Altered E2 glycoprotein of Sindbis virus and its use in complementation studies.

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0022-538X/78/0026-0126$02.00/0

Altered

E2

Glycoprotein

of

Sindbis Virus

and Its

Use

in

Complementation Studies

MOSHE BRACHAt ANDMILTON J.SCHLESINGER*

WashingtonUniversity Schoolof Medicine, Department of Microbiology and Immunology, St. Louis, Missouri63110

Received forpublication14November1977

We have detected a Sindbis virus variant that contains a

smaller-molecular-weight form of the viral glycoprotein E2. The molecular weight of the PE2

precursor and the glycosylation pattern of the smaller E2 are normal, thus indicating that this E2 is formed by an aberrant proteolytic cleavage. Thealtered E2 was detected in an

RNA'

temperature-sensitivemutantthat wasdefective in

proteolytic cleavage, but the abnormal PE2-to-E2 reaction could be separated

fromthe tsmutationand is notitselfatemperature-sensitivedefect. We used the variant E2 as a marker to monitor the complementation reaction between an

RNA'

and an RNA- mutant and discovered that complementation was not

reciprocal; the RNA defectwas corrected by theRNA' mutantgene products

but theRNA'defect was notcomplemented by any RNA- gene products.Other studies have shown that the smaller E2 is notpreferentially selected duringviral

maturation and budding. No significant changes have been detected in the

biological activity of virions with this altered E2 protein. Comparison of the

electrophoretic migrationof the El andE2Sindbis viralglycoproteinsin a

two-dimensional polyacrylamideslabgelsystemthat was first run inthe absence of

sulfhydryl-reducingreagent and then

with,B-mercaptoethanol

indicated that the

mobilityofEl,butnotthat ofE2,wassignificantly altered by reduction.

Two majorglycoproteins

(designated

Eland the E2 viral

glycoprotein

from a

glycosylated

E2) arefoundonthesurface ofSindbis virus,a precursorcalled

PE2,

and it is believedtooccur

smallRNA-enveloped virus of the Togaviridae closetothe cell'splasmamembraneduring

bud-family (24).Theyareformedinthe infectedcell dingofvirus from the hostcell(26).Someofthe

from larger-molecular-weight polypeptide pre- temperature-sensitive (ts) mutants isolated by

cursors viaaseries ofposttranslational proteo- BurgeandPfefferkorn (3) have been found

de-lytic cleavages and glycosylation steps (16, 22, fective in theseproteolyticevents, and precursor

23, 25, 26, 28,29).Aninitialproteolytic activity

polypeptides

accumulate in cells infected with

isbelievedtooccuronthepolyribosome of the the mutants atthenonpermissive temperature

infected cellaftertranslation of the viralstruc- (2, 13). During a survey ofviral proteins

pro-turalproteins has been initiatednearthe5'end ducedincells infectedwiththese different

mu-of the

viral-specific

26SmRNA, and this activity tants, we detected an alteration in theE2

gly-releases viral

capsid

polypeptides thataggregate

coprotein

formed

by

one of themutants when

with viral 42S mRNA to form

nucleocapsids.

grown atthe

permissive

temperature. We

con-Continued translation of the 26S mRNAyields sidered the possibility that thisphenotype

re-the twoproteins destined tobecome the enve- sulted from the same genetic lesion that

pro-lope glycoproteins.A secondproteolyticactivity duced the ts condition and proceeded to study

occursbeforeorshortlyaftersynthesisofthese thenatureandoriginofthealtered E2 glycopro-membranepolypeptideshas beencompletedon tein. In thisreport,wedescribehowthevariant

thepolyribosome,butpriortoglycosylation (8, E2

glycoprotein

appears, show that it is not

16). Failure to effect the initialcleavageresults related to the ts

defect,

andutilize the altered

in a protein of about 140,000 daltons (22, 23), E2asamarker for

following

eventsduring

com-andfailure of the later one leads to an accumu- plementation betweentwo tsmutants. lation of a polypeptide of 110,000 daltons (18,

23). A third proteaseactivityisrequiredtoform MATERIALS

AND

METHODS

Virus and cell culture. Sindbists2 mutant and

t Present address: Biochemistry Department, Tel Aviv the parental wild-type heat-resistant HR strain (3)

University,TelAviv,Israel. wereobtained from E.Pfefferkorn(Dartmouth Med-126

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ical School, Hanover, N.H.). Mutant ts5 was obtained beled glycopeptides from E2 glycoprotein.

Sec-from B. M. Sefton (Salk Institute, San Diego, Calif.). ondary CEF monolayers in 100-mm dishes were in-Preparations of primary chicken embryo fibroblasts fected at an MOI of 100 with either the HR or the (CEF) and titration of virus have been described (2). revertant Rts2a strain of Sindbis virus at 37°C. Six Isolation of ts2 revertant strains.Astock of ts2 dishes were used for each strain. After 1 h of adsorp-that had been reisolated from a single plaque was tion, MEM containing 10% the normal amou.it of titered at either 30 or 40°C, and plaques were devel- glucose and 3%FCS was added. A total of 30,Ciof oped after2days. The ratio of PFU at 40°C to PFU D-[1-_4C]glucosamine (55 mCi/mmol; New England at 30°C was 5.5 x 106. Single plaques were picked Nuclear Corp., Boston, Mass.) was added to HR-in-from theplates incubated at 40°C and transferred to fected cells and 300

