Densonucleosis Virus Structural Proteins

0022-538X/80/10-0224/12$02.00/0

Densonucleosis Virus Structural Proteins

D. C.KELLY,* N. F. MOORE,1C. R SPILLING,1 A. H. BARWISE,2 ANDI.0. WALKER'

Natural Environment ResearchCouncil,Instituteof Virology, Oxford,OX) 3UB,1 andDepartment of

Biochemistry, Universityof Oxford, Oxford, OX) 3QU,2 United Kingdom

The proteincoatsoftwodensonucleosisviruses(types1and2)wereexamined byavariety ofbiophysical, biochemical, andserological techniques. The viruses were24 nmin diameter, containedatleast fourpolypeptides, wereremarkably

stable toextremes ofpH and denaturing agents, andwere serologically closely related. The two virusescould, however, be distinguished serologically and by differencesinmigrationof theirstructuralpolypeptides. For each virus the "top component" (i.e., the protein coat minus DNA, found occurring naturally in infections) appeared to have acomposition identical tothat ofthe coat of the

virus andwasa morestablestructure.Electrometric titrationcurvesofthe virus particles and top components demonstrated that the DNA phosphate in den-sonucleosisvirusparticleswasneutralizedbycations other than basicaminoacid sidechains of theproteincoat.Circular dichroism studiesshowed thattherewas a conformational difference between the protein coats oftop components and virusparticles.

Densonucleosis viruses (DNVs) are small

icosahedralviruses,isolated from

invertebrates,

whichcontainlinear

single-stranded DNA,

and

they sharemanystructural features incommon

with both the defective and the nondefective

parvoviruses

isolated from vertebrates

(10, 17).

Structurally, the DNA contained

by

DNVs is

comparable

tothat of the defective

adeno-asso-ciatedvirusgenome and isorganizedas

comple-mentary, separately packaged, linear

single-stranded DNAwhich possesses inverted

termi-nalsequencesandalimited nonrandomcircular

terminal

permutation (1,

9, 13,

14).

Thegenome

is unlike the nondefective

parvovirus

genome,

which (as

typified by

minute virusofmice and

Kilham ratvirus) is

organized

asalinear DNA

molecule

(of

one

polarity)

with short

palin-dromicsequencesatthe ends

(2).

Little is known about the structure of the

protein coat ofDNVs.

Tijssen

et al. (33) have

reported thataDNV of themothGalleria

mel-lonella contains four structural

polypeptides

withmolecularweightsof 98,000,69,000, 58,500,

and49,000and that the98,000-dalton

polypep-tide isadimerof the

49,000-dalton

polypeptide.

We have

previously

shown that the

protein

coat of DNVs has amolecular weight of3.55 x

10'

and that themolecularweightofthevirus par-ticle (5.7x

106)

represents theapproximatesum of the protein coat, the genome (molecular

weight, 1.95x 106),and thepolyamines

(molec-ular

weight,

80,000) which constitute the

com-plete virus particle (13-15). In this study we

have examined theprotein coats of two DNVs

by exaning either the top components (i.e.,

the virusparticles minus DNA and polyamines)

orthecomplete virus particles.We have used a

variety of physical methodstoexaminethe

poly-peptide composition

ofthe coat, the secondary

structureofthe

protein,

andtheinteractions of thecoatwith the genome. Inaddition,we have

compared thetwoviruses serologically.The two

viruses studied wereDNVtype 1 (DNV1),

iso-lated from G. mellonella (24), and DNV2,

iso-latedfromthe

butterfly

Junonia coenia (29).

MATERIALS AND METHODS Virusproduction andpurification. DNV1 was originallyisolatedby G. Meynadier. DNV1 and DNV2 wereprovided by C. F. Rivers. DNV1 and DNV2 virus particles and corresponding top components were pre-paredaspreviously described (13, 20).

