Temperature-Sensitive Mutants of Complementation Group E of Vesicular Stomatitis Virus New Jersey Serotype Possess Altered NS Polypeptides

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JOuRNALOF VIRoLOGY, Aug. 1979, p. 325-333 0022-538X/79/08-0325/09$02.00/0

Vol. 31, No. 2

Temperature-Sensitive

Mutants

of

Complementation

Group

E

of

Vesicular Stomatitis Virus New Jersey

Serotype

Possess

Altered

NS

Polypeptides

D.EVANS, C. R. PRINGLE,ANDJ. F. SZILAGYI*

Medical Research CouncilVirologyUnit,Instituteof Virology, University of Glasgow, GlasgowGll5JR,

Scotland

Received for publication 29 March1979

Invesicular stomatitis virus NewJerseyserotypepolyacrylamide gel

electro-phoresiswasunabletodistinguishthepolypeptidesof thetemperature-sensitive

(ts) mutants ofcomplementationgroupsA,B,C, and F from those of the wild-type virus. However, the NSpolypeptide of therepresentativemutantof group E, tsEl, had asignificantlygreaterelectrophoretic mobilitythan that of the wild-typevirus NSpolypeptide.Theelectrophoretic mobilitiesoftheNSpolypeptides

ofthe three mutants of complementation group Evaried, being greatest in the caseof tsEl,slightlyless for tsE2,andonlyalittle greater than that ofwild-type

virus NS polypeptide in the case of ts E3. Since the NS polypeptides ofthe revertantclones ts El/Rl and tsE3/Rl have mobilities identicaltothatof

wild-typeNSpolypeptide,theobservedaltered mobilities of the group Emutants are

almost certainly the direct result of the ts mutations in the E locus. The

electrophoretic mobilities ofthe intracellular NS polypeptides of the group E

mutantswereindistinguishable from those of their virion NSpolypeptides. The

electrophoreticmobilities of theNS polypeptidesof the group E mutants

synthe-sized in vitro using mRNA synthesynthe-sized in vitro by TNP were identical to those of theNSpolypeptides of their purified virions. The NSpolypeptidesofall three mutants werelabeled with

'Pi

toapproximately thesame extent as wild-typevirus NSpolypeptide, indicatingthatgrossdifferences inphosphorylationof

thispolypeptide areunlikelytoaccountfor thealtered mobilities. We proposea

model in whichthe NS polypeptide consists ofatleast three loopsheld in this configuration by hydrophobic orionicforces or both andstabilized by phospho-diester bridges. If a mutation affects one of the amino acids to which the

phosphateiscovalently linked,thephosphodiester bridgecannotbeformed, and,

as aresult,in the presence of sodiumdodecylsulfate theaffectedloopopensand

thus the NSpolypeptidemigrates further into thegel.Suchaconfigurationmay

alsoexplainthemultifunctionalnatureof the NSpolypeptide.

Vesicularstomatitis virus (VSV)NewJersey

serotype consistsofasingle-strandedRNA mol-ecule and five polypeptides, L, glycosylated G,

N,phosphorylated NS,and M(12, 13,

15,22,28-30). PolypeptidesN,NS, andM,aswellas the

unglycosylated precursor to G, have been

syn-thesized in the rabbitreticulocyte lysate system

by usingRNAsynthesizedin vitroby the

tran-scribing nucleoprotein (TNP)complex (15).

The temperature-sensitive (ts) mutants of VSV New Jersey fall into six nonoverlapping complementation groups; the majorityisolated

belong to groups A and B, whereas group C

contains only four mutants, group E contains three, groupFcontains two,and groupD con-tainsonlyone(16, 17).

AttherestrictivetemperatureviralRNA

syn-thesis was defective in BHK-21 cells infected with mutants ofgroups A, B, and F, butnot in thoseinfectedwith mutants of groups C and D (17). Mutants of group E were heterogeneous; ts El and ts E3 did not synthesize viralRNA at

390C,

whereastsE2did (17). Viral protein syn-thesisat

390C

ininfected BHK-21cellsexhibited a similar pattern (30). Subsequent analysis

showed thatprimary transcriptionwasinhibited at39.5°C in cells infected with ts Bl and ts Fl but not incells infectedwith tsAl,tsCl,tsDl, andtsEl (9).