,Ci

ofD-[6-3H(N)]glucosamine (2

1ml ofminimal essential medium (MEM) containing Ci/mmol, New England Nuclear) to Rts2a-infected 3% fetal calf serum (FCS). Samples were diluted 10- cells after the adsorption period. Media were collected fold into phosphate-buffered saline containing 1% FCS after 12 h, cell debris wasremoved by brief centrifu-andretitered at 30 and 400C.Theratios of PFU at gation, and the virus sample was layered onto sucrose

400CtoPFU at 30°C for four plaque isolates were 0.6, gradients that consisted of 2 ml of 60% sucrose, 10ml 0.5, 0.47,and 0.82, respectively. Samples (0.2 ml) were of alinear gradient of 50 to 30% sucrose, and 12ml of diluted into2ml of phosphate-buffered saline plus 1% 10% sucrose. Sucrose solutions contained TNE and FCSand adsorbed to monolayers of CEF in 75-cm2 T 0.1% bovine serum albumin. After centrifugation at flasks. After 1h, the inoculum was replaced with 20 25,000 rpmfor3h at4°C inanSB110 rotor (Interna-ml of MEM containing 3% FCS, and thecells were tional EquipmentCo., Needham Heights, Mass.), frac-incubatedat37°C for21h.The media containing the tions werecollected, and radioactivity was measured revertantviral strains were stored at -70°. in smallsamples. The fractions containing the peak of

Labelingofviral-infectedcelis.Primary CEF in virus banding at 38%sucrose were pooled, diluted with

60-mmdishes were infected with either ts2 or ts5 virus twovolumes of TNE containing a sample of

nonradio-at amultiplicity of infection (MOI) of 200 and with ts2 active carrier virus (2x1011 PFU),and centrifuged at revertantsat anMOI of 100. After 1 h of adsorption, 50,000 rpm for90 mininaSpinco65 rotor at4°C. The

5ml of MEM containing 3% FCS was added to the pellets of virusweresuspended in0.4ml of 1%SDSin

plates, and the cellswereincubated at 30°C. At 9 h 0.5 MTris-hydrochloride (pH 9.0), reduced, and al-postinfection the medium was replaced with MEM kylated with iodoacetamide and the E2glycoproteins lacking amino acids butcontaining 3% FCS. For each isolated after electrophoresis on 10%polyacrylamide

virussample,twoplateswerekeptat300Candathird gels by the methods described previously (24). The platewasshiftedto40°C. After2h, the medium was eluted E2proteinsamplewaslyophilized, resuspended

replacedwith 1ml of MEMlacking amino acids and inasmallvolume of water, andprecipitated with 10

containing10,uCiofL-[3S]methionine (330 Ci/mmol; volumes of cold acetone. Bovineimmunoglobulin(200 Amersham/Searle, Arlington Heights, Ill.). After 20 ug)wasaddedascarrier materialpriortoadditionof minatthe abovetemperatures, mediumwasremoved, acetone.The driedproteinwassuspended in 0.5 ml of andoneplateat300Cwastreated with0.5ml of 2% 0.1MTris-hydrochloride (pH 8.0) containing 0.01 M sodium dodecyl sulfate (SDS) containing 1 M Tris CaCl2. Samples (25

pl)

of a 10-mg/ml solution of (pH 9.0). To the other twoplates was added 2 ml of Pronase (Calbiochem, LaJolla, Calif.,A grade), pre-MEM containing 3%FCSand four times the standard pared in thesamebuffer andpreincubated for 2 hat

amountof aminoacids. Theplateswereincubated for 37°C,wereaddedat24-h intervals overa3-day

incu-an additional 1 h, and cellswerelysed with SDSas bation periodat37°C. The entire sample wasloaded

above. ontoaBio-Gel P-6 column(0.9 by 120cm) and eluted

Preparation of

[3S]methionine-labeled

virus. according to the method described by Sefton and PrimaryCEF intworoller bottleswereinfected with Keegstra(28).Atotalof70fractions(1 ml each)were

either HRorts2virus in10mlofphosphate-buffered collected, and0.8mlwasaddedto8.0 ml ofaTriton saline and 1% FCS at an MOI of100. After 1 hof X-114-xylene(1:2 vol/vol) scintillation fluid. adsorption, each roller bottle received20ml of MEM Inhibition ofglycosylation withglucosamine.

lacking amino acids but containing 3%FCS. The HR PrimaryCEFwereinfected with HRorts2at anMOI bottlewaskeptat370Candthe ts2 bottleat300C. At of 100. At 7 hpostinfection at300C,the mediumwas 3 hpostinfection, the mediumwassupplementedwith replacedwithonelackingamino acids butcontaining

10%of thestandard amount of aminoacids and 100 3%FCSand,inone of eachpairofplates,20mM D-,uCi of[35S]methionine.Afteranadditional incubation (+)-glucosamine(SigmaChemical Co., St. Louis, Mo.). for16h,the mediumwasharvested and clarifiedby At 9hpostinfection, mediaonthe treatedplateswere

centrifugation atlow speedfor 20min. The volume replaced with 1 ml of MEM containing 20 mM D-wasreducedto 1mlbydialysisat40Cagainsta20% (+)-glucosamine, 10 MCi of [3S]methionine, and 106 solution ofpolyethylene glycol 6000 (Carbowax) in M unlabeledL-methionine. The control cells received