Determination of extinction coefficients and concentrations. DNAwasestimatedbythe diphen-ylaminemethod asmodified by Burton (3). Calibration curves were prepared with standard solutions of 2-deoxy-D-ribose. RNA was estimated by the orcinol method as modified by Mejbaum (23). Calibration curves were prepared with standard solutions of D-ribose.

Due to lack of material, it was not possible to determine the extinction coefficients of virusparticles and top componentsby the dryweight method. The extinctioncoefficients weretherefore measured indi-rectly, usingarefractive index methodtodetermine protein concentration and estimating phosphorus of virusparticles after dialysis into 0.1 MNaCl by the method of Jones etal.(12).

The estimation ofprotein involved measuring the areaofasynthetic boundary ofasample of top

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ponentphotographed withSchlieren optics in a Spinco model E analytical ultracentrifuge and comparing this with the area obtained by the same method at the same bar angle for a standard solution oflysozyme, assuming the refractive index increment of both pro-teins to be 0.193/g per ml at 435.8 nm (5). The method gave a measure of the protein concentration of the solution of top component. Since the refractive index increment of DNA at 436.0 nm is also 0.193 (27), a similar method was used with solutions of virus par-ticles and top component sedimented appreciably at the speeds used (usually 12,590 rpm), photos were taken at various time intervals, and the measured areas were extrapolated to zero time to obtain the initialarea.

Routine analysis for protein was by the Folin method (21) as modified by Eggstein and Kreutz (6). Bovine serum albumin was used as a standard, and a correctionwasapplied by using the previous refractive index method and the Folin method on the same sample. Extinction coefficients of virus particles and top components wereobtained by measuring the ab-sorbanceofasample ofknown concentration in a

1-cm-path-length cellat260and 277 nm, respectively. Results were expressed in optical density units per milligram per milliliter for a 1-cm path length. UV spectraof virus particles and top components were obtainedat25°Coneither a Cary14recording spec-trophotometer oraUnicanS.P. 500 spectrophotome-ter.The extinctioncoefficients for virus particles and top componentswere 9.49±0.32/mg perml at 260 nm and 1.39±0.11/mg per mlat 277nm,respectively.

Electron microscopy. DNV particles and top componentsnegatively stained with 2% (wt/vol) ura-nyl acetate were examined on an AEI 6B electron microscopeaspreviously described (20).

Analytical ultracentrifugation. Analytical ultra-centrifugationwasperformedaspreviously described (13), using aBeckmanSpinco analytical ultracentri-fuge equipped with both Schlieren and absorption opticalsystems.

Preparationof antisera. Antiserawereprepared inboth rabbits andguinea pigs.Preimmunesera were obtained from rabbitsbybleedingfrom themarginal earvein andfromguinea pigs by cardiac puncture.

Rabbits were immunized by injecting at weekly intervals 1 ml of virus (containing 1 mg of virus) emulsified with 1 ml of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.) subcutaneously, followedbyasimilar subcutaneousinjection with in-complete adjuvantandfinallyanintravenousinjection of1 ml of virus. Immune sera were taken4 weeks later.

Guineapigswereimmunizedbytwointraperitoneal injections (7 days apart) of1ml ofvirus (2 mg ofvirus) emulsifiedwith1mlofFreundcomplete adjuvantand werebled by cardiac puncture 3 weeks later to provide immunesera.

Gelimmunodiffusion.The reactions between

an-tibody and antigenwereexaminedby performing mi-crotests on glass slides (3 by 1 inch [ca. 7.6 by 2.5 cm]) coveredin0.75% (wt/vol) Ionagar 2 (Oxoid Ltd., London, United Kingdom) inphysiologicalsaline containing 0.5% (wt/vol) sodium azideand 0.001%(wt/ vol) trypan blue.

Polyacrylamide gel electrophoresis. Three polyacrylamidegel electrophoresis systems were used during these studies.

