Protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking.

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Vol. 33, No. 1 JOURNAL OFVIROLOGY,Jan.1980,p.152-166

0022-538X/80/01-0152/15$02.00/0

Protein-Protein

Interactions Within Paramyxoviruses

Identified

by

Native

Disulfide

Bonding

or

Reversible Chemical

Cross-Linking

MARY ANN K. MARKWELL* ANDC. FRED FOX

Departmentof Microbiologyand the Parvin Cancer ResearchLaboratories, Molecular Biology Institute,

University

of California,

Los

Angeles,

California

90024

Analysis of native disulfide-bonded protein oligomers in paramyxoviruses showed thatsomeviral proteinsareconsistentlypresent as covalent complexes. InisolatedSendai virus the hemagglutinating proteinHN is present in homodi-meric and homotetrahomodi-mericforms, and the minor nucleocapsid protein P exists

partlyas amonomerandpartlyas adisulfide-linkedhomotrimer. Similar disul-fide-linkedcomplexeswereobserved in Newcastle disease virus (strainHP-16), in which HNexistsas ahomodimnerandsomeof the majornucleocapsid proteinNP existsas ahomotrimer. Noncovalent intermolecular interactionsbetween proteins were studied with the reversible chemical cross-linkers dimethyl-3,3'-dithiobis-propionimidate and methyl

3-[(p-azidophenyl)dithio]propionimidate,

which con-tain disulfide bridgesand a 1.1-nmseparation between their functional groups. Thesameresultswereachieved with bothreagents.The conditions ofpreparation, isolation, and storage of the viruses affected the protein-protein interactions observed upon cross-linking. Homooligomers ofthe glycoprotein F, the matrix protein M, and the major nucleocapsidprotein NP were produced in both Sendai and Newcastle disease viruses after mild cross-linking of all viralpreparations examined, but NP-M heterodimner formation in both viruseswas mostprevalent in early harvest preparations that were cross-linked soon after isolation. The

abilityofNPand Mtoformaheterodimneruponcross-linkingindicatesthat the matrixproteinlayerlies in closeproximity (within1.1nm)tothenucleocapsidin the newly formedvirion. Some noncovalent intermolecularproteininteractions inSendai and Newcastle disease viruses, i.e., those leadingtotheformationof F, NP, and Mhomooligomers uponcross-linking, are more stabletovirus storage thanothers, i.e., thoseleading tothe formation ofan NP-M heterodimerupon

cross-linking. The storage-induced loss of the ability of NP and M to form a heterodimer isnotaccompanied byany apparentloss ofinfectivity.Thisindicates thatsomespacial relationshipswhich formduringvirusassemblycanalter after

particleformation andare not essential for theensuing stagesof the infectious process.

Sendai virus and Newcastle disease virus (NDV), members of the family

Paramyxoviri-dae,arecomposedofan outerlipoprotein

enve-lope and aninternal helical nucleocapsid. The

envelopewhich consistsofalipid bilayer,

exter-nal glycoprotein spikes, and an inner layer of carbohydrate-free Mprotein is acquired by the

virusesin theprocessofbuddingfrom the host

cell surface(29).At present,littleis known about thespatialrelationshipsofthe viralproteinsin thematurevirusparticleand therolesthatthese relationshipsmayplayin viralassembly and in theensuingstages of the infectious process.

Ev-idence available from selective extraction (34)

and phenotypic mixing experiments (20)

sug-gests that M isinvolvedintransmembrane

rec-ognitions between the spike and nucleocapsid

proteins. Direct interactionsofMwith the gly-coproteins or nucleocapsid proteins have not

been demonstrated in paramyxoviruses, but

have beendemonstrated invesicular stomatitis

virus withreversiblecross-linkingagents (7).

Our studiesweredesignedto testthe

hypoth-esis that M exists in close proximity to the interior nucleocapsid proteins andthe exterior spikeproteinsinparamyxoviruses.A

secondary

objective was to probe for information on the

spatial relationships of the individual proteins

within the viral membrane andnucleocapsidof

the maturevirusparticle by analyzing the pro-tein complexes present as a result of native disulfide bondingorformedbychemical

cross-linking.

The two cross-linking agents chosenforthis

152

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study both contain disulfide bridges and a

1.1-nmseparationbetweentheirfunctionalgroups.

Thehomobifunctional reagent dimethyl-3,3'-di-thiobispropionimidate (DTBP) is a

water-solu-ble imidoester with a half-life in the minute

range atslightly alkaline pH (26). The

hetero-bifumctional reagent methyl 3-[(p-azidophen-yl)dithio]propionimidate (PAPDIP)contains an

imidoester groupandalipophilicaryl azide

por-tion. Theimidoestergroupof DTBPorPAPDIP

reactswiththeprimary amine of a protein(Fig.

1,reaction 1). With DTBP (Fig. 1) the second

step (Fig. 1, reaction2) isthe sametypeof

site-specificreaction asthe first.

With PAPDIP (Fig. 2), reaction of the

imi-doester group with protein (Fig. 2,reaction 1) is

done in the dark to preventsimultaneous

acti-vation of the secondgroup. Thesecond group of PAPDIP, an aryl azide, is activated by pho-tolysis to the nitrene (Fig. 2, reaction 2a).

Ni-trenes donot require aspecific reactive group.

Theycaninsert atN-H and atC-H bonds in

proteins (Fig.2,reaction 2b) and have half-lives

in themillisecondrange(26).Protein cross-links

formed with either DTBP or PAPDIP can be reversedby reductive cleavageoftheirdisulfide bridges (Fig. 2, reaction 3).

(Parts of this workhave been described briefly before [16; M. A. K. Markwell, Fed. Proc. 37:

1773, 1978].)

MATERIALS AND METHODS

Virus. Sendai (also known as hemagglutinating

(H2N-Protein A)

1)

2)

3)

virusofJapan, HVJ) and NDV(strainHP-16)viruses werepropagated in 11-day-oldembryonatedchicken eggsincubated at38°C. Allantoic fluid was harvested at 22h(earlyharvest)or48h(lateharvest)

postinfec-tion. Viruswaspurifiedundersterile conditionsbya modificationof theprocedure of Samson and Fox (30); this modificationsubstantiallyshortenedthe time re-quired for purification. Centrifuge tubes and bottles weresterilized byovernight exposuretoethyleneoxide (Anprolene, H. W. Andersen Products, Inc.). Linear gradients were formed overnight by diffusionof se-quentiallayers(incrementsof5%)of sterile solutions of sucrose or Renografin. All procedures were per-formedat0 to4°Cunless otherwisespecified.Clarified (50,000g-min) allantoic fluidwaslayeredontopofa two-stepgradientconsistingof 20%(wt/vol) and 65% sucrose in 0.01 M Tris.hydrochloride-0.1 M NaCl-0.001 Mdisodium EDTAatpH7.4 (TSE).The gra-dientwas centrifugedfor 30 minat 19,000rpmin a Sorvall SV-288 verticalrotor. Virus concentrated at

the 20/65%sucrose interfacewasdiluted withTSE,

and bandedsequentiallyin linear 40 to 65% sucrose and 6to25% iodine equivalentRenografin gradients

bycentrifugationfor 1 hat19,000rpmin the vertical

rotor.The viruswaspelletedbycentrifugationfor 90 minat75,000xg inafixed-anglerotor, and thepellet

wassuspendedinTSEat aproteinconcentration of 5

mg/ml and used within 3 days or stored frozen at

-70°C. Thedye-bindingassayforproteins(2) under-estimated viralproteins bytwo- tothreefold inpurified

viral samples. Protein wasthereforeroutinely

meas-ured by a modification of the Lowry procedure for membrane andlipoproteinsamples(18).