0.05MTris (pH 7.5),0.1MNaCl,and0.001MEDTA thesamemediumlackingD-(+)-glucosamine. After1 (TNE).The virus waspurified bycentrifugationover- h,the cellswerelysedwith SDSasdescribed above. night through a composite velocityand equilibrium Mixed infection withts mutants at30°C. Sec-gradientasdescribedbyScheele andPfefferkorn(22). ondary CEF in 60-mm plateswereinfected atanMOI

Viruswaspelletedfrom thegradientfractionsbythe of 100 with ts2 alone, ts6 alone, or with an equal

water-dilutionmethoddescribedbySefton andKeeg- mixture of ts2 and ts6 virus. After 1 h ofadsorption,

stra(28). theplateswereincubated for 7 h at300C with 5 ml of

Preparation and analysis ofglucosamine-la- MEM containing 3% FCS and then with the same

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128 BRACHA AND SCHLESINGER J. VIROL.

medium lacking amino acids for an additional 2 h. E1 E2 *E2

This mediumwasreplacedwith 1 ml of MEMlacking amino acidsbutcontaining25MCiofL-[35S]methionine 9

and 106 M unlabeled methionine. After 1 h, the

medium wasremoved, and the cellswere lysed with 8

SDSasdescribedabove. 11

Mixedinfection with ts mutants at 40°C. Pri- 7 l mary CEFin 75-cm2T flaskswereinfected at400C

,-withts2, ts6, orboth,asdescribed above. Between3 6

and 4 h postinfection, the cells werewashed exten- § l

sively severaltimeswith medium andincubatedwith 5S

4ml of MEMcontaining10%of thenormalamounts c I

of amino acids, 1% FCS, and50MCi ofL-[35S]methio- 4 nine. After an additional 5 h, the medium was re- 3

moved, andvirus waspurifiedasdescribed above.

SDS-polyacrylamide gel electrophoresis. Cell 2

extracts were reduced andalkylated priortoelectro- l

phoresis

(24).Purified viruspreparations weremixed 1 -with a loading bufferconsisting of 20 mM Tris (pH

7.4), 2% SDS,5%2-mercaptoethanol,10% sucrose, and 2

bromophenolblue. Themixtureswereboiled for 5 min DISTANCE

andappliedto discontinuous slabgels prepared with

DiStAnCE

7.5or10%acrylamideaspreviously described(24).In FIG. 1. Electrophoretic pattern ofproteins from

general, 20,000 to 40,000 cpm wasadded togel slots. purified virions of ts2 and its parental HR strain. Two-dimensionalgel electrophoresis. Samples Samples of35S-labeled virus were denatured and

(20jl) ofpurified 35S-labeledHR and ts2 virus were subjected to electrophoresis in SDS-polyacrylamide mixed with an equal volume of the loading buffer slab gelsasdescribed in the text. Autoradiogramsof

described above butlacking2-mercaptoethanol. The the dried gelswerescanned inaGilford

spectropho-samplewereboiled for4minandsubjectedtoelectro- tometer at 600nm.( ) HR;

W---)

ts2.

phoresisin a 7.5%acrylamide discontinuous slab gel

at 20Vfor13h.Strips containing viral proteinswere

immediatelycut from the slab gelandplaced across be expressed at 30°C as an aberrant cleavage thetop of a 10%acrylamide slab gel apparatus that thatproduces the E2 protein. To test this

model,

contained only the "running" gel. A layer of 7.5% we isolated temperature-resistant revertants of

acrylamide "stacking" gel containing 10%2-mercap- we andas nledanother ts mutant om

toethanoland 1%agarosewaspouredto fillthespace ts2 and also analyzed another ts mutant from

(1 cm) betweentherunning gel andthestrips. After complementation groupC. Thepatternsof

viral-electrophoresis at 120 V for 3 h, the gel was dried specific proteinsformedbytwo ofthe revertants under vacuum and exposed toKodak No-Screen X- showed that the E2 phenotype was retained

rayfilm. while thep140 protein, which ischaracteristicof

groupC mutants grownat40°C,disappeared in

RESULTS

cells

infected with the revertants (Fig. 2).

Cells

Identification ofanaltered E2 glycopro- infected with ts5, another group C mutant, made

tein and its relationto the tsmutation. In p140 at the higher temperature and formed a

the process ofstudyingthe formation ofviral- normal E2 protein at permissive temperatures

specific proteins in cells infected with Sindbis (Fig.2). Thus, the mutation affecting

tempera-virus ts mutants, weobserved that virions pro- turesensitivitywasdistinctand separablefrom ducedbythe ts2mutant atpermissivetemper- thatleadingtothe E2phenotype.

aturesdisplayedaprotein pattern distinct from Origin of *E2. Despite an easily detectable that of theheat-resistantparental strain (HR). difference inelectrophoretic mobility of *E2, its The ts2 virions contained an E2 glycoprotein immediate precursor, PE2, displayed a mobility

(designated*E2) withasignificantly greater mo- in SDS-polyacrylamide gels identical to that of

bility inSDS-polyacrylamidegel electrophoresis thewild-type virus (Fig. 2 and 3). It is known, than was observed for the HR strain (Fig. 1). however, that glycoproteins have atypical mo-The ts2 strain belongs to complementation bilities inSDS-gelelectrophoresis (12, 30); there-group C mutants (4) and is defective in the fore weexamined the mobilities of the

nongly-posttranslationalprocessing of a high-molecular- cosylated viral membrane proteins. When

glu-weight protein (designated pl40) that contains cosaxnine was used to inhibit glycosylation, the

the sequences of the three viral structural pro- patternsofproteins from the ts2 and wild-type teins(23). At40°C,almostnoproteolytic cleav- viral-infectedcells were identical (Fig. 3, lanes 2 ageofp140isdetectedincellsinfected with ts2, and 4). Conversion of PE2 to E2 also involves

andwepostulatedthat thesamemutationmight changesinglycosylation (28), and it was possible