Recently, Szilagyi and Pringle (26) showed

that in vitro RNA synthesis at

390C

by the RNA-negative ts Al and the RNA-positivets Cl and tsDlwas similartoRNA synthesis in wild-type virus, whereas the RNA-negative ts Bl

325

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326 EVANS, PRINGLE, AND SZILAGYI

synthesizedonlysmallamounts of RNA in vitro

at 39°C. Mutant ts El synthesized very little RNA,tsE2 synthesizedmoderate amounts, and ts E3 was not inhibited. The group F mutants werealso dissimilar;tsFlsynthesized only very little RNAin vitro at39°C,whereas ts F2 syn-thesized as much RNA as did wild-type virus. We haveanalyzedthepolypeptidesofsomeof these ts mutants of VSV NewJersey to

deter-mine whether differences in theelectrophoretic mobilities of any of the structuralpolypeptides areassociated withanyof thecomplementation groups. Initially, we examined one representa-tive mutant from each complementation group and found that the mobility of the NS poly-peptideof ts El differedfrom that ofwild-type NS polypeptide. The present paper describes ourconsequent studyof the NSpolypeptide of all threecomplementation group Emutants.

MATERIALS AND METHODS Mutants. The tsmutantsof VSV NewJersey de-scribed previously by Pringle etal. (17) were used. The revertant clone ts El/Ri has been described previously bySzilagyiandPringle (26),andtsE3/R1

was isolated from BS-C-1 monolayers incubated at

390C.

Growth and purification of virus. Virus was grown inBHK-21 clone13monolayersandpurified by

the meothod of Sziligyi and Pringle (24). Purified virus was suspended in 20 mM Tris-hydrochloride

buffer, pH 8.0, and theproteinwasdeterminedbythe method ofLowryetal.(10).

Preparationofradioactivelylabeled virus. Vi-ruswasgrown in 80-ounce (2,400-nil) Burrler bottles

asdescribed above except that (i) the medium con-tainedonly20%oof itsnormal amino acidcomposition

when theviruswas tobelabeled with[3S]methionine,

or(ii)the mediumwasphosphate-freewhen the virus wastobe labeled with32P.[35S]methionineor32Pi(200

ytCi in35 ml of medium perbottle) wasadded 6h

postinfection. Afterharvesting, the medium contain-ing the virus wasclarifiedby centrifugation at 2,500

rpmfor 30 minto removecellulardebris,andthen the virus waspelletedbycentrifugationat21,000 rpm for

90minthrougha30%glycerolcushioncontaining20

mMTris-hydrochloride,pH8.0,1mMEDTA,and0.1

MNaCl.Thepelletswerethenresuspendedinabuffer consistingof 20 mMTris-hydrochloride buffer, pH8.0,

1mMEDTA, and 100mMNaCl.

Preparation ofintracellular

[35Slmethionine-labeled polypeptides. Throughout theexperiment

the medium contained only20% of its normal amino acid composition. Each BHK-21 cellmonolayer ina Burrlerbottlewasinfectedwithalowmultiplicityof purified virus (24) andincubated at310Cfor22h in 35 mlofmedium. After this incubationthemedium was removed, and the surface of thecell monolayer

waswashed twicewithfresh medium. Then 20ml of medium containing 300 ,uCi of [3S]methionine was added, and incubationwascontinued at310C. After

90minofincubation, themediumwasremoved, and thecell surfacewaswashed twice with fresh medium.

Finally, 15 ml of 20 mM Tris-hydrochloride buffer,pH 8.0, wasadded, the cell monolayer was removed by sterileglass beads, and a homogeneous suspension was obtainedby extensive sonication.

Amock-infected cell suspension was obtained in the sameway except that the virus was omitted.

Preparation of TNP. TNP complexes were pre-pared by the method ofSzilagyi and Uryvayev (27), incorporating the modifications of Szilagyi and Pringle

(25).