Celis. Monolayercultures ofbabyhamsterkidney

(BHK-21) celLsweregrowninamediumconsistingof

10%tryptose phosphate broth, 10%fetal calfserum,

NH2 NH2

11 I1I

+ H3COCCH2CH2SSCH2CH2COCH3

[DTBP]

NH, ' , NH2

(Protein A)-HNCCH2CH2SSCH2CH2COCH3

(H2N-Protein B)

0

e

NH2 NH2

11 eif

11

(Protein

A)-HN`CCH2CH

2SSCH2CH2CNH-

(Protein B) (reducing agent)

e D

NH2 ' NH2

HSCH2CH2CNH-(Protein

A) +

HSCH2CH2CNH-(Protein

B)

FIG. 1. Cross-linkingofproteins with DTBP.

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154 MARKWELL AND FOX

(H2N-Protein A)

NH2

11Cii)N3

H3COCCH2CH2SS

PAPDIP]

ID_NH2

(Protein A)-HNCCH2CH2SS N3

NH hv

(Protein A)-HNCCH2CH2SS-N

e j (Protein B)

NH2

(Protein A)-HNCCH2CH2SS

iQN]-(Protein

B)

Q | (reducing agent)

NH

[image:3.504.115.409.65.361.2]

HSCH2CH2CNH-(Protein A) + HS(N]-(Protein B)

FIG. 2. Cross-linking ofproteinswith PAPDIP. and 80% minimum essential medium from (GIBCO

Laboratories,GrandIsland, N.Y.).Thecells wereused for virusadsorption studies within 24 hofattaining confluency.

lodination and cross-linking conditions. The externallydisposedproteinsof thevirusenvelopewere

iodinated with 1,3,4,6-tetrachloro-3a,6a-diphenylgly-coluril (chloroglycoluril, available as Iodo-gen from

Pierce ChemicalCo.) under conditionsoptimizedfor surface-specific labeling (17).Portionsofintact virus (100jigofprotein)werereactedfor 10minat0°Cwith 0.5 mCi ofNaI251 (carrier free,Amersham Corp.) in

thepresenceof10,Agofchloroglycoluril.The iodinated virus preparation wastransferred from the reaction vesseltoatesttubecontaining25,umolof carrierNaI in TSEand usedimmediatelyforcross-linkingstudies. Interioras well asexternallydisplayed proteinsof the viruswereiodinatedbyanonvectorial

chlorogly-colurilmethod which includesdetergenttodisruptthe virus (17).Controlandcross-linkedsampleswere sol-ubilized in2% sodium dodecylsulfate (SDS) by

im-mersion in boilingwater for 2minand then reacted withNa'25I in thepresenceofchloroglycolurilas

de-scribed above. Thesampleswereusedforgel

electro-phoresisafter the addition of 25,umolof carrierNal. Paramyxoviruses were cross-linked with DTBP (Pierce Chemical Co.) by the procedure described previouslyfor NDV(21)exceptthat thenative disul-fidebondsofthe viruswerenotreduced before

cross-linkingandthecross-linkingreactionoccurredat0°C. The viralsuspension(100ugofproteinin 25plof 0.2

MtriethanolaminehydrochlorideatpH 8.5)wasmade

3 mM in DTBP and incubated for 30minat0°C. The reactionwasterminatedbythe additionof ammonium acetateandfreshlyprepared N-ethylmaleimide, both

atafinalconcentration of 50mM,andincubatedfor

30minatroomtemperature.Sampleswerethen made 2% in SDSandimmersedfor2mininboilingwaterin preparationforgel electrophoresisornonvectorial io-dination.

Thesameinitialincubation conditionwasused with theheterobifunctional cross-linkingreagent PAPDIP (5), but in the darktopreventphotolysisof thearyl azidegroup.Afterthe30-min incubationin ammonium acetate and N-ethylmaleimide, the virus was

sedi-mented for 90minat75,000x gthrough20%sucrose

ontoa65%sucroseshelf,and thePAPDIP-derivatized virus that concentrated attheinterface wasdivided into two samples. In the first sample, the second reactivegroupof PAPDIPwasactivatedbyexposure

to a UV lamp (Blak-ray UVL-21, peak of 366 nm,

Ultra-violetProducts, Inc.) for 3minatadistance of

5 cm from the sample. The rest of the PAPDIP-derivatizedviruswasdiluted with minimum essential

mediumtoaproteinconcentration of 0.5mg/mland

allowedtoadsorbto BHK confluentmonolayersfor 30 min at 4°C. Themonolayers were washed three

times with ice-cold minimum essential medium to

removeunattachedvirusand thenexposedtoUVlight for 3min.

Fractionation ofBHK cells and cross-linked Sendaivirus. The cellularpreparationwasenriched

for virus cross-linkedtoplasmamembranebya mod-ification ofthe zinc ion method used to isolate the 1)

2a)

2b)

3)

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surface membranes of L cells (38). BHK cells were

scraped with a rubber policeman from three 10-cm

dishes into 15mlof 0.025 MTris.hydrochloride-0.14 M NaCl atpH7.4andcentrifugedat500 xgfor 15 min. Thepelletwas gently suspendedin 3 ml of 50

mMTris-hydrochlorideatpH7.4containing1mgof

soybean trypsin inhibitor per ml. Thecellswere

al-lowed toswell for 15min at roomtemperature. An equal volume of 1 mM ZnCl2 was added, and the

suspension was incubated foran additional 10 min.

Thesuspension wascooledto4°C and homogenized with50to60strokes ofatypeB, tight Dounce pestle. Thehomogenatewaslayeredontopofatwo-step(20

and65%sucroseinTSE) gradient and centrifuged for

30minat19,000rpminaSorvall SV-288verticalrotor.

The membranefraction which concentratedatthe20/ 65% sucrose interface was collected, diluted 10-fold

with TSE, and centrifugedat 6,000 x gfor 15min. The resulting pelletwas solubilized in 0.5 M

Tris-hydrochloride-2% SDS atpH 6.8forgel

electropho-resis.

Paramyxovirus preparations cross-linked with either DTBPorPAPDIPweresubdividedinto

enve-lope and nucleocapsid fractions by the selective

ex-traction procedures of Scheid andChoppin (31) and RaghowandKingsbury(28)butsubstitutingNonidet P-40(NP-40) (Shell Oil Co.)for Triton X-100. Suspen-sions of the cross-linked virus(2.5mgofproteinin 2.5

ml)weredialyzed against1MNaCl in 0.01 M sodium phosphate buffer at pH 7.2. NP-40 wasadded to a

final concentration of2%(vol/vol). The virus

prepa-rations wereincubated for 2 hat roomtemperature

(21to 22°C), then iodinatedfor 10additional minat

roomtemperaturewith 2.5 mCi ofNa251Iand 25ytgof chloroglycoluril. CarrierNaI (1.25 mmol) wasadded

to the iodinated virus. The viralsuspension was

cen-trifuged for 30 min at 10,000 x g to separate the solubilized membrane proteins HN,F, and M from the insoluble nucleocapsid fraction. The pellet was

sus-pendedin 2 ml of 1 MNaCl in 0.01 M sodium phos-phate buffer at pH 7.2 and extracted for 30min at

room temperature to selectively remove the minor

nucleocapsid proteins (28). Thesuspensionwas

cen-trifugedfor 90minat75,000xg.Theresultingpellet, containingNPcomplexedwith viralRNA,was solu-bilizedin 0.5M Tris hydrochloride-2% SDSatpH6.8 forgelelectrophoresis.