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WT

ts

2 REVERTANTS

t

5 WT

ts2

~A

B

t

P 140

E

E2

9 ,*-...%. e,

a

b c

a

b

c

a

b c

a b

c

FIG. 2. Viral-specific proteinsin cells infected with mutants ts2 and ts5 and revertants ofts2.Procedures

forelectrophoresis of samplesinSDS-polyacrylamideslab gelsand preparation of autoradiograms are in the text. (a)20-minpulseat30°C;(b) pulsefollowedby1-h chase at30°C;(c) 20-min pulse and1-h chase at 40°C.

that E2wasdeficient inglycosylgroups,thereby esis that *E2 is ashorterpolypeptide chainas a

increasingitselectrophoretic mobility. result of an aberrantproteolytic cleavage from

Glycopeptidesfrom E2and *E2 wereanalyzed anormal-sizedPE2 precursor.

after Pronasedigestion of theglycoproteins that Mobilities of viral glycoproteins in the

werepurifiedfrom virionsgrownin cellslabeled nonreduced conformation. The

electropho-with

["C]-

or

[3H]glucosamine.

Theelutionpro- retic mobilities offully reduced and denatured

files ofglycopeptidesfromaBio-Gel P-6column proteins in SDS-gels can be used to calculate

werevirtually identical forE2and *E2(Fig. 4). molecularweights (9,31);weestimated that*E2

The three peaks eluted first (designated else- is smallerthan E2by 3,000 to4,000 daltons. A where as

Si,

S2, and S3) representthecomplex loss of about 30amino acids might havea

sig-oligosaccharide

with varying amounts ofsialic nificanteffectonvirusstructure,but thereisthe

acid (14), and the last fraction isthe

high-man-

possibility that *E2 retains this fragmentinits

nose"simple" oligosaccharide.The

slight

differ- native state through a disulfide linkage. We

ence inrelativeamountsof

Si

and S2 in *E2 as couldtestthismodel bycomparing the

electro-compared

with E2 could represent more sialic phoretic mobilities of E2 in its reduced and

acidin *E2. However,therelativemobilities of nonreducedstatesandatvaryingconcentrations

the E2 and *E2

proteins

in

gel

electrophoresis

ofacrylamide. Here,wedescribe the results ob-were notaltered after

treating

virionswithneur- tained witha two-dimensionalgelsystem (Fig.

aminidase, even though all bands showed a 5). In their nonreduced forms, there is poor

slightly increased mobility as a result of the resolution between Sindbis virus glycoproteins

neuraminidase treatment(datanotshown).We Eland E2 of the HR strain(Fig.5,1D),and the

alsoassayedtherelativeamountsof

galactose

in second dimension of the

gel

shows that the

the glycoproteins of the variant and wild-type

faster-moving

component of the

partially

re-virions. The E2 protein contained 45% of the solved

glycoproteins

in anonreduced

gel

has a

total

galactose

recoveredin ElandE2,andthis slower

mobility

after reduction

(Fig.

5,

arrow).

value was obtained for both E2 and *E2proteins. Thus,for the normal viral

glycoproteins

in the Allofthese dataareconsistentwith thehypoth- nonreducedstate, El hasafaster

mobility

than

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2 3N

4

' cal gels andrerunning the leading and

trailing

edges of the band after reduction with 2-mercap-toethanol.The Elglycoproteinapparently takes

on adifferent conformation after

reduction,

and

the native nonreduced form of El may be a

highlycompactdisulfide-linkedstructure.

The mobilities of nonreduced El and *E2

fromthets2strain arevirtually identical (right

portion ofFig. 5), butareclearly resolved in the

second dimension after reduction. From these

dataweconclude thatE2and*E2donot

change

their conformation significantly between the

nonreduced and reduced states and that *E2

doesnotretainthe cleaved

polypeptide

fragment

in a covalent form.

The aberrantputativeproteolytic

cleavage

of

PE2inthets2andrevertantstrainscould arise

byamutational

change

inthe

cleavage

site in

PE2,oritmight be the resultof a

mutationally

altered

viral-specific

protease. On the basis of

results described

below,

we consider thelatter

explanationunlikely.

2 Useof *E2as amarker in

complementa-tion. The *E2

phenotype

has

provided

auseful

,.-

_=

~

marker formeasuring the expression ofts2viral

E

I structural genes

during

a

complementation

re-2 action between ts2 and other ts mutants. For

*---

E

this

study

wechose

ts6,

anRNA-tsmutant

(3)

____* E that

complements

verywellthe

RNA'

ts2 defect

*E

, (4). We first examined the viral proteins from

cellscoinfected with

equal

numbers ofthe

mu-tant virions and incubated at the permissive

2000- -200

500 I'I' 15..1

S 6

T-f

000o

oo0

FIG. 3. Effect of glucosamine on

electrophoretic

/Oo

is

patternofviralproteins in cellsinfectedwith wild-typeorts2 virus.Experimentaldetailsarein thetext.

(1)Nornal cellsinfected with wild-type virus;(2) cells

treated with 20 mMglucosamine and infectedwith

wild-type virus; (3) normal cells infected with ts2 40 50 60 70

virus; (4)glucosamine-treatedcells infectedwith ts2 FRACTION NUMBER

virus. Thenonglycosylated proteins are located be- FIG. 4.

