

tsV45, tsV 57, tsV110,and

tsMf

89.

Chickenembryocellsweregrown inEagleminimal essential medium buffered with Tris and

supple-mented with 6% calf serum and 8% tryptose phos-phate.

Chemicals. Calf serum, Eagle minimal essential

medium, and special Eagle minimal essential me-diumwithout leucinewerepurchasedfrom Laborato-ries Eurobio. Ultrapure Tris was obtained from

Schwartz/Mann. [8H]leucine (30 Ci/mM) and

[14C]amino

acid mixture(50mCi/mg)wereobtainfed

from Commissariat a l'Energie Atomique (CEA-France). Actinomycin D was a generous gift of Merck, Sharp and Dohme (French branch).

Labeling of chicken embryo cells orvirus. Mono-layers of chicken embryo cells in 9-cm petri dishes were infected at a multiplicity of 40 to 150PFU/cell. Virus was adsorbed for 45 min at 17 C, and the cells were incubated at 30 C in Eagle medium. Ac-tinomycin D (1

lAg/ml)

was used when themultiplicity ofinfectionwas less than 100PFU/cell. Three hours 1220

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after infection, the medium was removed, and

pre-warmed special minimal essential medium without unlabeled leucine and containing 10 uCiof ['Hlleu-cine perml wasadded. Plates wereimmediately set either at 39.6 or at 30Cforthe labelingperiod.

Cell fractionation. Whereas four to eight petri dishes ofcellsaresufficient to give enough radioactive

material, in order to obtain visible light-scattering

bands, carrier unlabeled cellswere added. For this reason, 15 monolayers were used foreach gradient.

The procedure of fractionation was basically that of

CaliguiriandTamm (3), already appliedtovesicular stomatitis virusby Wagneretal. (22).Afterwashing

with cold Tris-buffered saline, cells were scraped,

centrifuged, washedagain, and recentrifuged at 1,500 x g for 5 min.The pelletwassuspended in 5ml of reticulocyte standard buffer, consisting of 0.01 M

Tris, 0.01 M NaCl, and 0.0015 M MgCl, (pH 7.4). Afterallowingthe cells to swell for 20 min, thecells weredisrupted with20 strokes of a Dounce

homoge-nizer. Nuclei and large celldebriswere removedby centrifugationat800 x gfor 5 min. Thecytoplasmic suspension, containing membrane fragments, was

fractionated by centrifugation in a discontinuous sucrosegradientofthefollowingcomposition: 3 ml of 60% sucrose in reticulocyte standard buffer, 7 ml of 45%, 7mlof 40%, 10mlofthecytoplasmicsuspension in30%sucrose, 7 ml of 25%,and3 ml ofreticulocyte standard buffer. Gradientswerecentrifugedat86,000

xgfor 18 h inaSpincoSW27rotor. Four light-scat-tering bands appeared at the interfaces of the suc-cessive layers, except between the 0 to 25% layers. The bands were collected from the side of the tube with a syringe. Thepelletfraction was also recovered. Each fraction diluted withreticulocytestandardbuffer

wasrepelleted by centrifugationat80,000 x g for 90 minin aSpincotype 30 rotor.Solubleproteinsdid not migrate in the gradient and were recovered by

tri-chloracetic acid precipitation from the 30% sucrose

layer andfromthesupernatantsoftherepelleted frac-tions 1and2.

Extractionandanalysisofproteins.Cytoplasmic extracts and virions were treated by the Maizel

method (18). After adding0.1 vol of glacial acetic

acid, thesuspensionwasmade 0.5M with respectto urea and1%withrespecttosodiumdodecylsulfate.

After incubation for 1 h at 37 C, samples were

dialyzedovernightatroomtemperatureagainst 1,000

vol of 0.01 M phosphate buffer (pH 7.2) containing

0.1%sodiumdodecylsulfate,0.1%2-mercaptoethanol,

and 0.5 M urea.Cytoplasmicsolubilizedproteinswere

mixedbeforeelectrophoresis with10ilitersofmarker

"ICproteins extracted frompurifiedvesicular stoma-titis virions (21). Electrophoresis was performed as

previously described (16), except that a higher gel concentration (7.5% acrylamide) was used. Frozen

gels, sliced into 1-mm disks, weredepolymerized in 0.2 ml of 60 vol ofH,O,at60C for4hpriorto the addition of Bray scintillation fluid (2). Vials were counted in an Intertechnique SL40 assisted by a double-labelingcomputer program.