Gel electrophoresis in SDS. Reagents for gel electrophoresiswerepurchasedfromEastman Kodak

Co. SDS (lauryl, sequanal grade)wasobtainedfrom Pierce Chemical Co.SDS, glycerol,andbromophenol blue were added to samples for electrophoresis to

achieve final concentrations of2, 10,and0.008%,

re-spectively, in a sample buffer of 0.0625 M

Tris-hy-drochloride, pH6.8.Reducedsamples contained 5% 2-mercaptoethanol. The molecularweightmarkers mol-luschemocyanin, erythrocyte spectrinbands 1and2, Escherichia coli,-galactosidase, phosphorylasea,

bo-vine serum albumin, ovalbumin, soybean trypsin

in-hibitor, myoglobin, and lysozyme were used in

con-structingcalibrationcurvesfor the 5to12.5%gradient gels.Hemocyaninwasthe gift ofP. S. Linsley,

Uni-versity ofCalifornia, Los Angeles. Spectrin bands 1

and2wereisolatedasreferenced (24).

,B-Galactosid-ase,phosphorylasea,lysozyme, andtrypsininhibitor

werepurchased fromWorthington BiochemicalCorp.,

bovine serum albumin, and myoglobin were from Sigma Chemical Co., and ovalbumin was from Schwarz/Mann.

Theprocedure for two-dimensionalgel electropho-resis isamodificationof theone-dimensionalLaemmli system(13) and has beenpreviously described foruse with erythrocyte ghosts (17). Samples were electro-phoresed in the first dimension without mercaptanon a 1-mm-thick 5 to 12.5% (wt/wt) acrylamide linear gradient resolving gel.The first-dimensional slab gel

was thensliced intoitsrespectivelanes,incubatedfor 15 min in reducing solution (0.0625 M Tris.hydro-chlorideatpH6.8,3% SDS,and 3%2-mercaptoethanol

or 5 mMdithioerythritol), andaffixed with 1.5% aga-rose inreducing solutionontoanother5to12.5%slab gel (1.25 mm thick) for the second dimension. To improve the resolution between the F, and NP pro-teins ofNDV, a7.5 to10%gradient gelwasused in thesecond dimension.

Gelswerefixed,stained forprotein,destained,and

soaked in the aqueous solutions as described in the outline which follows. The gelswere gently agitated on ashakerat roomtemperaturefor the stated times

intightlysealed containers. At least 500 ml of solution

pergelwasusedfor each step. (i) Fixingsolution was composed of 25%isopropanol-10%acetic acid(60 min).

(ii)Stainingsolution consisted of 0.1%Coomassie

bril-liantblue R-250, 25%isopropanol,and 10% acetic acid (30 to60min).(iii)Destainingsolutionwascomposed 5% methanol-7% acetic acid to which were added several whiteplastic foamplugs (diSPo plugs, Scien-tificProducts)toadsorb the staineluted during over-night destaining. (Gels can be soaked forat least 1 week withoutlosingdyefrom the protein bands.) (iv) Soaking solution Awas10%methanol-2%glycerol(3 h).(v)Soaking solution Bwas20%methanol-2% glyc-erol (1h).ThegelswerethentransferredtoWhatman no. 1 chromatography paper and dried under house vacuumovernightatroomtemperature.Kodak single-coatedSB-5X-rayfilmwasused forautoradiography. To determine the percent distribution of radiolabel

incorporated byindividual viralprotein species,

Coo-massie brilliant blue-stained spotswere cutfrom the driedgeland counted inaPackard gamma scintillation spectrometer.

RESULTS

Resolution ofSendai viral proteins. The

majorpolypeptide speciespresent in Sendai

vi-rusand theirlocationwithinthevirionare

pre-sented in Table1. Identification of each species

is based onapproximate molecular weight,

ac-cessibility tosurface-specific labeling,and

frac-tionationby selective extractionprocedures. F2

hasbeen shown to existin disulfide linkage to

F1.to formthe active Fprotein (33). The

glyco-proteins HN and F arethe only viral components

readily accessible to surface-specific iodination

(15, 17). During sequential selective extraction

procedures themembrane proteinsHN, F, and

M(fraction 1) andminornucleocapsid proteins

L and P(fraction2) arestrippedfrom the virus,

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TABLE 1. Proteins of Sendai virus Accessi- Selective

Poly- Molwt biltyto

extrac-peptide'