In vitro RNA and protein synthesis. Comple-mentary RNA wassynthesized in vitro at310Cfor 90 min in a 0.2-ml incubation mixture containing 0.02 ml ofTNP,0.05 ml ofreagent mixture (containing 400 mMTris-hydrochloride buffer, pH 8.0,400 mM NaCl, 3.2Mig of actinomycin D per ml, 2.56 mM ATP, 2.56 mMCTP, 2.56 mM GTP, 0.256 mM UTP, and 12,tCi

of[5,6-3H]UTP), 3.5 mMdithiothreitol, 2 mM addi-tionalATP, 25,iM S-adenosylmethionine, and 1 U of ratliver RNase inhibitor (Amersham/Searle) per ml. An unfractionated rabbit reticulocyte lysate was prepared as described by Preston and Szilagyi (15). Theendogenous RNA from the lysate was removed by treating it with micrococcal nuclease as described by Pelham and Jackson (14).

The cell-free translation system (25 ,u) contained 100 mMHEPES (N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid) buffer (pH 7.7), 40 mMKCl,0.8 mMspermidinetrihydrochloride, 1 mM ATP, 0.1 mM GTP, 4mg of creatinephosphate per ml, 200 Mg of creatinephosphokinaseperml, each of the 19 common amino acids except methionine at a concentration of 50

AM,

500

ACi

ofL-[35S]methionine, 12.5% lysate,and

200MuMhemin.Finally,2.5,l of the incubation mixture wasadded, and it was incubated at310Cfor 60min. Protein synthesis was terminated by the addition of 25 Il of a solution containing 100 mM EDTA, 2% methionine and 300 ,ug of RNase A per ml, followed

byafurtherincubation at310Cfor 15 min.

Polyacrylamidegel electrophoresis. Samples of

purifiedvirus or cell-free translation system were an-alyzed by discontinuous sodium dodecyl sulfate

(SDS)-polyacrylamidegradient gel electrophoresis as described by Marsden et al. (11). To obtain greater separationof the N and NS polypeptides, the electro-phoresis was continued for an additional 2 h after the bromophenol blue dye front had reached the bottom of thegel. Kodirexfilm was used for the autoradiog-raphy.

RESULTS

Polypeptide composition of the virions of thets mutantsof VSV NewJersey.We chose arepresentative mutantfrom eachofthe com-plementation groups A, B, C, E, and F and analyzedtheirpolypeptide compositions by dis-continuouspolyacrylamide gradient gel electro-phoresis. Group D was excluded for reasons outlinedbelow.

Figure 1shows thatwild-typeVSV New Jer-seycontains fivepolypeptides, L, G, N, NS,and M, although at least two minor bands (MB1, MB2) of unknownoriginwerealso detected. The

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NS POLYPEPTIDES OF VSV ts MUTANTS 327

polypeptides of themutantstsAl,tsBl,tsCi, andtsFl had electrophoretic mobilities identi-caltothose of the wild-type virus polypeptides. However, the NS polypeptide oftsEl hasa

significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide, whereas the mobilities of the other polypeptides

werenotdifferent (Fig. 1). This indicated that

the ts El mutation might have affected the electrophoretic mobility of the NS polypeptide. Polypeptide composition of thevirionsof

the group E mutants. Since the NS poly-peptideof tsEl hadanalteredelectrophoretic

mobility inapolyacrylamide gel,wedecidedto

examine allthreemutantsof complementation

group E,as wellastherevertantclones tsEl/

Rl andtsE3/Rl.

01)

-CL

3 <2

n 4U:

0) Q)

X -_ i. LL

U) 0 0

4- 4-1 4- 4t

.. :. :: .... :. :}:.:..: .:

^.=

Ls_w_-

. rg

*.:...::...

.... .w *.:hi,s. s i . ''

... ... ... .. ..

:. .: ::

.D ..

..:...:::::::::

:: :: .:: ::. :: .. : ...

.. .. :.

:. ... .. ..

.:

...:...:.

....,S,.--;-'

.*...:

.. . . ..

.: .: .5 .... ...

.,

.. .: :: .. ....