(i) Polypeptides were analyzed on 16-cm-long 12.5% polyacrylamide slab gels, using the buffer system of Laemmli (19) as previously described (7). Samples weredenatured by boiling in 0.1 M Tris-hydrochloride (pH 6.8)-2% (wt/vol) sodium dodecyl sulfate (SDS)-2% (vol/vol) 2-mercaptoethanol-10% (vol/vol) glyc-erol-0.01% (wt/vol) bromophenol blue. ,B-D-Galacto-sidase(molecular weight 132,000), phosphorylase (mo-lecular weight, 97,200),transferrin(molecularweight, 90,000), bovine serum albumin (molecular weight, 69,000), and ovalbumin (molecular weight, 46,000) wereusedtocalibrate thegels for molecular weight determinations.

(ii) Polypeptideswereanalyzedon10and 7% SDS-polyacrylamide cylindrical gels(6 by 100mm),using acontinuousphosphate-bufferedsystem aspreviously described (16). Thesamefive standardswereused for molecular weight determinations.

(iii) Native virus particles and top components wereanalyzedon3%polyacrylamide gels (5 by75mm) made up in 0.1 M Tris-hydrochloride, pH9.0, inthe absenceof SDS. Reservoir buffers contained0.025M Tris-glycine,pH9.0. Samplesin reservoirbufferplus 10% (wt/vol) sucrose were applied directly to gels. Electrophoresiswas at5mA pergelfor about70min. Nonreducing gelswere runby omitting mercapto-ethanol from the systemaspreviouslydescribed(25). Sugar residues were detected on proteins by Schiff stainingasdescribedby Fairbanksetal.(8).

Proteinswere located byCoomassie blue staining

aspreviouslydescribed.

'"I-labled

proteinwaslocated byautoradiography, using Kodirex X-ray film. Coo-massie blue-stained gels and autoradiographs were scanned inaJoyce-Loeblmicrodensitometerequipped withathin-layerattachment.

lodination ofpolypeptides. Iodination of poly-peptideswascarried outessentially asdescribed by Mooreetal. (26). Intact virusortop componentwas iodinatedchemicallywithchloramine-Tor enzymati-callywith lactoperoxidase. Virus components previ-ouslydenaturedby boiling in1% (wt/vol) SDS for2 minwerealsoiodinatedbythe chloramine-Tmethod. Iodination catalyzed by lactoperoxidase was per-formed by adding 10 ILI (100

ACi)

of carrier-free 125I (Radiochemical Centre, Amersham, United King-dom),5

pl

oflactoperoxidase (1 mg/ml)(Sigma Chem-ical Co.Ltd.,London,UnitedKingdom),and10

pl

of freshlydiluted 0.05%(vol/vol) H202to 100

pl

of phys-iologicalsalinecontaining20 ugof virusortop

com-ponent.The incubationwascarriedoutat room tem-peraturefor30minandstopped by dilutingthe

reac-tion40-fold withphysiologicalsaline.

Iodinationbychloramine-Twasperformedby add-ing 10,ul (100

jCi)

of

"2I

to100

pl

of intactor SDS-disrupted virus(20Agofprotein),followedby10

pl

of chloramine-T (14mg/ml). The reactionmixturewas

vigorously shaken for30s, andthereactionwas

ter-minatedbytheadditionof20

pi

of sodium metabisul-fite(28mg/ml).

All iodinatedsamplesweredialyzed againstseveral 1-literchanges ofphysiologicalsaline before electro-phoresis. Intact virus componentswere rebandedon

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20 to50%(wt/vol)sucrosegradients made up in 7 mM sodiumphosphate(pH7.2)-0.1MNaCl. The gradients were run at 35,000rpmfor16hin a swing-out rotor (6 by 16.5 ml; M.S.E. Ltd., Crawley, Surrey, United Kingdom).

Circular dichroism spectrometry. Circular di-chroism spectraof DNV particles and top components wererecorded at200Cin aRousellJouan Dicrographe III, using cells of 0.01-, 0.1-, or 1.0-cm path length. Circular dichroism results arereported in terms ofe,

the mean residue ellipticity in unitsofdegrees per square centimeter per decimole. The mean residue weightof an amino acid residue was 108 as determined from amino acidanalysis(20).