(XlO_3

b Positioninvirion surface- tion frac-peptidea(x10)

~~~~specific tionld

________ ~~~~labeling ____

L 215 Nucleocapsid _ 2

P 78 Nucleocapsid _ 2

HN 70 Membrane exterior + 1

NP 60 Nucleocapsid _ 3

F, 47 Membraneexterior + 1

M 34 Membranematrix 1

F2 15 Membrane exterior + 1

aNomenclature forparamyxovirus proteins

descrbed

by

ScheidandChoppin(32).

bApparentmolecular weights of Sendai viral proteins were calculated from molecular weight standards (see Fig. 3) coe-lectrophoresed with the viral proteins on 5 to12.5%gradient slabgels.

cAportion of intact virus (100 pgofviral protein) was

reacted for 10 min at 0°C with 0.5 mCi ofNalnI in the presence of 10pugofchloroglycolurilasdescribed in the text. dSendai viralpreparationswere subjectedto sequential selective extraction steps to fractionate the virus into its components asdescribedinthetext.FractionI, supernatant fraction of the 2% NP-40-1 M NaCl in 0.01 M sodium phos-phate extraction step; fraction 2,supematant fraction of the 1M NaCl in 0.01 M sodium phosphateextraction step; fraction 3,pelletof the 1 M NaCl in 0.01 M sodiumphosphate extrac-tionstep.

leaving NPcomplexedtoviral RNA(fraction3). Because of the greatly differing sizes of the

polypeptides,agelsystemwasdevelopedwhich

couldbeusedtoapproximatemolecularweights

ofpolypeptidesand theircomplexesover awide

range ofvalues. The Laemmli SDS gel system

(13)wasmodifiedtoincludea5 to12.5%linear gradient resolving gel (17). Molecular weight standards from 14,000to290,000were runwith eachslabgeltoconstructmolecularweight cal-ibrationcurves(Fig. 3).

By using the5 to 12.5% gradient gelsystem, all theproteinsof thereduced, SDS-solubilized

viruswere resolved (Fig. 4A). The high degree ofintegrity of the viral preparation is demon-stratedby the fact thatonlythe external mem-brane polypeptides HN, F1,and F2are labeled

bysurface-specificiodination of the intact virus

(Fig. 4B).Thissurface-specifictechnique proved usefulindistinguishingbetween NP and F which have similar electrophoretic mobilities after SDS solubilization from the nonreduced virus (Fig. 4C and D).

NativedisulfidebondinginSendai virus. Reduction of Sendai virusdestroys allits biolog-ical activities:infectivity, cell fusion, hemagglu-tination, neuraminidase, andhemolysis (23, 25, 33). Therefore, the polypeptides which

partici-pate innative disulfidebondingwereexamined.

Ozawa et al. (25) had previously reported the

presence of disulfide-linked

high-molecular-weight oligomers of HN and P in egg-grown

Sendaivirus (Z strain), butwereunable to ac-curately estimate their size. Under nonreducing conditions, threehigh-molecular-weight species inadditiontoLwereobservedonthe5 to12.5% gels (Fig. 40). Two of these(273,000and140,000 molecular weight) were accessible to surface-specific labeling; the third (238,000 molecular weight)was not(Fig. 4D).

Two-dimensional slab gel electrophoresis sys-tems facilitated identification of these species.

Polypeptidechains existinginnativedisulfideor cross-linkedcomplexes will exhibitagreater

mo-lecular weight inanonreducing firstdimension than in a reducing second dimension. They

therefore appear as spots below the diagonal

formed by polypeptideswhich have equal

mo-lecularweightsin both dimensions.Thespecies migrating at 273,000 and 140,000 in the first

(nonreducing) dimension migrated as a single polypeptide of 70,000 in the second (reducing) dimension(Fig. 5).Themigrationcharacteristics and theaccessibility tosurface-specific labeling

(Fig. 4D) indicated that thesespecieswere

tetra-meric anddimeric forms of the membrane

gly-300F _HEMOCYANIN

200-1501

In

I0

x

3

<,r

I-0

w 1oo0

7

50

PHOSPHORYLASEA

\ VINESERUM

OLALBUMIN

t OVALBUMIN

30-20- SOYBEAN TRYPSINi

LYSOZYME-20 40 60 80 100

%MOBILITY

FIG. 3. SDS-polyacrylamide gel electrophoresis of molecularweightstandards. A standardconsisting of mollusc hemocyanin (290,000), erythrocyte spectrin bandsIand2(240,000 and 215,000),E.coli

Il-galac-tosidase (130,000),phosphorylasea (100,000), bovine serumalbumin(68,000), ovalbumin (43,000), soybean

trypsininhibitor(21,500), myoglobin(17,200), and

ly-sozyme(14,300) waselectrophoresedon a 5 to12.5%

acrylamidelineargradientgel.

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coprotein HN. The homotrimeric form of HN was notobserved. The incompletereduction of the HN tetramer ofSendaivirus to the dimeric form suggests that notall the disulfide linkages

_ _ am 300

_ - 200

_ I _ .150

MO 0

- 100

-70:

4 a - * 60 !

C D E F

FIG. 4. Comparisonof reduced, nonreducea, and

cross-linkedproteinsofSendai virus by

one-dimen-sional gelelectrophoresis.A100)-pgsample ofreduced egg-grownSendai virus waselectrophoresedon a 5

to12.5%acrylamide linear gradient gel and stained

with Coomassie brilliant blue (A). Samples of the

sameviruspreparationwithout reducing agent (C)br

aftercross-linkingwith 3 mM DTBP (E) are shown.

Theautoradiographs ofsamples A, C, and E which

had beensurface-specifically labeledby the

chloro-glycolurilmethodfor'2Iarerespectively shown in B,

D, and F. Lettersdesignatethepositionsof the major Sendai viral proteins and their complexes, theorigin

(or)ofthegelandthetracking dye (td)front.

haveequivalent sensitivity to reduction and that the tetramer maybe formed from thelinkage of dimers during viral assembly. When the first-dimensional gelwasincubated inreducing solu-tion(0.0625MTris-hydrochlorideatpH6.8, 3% SDS, and 3% 2-mercaptoethanol or 5 mM dithi-oerythritol) for 30min instead of the usual 10 mi, total reductionwasachieved in the second dimension.

In theisolated nonreduced virionmostof HN existed in disulfide-linked dimeric and tetra-meric forms (54 and 38%, respectively, of the total radioactivity incorporated by HN); only8% was detected in monomeric form. In the nine different batchesof egg-grown Sendai virus ex-amined, the percentage of HN existing as a dimeror tetramervariedbyless than 5%. For-mation of the disulfide bond by autooxidation

duringsolubilizationasreportedfor H-2 (9)can

probably be excluded, since treatmentof virus withfreshly prepared50mMN-ethylmaleimide before solubilization did not alter the electro-phoreticprofileof the viralproteins.

The third high-molecular-weight

disulfide-bondedspeciesmigrated at amolecular weight

of238,000 in the first dimensionandcomigrated

with P monomer (78,000 molecular weight) in the second dimension. This and its lack of

ac-cessibility tosurface-specific labeling indicated that itwas atrimericform of minornucleocapsid

protein P. Approximately equal amounts of P

1stD-nonroduced

X MWM x1-3

b 300:or~ ° X0

t2001

1501 S *

70-*

3

60.]

a 45j,;

304 9

201

td'

A

FIG. 5. Two-dimensionalelectrophoretic analysisofproteins and theirnative disulfide complexespresent

inegg-grownSendai virus.Approximately50pgofviral protein which had beeniodinated in thepresenceof

SDSwerefractionatedinthefirst dimension withoutreducingagenton a5to12.5%polyacrylamideslabgel.

Thefirst-dimensionalgelwasslicedintoitsrespective lanes,reduced with2-mercaptoethanol, and affixed

ontoanother 5to12.5%slabgel for theseconddimension. Theautoradiograph of the gel isshownonthe left,

and itscorresponding schematic diagramisshownontheright. RefertoTable2for identificationofproteins

and their nativedisulfide complexes.

or

L

p

HN _ _

F NP-m

M _

F2 td

A 8