G ^6i__wlw _ _- t_ __

N NS

MB2 >4; *4

MB1

M e4 f

FIG. 1. Virionpolypeptides of the ts mutants of VSV New Jersey. [ Sjmethionine-labeled purified

virionsofwild-typevirus andts mutantswere

ana-lyzedon a discontinuousSDS-polyacrylamide

gra-dientgelasdescribed inthe text. Each track

con-tainedapproximately60),000cpmof[3Slmethionine.

ThepolpeptidesL,G, N, NS,andM,as well astwo

minor bandsofunknown origins (MB1, MB2), are

indicated. Toemphasizetheposition ofthe NS poly-peptides, theyaremarkedbyopentriangles.

Figure 2 shows thatthe mobilities of

polypep-tides L, G, N, and M of each mutant and the

two revertants wereindistinguishable from those of wild-type virus polypeptides although the NS polypeptides of the threetsmutantshad differ-entmobilities.

Because theelectrophoretic mobility of N was

identical in eachcase, it was convenient to

ex-presstherelative mobilities of the NS polypep-tides of the mutants by comparing theextentof theseparation of their N and NSpolypeptides

with theseparationobserved inwild-typevirus.

Theseparation ofpolypeptides Nand NSwas

approximately 1.6timesgreaterfor ts El than

for wild-type virus (Fig.2, tracks 1 through3),

it wasslightly less in the case of ts E2

(approxi-mately 1.4 times greater, tracks 5 through 7),

and in tsE3 the separation was only 1.3 times greaterthan inwild-type virus (tracks 8 through

10).

Thedifferencesintheelectrophoretic

mobili-ties of theNSpolypeptides ofthe three ts mu-tants wereclearly resolvedin thosetracks where

mutantsandwild-typevirusweremixed in

var-ious combinations (Fig. 2, tracks 13 through 15).

Thus,the order of the mutants, according to

the extentofmigrationof their NSpolypeptide,

is tsEl, ts E2, ts E3.

Theelectrophoretic mobilitiesof theNS

poly-peptides of the revertant clones ts

El/Ri

and ts

E3/Rlappear tobe identicaltothat of the NS

polypeptide ofthewild-typevirus(Fig. 2, tracks

4, 5, 11and 12). The simultaneousreversion of the tsphenotypeand theelectrophoretic mobil-ity ofthe NSpolypeptide stronglysuggeststhat the ts mutation is responsible for the altered

mobilityof the NSpolypeptidesof themutants

ts El and tsE3,andprobablyof ts E2 aswell.

Polypeptide synthesis in

cefls

infected

with the group E mutants. BHK-21

celis

were infected with the three group E mutants and the two revertants, and the intracellular

polypep-tideslabeled duringa 90-minpulse of

[3S]me-thionine were analyzed by polyacrylamide gel

electrophoresis (Fig. 3).

Actin was the predominant labeled

poly-peptide in mock-infected cells (Fig. 3, tracks 2

and 13). In cells infected with the wild-type

(tracks3,7, 8, and11),mutant(tracks4

through

6), andrevertant(tracks10and11)virusesthere were,besides theactin, sixmajorlabeled

poly-peptides. These were the viral polypeptides L, G, N, NS, and M, as well as a further

poly-peptide,pG,which ispresumablyaprecursorof G.Ineach casepolypeptides L,G, pG, N,and M were

indistinguishable

from thepolypeptides of the wild-type virus(tracks1 and 14). However,

theelectrophoreticmobilitiesofpolypeptideNS

in cellsinfectedwith thethree mutants(tracks

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a 4r,4)

e- r- ._ N

LU IL L - wU

U,0 U- 4t

4-1 2 3 4 5

* ::

L i r¢z.

+ 4- + \

X - L UJ L

u) : z u: u

6 7 8 9 10 11 12 13 14 1516

*:.

0% 44^_

N

S

NS t

Cr~*"

~~

D

[image:4.504.114.404.72.414.2]

M -~ up_ .qoe- e-4I~

FIG. 2. Virionpolypeptides ofthetsmutantsofcomplementationgroupE.[35SJmethionine-labeledpurified

virions ofwild-type VSV New Jersey (wt), the threets mutants(tsEl,tsE2,tsE3) ofcomplementationgroup

E, and therevertant clones (ts El/RI, tsE3/RI) wereanalyzedon adiscontinuous SDS-polyacrylamide

gradient gelindividuallyorin mixedtracks,asindicated.Each track contained approximately60,000cpmof

[35S]methionine. Therefore, 60,000,30,000,or20,000cpmof[S]methionine for each viruswasplacedonthe

tracks,dependingonwhether the viruseswerealoneorincombination.Thepositionsof the NS polypeptides

areindicatedbytheopentriangles.