Profile of glycopeptides from

E2 and *E2

tweenthe normalglycosylatedEl and E2.

glycoproteins.

Pronase-digested

sampleswere sepa-rated on a Bio-Gel P-6 column according to the

procedure of Sefton and Keegstra (28). Details of E2,but Elisslower than E2 afterreduction.

labeling

the proteins with

[14C]-

and

[3H]glucosa-We have confirmed this property ofEl by mine are in the text. E2 was labeled with

'4C-

*E2

elutingthepoorlyresolvedbandsafterelectro- was labeled with

[3HJglucosamine.

About 10 times

phoresisof thenonreducedproteins in cylindri- more 3H counts were used than 14C in the analysis.

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1D

HR

ts2

04 El

E2

~~~~~~~~~~El

~E

CCM

c~~~~~~~~~~~~~~~~~~

go

FIG. 5. Autoradiogram ofa two-dimensionalSDS-polyacrylamide gel electropherogram containing

'S-labeled virionproteins. Preparation of samplesandproceduresforthisgelsystemaredetailed in thetext.

Thefirst dimension(ID)contains nonreducedproteins. The second dimension (2D)wassubjectedto

electro-phoresisin thepresenceof mercaptoethanol. On thefar leftisthepattern forasetofreduced andalkylated

viralproteins.

temperature.

Equivalent

amounts ofboth viral Thecis-dominance andnonpreferential

selec-structuralgene

products

werefound

(lane

c,

Fig.

tion in viral budding of the *E2 gene product

6), while cells infected with

only

one of the allowedus to

analyze

the expression of the ts2

mutants revealed the

appropriate

phenotype

genomein cells coinfected with

complementing

(lanesaand

b, Fig.

6). Therewasnorestriction mutantsatthe

nonpermissive

temperature.

La-on ts2 or ts6viralRNA

expression

inthe

mixedly

beled virus

particles

wereisolated from the

me-infected cells

but,

more

importantly,

the aber- diumof the coinfectedcells, and thepattern of

rant

cleavage

reaction

yielding

the *E2was co- viral proteins was displayed in

SDS-polyacryl-dominant with that

producing

E2. Ifa

change

amide gels(Fig.7).Of thelabel foundinthetwo

had occurredin a

viral-specific

protease coded viral E2

proteins,

morethan 90%appearedas a

by the ts2 genome,we

might

have

expected

all

nornal

migrating

E2band

(lane

c,

Fig.

7).

In the

E2

protein

to be altered in thecoinfected cell. coinfectedcells at

400C,

more than 80% ofthe

Thus *E2acts as acis-dominant trait that

prob-

labelwasfound in

p140 (data

not

shown),

indi-ably

reflects a

change

in ts2 PE2

protein

se- cating that the

ts2-specific

mRNA was

trans-quence. In

addition,

we have examined cells latedat

400C.

Thus, themostlikely explanation

coinfected with the HR and Rts2a Sindbis virus for the failuretodetect

larger

amountsof*E2is

strains todetermine whether there was a

pref-

that the

complementation

between ts2 and ts6

erential selectionforthe E2or*E2

glycoproteins

is

nonreciprocal.

The ts2 genome can

provide

during

viral maturation and

budding.

Coinfected the function

missing

in ts6

(possibly

an RNA

CEF

monolayers

at

370C

werelabeled with

[35S]

polymerase),

butts6cannotcorrectthe

temper-methionine a 4 h

postinfection,

and virus and ature-sensitive defect in the ts2genome;

other-cellswereharvested4hlater. Proteins extracted wise,the structuralproteinsencoded

by

ts2

(ex-from the cells and from

purified

virions were hibited by *E2) would have been found in the

separated

by

SDS-polyacrylamide gel

electro- virions secreted from the coinfected cells

(Fig.

phoresis,anddensitometricscanswere

prepared

8).

The small amount of *E2 is attributed to

from

autoradiograms

of the slab

gel electropher-

leakage

ofthetsmutation. ograms. The ratio oflabeled E2 to *E2 in the DISCUSSION virions wasalmost identicaltothatmeasured in

thecells(0.51and

0.53,

respectively).

These data Wehave described here anaberrant form of showthat neither E2nor*E2was

preferentially

the Sindbis virus E2

glycoprotein

that differs

utilized

during

the

budding

process. fromthe

normal

protein

by

anincreased

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ridechains (28). Our data on the glycopeptides in E2 and *E2 indicate that both contain the same amount of the Aand B type oligosaccha-rides.

.n4 Theglycosylated and nonglycosylated

precur-P

14'

sorsto*E2 in thets2strainhad

electrophoretic

mobilities identicaltothose of theparentalHR strain. These data leadustoconclude that *E2 results fromamutation that has altered the site for proteaseactivity onthePE2 precursor.The mutation is distinct from the one that confers

temperaturesensitivity to thereplication ofts2

virus, eventhough the ts mutation itself involves

adefect in another

proteolytic cleavage step

in

viral structural-protein formation (22). The

other product of the PE2 cleavage has been

designated asE3 for the closelyrelated Semliki

Forest virus (11), but has not been detected in

Sindbis virions. It appears in the medium of

aU

:<

1E-1

~muII