Continuous electrometric titrations.

Continu-ouselectrometic titrationswerecarried outin a Ra-diometermicrotitrationassembly,consisting of a ther-mostatically controlled glass titration cell and mag-netic stirrer.HCl (0.1 N) or0.1 N NaOH was added fromanAgla glassmicrometersyringe.pH was mea-sured with anE.I.L. direct-reading pHmeterwith a glass electrode and a calomel electrodejoined by a KCl-saltbridge. Before titration a stream of nitrogen wasbubbledthroughthesample until the pH became constant,to removedissolved carbon dioxide. During thetitrationastreamofnitrogenwasblown over the sample.All titrationswerecarriedoutat25°C. Titra-tionswereperformed ontop component (2.5 mg/ml) in0.1 M KClandonvirusparticles (3.75 mg/ml) in 0.1MKClbelowpH5.7and in0.001 MKClabove pH 5.7.

The titration curve of top components or virus particles wasconstructed by subtracting the solvent titrationcurve(measured separately and corrected for dilution) from the sample titration. The results are expressedasmoles ofH+dissociatedorassociated per 100mol of amino acid residues. Todetemnine if titra-tionswerereversible, thesampleswere immediately retitrated from highorlow pH back to about pH 6.0 with0.1NHClor 0.1NNaOH.

RESULTS

Electron

microscopy.

Preparations of pure

virus

particles

and top components of DNV

stained with 2%uranylacetateshowed that all

preparations comprised small, spherical,

pre-sumably icosahedral,

particles about 24nm in

diameter (Fig. 1). Some of the virus particles

werepenetrated byavarietyofstain, including

uranyl acetate, ammonium molybdate, and

so-dium tungstosilicate. The extent ofstain

pene-tration oftop components varied from

experi-ment toexperiment,but an appreciable number

of particles remained inviolate. Structure was

observed, but not in sufficiently fine detail to

enumerate capsomeres with confidence. Such structureis shown in Fig.1.

Serology. Relationships betweenDNV1 and

DNV2 were examined by using antisera

pre-paredinrabbits andguineapigs against DNV1

andin guinea pigs againstDNV2.

Microimmu-nodiffusion

tests were

performed

to conserve

antigens and antisera.

Suspensions

of both

vi-ruses were

placed

in

adjacent wells,

and their

reactions with the three antisera were

moni-tored.After 48 hadjacentbands ofprecipitation

in the gels became contiguous. When the

sus-pensionsweretestedagainstantiserumprepared

inrabbits,atinyspurwasformed where thetwo

bandsofprecipitationagainstDNV1 and DNV2

met (Fig. 2A). This spur was acontinuation of

the

precipitation

band

formed

by

DNV1. To

show that this was not an

artifact,

the same

virus

suspension

was

placed

in

adjacent

wells;

therewere nospursontheprecipitationbands

produced

(Fig.

2Band

C).

When the antiserum

prepared in guineapigs

against

DNV2wasused

tocompareboth

viruses,

itwasfound that

single

bands of

precipitation

became

contiguous,

with

the band formed

by

DNV2

continuing

into a

large spur

(Fig. 2D).

The

guinea pig

antiserum

prepared against

DNV1 wasof low

titer,

but it

stillgave avisible spuronthe band created

by

DNV1

(Fig. 2E).

The virusparticles of DNV1 andDNV2were

serologically identical to their

respective

top

components

(data

not

shown).

Polyacrylamide

gel electrophoresis

of

DNV

structural

polypeptides.

Polyacryl-amide

gel

electrophoresis

revealed that the virus

particles contained four main structural

poly-peptides,

designated

in

decreasing

molecular

weightasVP1

through VP4,

asshown in

Fig.

3.

Adeno-associated

virustype 2

polypeptides

are

shown for

comparison.

The molecular

weights

of

these

polypeptides

areindicated in Table 1.