RESULTS

Thermolability.

Amongall thets mutantsof

the Orsay collection, the virions of tsV 45 and

tsV 110arethemorethermolabileinvitro. Both

belong to complementation group V. Thermal

inactivation of tsV 45 has already been

de-scribed by Deutsch and Berkaloff (9). Thetwo

othermutantsclassified ingroupV, tsV44and

tsV 57, show the same thermolability as the

reference wild-type strain (Fig. 1).

Viral proteins in whole cytoplasmic

sus-pensionof infected chicken embryo cells. The

distribution in asodiumdodecyl

sulfate-polya-crylamide gel of the viral proteins synthesized

at 39.6C in cells infected either with the

wild-typeorthe tsV 45 strains are shown in Fig.

2. As already noted by Printz and Wagner (21)

and by Wunner and Pringle (26),nosignificant

differences appear between the two profiles.

Under these conditions, proteins N and NS

migrate very closely and are not generally

resolved as twowell-separated peaks. In Fig. 2

the NSpeak is separatedby only one point from

tsV 45 and is notdistinct from the N peak for

the wild type. Such a difference is not

signifi-cant. As noted by Kang and Prevec (13), the

virion marker G protein migrates slower than

the intracellular G. Compared to results

ob-tained at 37 C with L cells (13, 25) or BHK-21

cells (26), we regularly find a smaller relative

amount of M protein. Cells infected at 39.6 C

with tsV 57, tsV 110, and tsllI 89 give patterns

identical to those ofFig. 2.

Distribution of viral proteins in cytoplas-mic fractions from infected chickenembryo

-0

1 2 4

hours

FIG. 1. Thermolability of tsV mutant strains.

Samples (1ml) of fresh virus stocks wereimmersed in awater bath at 40 C 4 0.1 andperiodically assayed

for survival of plaque-forming ability on chicken

embryo cells at 30C. Symbols: U, ts+; 0, tsV44; A.tsV45;0,tsV57;0,tsVlIO.

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LAFAY

peratr nciknebyel

infce

ihrwt

L G

the ts+ wild-type or the tsV 45 strains. Chicken

embryocellsareinfectedatamultiplicity ofinfection

of80PFUforthe wildtype(a_0)andof120PFUfor

tsV45(O--O).Afteranad-sorption period of45min

at 17C, cells were incubated at 30C for 3 h in

medium without (tsV 45) or with (ts+) I jug of

actinomycinD per mi. [3H]leucine was then added

andtheinfectedcellswerefurtherincubatedat39.6C

for3h.Cellswerethenwashed,scraped,pelleted,and

disruptedinreticulocytestandardbuffer.Cytoplasmic

extractswerepreparedasdescribed inMaterials and

Methods. Electroptwre8i8 wasperformed for5 h on 7.5%6sodiumdodecyl -sulfatepolyacrylamidegels.The arrows show thepeakg position ofthe

"4C-coelectro-phoresedvesicularstomatitisstructuralproteirs.

cells.Thedistributionof viral

proteins L, G, N,

and

M,

synthesized

at39.6C in five

cytoplas-mic fractions of infected cells

separated

on a

discontinuoussucrose

gradient,

isshown in

Fig.

3. In the

wild-type

infection, G

protein

is the

major

viral component of band 1, which

con-tains 20% of theradioactive-insolubleG

protein.

N

protein

is

regularly

presentinsmallamounts,

whereas M

protein

isnot

always

found.G is also

predominant

inbands 2 and3, wherethe three

other

proteins

are

always

found. N is

predomi-nant in band 4 and in the

pellet, which,

together,

containtwo-thirds of the

radioactivity

associated with sedimentable material. We do

not find M in the

pellet.

The distribution of each

protein

inthe different fractionsand of the

different

proteins

in each fraction is

given

in

Table 1, which includes results for soluble

material.

Extracts of cells infected either with ts+ or

with tsV45, tsV57,ortsV 110 andincubatedat

30Cgivesimilar distribution.