0

O

c

P tomer

aW N

Orw _mer

FQ1

p_

F N

r--B

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existed in the monomeric andtrimeric forms(49 and 51%, respectively), and this distribution var-iedby less than5% among thedifferentbatches ofSendai virus examined. Disulfide-linked hom-odimers ofPwerenotobserved.

Reduction of the disulfide-linked F protein (60,000molecular weight)toitscomponent poly-peptides F1 (47,000 molecular weight) and F2 (15,000 molecular weight) isclearly definedon the two-dimensional 5 to 12.5% gradient slab gels. Several minor proteins were sometimes

seen on orbelow thediagonal. These differed in

intensity frombatch tobatch ofegg-grown virus andcould be cellular contaminantsor proteoly-sisproducts of the viral proteins.Asummaryof theproteins and their native disulfidecomplexes

presentin egg-grown Sendai virusis presented

inTable2.

Cross-linking of Sendai virus. Noncova-lent protein-protein interactionswereelucidated with reversible chemical cross-linkers. Intact, nonreduced preparations of Sendai virus were cross-linkedasdescribedinMaterialsand Meth-ods with either DTBP or PAPDIP. Both

re-agents contain disulfide bridges and a 1.1-mn

separation between functional groups. The in-dividualcomponents of cross-linked complexes formed with these reagents appeared as new

spots below the diagonal on two-dimensional

[image:7.504.64.259.383.577.2]

gels.

TABLE 2. Proteins and their native

disulfide

complexes present in egg-grown Sendai virus

Present in: Access-Molwt Nonre- Re- ible to

(X1O')a ore e surface-

Identity'

duced duced specific gels gels

labelingb

273 + + HNhomotetramer

238 + _ - P homotrimer

215 + + - L monomer

140 + - + HNhomodimer

78 + + - Pmonomer

70 + + + HNmonomer

60 + - + Fmonomer

(F1+ F2oligomer)

60 + + - NPmonomer

47 - + + F1monomer

34 + + - Mmonomer

15 + + F2monomer

aMolecularweights ofSendai viralproteinswere

calculated from molecularweightmarkers(seeFig.3)

coelectrophoresed on5 to 12.5% slabgelsinthefirst

(nonreduced) and second (reduced) dimension. bA100-,ugportion of intact viruswasreactedfor10 minat0°Cwith0.5mCi ofNaI261inthe presenceof10 ,ug ofchloroglycolurilasdescribed inthetext.

'Theindividual components ofproteincomplexes wereidentifiedbytheir molecularweight,accessibility

tosurface-specificiodination,and selective extraction

procedures.

J. VIROL.

Experiments were performed to ensure that the off-diagonal spots arosefromthe reductive cleavage ofcomplexes stabilized by native disul-fide bondsorbydisulfide-containing cross-links. No off-diagonal spots were observed with control

or cross-linked samples, when reducing

condi-tions (3%2-mercaptoethanol or5 mM

dithioe-rythritol) or nonreducing conditionswereused in both dimensions of the two-dimensional gel. Additionalexperimentsweredesignedto estab-lish that theappearanceofnew high-molecular-weight bandswasthe direct result of cross-link-ingby the chemicalreagentintroduced. All the

newcomplexes formed in the presence ofDTBP

orPAPDIPwerereductivelycleavedbyboth

2-mercaptoethanolanddithioerythritol.A10-min incubation ofDTBP or PAPDIP with 20mM ammoniumacetatebefore the addition of virus prevented theappearanceofnew

high-molecu-lar-weight complexes. These complexes were alsonotformed when viralsampleswere irradi-ated for3 minwith UVlightin the absence of PAPDIP. When PAPDIP-derivatizedviruswas solubilized in 2% SDS before photolysis, no

cross-linking was observed.Therequirementfor

photolysis eliminated the possibility of

cross-linking by monofunctional imidoesters (3,26,36)

or by DTBPproduced from PAPDIP through

an exchange reaction: 2PAPDIP - DTBP +

4,4'-dithiobisphenylazide.

Disulfide reagents (R-SS-R) such as DTBP and PAPDIP are susceptible to SH-SS inter-change (26).Duringincubation of suchreagents with virusatpH 8.5 anexchangereaction (pro-tein-SH + R-SS-R protein-SS-R + R-SH)

can occurbetween thereagentdisulfide and free

sulfhydrylgroupsofviralproteins.The

require-mentforphotolysistoobtaincross-linkingwith

PAPDIP suggests that significant amounts of

e

NH2

11

protein-SS-CH2CH2COCH3 had not been

formed bymercaptan-disulfide interchange

be-tweenprotein andreagent. Also, incubationof

viral proteins with 50 mM N-ethylmaleimide

beforecross-linkingdid notalterthe

cross-link-ing results.Thesecontrols indicatethat none of

the cross-linkedproductswasformedby SH-SS

interchange.

Chemical cross-linked homodimers of F

and NP. DTBP treatment ofSendaivirus

prep-arations stored frozen for 3 to 12 months at

-70°C before cross-linking produced the

one-dimensionalpatternseeninFig.4E. Under the

mild cross-linking conditions used, little or no protein remained at the origin ofthe

first-di-mensional gel. The sameresultswereobtained

whencross-linkingwasperformedat 0, 11, 22, or

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37°Cover arangeof DTBPconcentrations(0.5

to 10 mM) or when PAPDIP (0.1 to 0.5 mM)

was used as the cross-linking reagent. A new

band(120,000molecular weight) appeared in the

one-dimensional profile as the result of

cross-linking.Thiscomplexwasselectively labeledby surface-specific iodination, indicating the pres-enceof HNorF (Fig. 4F).

Two-dimensional analysis indicatedthat the 120,000-molecular-weight bandcontains NP,Fl,

andF2 (Fig. 6A).From molecular weight consid-erationsalone, the band could be explained by the formation ofaheterodimerof the two 60,000-molecular-weightproteins NP and F or by their

homodimers. Thefact that theF1spot at

molec-ular weight47,000containedmoreradioactivity than the NP spot at molecular weight 60,000 suggested the homodimer explanation. Also, a species just below the diagonal is visible, mi-grating atmolecularweight 120,000 in the first

MWx1cr3 Ott) 00 OtLo

N.- - t- IDl

C

a

a a,

STORED VIRUS

0 0

0

GQoj

t)(

NPdin,ner a

0

Fdimer Mdimer

0 0

A B

MWxio-3

0 0 0 0

Ott)Ln

0

8-o O 0O ° e 00m

.o

.-0

aS

4

00 00

00

0 0

NP-M dimer

oC; e

20

15 0

td

C D

FIG. 6. Reversible cross-linkingofproteins inegg-grownSendaivirus by DTBP. Sendai virns (100pgof protein) wastreated with 3 mM DTBPfor30minat0to4°C.Thesamepreparation of earlyharvestSendai

viruswasusedinFig.4A and4C,but the stored virusof4Awaskept frozen3monthsat-70°Cbefore

cross-linking. Thefresh virus of4C waskept at 4°Cand usedfor cross-linking experiments within 3days of isolation. Cross-linkedproductswere iodinated inthepresence ofSDSand analyzed by two-dimensional

polyacrylamide electrophoresisasdescribed inFig.5.

ionreduced

0

1stD-n4

-0

0

n or

v 300

t 200 150

100

I?

O 70

x 60

45 30 20 15 td

Kb

300 200 150

FRESHVIRUS

100

0

70-3 60

45

30

Ab a

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dimension and molecular weight 97,000 in the second dimension. From its molecular weight and accessibility to surface-specific iodination (datanotshown), itappearedtobeahomodimer of F1, whichwas nottotallyreducedtoits mono-mer form. The presence ofan F1 homodimer

supports the homodimer explanation of the

120,000-molecular-weight species.The presence ofsomeHNin tetrameric and dimeric forms in the second dimension further indicates incom-plete reduction of the first-dimensional gel. A 30-min ratherthan the usual10-minincubation of thefirst-dimensional gelinreducing solution completely reduced the F1 homodimeric and HN tetrameric and dimericforms.