Es-timates of molecular

weight

varied with the

buffersystemand

gel

concentrationsused. The

polypeptide

compositions

of the virus

particles

andtop componentswereidentical for each virus

(Fig. 4),

though

the relative amounts of each

polypeptide

varied

slightly (Table

2and

Fig. 4).

Additional

polypeptides,

intermediate in molec-ular

weight

between VP1 and

VP2,

were

some-times detected

by

Coomassie blue

staining

though they

were more

readily

detected

by

io-dination

procedures.

Omission of a

reducing

agent from the

gels

failed to alter the

polypeptide profiles

of the

components,

showing

that

disulfide

bonds are

not involved in

interpolypeptide

interaction.

Schiffreagent failed to

positively

stain any of

the

polypeptides;

this is tentative evidence that the

polypeptides

are not

glycoproteins.

lodination

ofDNV polypeptides.

Iodina-tion of DNV particles and top components was undertaken to explore the location of the various

structural polypeptides within these virus

par-ticles. The data presented here are for DNV1 components, though

similar

results were ob-tained with DNV2 components.

AR

four main J. VIROL.

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-_ -L_w---t aM-* - .*-,_i

FIG. 1. DNVparticles negativelystainedwith 2%uranylacetate,showing generallyfeaturelessstructure. (a) DNV1particles; (b) DNV2particles; (c) DNVI top components; (d)DNV2 top components; (e) DNV2 particlesgreatly enlargedtoshow tentative substructure. Note stainpenetrationin some, butnotall, top

components. Bar=100nm.

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FIG. 2. Serologicalrelationshipsbetween virusparticlesofDNV1 and DNV2 testedby microimmunodif-fusion. Wells: 1, rabbit antiserum againstDNVI; 2, guinea pigantiserum against DNV2; 3, guinea pig antiserum against DNV1;g,DNV1 antigen;j,DNV2antigen.Note the small butwell-definedspurin A.

A

B

C

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_ -_

FIG. 3. SDS-polyacrylamide gel electrophoresis of the structuralpolypeptidescontainedby DNVI (A), DNV2(B),and adeno-associated virus(C). The pro-teinswereresolvedon a12.5% slabgel.

structural polypeptides and the minor, fifth polypeptide were efficiently radiolabeled with 125I whenchemically iodinated with chloramine-T after disruption with SDS (Fig. 5), and the profilewassimilartothe Coomassieblue stain profiles. Both lactoperoxidase and chloramine-T radiolabeled the two high-molecular-weight

TABLE 1. Molecularweights ofstructural polypeptidescontained byDNV1,DNV2, and

adeno-associated virus type2(AA V2)a Mol wt(+standarddeviation)of: Virus Individual polypep- Total poly-tide peptides DNV1 107,300±300 281,900

70,900±400 61,100±100 42,600±300

DNV2 109,600 ±200 281,000 70,800±300

58,700±300 41,900±400

AAV2 107,300±200 263,300 89,100±200

66,900±100

aThe peptides were resolved on 12.5%

polyacryl-amide gels. ,B-D-Galactosidase (molecular weight, 132,000), phosphorylase (molecular weight, 97,400), transferrin (molecular weight, 90,000), bovineserum

albumin (molecular weight, 69,000), and ovalbumin (molecular weight, 46,000)wereusedasstandards.

polypeptides (VP1 and VP2) predominantlyand

aminorpolypeptide intermediate in molecular

weight between VP1 and VP2 of both virus

particles and top components. This

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DNV STRUCTURAL PROTEINS 229

MIGRATION DISTANCE (mm)

FIG. 4. Densitometric traces of the polypeptides contained by DNVparticles and top components

stained with Coomassie blueafter electrophoresison a10%SDS-polyacrylamideslabgel. (A) DNVIvirus particles; (B) DNVItop components;(C) DNV2virus

particles; (D)DNV2 top components.

stratesthatVP1, VP2, and the minor polypep-tide have accessible surface tyrosine residues, which may mean that these polypeptides are located predominantly at the exterior of the components. VP2 wasiodinated less efficiently invirusparticles than intop componentswhen lactoperoxidase wasused to iodinate, and this

may indicate a conformational difference be-tweenvirusparticlesandtop components.