Strikingly

differ-entpattemsareobtained for G

protein

synthe-sized at 39.6C in cells infected

by

the tsV

mutantstrains, tsV110

(Fig.

3), tsV45

(Fig.

6),

and tsV57 (not shown).

Very

little G

protein

is

found in band 1, andthe proportion of G protein

in band 2 is often reduced. Results obtained

from cells infected at 39.6 C by the

tsllI

89

strain aresimilar tothose observed with the ts+

wildtype (Table 2). Thus, it would appear that

the abnormal distribution of G protein is a characteristic of group V mutants.

Distribution of viralpulse-labeledproteins

in cytoplasmic fractions. In order to provide

information on the virus biogenesis, we have

studied the evolution of ts+ wild-type, viral pulse-labeled proteins in the cytoplasmic

frac-tions of adiscontinuous sucrose gradient.

At the end of a 10-min labeling period with

['H]leucine (10

,uCi/ml),

the majority of the

radioactivity found inthe cells is not

trichloro-acetic acid-precipitable. This soluble

radioac-tivity decreases veryrapidlyduring the first10

minofincubation in Eaglemedium containing

100

lAg

ofnon-radioactive leucine per ml. It is

clear from Fig. 4 that the bulk of the soluble radioactivity in the cells at the end of a pulse is

notultimately incorporated in thetrichloracetic

acid-insoluble material, attesting to the chase efficiency.

Fractionation in sucrose gradients of

cyto-plasmic extracts was performed immediately

afterpulse-labelingfor 20 min(to) and after 20

(t,0)

and 45 min

(t4,)

ofincubation at 37 C in

chase medium. The virions presentin the

cul-ture supematant and the soluble proteins

re-covered from the gradient were also analyzed.

The results are summarized in Fig. 5. The virion-associated radioactivity increases from 5%ofthe totallabelattoto15%att20 and 33%

at

t4,,

but the kinetics of incorporation of

radioactivity into the virions is very different

from oneprotein to another. Half of thelabeled

Mprotein isalreadyincorporatedinto virions at

to, 80% at t2o, and almost 100% at

t45.

No

detectable labeled G protein is found in the

virions attoandt2oandonly12%isfoundat

t45.

The proportion oflabeled Gprotein increasesin

band 1from2%attoto 4%at

t,o

and10% at

t43.

Nosignificant variation in the proportion ofGis

observed inthepelletand infractions 2, 3,and

4.Solubleproteins, whichareessentiallyN and

NS,decreasefrom 20% attoto5 to 6%att2oand

t,4.

Concomitantly, the proportion of N label

increases first in the pellet and later in the

virions.

Distribution among cytoplasmic fractions

of the tsV viralproteinssynthesizedat 39.6 C

after shift down to 30 C. Cells infected with

tsVmutants wereincubatedat39.6 Cfrom3 to

6 h after infection inmediumcontaining 10 uCi

of [3H]leucine

per

ml.The distribution of viral

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lb 20 3b 40 1b 2'0 30

FRACTIONS 40

ib 20 306 4'0

2

2

x

10,

FIG. 3. Distribution of viral proteins in cytoplasmic fractions ofinfected cells separatedondiscontinuous

sucrosegradient. Aftera45-minperiod of adsorptionat17C, infected cellswereincubatedfor 3 hat30 C in thepresence of actinomycin D(1 pg/ml). ['HJLeucine (10/Ci/ml) was then added, and cells were further

incubatedfor 3hat39.6C. The cellswere thendisrupted andcentrifugedat800xgtoremovenuclei. The supernatantwascentrifugedfor 18hat86,000xginadiscontinuous0to60%sucrosegradient. Light-scattering

bands, locatedattheinterfaces of the successivesucroselayers, werecollected andpelleted by centrifugationat 80,000 xgfor 90 min. Fraction1 is locatedatthe25to30%interface; fraction 2atthe 30to40%interface; fraction 3atthe 40to45%interface, and fraction4atthe 45to60%interface.Left panel: wildtype(multiplicity

of infection [MOI]- 60); middlepanel: tsIII89(MOI - 30);rightpanel: tsV 110(MOI = 40).

proteins labeledat39.6Cwasanalyzedafter0,

20, and 40 min ofincubation at 30 C in

non-radioactive chase medium (Fig. 6). Duringthe

labeling period, therewas noreleaseofPFUto

the supernatant. After the temperature shift

down, virion releasebegan within 30 min, and

extracellular particles accumulated rapidly (Fig. 7).