4 through 6) were different from that of NS

polypeptide synthesized in cells infected with

wild-type virus(tracks 3,7, 9,and 12) andwere

alsodifferent fromeach other. Theseparations ofthe Nand NSpolypeptides forts El,ts E2, and ts E3 were 1.6, 1.4, and 1.3 times greater, respectively, than the separation for wild-type virus. Thus, the mobilities ofthe intracellular NS polypeptides ofthe group E mutants were

indistinguishablefrom thoseof thepurified

vir-ionNSpolypeptides.

The intracellular NSpolypeptidessynthesized in cellsinfected with the revertant clones(Fig. 3,tracks 10 and11)wereindistinguishablefrom

the NSpolypeptideofthewild-typevirus(tracks

9and12), which strengthensthesuggestionthat thealteredelectrophoreticmobilitiesofthe NS

polypeptidesofthemutantsarethe result ofthe

tsmutations.

Invitro-synthesizedpolypeptides. Asthe aberrant electrophoretic mobilities of the NS polypeptidesingroupEappeartobe the result of mutation, these differences should also be observed in polypeptides synthesized in vitro. We tested this bysynthesizing mRNA'sinvitro, using TNP complexesandtranslatingthem ina

rabbitreticulocytelysate cell-freesystem.

Figure 4shows that four major polypeptides

were translatedwhen the RNA synthesized by

wild-typeTNPwasused. Three of these

corre-spondtothe N, NS, and M polypeptides of the virion, whereas the fourth polypeptide (pG) is presumably the unglycosylated G polypeptide (Fig. 4, tracks 1, 2, 11, and 12). In

vitro-synthe-N

-a

_13-

LU₊

r-LUJ

LU LLU

+ + 7

LU

NU

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Q .

I._

-

=-r.L CL cc ClC rn CL)m

>- t. >. w + : *,\ \ - .C -T- N M r-M X Y

0 ._ E 3

1 2 3 4 5 6 7 8 9 10 11 12 13 14

m PM _ _ _ _ _ _ _

pG

X o

Le-

" ="M a

N do_^, am___ Om am _

actin __ ,__

NS

FIG. 3. Intracellular polypeptides ofcelsinfected

with thegroup E mutants. [55],methionine-labeled infected cellextractswereobtainedandanalyzed by

SDS-polyacrylamidegradient gel electrophoresisas

described in thetext. Eachtractcontained

approxi-mately5,000cpmof labeledcellextract.For

compar-ison, [35S]methionine-labeledpurified wild-typeVSV

NewJersey virions (wt virus)or amock-infected ceU

preparation wasused. The NSpolypeptidesare

in-dicatedby theopentriangles.

sized polypeptides pG, N, and M of all three

mutantswereidenticaltothose of the wild-type virus (tracks3, 5, and 9).

Theelectrophoretic mobilities of the in vitro-synthesized NS polypeptides oftsEl andtsE2

were significantly greaterthan the mobility of

wild-type virus NS polypeptide (tracks 3 through 6); the mobility of the NS polypeptide oftsE3, although lower, wasstill greaterthan the mobility of the wild-type NS polypeptide (tracks9and 10).

The differences in theelectrophoretic mobili-ties ofthe NS polypeptides of themutantsare

easierseenherethan inthevirionanalysis,since

the NSpolypeptide isamajor product of the in

vitrotranslation. The in vitro results fully

con-firm the differences in the electrophoretic

mo-bilities ofthe NS polypeptides of the group E

mutants.

Since themobilitiesofthe NSpolypeptides of thevirions andinfectedcells,aswellasofthein

vitro-synthesized NS polypeptides of all of the mutants, were identical, it seems unlikely that the altered mobilitiesof the NS polypeptides are due to aposttranslational cleavage. The lack of low-molecular-weight cleavage products ob-served ingels where electrophoresis was stopped before the dye front ran off the gel is further evidence that cleavage does not take place.