Acrylamide gelelectrophoresis ofintact

virus particles and topcomponents. Electro-phoresis of mixed DNV1 particles andtop com-ponentson3% acrylamide gelsatpH 9.0showed

two well-separated bands (Fig. 6), the faster moving with twice the mobility of the slower. Purified virus particles andtop components, re-spectively,comigrated with the bands migrating rapidly andslowly towards the anode; electron microscope observation of particles recovered from thebands detected inanatural mixture of virus andtop componentsconfirmed that virus particles migrated more rapidly. The rate of migrationwasdirectly proportional to the cur-rent applied. It also appeared that the propor-tion of topcomponent tovirus in partially

puri-fiedpreparationswas3:1 (as assessed by scan-ning negative photographs of Coomassie blue-stained gels), confirming earlier estimates (20). Thus, both virus andtop componentwere neg-atively charged at pH 9.0, the former having twice thenetcharge of the latter.

Dissociation of virus particles and top components of DNV1.Experiments dissociat-ing DNV1 particles and top components were carriedout togive information about forces sta-bilizing virus particles andtop componentsand toenablemeaningfultitrationstudiestobe car-riedout(seebelow). The virus proteinwas dis-sociated by incubating the material with an equal volume of twice-concentrateddenaturant at30°C for 24 h. Table 3 showsasummaryof

theeffects ofavariety ofdenaturing conditions onthe sedimentationproperties.

2.5% SDS, 2.5 M NaCl, and 3 M LiCl-4 M urea failed to break down DNV particles and top components and so were not suitable for preparing protein subunits from the virus.

Treatment with either 66% acetic acidor6 M guanidine hydrochloride-0.1 M 2-mercaptoeth-anolsuccessfullydissociatedtop componentsto

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low-molecular-weightcomponents(Table 3).

TABLE 2. Calculationofpercentageof each

polypeptideinDNVIand DNV2 andtheir

respectivetopcomponentsa

%ofprotein(±standarddeviation)in:

Pro- DNV1 DNV2

tein

Particles Top_ponentcom- Particles Top com-_ponent VP1 11.9 ± 0.2 13.6 ±0.1 15.2±0.2 11.4±0.2 VP2 28.9 0.3 28.5±0.3 22.7±0.3 27.6±0.3 VP3 22.1 ± 0.3 21.0±0.3 24.1±0.4 22.4±0.3 VP4 37.1 ± 0.3 26.9±0.5 38.2±0.4 38.6±0.4 'The proteins were separated by polyacrylamide gelelectrophoresis,detectedbyCoomassie blue stain-ing, and scannedonaJoyce-LoeblChromoscan with anintegratorattachment.

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A

B

,,

ii

III iii

RELATIVEMiWATICN

FIG. 5. DensitometrictracesofDNV2

125I-Iabeledpolypeptides

resolvedbyautoradiographyof10% SDS-polyacrylamidegels ofvirusparticles (A)and top components(B)iodinatedbychloramine-Tafterdisruption

byheatinginSDS(i),lactoperoxidaselabelingofintactvirus(ii),and chloramine-Tlabelingofintact virus (iii).Note thatthe three

high-molecular-weightpolypeptides

wereradiolabeled in intactvirus.

A

B C

FIG. 6. Polyacrylamide gel electrophoresis of

in-tactDNVIparticlesandtop componentson3% cylin-dricalgels. (A) Virusparticles; (B) top component andvirusparticles;(C)top component.