The maturation of G protein synthesized at

nonpermissive temperature during the chase

period at 30C is summarized in Fig. 6. As

alreadyseen,verylittlyradioactiveG ispresent

in band 1atthe end of the 3-hlabeling periodat

39.6 C. The amount of labeled G in band 1

increasesprogressively during postincubationat

30C andappearsinvirions.Thus,the Gprotein

synthesized at the nonpermissive temperature

in cellsinfected bytsV mutantsisable, aftera

temperatureshiftdown, tobeincorporatedfirst into thecellularfraction 1 and later into virions.

Appearance of proteins synthesized at

39.6C in taIII and tsV virions after shift

down at30 C. In order tosee ifradioactive G

protein synthesized atthe nonpermissive

tem-perature is normally incorporated into virions after a temperature shift down, we have

com-paredthe 'H-C140ratio for each structural

pro-tein ofgroupsmandVtsmutants. ['H

]leucine

0;

WIC

a.

I

2

5

I~~~~~

Pellet~~~~~~~~~~~~~~~~~~~~~~~~~~~~

20

____ . . * . . ~~~~~~~~~~~~~~~~~~.1

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I I

-4k-I

4

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I v I

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TBmLE 1. Distributionoftheradioactivityassociated withG,N(+NS forthesolublefraction),andMproteins in thefractionsisolatedfromadiscontinuous sucrosegradienta

Protein"

G N M Labelineach

Band fato

Distribution Proteinin Distribution Proteinin Distribution Proteinin fraction (%) in sixfrac- eachfrac- in six frac- eachfrac- insix frac- each

frac-tions(%) tions(%) tions(%) tion(%) tions(%) tion(%)

1 19 95 0.2 2 5 3 6

2 27 58 6 33 36 9 13

3 7 61 1 28 10 11 3

4 22 31 17 60 50 8 20

Pellet 8 18 14 82 NDC ND 12

Soluble 16 9 61 91 ND ND 47

aChickenembryocells wereinfectedwith wild type. TheexperimentalprotocolisgiveninthelegendtoFig. 3.

bPercent oflabel in eachprotein: G,27;N, 70;M, 3.

cND, Notdetectable.

3

°t

2

0

:E~_

~

10 20 30 45

[image:5.498.72.246.324.575.2]

MIN AFTER CHASE

FIG. 4. Efficiency ofthe chase medium. Cells

in-fectedwith ts+ wildtype (multiplicityofinfection = 100) were incubated at 37 C. ['HJleucinewasadded 4 hafterinfection. Ten minutes later, cells were washed severaltimeswith cold l7risbuffer andcoveredwith

Eaglemediumsupplementedwith 100 sgofunlabeled

TABLE 2. DistributionofGproteinsynthesizedat

nonpermissivetemperature indifferent cytoplasmic

fractionsa

Complementationgroup

Band ts+ tsV 45

ts1189 tsV110

1 2 1 2

1 18 23 18 2.5 2 3

2 38 32 31 17 22 40

3 11 9 20 12 9 14

4 17 26 17 31 33 30

Pellet 17 10 17 37 33 13

Fractionb 35 35 25 16 25 15

aExpressed as the percentage of G in each frac-tion. Experimental procedures were as described in

legendtoFig. 3.

b Fractionofthetotalradioactivity incorporated in the viral proteins associatedwith G.

leucine per ml. The zerotimesample was takenat this point, and subsequent samples were taken at 10-minintervals. Eachsamplewaswashedcarefully,

and cells were treated with 5% trichloroacetic acid. The amounts ofradioactivity in the trichloroacetic

acid-soluble (0- -0)andprecipitable (0-O)

frac-tions were determined andplotted.