FIG. 6. Viralproteins incells cointectedwith ts2 andts6 virus at 30°C. Details ofcelllabeling and

preparation of the autoradiogram after

SDS-poly-acrylamide gel electrophoresis are in the text. (a)

Infection withts2alone; (b) ts6alone; and (c) both

together.

phoretic mobility in SDS-polyacrylamide gel.

This trait isgenerallyattributed to a decrease in the size ofapolypeptide, althoughit could also indicate alower amount ofcarbohydrate in the

glycoprotein.Based onthe difference in

mobili-ties, *E2 was estimated to be 3,000 to 4,000 daltons smaller than E2. Can thisdifference be attributed to only a change in carbohydrate? Sindbis virus glycoproteins deficient in carbo-hydrateby virtue of growth incellsaltered in a

glycosyl transferase activity (25) or treated with ja b

C.

inhibitors ofglycosylation activities (8, 16) show

increasedmobilities,and in the former case the FIG. 7. Structuralproteinsfromvirionsformed by alteredproteinwasestimated todiffer in molec- cells coinfected with ts2 and ts6 virus at40°C. Refer

ularweight by800. The totalamount ofcarbo- to the text for the preparation of these 35S-labeled

hydratein eachglycoproteinofSindbis virus has samples and the gel electropherogram. (a) HR

paren-been estimated to be 4,000 daltons (28), and tal

virus;

(b) ts2

virus

formed at30°C; (c) virus from

analyseshaveshownthtvs

gcoinfection.

Plaque assays gave thefollowing virus

analyses have shown that virus grown in chick titers after 10 h of

infection

at40°C: ts2, 4.2 x

10i

cellshas E2 glycoproteins that contain a"com- PFU/ml; ts6, 7.5 x

10i

PFU/ml; ts2 +

ts6,

1.4 x 109

plex" A type and "simple" B type oligosaccha-

PFU/ml.

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ts 2: ' ' ' which led to noninfectious, atypical, and

heter-i

StI

ogeneous particles (21). Results from tryptic

Replicase 2

StructuralPr(4O)

peptide

analyses

of the altered

proteins

estab-p lished that the ts defect caused an incorrect

le A cleavage ofthePR76 precursorprotein, but the

ts 6: I I precise nature of the mutation was not

deter-t

+ mined.

Replicase 6 Structural Protein Variations in the

posttranslational

cleavage

(C, El, E2) products have also been

reported

fortsmutants

FIG. 8. Modelfornonreciprocalcomplementation.

of

poliovirus

(6). A putative alteration in a

pro-The ts2 virus supplies polymerase complex for both teolytic cleavage site was found in reovirus ts ts2 and ts6 26S RNA production, but ts6 cannot mutants, but the

genetically

stable defect pro-correct the ts2defect in cleavage.

ducing

an

electrophoretically

altered,2 viral

structural protein could be geneticallyseparated

from the ts mutation inseveral of the reovirus infected cells (D. Brown, personal communica- mutantclassesandshoweditselfnot to be a ts

tion).Wehavenotdetectedthepredicted larger defect(7).Thisnondefective alterationprovided

E3from themutantstrain. Crossand Fields(7) witha thirdgenetic marker

The shortened *E2 glycoprotein does not for evaluating interactions between gene

seg-seem tohave grossly affected the structure or ments during reovirus

replication.

Analyses of function of the virion. ts2virus wasreportedto thetemperature-resistant"recombinant" clones

be slightlymore thermolabilethan ts5 (3), and for the,u2 phenotype provedthatreassortment

wefoundnodifferenceinsensitivitytorepeated of RNA segments during reovirus replication

freeze-thawingin ts2 ascomparedwith theHR was arandomevent.Further studies located the

orwild-type strains

(unpublished

data).Asingle- ,u2 changeto the RNA A segment, but no

addi-step growth cycle ofts2 was identical to other tional information has been obtained on the

Sindbis strains when tested at

300C

(unpub- biochemical nature of the defect. Another

reo-lished data).Wehavenotcarefully analyzedthe virusmutant had an

electrophoretically

altered

host range or antigenic behavior of the *E2 viral protein,

X3.

Interestingly, both

X3

and,u2

variant, but no differenceswere seen inplaque aremajor surfacecomponents ofthevirion,and

size on CEF cells. BHKcells infected with ts2 these

capsid polypeptides

mayalso differamong

producedan*E2 thatwasindistinguishablefrom thereovirus serotypes(20).

*E2 from the chick fibroblasts. On the virus In amanner somewhat analogous to the

reo-surface, E2 glycoprotein appears to be more viruswork noted above, we have used the

elec-exposed than El (29). Our analyses of thenon- trophoretically distinct *E2 mutation as a

phe-reduced forms of these glycoproteins suggest notypic marker in examining gene expression

thatEl maybein a

"tighter"

conformationin

during

the

complementation

reaction between

its native state, possibly the resultofdisulfide two tsSindbis virus mutants.Ourresultsshowed

loops. that complementation wasnonreciprocal-that

Abnornalproteolyticcleavages have beende- is, theRNA'ts2 mutant couldcorrect the defect

tectedinthereplicationofseveral animal virus of the RNA- ts6, but ts6 gene products were