Phosphoprotein composition ofthe group E mutants. The mutants were grown in the presenceof32Pito determine whether the alter-ations in the electrophoretic mobilities of the

NS polypeptides of the group E mutants were

due todifferencesinthe degree of phosphoryl-ation. Equal amounts of protein were placed on each track todetermine whether there were any differences in the degree of phosphorylation of the mutants.

Figure5shows thatonly NS was

phosphoryl-ated(tracks1, 2, 8, 12, and 16).One of the minor

polypeptidesofunknown origin (MB2) was also

phosphorylated. The 3P-labeled NS

polypep-tidesof the three mutants showed differences in

mobilities similar to those obtained previously

with[3S]methionine-labeled virus.

The chief conclusion from Fig. 5 is that the NS polypeptides of all three mutants and the two revertants arephosphorylated.The degree

ofphosphorylationof the NS polypeptide of the

individual mutants is approximately of the same order ofmagnitudeasthatof thewild-typevirus

NS polypeptide. This is clearly illustrated in

mixed tracks whereequalamountsof themutant

and wild-type virus were present (tracks 4, 7,

and10).

DISCUSSION

Polyacrylamide gel electrophoresis was

un-abletodistinguishdifferences between the

poly-peptidesof thets mutantsofcomplementation

groups A,B, C, and F andthe polypeptides of

the wild-type virus. Previously Wunner and

Pringle(31)reportedthat the G and N

polypep-tides ofts Dl, the onlymutantof

complemen-tation group D, exhibited aberrant

electropho-reticmobilities and that theNpolypeptide de-fect was associated with the ts lesion. Subse-quent work suggests that neither polypeptide

defect may beresponsiblefortemperature sen-sitivityoftsDl.This workwillbe thesubjectof aseparate report andistherefore excluded from this discussion.

Theexperiments describedinthispaper show that the NS polypeptides ofall threemutants

have altered electrophoretic mobilities in rela-tionto oneanother andtowild-typevirus.Since the electrophoretic mobilities ofthe NS

poly-peptides ofthe revertantclonestsEl/Rlandts

L

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a a 2

.-4 N N

> + + > VI

C) - Li LU LU LU

LO g TO !In 0 S _

1 2 3 4 5 6 7 8

LU Ln- '7

9 10 11 12

L

G pG

N '-NS cI'

-.4S.

_;uif

[image:6.504.109.402.71.447.2]

m om o

FIG. 4. Polypeptidessynthesized in vitrousingRNAproduced in vitroby TNP complexes of the group E mutants.The preparationofthe TNPcomplexes and the in vitro RNA and protein syntheses aredescribedin the text. Tracks 1, 7, and12contained[35S]methionine-labeledpurified virions ofwild-typeVSV New Jersey

(35Svirus). Track8containedcell-freetranslation systemfromwhich the TNPwasomitted. The other tracks containedcell-freetranslation systemsofthevarious mutants andwild-type virus,asindicated.Singletracks contained 20 ttl. Mixed tracks contained 10 ul from each of the cell-free translation systems. The NS

polypeptidesareindicatedby the opentriangles.

E3/Rl wereidenticaltothat of

wild-type

virus

NSpolypeptide,the alteredmobilitiesof the NS

polypeptidesofthemutantstsEl andtsE3are

almost certainly the result of the ts mutation.

Presumablythis isalsotrueforts

E2, although

as yet we have been unable to obtain a stable spontaneous revertantfromthismutant. Thus,

theinhibitionof virusdevelopmentof thegroup E mutants at the restrictive temperature is

al-mostcertainlyduetoinabilityoftheirmutated

NS polypeptides to function normally at this temperature.

Recently,SzilagyiandPringle(26)foundthat thevirion-associatedtranscriptase

activity

ofts

E1 is temperature sensitive, although in vitro RNA synthesisis not inhibitedat 390C in the

caseoftsE2 andtsE3. In vivo studies showed thatts E3 isdefective in thereplicationofthe virion RNA (17), whereas in the case of ts E2 replication did notappear to beinhibited (17).