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[image:5.498.264.456.345.553.2]

A B C

~~0t L G N M L G NM L GN M

VIrin

.1

0_

.8

[1 1

jjJJj

0

3J

jfJj

0

L..08

.05 0 7 .05 .02 0 M

.05

0

2

3

-~

~~~

]mM3n4 .2 0

F4]

Fl3

4

1171

Pelt

12369

7

1

19708

3

206312

FIG. 5. Distributionof theradioactivityassociated withL,G,N(+ NS forthe solublefraction),and M in the

fractions (frctn) ofa sucrosegradient and in the virions. Eachfigurerepresents thepercentage ofthe total

radioactivity recovered in eachgradient. Chickenembryocellswereinfected with wildtypeandpulse-labeled for20min. at 4hpostinfection. Incubationtemperature was 37C.(A)Chase0min;(B)chase20min;(C) chase

45min.

waspresentduring incubation at39.6 C (3to6

h postinfection), washed outjust before shift

down at 30C, and replaced by a

[14C]amino

acid mixture. Extracellular virions were

col-lected after 1.5h of additional incubation.The

tsM mutant was abettercontrol than ts+ wild

type, because maturationwas blockedforboth

groups at 39.6C and began within the same

time after shift down to 30C. The relative

incorporation into virions of proteinsG, N, and

M synthesized at the nonpermissive (3H la-beled) and at the permissive ("IC labeled)

temperature is shown inFig. 8.The 3H-14C

ra-tiosinproteinsG andN areidenticalforthetwo

mutants. Thus, the G protein synthesized at

39.6C in cells infected by tsV mutants is

therefore incorporated into virions in normal

amounts after shift down at 30C. The M

protein is synthesized at 39.6 C by tsII

89-infected cells andeasily detectedincytoplasmic

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Chase*min0 ChaseZs min Chase 40min VIRION

______ L

3 2 1

G

I N

L I m1

I

I

N L M

I i

-

s

w I I

*I||, t

I

I~

I

I I I $

I.III

-I~~~~~~~~~

I

I-3

2

[image:7.498.48.463.52.491.2]

FRACTIONS

FiG. 6. Evolution afterashift downat30 Cof the distributionamongcytoplasmic fractions of the tsV45 viralproteins synthesizedat39.6 C. Cellswereinfected with tsV45 (multiplicity of infection =120) in thesame

wayasdescribed in Fig.2.After the 3-h labelingperiodat39.6C, the cellswerefurther incubated in unlabeled

chasemediumat30Cfor20or40minbefore collection. Labeled virus, released into thesupernatantonly after 40 min of incubationat30C, were also collected. Fractionation was carried outon sucrose discontinuous

gradientsasdescribed in Fig.3.

extracts (Fig. 3), but it is not recovered into

virions produced after the temperature shift

down,as already reportedfor tsM 23(16). The

high valueofthe 5H-14C ratio ofM protein in

the tsV 45virions is also regularly observed in wild-type infection for such labeling periods. This maybe duetothe fact that protein M is

incorporated into virions very quickly after

synthesis, as already shown in Fig. 5 and by

Kangand Prevec (13).

DISCUSSION

The viralproteins L, G, N, and M arefound

inthesameproportion in the whole cytoplasmic

extracts from cells infected at permissive or

nonpermissive temperature either by the

wild-3

2

0.8 0.6 0.4 0.2

1.5

I1

cx30.5

a.

3 2

lx

0,

0.5 '

4 3 2

1

20

10

el

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VESICULAR STOMATIS VIRUS ENVELOPE PROTEINS

1op

I10.6

a.

105

3.1&

103

AP- ---_. . . .-A

1 2 3 4 5 6 7 8

[image:8.498.49.236.52.253.2]

HOURS

FIG. 7. Growthcurvesat30, 39.6,and 30-39.6-30C shift of mutant tsV 45. After a 45-min period of

adsorptionat17C, cells infected with tsV45 (multi-plicity of infection=5)werewashed with Tris buffer.

Fresh medium wasadded and petri disheswereheld

at 30 or 39.6C. One hour later the medium was

discarded andreplaced by prewarmed medium; 3 h after infection oneplateat 30Cwas transferred to 39.6 Cfor 3 h andfinally set back at30C. At the indicated times samples were taken for titration.

Symbols: 0,30Cthroughout; A,39.6Cthroughout; 0, 30-39.6-30C; -, 30C incubation; - -, 39.6C

incubation.

typeorby tsmutantsofgroupsHI and V. This

is not unexpected, since thermosensitivity is