To obtain conclusive evidence of the identity of the 120,000-molecular-weight complex(es),

cross-linked viral preparations were extracted firstwith 2% NP-40 in1MNaCl, then with1M NaCl aloneasdescribedinMaterials and Meth-ods. The extractedpellet consisted ofNP mono-mer (60,000 molecularweight) and homodimer (120,000molecular weight) (Fig. 7A).A 120,000-molecular-weightcomplex containingonlyFwas found in the NP-40-NaCl supernatant fraction (datanotshown). No evidence forNP-F heter-odimer formation was observed in any of the extracted fractions.Therefore, the

120,000-mo-lecular-weightspeciesactuallyconsisted oftwo

1st0-nonreduced

u

0

a

L

ff

different types of protein complexes: an NP homodimer and an F homodimer which comi-grated.

Chemicalcross-linkedhomodimers ofM. Two additional products of cross-linking were detected as off-diagonal spots on two-dimen-sionalgels. These migratedatmolecularweights

67,000and63,000in thefirstdimensionand thus

wereobscured by the F and NP bandson one-dimensionalgels.Inthesecondreducing dimen-sion, they both migrated at molecular weight

34,000.Bothspecieswereinaccessibleto

surface-specific labeling. Their molecularweights in the first dimensionplus comigrationuponreduction with the monomer of the M protein strongly suggested thattheyarehomodimersof M. When cross-linked preparations of Sendai viruswere extracted with 2% NP-40 in 1 M NaCl as de-scribed in Materials and Methods, the 67,000-and63,000-molecular-weiiht sDecieswerefound in the supernatant fraction along with the M

monomer. These selective extraction

experi-mentsconfirmed their identificationas M

hom-odimers. Thetwodifferent molecular weightsin the firstdimensionmaybe dueto adifferencein theiraxesofcross-linkingorthe 63,000-molecu-lar-weight species may have intramolecular as wellasintermolecular cross-links.

Additional species formed by chemical

MWx1O-3

0

x3o

0 O ° ° C

4 r

v-300 200 150

90 60

:E.

_e 4

0 0",

__ 4

-M

20 15 toi

A B

FIG. 7. Selective extractionofcross-linked Sendai virus. The stored(A)andfresh (B) virussamplesthat had beencross-linkedfor Fig. 6A and Cwereextractedfor2hat roomtemperaturein 2%NP-40-1 MNaCi

in0.01 Msodiumphosphate buffer atpH 7.2and then iodinated in the extraction buffer. The insoluble

nucleocapsidfractionwas thenfurtherextractedwith 1MNaCi. Theresulting pelletwasanalyzedby

two-dimensionalpolyacrylamidegelelectrophoresisasdescribed inFig.5.

J. VIROL.

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cross-linking. Production of homodimers of

NP, F, and Mupon cross-linkingwas observed

with six independent batches of late harvest

Sendai virus and three independent batches of

early harvest Sendai virus. No differences in

cross-linking productswerenoted betweenearly

and late harvests after 3to12 months of storage

at -70°Corin the cross-linked complexes

pro-ducedbyDTBP and PAPDIP. Evidence for the

formation of small amounts of M trimeric

(105,000 molecular weight), F tetrameric

(245,000 molecular weight), NP trimeric (178,000

molecular weight) and tetrameric (250,000

mo-lecular weight) forms was found when the gel

which produced the autoradiograph shown in

Fig. 6Awasexposed for several weeks (datanot

shown).

Effect of storage on cross-linking:

for-mation of an NP-M heterodimer upon

cross-linking of freshly harvested

prepa-rations. When freshly harvested (not frozen,

storedno morethan 3daysat4°C) Sendai virus

preparationsweresubjectedtocross-linking,an

additional bandwasobservedinthefirst

dimen-sion at molecular weight 97,000 (data not

shown). This bandwas morepredominant in the

twoearly harvest preparations examined than in

thetwolate harvest preparationsgrowninthe

samebatches ofeggsastheearlyonesbut

har-vested 26 h later. Reduction of the 97,000-mo-lecular-weightcomplex inthesecond dimension

producedtwospotswhichcomigrated with NP

(60,000 molecular weight) and M (34,000

molec-ular weight), indicating the possible formation

ofaNP-Mheterodimer (Fig. 6C).

A selective extraction method was used to

demonstrateconclusivelythatthe

97,000-molec-ular-weight species was aheterodimer and not

homooligomers of NP and M with similar

elec-trophoretic mobilities. Cross-linked

prepara-tions were extracted with2% NP-40 plus 1 M

NaCl and then with 1 M NaCl as previously

described in Materials and Methods. The

insol-ublepelletcontained the NPmonomerand

hom-odimer plus the NP-M heterodimer (Fig. 7B).

The monomer and homooligomers of M were

notpresentinthepellet,havingbeen previously

extracted into the 2% NP-40-1 MNaCl

super-natantfraction.

Theability of NP and Mtoforma

heterodi-merupontreatmentwithcross-linkingreagents

diminisheduponfreezing and prolongedstorage

of the sample. The same preparation ofearly

harvest Sendai viruswasused inFig. 6A and C,

but the sample in 6Awasfrozenand storedfor

3 monthsat-70°C beforecross-linking. In Fig.

6C the amount of NP-M heterodimer formed

wasabout thesame astheamountof NP

hom-odimerformed. InFig. 6Aonly NP homodimer formation wasobserved.

Cross-linking of Sendai virus was also per-formed after its adsorption to host cells. Both freshly harvested and stored Sendai virus prep-arationswerederivatized in the dark with PAP-DIP and then adsorbedtoBHKmonolayers as described in Materials and Methods. Unad-sorbed virus and excess cross-linking reagent were removed before photolysis. Cross-linking ofstored Sendai viruspreparations adsorbedto BHKcellsproduced thesameviralprotein com-plexes as seen in Fig. 6A, i.e., homodimers of

NP, F, andM.Cross-linkingof thefreshly

har-vestedvirus preparation adsorbed tohost cells additionallyproduced the NP-M heterodimeras inFig. 4C. These results indicate that PAPDIP-derivatized virus retained itsabilitytoadsorbto host cells and that the cross-linked products

obtained with isolated viruswerealso obtained with virus adsorbedto hostcells. No new band formed togivean indication of cross-linking of

HNor F tohost membraneproteins.

Protein-protein interactions inNDV. Pro-tein-protein interactions within NDV, another member of the paramyxoviruses, were investi-gated with thesameapproachused with Sendai virus. The major polypeptide species of NDV have thesamenomenclatureasthoseofSendai virus but differslightlyinmolecular weight (see Table 3). The most noticeable differences

be-tween the Coomassie patterns of the reduced

viral proteinsweretheabsence of P and greatly reduced amount of F1 in NDV. (Compare Fig.

TABLE 3. Proteins and their native disulfide complexes present in egg-grown NDV (HP-16)

Present in:

Accessi-Molwt bleto

(xlO-)a Nonre- Re- surface- Identityc duced duced specific

gels gels labelingb

220 + + - Lmonomer

154 + - + HNdimer

147 + - - NP trimer

74 + + + HNmonomer

61 + - + Fmonomer

(Ft+F2oligomer)

55 + + - NPmonomer

53 + + + F,monomer

34 + + - Mmonomer

15 - + F2monomer

Molecularweightsof NDV viralproteinswerecalculated from molecularweight markers(seeFig.3)coelectrophoresed on5to 12.5% slabgelsin the first(nonreduced)andsecond (reduced) dimension.

bA 100-ug portion ofintactviruswasreacted for10minat

00Cwith0.5mCi ofNa'251Iinthe presence of 10 yg of chloro-glycolurilasdescribed inthetext.

'The individual components ofprotein complexes were identifiedbytheirmolecularweight, accessibilityto surface-specificiodination,and selective extractionprocedures.