systems, and

apparently they

are widespread. unable to correct the RNA' defect. Onlytrace

The ambiguities detected in posttranslational amounts of the ts2 viral gene products were

cleavageof

picornavirus polyprotein

precursors detected in virions formed

during

complemen-may accountfor differencesinserotyping of the tation at the

nonpermissive

temperature. Our viruses. For example, type 1 Brunhilde

polio-

analysesofcells

containing

both thets2and ts6

virus is distinguished from type 2 strain by a viralgenes showed that ts2mRNAwas

function-differenceinthe sizesof

capsid proteins

VP2 and ingandproducing the 140,000-daltonprecursor.

VP3(1). These

proteins

are

products

ofacleav- Thus, thetsdefectisacis-dominant trait.Ifthe

age in precursor3a, andtype1iscleavedto

give

ts2

temperature-sensitive

defectwere inaviral

asmaller VP3 anda

concomitantly larger

VP2 codedprotease,we

might

have

expected

thetrait

thanfoundinthetype2virus. Alterationswere to be recessive in a trans

configuration,

anda

alsodetected in those

cleavages

that

yield

non- nondefectivets6protease should have been able

structural

polypeptides

in thetype2strain. Var- to cleave the ts2 precursor. We have made an

iations have been found in the

pathways

of analogousargument for the *E2 mutation since

posttranslational processing of rhinovirus spe- thisdefectwasretainedin amixedinfection of

cificproteins(17).A tsmutantof aviansarcoma twomutants, oneof which carriedanormal E2. virus produced a different set of viral "core" cis-dominant mutations here are most

easily

proteins at the nonpermissive temperature,

explained by

alteration ofsequence and

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tureof precursor

polypeptides.

Our results differ and evidence foracomplex ofPE2-E1 viral

glycopro-fromthose

recently

published by Scupham

etal. teins.

Virologyr

74:441-449.

3.Burge, B.W., and E.

R.

Pfefferkorn.

1966.

Isolation

(27), who notedadisappearanceof the ts2pro- andcharacterization of conditionallethal mutantsof

tein incells coinfected with thecomplementing Sindbis virus.Virology30:204-213.

ts2O mutantand

suggested

that a

viral-specific

4. Burge, B. W., and E. R. Pfefferkorn.1966.

Comple-protease from the ts2O was acting on the ts2 mentation between temperature-sensitive mutantsof

polypeptid.Wewouldarguethat theirputative 5. Sindbis virus.Virology30:214-223.

polypeptide. wewoula argue tnat tnelr putatlve 5. Cancedda, R., R. Swanson, and M. J.Schlesinger. proteaseis unableto convert ats2precursorto 1974. Viral proteins formed in a cell-free rabbit

reticu-properly

sized viral membraneproteins. For the locytesystemprogrammed with RNAfroma

tempera-ts2 temperature-sensitive defect, the cis-domi- ture-sensitive mutant of Sindbis virus. J. Virol.

nantcaracteisticsconsstent ith th dem- 14:664-671.

nant characteristic is consistent with the dem- 6. Cooper, P. D., D. F.

Summers,

and J. V.

Maizel.

1970.

onstration that the mutation was expressed in Evidence for ambiguity in post translational cleavage of

an in vitro protein-synthesizing system (5, 32). poliovirusproteins.Virology41:408-418.

However, one intriguing model that our data 7. Cross, R. K., and B. N. Fields. 1976. Use of anaberrant

exclude is a cis-dominant sf polypeptideas amarker in three-factorcrosses:further

cannot totally evidence for independent reassortment as the

mecha-proteolytic activity expressed by the virus capsid nism of recombination between temperature-sensitive polypeptide as it is translated from the 26S mutantsof reovirus type 3.Virology 74:345-362.

mRNA. If thistype ofactivitywere altered in 8. Duda, E., and M. J.Schlesinger. 1975. Alterationsin

,thenafunctional self-protease in ts6 capsid

Sindbis

viralenvelope proteins bytreatingBHK

cells

ts2, tne a nclonl slrproeas mtsocapla withglucosamine. J. Virol.15:416-419.

mightactpoorlyifatall withthenascentpoly- 9. Dunbar, A.K., andR.R.Rueckert.1969. Observations

peptide translated from the ts2 mRNA. There onmolecularweightdeterminationsonpolyacrylamide

are now several reports

suggesting

that post- gels. J.Biol. Chem. 244:5074-5080.

translational cleavage products ofvirus genes 10

Fennell, RI,

andB. A.Phillips.1974.Polypeptide

com-position ofurea-andheat-resistantmutants of

polio-haveproteolytic activity (15, 33). virus types 1 and 2. J. Virol.14:821-833.

The nonreciprocal contribution of the differ- 11.Garoff,HI,K.Simons,and0.Renkonen. 1974.

Isola-ent genomesdiscovered herecanofferanexpla- tion andcharacterizationof the membraneproteinsof

nation foraresult described

by

Pfefferkor and _12. _{Grefrath, S. P., and J. A.}Semliki Forest virus.Virology_{Reynolds. 1974.}

61:493-504.

_The

molec-Burge (19)intheir initial studiesoncomplemen- ular weight of the major glycoprotein from the human

tation with the tsmutants.Theyobservedthat erythrocytemembrane.Proc. Natl.Acad. Sci. U.S.A.

the yield of virus obtained fromamixed infec- 71:3913-3916.