usually theconsequenceofmis-sense mutations

which havenoeffectonthegenetranslationand

whose expressionatthepolypeptide level isthe

substitutionofoneaminoacidbyanother. Such

a substitution, which could affect the protein

configuration and function at nonpermissive

temperature, is not expected to modify the

migration properties of the polypeptide in a

sodiumdodecylsulfate-polyacrylamide gel.

We interpret the results of our pulse-chase

experiment with wild-type-infected cells as

meaningthat the vesicular stomatitis Gprotein

migratesfrom the heaviestfractionstothe band

1 before being incorporated into the virions.

Migration for influenza glycoproteins through

thecellular fractionsseemsquitesimilar(7, 14).

As already notedby KangandPrevec (13), we

observe that newly synthesized M protein is

very quickly incorporated into the virions. In

accordwithWagneretal.(25), wefound thatN

protein migratesfrom the soluble tothe

heavi-estfractions, probablyas aresultof

nucleocap-sid formation.

Thiswork demonstrates that association ofG

protein with the lightest insoluble fraction

which can be resolved by discontinuous sucrose gradients is blocked atthenonpermissive

tem-perature by all four mutations classified in

complementation group V by Flamand (10). It

isimportant to notice that identical results are

obtainedeither withgroup V mutants whichare thermolabile in vitro or with those which are

not.The simplest interpretation of these

obser-vations is to assume that incorporation of G

protein in cellularfraction 1 is a necessarystep

ofvesicular stomatitis virus maturation.

After transfer tothe permissive temperature,

the G protein synthesized at the nonpermissive

temperature becomes associated with the light

cellularfraction and released virions. This result does not allow a choice between two hypotheses:

either gene V is the structural gene for the G

protein or gene V encodes a minor protein

necessary for G incorporation in cellular

frac-tion 1. If gene V isthe structural gene of protein

20 '5 5-~~ ~~~~~I 1

a.9

o U

a.

?: 0

5 I

16 20 30 410

FRACTIONS

FIG. 8. Polyacrylamide gel electrophoresis oftsIII 89and tsV 45virionsreleased afterashift down at 30C. Cells infected with tsV 45 (multiplicity of infection [MOIJ = 20) or tsffI89(MOI = 15) were

incubatedat 39.6C from 3 to 6 h afterinfection in

Eaglemedium containing2.5MCiof[3H]leucineper

ml. Cells were washed, and minimal essential

me-diumdiluted10-foldinTris-saline containing2.5uCi

of ["'CJamino acid mixture per ml and 100 gg of

unlabeled leucine per ml was added. Virions were collectedafter1.5hofincubationat 30C andpelleted

at80,000 x gfor60min,andproteinswereextracted as described in Materials and Methods. Top panel:

tsV45; lowerpaneltsHI89.Symbols:*, 3Hlabel;0; "4C label.

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J. VIROL.

G, we mustconclude that alterations produced by the four tsV mutations in the G protein

synthesized at 39.6C are reversibleupon shift

downor, alternatively, that the Gprotein

syn-thesized after shift down allows G protein

synthesized at 39.6 C to be incorporated into

fraction 1.

Protein M, synthesized at39.6 C bytsIII 89,

isreadily detectedincytoplasmicextracts, but,

in contrast to group V mutants, it is not

incorporated into virions after transfer to the

permissivetemperature. These results strongly

confirmourprevious hypothesis thatgene III is

thestructural geneofthe M protein (16, 17).

ACKNOWLEDGMENTS

Thisresearchwassupported by the Centre National dela

Recherche Scientifique through the L. A. 86and 136, the

Fondation pourla Recherche M6dicale Francaise, and the Commissariat al'Energie Atomique, Saclay,France.

I thank Ph.Vigier and A. Berkalofffortheirinterest inthis

work and formanyhelpful discussions. I thank L. Prevec for

criticism ofthe manuscript. I am greatly indebted to B.

Jaillard, D. Pese, and A. Friedman for technical assistance. LITERATURE CITED

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