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[image:10.504.249.441.432.562.2]

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4A to Fig. 8A.) Although NP and

F,

have similar electrophoretic mobilit wereeasily distinguished by

surface-sj

beling (Fig. 8B). Most of the radiol incorporated into the major glycopro and therest wasincorporatedintoFl.'

resultswereobtainedfor NDV (L.Kan lactoperoxidase-catalyzed iodination (

NDV, unlike F2 of Sendai virus, inc(

little radioiodine.

Under nonreducing conditionsthe F observedjust above NP (Fig. 8Cand ]

F1wasalsopresent inthenonreduced

tion. Two bandswerepresent in the

hig

ular-weight region of the nonreduced

80). The one at molecular weight 154 accessibletosurface-specificiodinatior

atmolecular weight 147,000 was not ( Two-dimensional analysis (Fig. 9A) thatthe154,000-molecular-weightspec homodimer of glycoprotein HN. Most

HN was present in the dimeric form;

or--L!

HN Q

F-F,

2

m....t

"I

so

J. VIROL.

ofNDV (5%) was present as a monomer. The

147,000-ties, they molecular-weight specieswasidentifiedas

hom-pecific

la- otrimerof NP. Most(92%) of NP existed in the

label was monomeric form in thenonreduced virus, buta itein HN, significant amount (8%) formed a

disulfide-The same linked homotrimer inthe sixviral preparations

isas)with examined.Noevidence forahomodimer form of

15). F2 of NP wasobserved. The 5 to 12.5% gradient gel

Drporated employed in these studies resolved the

homo-oligomersof HN andNP into twodiscretebands,

bandwas but on a 7.5%gel such as thatused previously

D). Some for cross-linking studies in this laboratory (21,

prepara- 37), the two homooligomers comigrated at an

gh-molet-

apparentmolecular weight of140,000.

gel (Fig. DTBP-induced cross-linking of freshly

har-4,000 was vested (notfrozen,storedno morethan3 days

i;theone at400)

preparations

of NDVproducedthe

two-(Fig.

8D). dimensional pattem observed in Fig. 9C. The

indicated same results were obtained when cross-lilking

-ieswas a wasperformedat0, 11, 22,or

370C

over arange

(95%) of of DTBP concentrations(0.5to10mM)orwhen the rest PAPDIP (0.1to0.5mM) wasusedasthe

cross-linkingreagent.Thehomodimersof F, NP, and Mproducedby cross-linkingwereidentified by theirmolecularweight and accessibilityto

sur--300 face-specific labeling (data not shown). NP-M

heterodimer formation was furtherverified by -200 selective extraction

experiments

described

pre-viously

foridentification of the NP-M

heterodi--150 mer in Sendai virus. There wasslightly greater K heterodimerformation inearlyharvest than in > late harvest preparations ofNDV, but the

dif-__1Q(

X ference betweenearlyandlate harvestwas not

o

nearly

so dramatic aswith Sendai virus. NDV

_70

C whichwasstoredfor3monthsormore at-70°C beforecross-linkingshowedlittleor noevidence 60 of NP-M heterodimerformation.

DISCUSSION 30

20

15

[image:11.504.74.260.294.526.2]

A kvi D

FIG. 8. Comparison ofreduced and nonreduced

proteins ofNDVbyone-dimensionalgel electropho-resis. A 100-pug sample ofreduced egg-grownNDV

waselectrophoresedon a5 to 12.5%lineargradient resolving geland stained with Coomassie brilliant

blue (A). A sample ofthe same viruspreparation withoutreducingagent is shown in C. The

autora-diographs of samples A and C which have been

surface specifically labeled by the chloroglycoluril methodfor125Iare shown inB andD, respectively. Letters designate thepositions of the major viral

proteins. RefertoTable 3for identificationofproteins andtheir nativedisulfide complexes.

Theprotein-proteininteractions identified in these studiesexpandourconceptual knowledge

of the molecular architecture of the paramyxo-viruses. Analysis of native disulfide bondingin isolated egg-grown viruses demonstrates that someof the proteinsof Sendai virusandNDV consistentlyform covalentcomplexes. Thesize

of thecomplexand the percentage of the

partic-ular protein found in complex form appear to

depend on the infecting virus rather than the

hostcell. The HN ofegg-grownSendaivirus is

found mostly in tetrameric anddimeric forms,

withonlyasmallamountpresentas a monomer.

Thissamedistributionof HNincomplexeswas

observedwith Sendai virusproducedbyMDBK

cells(unpublished data,MarkwellandFox).The

HN ofegg-grownNDV,strainHP-16,ispresent

mostly as adisulfide-linked dimer,but the HN of arelatedstrain ofNDV,L-Kansas,ispresent

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1stD-nonreduced

's MWx1,03

a

i 2001

120-1

>ci

cp 75;t

3 60' '

451

30-1

A MWx1Oe

O-

9 8v

IC-

, rne 8 8 a

I

a

~6 i

@0 9

C

O

o

0

NMd mr,

rd°*-d

(S?

__i

N-dOr _~:bo

FIG. 9. Two-dimensionalanalysis ofnativedisulfideandchemically cross-linkedprotein complexes of

egg-grownNDV. Approximately50pgof viral protein from untreated (A)orDTBP-cross-linked (C) viruswere

iodinated in thepresenceofSDS andthenfractionatedin thefirstdimension withoutreducingagentona5

to12.5%polyacrylamideslabgel. Thefirst-dimensional gelwasslicedinto itsrespective lanes,reduced with 2-mercaptoethanol, andaffixedonto7.5to10% slabgel forthe second dimension. Theautoradiograph ofthe gelis shownontheleft,and itscorrespondingschematicdiagramis shownontheright.

onlyas amonomerwhenproducedin

embryo-nated eggs (unpublished data, Markwell and

Fox).To what extentthesenativedisulfide

link-ages influence the number of HN polypeptide

chains which assembletoformthe HNspike is

notcurrentlyknown.

At theonsetofthesestudies, itwasrecognized

that thevalidityofthecross-linking results

ob-tained would depend to a large extent on the

integrity of the virus preparations used. From

hemolysis studies,Homma et al. (10)concluded

that Sendai virusobtained from harvestsjustat the end of theone-stepgrowth cycle (early

har-vests) hadmoreintactenvelopesthanvirus

ob-tained from late harvests. Acomparisonofearly

and late harvest preparationswastherefore in-cluded in thepresentstudies.Use of the SV-288

vertical rotor greatly shortened the time

re-MP -0

trimer: p

201

15;

td

a

3004

200-15011

120-_ 75

x

60-30 20H,

15 tdK

D

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[image:12.504.98.401.56.498.2]

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quired for viruspurificationandproduced prep-arations of virus thatweremoreintactasjudged bysurface-specific iodination than theprevious procedure (30) with swinging bucketrotors and a number of pelleting steps. Sterile, purified virus preparations that were stored for longer than3dayswerefrozenathigh protein concen-trations (5 mg/ml) at -70°C and thawed only

once, immediately before use. The importance

of native disulfide bondingin Sendaivirus has beenemphasizedbyreportsof the loss of infec-tivity,cellfusion,hemagglutinin, neuraminidase, andhemolyticactivitiesupon treatmentof Sen-dai virus with reducing agents (23, 25, 33).

Therefore,viruspreparationsused inthepresent

cross-linking studies were not pretreated with

reducing agent asdescribed inpreviousreports (21,37), andcross-linkingwasperformed under conditionsdesignedtopreservenative disulfide bonding. Moreover, under the mild cross-linking conditions used in thisstudy,littleor noprotein

remained atthe origin of thefirst-dimensional gel. This indicates that we are looking at the

initial products of cross-linking and that the