Therefore, Szilagyiand Pringle suggested that

the polypeptide affected by the mutation in group E is a multifunctional polypeptide in-volved intranscription, replication,and possibly

somelate stage of virusdevelopment (26).Since the results presented in this paper showthat the mutatedpolypeptideisalmostcertainlyNS, we conclude that NS is the multifunctional

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4)(1-' -4-'cn ) tt Ut

L- a>

t-L-> I= ,- L > + + +

> + 3 ~~~~~~N~ >~

-~~~~~- ~~~ N N CL WL Uii

Cl) LWLLJ LX LAU)LL X U- + + + l LO >LOU) XJa U) V1 U:*-, N LO

5 6 7 8 9 10 11 12 13 14 15 16

L

NWS

[image:7.504.97.399.72.421.2]

MB2 >HJ

FIG. 5. Phosphoproteincomposition of complementationgroupEmutants. 32P-labeled purifiedvirionsof

thegroupEmutantsandtheirrevertantswereanalyzed bydiscontinuousSDS-polyacrylamide gradient gel

electrophoresisasdescribed in thetext.Each trackcontained 9 ,ugofvirusprotein.Intracks whichcontained

morethanonevirus,theamountsofeach viruswereproportionately reduced in ordertomaintain thetotal

protein concentrationataconstantlevel. The NSpolypeptidesareindicated by theopentriangles.wt, wild-typevirus.

peptide. That the NS polypeptide of VSV

Indi-anaisinvolved intranscription has already been

shownby dissociationandreconstitution

exper-iments (4, 7). Our evidencesuggeststhat the NS polypeptide is also involved in thereplicationof virionRNA, althoughwecannotsaywhether it

isthereplicaseitselforpartofa replicase

com-plex. Since viral RNA synthesis takes place ints

E2-infectedcellsattherestrictivetemperature,

the involvement of NS polypeptide in a late

stage ofvirusdevelopment is suggested. Previ-ously, Szilfigyi and Pringle (26) showed that the heat stability of the transcriptase ofts El was

dependent on an interaction between the viral

coatandthecore.

At present it is not certain whatcauses the

marked alterationintheelectrophoretic mobil-ities of theNS polypeptides of the threegroup

Emutants. Twopossible explanations arise: (i) either the NSpolypeptides of thegroup E

mu-tantsareshorterthan theNSpolypeptide of the

wild-type virus, or (ii) the phosphorylation of

mutant NS differs from that of the wild-type NS.

The occurrence of a shortened polypeptide canbeexplained in severalways,although there

areobjectionstoallof them.(i) A section of the

NS cistronmayhave been deleted by the

mu-tation. However,weconsiderthisunlikely since

the base analog 5-fluorouracil, which favors point mutation, was used as the mutagen and

sincerevertantswereobtained fromtsElandts

E3athigh frequency.

(ii) The mRNA for NS may have been

pre-maturely terminated. However, all of the evi-dence available suggests that transcription is

1 2 3 4

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initiated only at the 3' end ofthe genome (1, 2). Thus,premature termination of the mRNA syn-thesiswould producepolareffects whereby the subsequent messengers for the M, G, and L polypeptides would notbe transcribed. To ex-clude this possibility, the mRNA's of the mu-tantswill have tobecomparedwith those ofthe wild-type virus by gel electrophoresis and

oli-gonucleotidefingerprinting.

(iii) The mutant mRNA may contain an early termination or late initiation codon resulting in a shorterNSpolypeptide.To determinewhether this isthe case, thesequence of themRNA's of

the various group E mutants will have to be

determined.

However, itisdifficulttoreconcile early ter-mination or late initiation of transcription or

translation with the loss of functions of the

various group E mutants.

(iv) Another possibility is that mutation al-tered the NS polypeptide insuch a way as to render it moresusceptibletocleavage by

prote-olyticenzymes.This is alsounlikely since the in

vitro-synthesized NSpolypeptideshadsimilarly alteredmobilities. Also, it isgenerallyheld that in the rabbit reticulocyte lysate system the in

vitro-synthesized polypeptides are not cleaved

(3, 18, 21), and there was no evidence for the presence of a low-molecular-weight cleavage

product. Furthermore, the mobilitiesof the

in-tracellularNSpolypeptides of the group E

mu-tants were indistinguishable from those of the purified virion NS polypeptides,making prote-olytic cleavage even more unlikely.