tinofRNA'and RNA- mutantwasverysen- 13. Jones,K.J.,M. R. F.Waite,and H. R.Bose. 1974.

tion of RNA andRNA mutant was very sen- Cleavageofaviralenvelope

precursor

duringthe

mor-sitive to the MOI of the RNA- but insensitive phogenesis of Sindbis virus. J. Virol.13:809-817.

tothe MOI of theRNA'.Fromourresults, only 14. Keegstra,K., B.Sefton,and D. Burke.1975.Sindbis the RNA- isprovidingthestructuralgeneprod- virus glycoproteins: effect of the hostcellonthe

oligo-ucts, and alimitation onthe amount of these

saccharides.

J.

Virol.

16:613-620.

15.Lawrence,C., andR. E.Thach.1975.Identification of

structural proteins would be expected to limit a viral protein involved inpost-translationalmaturation

virion production. In contrast, RNA' mutants of theencephalomyocarditisviruscapsidprecursor.J. are

presumed

to

provide

gene

products

thatact Virol.15:918-928.

catalytically

during complementation.

16. Leavitt, R., S. Schlesinger, and S.

Kornfeld.

1977.

Tunicamycin

inhbits

glycosylation and

multiplication

More detailed structuralanalysesofthe *E2 of Sindbis and vesicular stomatitis viruses. J. Virol.

variant may allowus to identify the region in 21:375-385.

the

polypeptide

of normal E2 that has been 17. McLean, C., T. J. Matthews, andR. R.

Rueckert.

1976.

changed,

and may further help to identifythe Evidenceof ambiguous processing and selective

degra-changed, dation in thenoncapsid proteinsofrhinovires 1A. J.

substrate

specificity

for the

important

proteo-

Virol.

19:903-914.

lytic cleavage that is essential for budding of 18.Pfefferkorn,E. R., and M. K. Boyle. 1972.Selective

Sindbisvirus. inhibition of thesynthesisof Sindbisvirionproteinsby aninhibitorofchymotrypsin. J. Virol. 9:187-188. ACKNOWLEDGMENTS 19. Pfefferkorn, E.R., and B. W. Burge. 1967. Genetics We thank Sondra Schlesinger and Janice Brielmeier for and biochemistry of arbovirus temperature-sensitive assistanceinpreparation andanalysis of the glycopeptides. mutants, p. 403-426. In J. S. Colter and W.Paranchych Thisinvestigation wassupported by Public Health Service (ed.), The molecular biology of viruses. Academic Press grants CA-14311-04 and CA-16217-03fromtheNational Can- Inc., New York.

cerInstitute. 20. Ramig, R. F., R. K. Cross, and B. N. Fields. 1977. Genome RNAsandpolypeptides of reovirus serotypes LITERATURE CIMD 1, 2, and 3. J. Virol. 22:726-733.

21. Rohrschneider,J.M., H.Diggelmann, H. Ogura, R. 1. Beckman,L.D., L A.Caliguiri,andL.S.Lilly.1976. R.Friis,and H. Bauer. 1976. Selective cleavage of a Cleavage site alterations inpoliovirus-specific precur- precursor polypeptide in atemperature-sensitive mu-sorproteins.Virology73:216-227. tant ofavian sarcoma virus. Virology 75:177-187. 2. Bracha,M., and M. J. Schlesinger. 1976. Defects in 22. Scheele, C. M., and E. R. Pfefferkorn. 1970.

Virus-RNA'temperature-sensitive mutantsofSindbis virus specific proteins synthesized in cells infected with

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RNA' temperature-sensitive mutants of Sindbis virus. Sindbis virus:preliminary characterization of the oli-J. Virol.5:329-337. gosaccharides. J. Virol. 14:522-530.

23. Schlesinger, M. J., and S. Schlesinger. 1973. Large- 29. Sefton, B. M., G. G. Wickus, and B. W. Burge. 1973. molecular-weight precursors of Sindbis virus proteins. Enzymatic iodination of Sindbis virus proteins. J. Virol.

J.Virol.11:1013-1016. 11:730-735.

24. Schlesinger, M. J., S.Schlesinger, and B. W. Burge. 30. Segrest, J. P., R. LJackson, E. P.Andrews, and V. 1972.Identification of a second glycoprotein in Sindbis T.Marchesi. 1971. Human erythrocyte membrane gly-virus.Virology 47:534-541. coprotein: areevaluation of the molecular weight as 25. Schlesinger, S., C. Gottlieb, P. Feil, N. Gelb, and S. determined by SDSpolyacrylamide gel electrophoresis.

Kornfeld. 1976.Growth ofenveloped RNA virusesin Biochem.Biophys. Res. Commun. 44:390-395. alineof Chinesehamsterovarycells with deficient N- 31. Shapiro, A. C.,E.Vinuela, and J. V. Maizel. 1967. acetyl-glucosaminyltransferase activity. J. Virol. 17: Molecularweight estimation ofpolypeptide chainsby

239-246. electrophoresis inSDS-polyacrylamide gels. Biochem.

26. Schlesinger,S., and M. J. Schlesinger. 1972. Forma- Biophys. Res. Commun. 28:815-820.

tion ofSindbisvirusproteins: identification ofaprecur- 32. Simmons, D. T., and J. H. Strass. 1974. Translation of sor for one of the envelope proteins. J. Virol. Sindbis virus 26S RNA and 49S RNA in lysates of 10:925-932. rabbit reticulocytes. J. Mol. Biol. 86:397-409. 27. Scupham, R. K., K. J.Jones,B. P.Sagik,and H. R. 33. VonderHelm, K.1977.Cleavage of Rous sarcoma viral

Bose, Jr.1977.Virus-directedpost-translational cleav- polypeptide precursor into internal structural proteins agein Sindbis virus-infectedcells.J.Virol.22:568-571. in vitro involves viralproteinp. 15. Proc.Natl. Acad. 28. Sefton, B.M., and K. Keegstra.1974.Glycoproteinsof Sci. U.S.A.74:911-915.