cross-linking process has only miniimally per-turbed the nativestructureofthe virion.

The sameproductswereformed when

cross-linking was induced with DTBP or PAPDIP.

Both reagents have an 1.1-nm separation

be-tween theirfunctionalgroups (4, 26),andboth

diffuse across the viral membrane as demon-stratedbytheirability toformhomodimers of NP. The imidoester group of both reagents is

specificfor primary amino groups (26); the

ni-trenegenerated by photolysis of the arylazide

group of PAPDIP does not require a specific

reactive group. Recent evidence suggests that theimidoesters and nitrenespreferentially inter-actwith extrinsic membranecomponents(1, 11). Ourstudies indicate that theprotein-protein interactions identifiedbyreversiblecross-linking

areinfluenced bythe conditionsofproduction, isolation, and storage of these

membrane-enve-lopedviruses. Some of the

interactions,

namely

theabilitytoformhomooligomersofF, NP, and Mupon cross-linking, appeartobevery stable

andcanbe observed inpreparations that have

beenstoredat-70°Cforaslongas2years. This typeof interaction has beenpreviously reported

for theparamyxoviruses (22, 28).Other

interac-tionssuchasthosebetween NP and Marebest

observed in preparations with the greatest de-gree of biological integrity, i.e., early harvest

preparationsisolated bytheverticalrotor pro-cedure and cross-linked immediately after iso-lation. This type of interaction has not been demonstratedpreviously by cross-linkingin

par-amyxoviruses.The

ability

of NP and Mtoform aheterodimerappears tobe thereflection ofa

stable complex rather than a transient one formed momentarily by diffusionof components for a numberofreasons. Comparable complex formation was demonstrated at 37 and 0°C,

wherediffusionis severely limited, and also with

aphotoactivated reagent which yields a reactive

species witha lifetimeon the order of millisec-onds (6).

This report of NP-M heterodimerformation

in paramyxoviruses demonstrates that the M

protein layer is in close proximity (1.1 nm or less) to thenucleocapsid and could serve as its recognition siteduring viralassembly. The pro-gressive loss of the ability of NP and M to form a heterodimer as the virus is stored without a corresponding loss in infectivity suggests that thisspatial relationship alters afterparticle for-mation and is not necessary for the ensuing

stagesof the infectiousprocess. Thisstructural

changeis notsimplydue to freezing and thawing. Viral preparationsthawed after 1 week of

stor-age at-70°C still formedsome NP-M

hetero-dimer upon treatment with cross-linking

re-agents.Rather, itappearsthat thepreparations

age upon storage even at-70°C. Theprogressive

lossof the interaction betweenNP and M may reflect eitherageneral breakdownof weak, non-covalent interactions between proteinsor inac-tivation ofparticular binding sites between the nucleocapsid and membranecomponentsof the virus. A recent electron microscopic study of Sendai virus (12) indicates that as the virions ageinovo(late harvest preparations)orinvitro, the nucleocapsid becomes irregularlyfolded and detaches from the viral envelope.

F-FIG. 10. Astructural modelofprotein-protein in-teractions within theSendaivirion.RefertoTable2

and textfor description of protein complexes

pro-ducedbynativedisulfidebondingorreversible

chem-icalcross-linking.

J. VIROL.

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Figure10depictsourmodel of the topograph-ical organization of proteins within the para-myxoviruses,specifically Sendai virus, basedon theprotein-proteininteractions discussed in this

paperandcurrentknowledgeofparamyxovirus

structure. The glycoproteins HN and F form

separatespikesontheoutersurface of the

mem-brane envelope. Each spike appears to be an

oligomer whose component polypeptides asso-ciatethrough noncovalent interactionsor disul-fide bonding or both. Shimizuet al. (35) have

separated two types ofdumbbell-shaped spikes

(HN+ and HN-) from detergent-solubilized

Sendai virus that differed in their biological,

serological, morphological, and chemical prop-erties.

Theglycoproteins ofmembrane-enveloped

vi-rusesappear to associate with thelipid bilayer

through a relatively small, predominantly

hy-drophobic peptide (14). Cross-linking studies with SemlikiForestvirus (8) and vesicular sto-matitis virus(7)suggestthat in these lipid-con-taining viruses the anchorpeptide tailspansthe bilayer. The selective extractionexperiments de-scribed in this paper suggest that HN and F of Sendai and Newcastle disease viruses are

embeddedinthelipid bilayer. However,no evi-dence for transmembranecross-linking between the glycoproteins andM wasfound for thetwo paramyxoviruses examined in this study, and previous reportsfrom this laboratory of trans-membranecross-linking betweenHNandNP in NDV (HP-16) (21, 37) wereshowntobea mis-interpretation of data due to the inadequate resolution of the two-dimensional gel system used. The lackofcross-linking between HN or

FandMbyno meanseliminates the possibility

thatthe glycoproteins span the bilayer. Cross-linkingreagents with different site specificities than those usedinthisstudy might demonstrate this topographicalrelationship.

Our cross-linking data indicate that the nu-cleocapsidlies inclose proximitytothe M pro-teinlayerinfreshly harvested paramyxoviruses.

The ease of extraction of P and L from the

nucleocapsidsuggests a moresuperficial location

in or weaker associationwiththe nucleocapsid

forthese twoproteinsthan the

firmly

attached

NP. Both thePprotein ofSendaivirus and the

NPprotein of NDV (HP-16) exist as monomers

and as disulfide-linked homotrimers. A

func-tionalrole(s) for these trimers is not known.

Cross-linking of intact virus revealed little

more about the organizationof the

paramyxo-virus nucleocapsid beyond the relationship of

NP as nearestneighbor to itself. No cross-linking

of PorLwhich are alsopart of the transcriptive

apparatus of Sendai virus (19) was observed.

Whether P and L interact with NP ordirectly

with viral RNAorboth is stillopen toquestion. Cross-linking of isolated Sendai virus nucleocap-sids with DTBP,tetranitromethane,ordimethyl suberimidate has been previously reported to produce homooligomers of NP andto convertP andL tohigh-molecular-weightcomplexes (27,

28).In ourstudy,cross-linkingof intact virus led

toformation ofverysmallamountsof NP hom-otrimerandhomotetramer, butnot to high-mo-lecular-weight complexes of PorL.Thismaybe

duetothe presenceof the viral membrane which

could either limittheaccessibilityof

cross-link-ing reagents to the nucleocapsid or subtly

change thestructure of thenucleocapsid by as-sociating with it during the processof viral as-sembly.

ACKNOWLEDGMENTS

Wegratefully acknowledge the assistance of Rochelle Weiss inpreparing the egg-grownSendai virus and of Tokichi

Mi-yakawafor synthesizing thecross-linking reagent PAPDIP used in thisstudy. We also wish to thank Debi P.Nayak and Joseph T. Seto for theirhelpfulcommentsontheproposed structural model of Sendai virus.

This workwassupportedby Public Health Research grant GM-18233 from the National Institute of General Medical Sciences and by a grant from the Muscular Dystrophy Asso-ciation,Inc. M.A.K.M.is therecipient of postdoctoral fellow-ship grant DRG 118-FT from the Damon Runyon-Walter Winchell Cancer Fund.

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