SinceNS is aphosphorylatedpolypeptide, the secondpossibility is that the aberrant mobilities of this polypeptide in thevarious group E mu-tants are aconsequence of the differences in the extentofphosphorylation. However, the exper-iment with32P-labeled virions indicated that, in the case ofall three mutants,polypeptide NS is

phosphorylated toapproximately the same

ex-tent asit is inwild-type virus. Therefore, gross

differencesin the level ofphosphorylationinthe

variousmutants areunlikelyto bethe cause of thedifferencesinthemobilitiesofthe NS poly-peptide.

Wepropose a model which couldexplain both thealteredmobility of the NSpolypeptide and the nature ofthegroup Emutation.According tothismodelthe NSpolypeptide chain is folded into atleast three loops. Each loop is held to-gether by ionic orhydrophobic bonds or both

and stabilized in this configuration by a

phos-phodiester bridge. Phosphate diesters cannot be hydrolyzed by alkaline phosphatase (6, 19), which explains why polypeptide NS was not dephosphorylated by this enzyme (12, 22; un-published observations on VSV New Jersey).

J. VIROL.

Therefore, the structure ofpolypeptide NS can-not bedenatured by boiling in SDS and 2-mer-captoethanol during preparation for polyacryl-amide gel electrophoresis, and hence it would notmigrate asfar into the gel as afully dena-turedpolypeptide. This would also explain why the NS appears as ahigh-molecular-weight poly-peptide inpolyacrylamidegels despite the rela-tivelysmall coding capacity of its mRNA and

also would account for the differences in the

mobilitiesof the NSpolypeptideindifferentgel

systems (5,8, 20,23).

Ifmutation results in the substitution ofone of the amino acids to which the phosphate is

covalently bonded, the phosphodiester bridge

cannot be formed and the stabilization of the

loop doesnottakeplace. Therefore, under

de-naturing conditions this loop opens, and the

mobility of the NS

polypeptide

in the gel

in-creases.The extentofthis increasepresumably

dependsonthe size of theloopaffected bythe

mutation,whichwouldexplain the differing

mo-bilities of the NS polypeptides of the group E

mutants.

Our model can alsoexplain the ts nature of

themutation.Atthepermissivetemperature the

ionic and hydrophobic bonds alone are

suffi-cient, but at the restrictive temperature the

phosphodiester bond is required to hold the

configuration of the loop. Thus, a mutation

which results in the breaking of the

phospho-diester bridgewill be temperature sensitive for the function of that loop. Mutations affecting

any other amino acid notlinked by the

phos-phodiester bond will presumably be either a

silentor alethalmutation.

Accordingtothismodeleachloop corresponds

to a different function of the NS polypeptide.

One of theloops (loop ts

El)

isinvolved in the

transcription of mRNA's, the second (loop ts

E3) isinvolved in thereplicationof virusRNA,

and the third (looptsE2)isinvolved in the late

functioninwhich themutant tsE2is defective.

The model would explain why there is no

correspondencebetween the loss of functionsby

the mutants andthe differencesin the electro-phoreticmobiitiesof theNSpolypeptide.

Althoughatpresentwehavenoexperimental

evidence tosubstantiate our model,weare ac-tivelyengagedinthedetermination of the struc-ture ofthe NS polypeptide.

Also,

we willhave to isolate morerevertantclones,especiallyfrom tsE2,sinceattemptstoisolate stablerevertants from thismutanthave sofar beenunsuccessful.

ACKNOWLLEDGMENTS

We thank J. H.Subak-Sharpeforacriticalreadingof the manuscript. We also thank C.Cunninghamfor hisexcellent technicalassistance, C. M.Prestonforhelping with the in

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vitrotranslation, and J. T. Poyner for helping with the prep-arationofthe manuscript.

D.E. was arecipient ofaScience Research Council scholar-ship for training in research methods.

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