Ahigh pHhad a moremarkedeffect on virus

particlesthanontop components. Thetwo

en-tities (at =1mg/ml) weredialyzedinto 0.05 N

NaOHatpH 12.7or0.05NNaOH-1.0 M NaCl

atpH 12.3 at40Cfor48 h.The productswere

thenanalyzed byanalytical ultracentrifugation,

using Schlieren optics. Inlowsalt athigh pH,

thetopcomponent sedimentedas asymmetrical

peak with a sedimentation coefficient of 61S,

suggesting that it was

essentially

unchanged,

whereas the virus particles sedimentedasthree

peakswithsedimentation coefficientsof9,119,

and 122S. In 1.0 MNaClandhighpH, thetop

componentsedimentedastwocomponentswith

sedimentation coefficients of 2 and 47S (the

latter wasthemajor component),andthe virus

particle sedimented as a single peak of 12S.

Thus, thetop components werefairly stableto

alkalineconditionsupto

pH

12.5, whereas under

similarconditions ofhighpH and ionic strength,

virusparticleswereless stable thantop

compo-i I

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DNV STRUCTURAL PROTEINS 231

TABLE 3. Dissociation ofDNVparticlesbyvariousdenaturing solvents: effects onsedimentation

coefficienta

Sedimentation coefficient Approximates20,w

SolventTocop-Tpom

_{Top com}

_Vo-

_virus

_particle

_Top

_con-

_Virus

_particle

2.5%SDS, pH 7.0 50S 97S 50 97

2.5%NaCl,pH 7.0 29S 63S 51 102

3MLiCI-4 M urea, pH 7.0 16S 29S 56 90

0.05 NNaOH, pH 12.7 61S 9S, 119S,122S 61 9, 118, 122

0.05NNaOH-1 M NaCl, pH 12.3 2S, 47S 11S 2, 47 14

0.05 NHCl-1MNaCl, pH 1.45 48S 95S 59 112

0.05 NHCl,pH 1.45 61S 122S 61 122b

66%acetic acid 0.45 NDC 1 ND

6Mguanidinehydrochloride-0.1 M 0.95 ND 2 ND

mercaptoethanol

aThe sedimentationcoefficients of virus particles and top components in 0.01 M phosphate buffer-0.1 M

NaCl,pH7.0, are117and 59 respectively (8). b Trace amounts detected.

cND, Not done.

nents. Thestabilities of both virus particles and

top components at a particular high pH

de-creased with increasing ionic strength. The

sta-bility oftopcomponent wasthuslittle affected

by the titration of basic groups onthe surface,

whichcaused theparticletohave a netnegative

charge. It is

likely

thatsomeof the stability of

topcomponentarises fromstrong

hydrophobic

protein-protein interactions. The virus maybe

less stable thantop component at apHgreater

than12duetotheionization of the bases in the

DNA.

Samples

of virus

particles

andtopcomponent,

each

containing

1.2

mg/rnl,

were

dialyzed

at

40C

for 60 hinto 0.05 N HCl, pH 1.45, and0.05 N

HCl-1.0 M NaCl at pH 1.45. The samples of

virusparticles and the corresponding samples of

top component werethen sedimentedinpairs in

theanalytical ultracentrifuge.AtlowpH and in

high

salt,

virus

particles

andtopcomponentboth

sedimented as single

symmetrical

peaks with

sedimentation coefficients of 112 and 59S,

re-spectively. Littleornodissociationhadoccurred

in either virus particles or top component. At

lowpH and in low

salt,

topcomponentshowed

somedissociationto

slow-sedimenting material,

but the main component was still

apparently

intact top component, with a sedimentation

coefficient of 61S. In contrast,

only

a trace of

intact virus

particles

was present in the

corre-spondingvirus

particle

sample. Thus,top

com-ponent isstabletoacidic conditions down topH 1.45. Under similar conditions of low pH and ionicstrength,virusparticlewasnomorestable

and

probably

less stable than top component.

The stabilities of both virus particles and top

componentatalowpH decreasedwith

decreas-ing ionicstrength. The stability of top compo-nent waslittle affectedbythetitration of

side-chain carboxyl groups on the surface, which

caused the

particle

tohavea netpositive charge.

Again,someof thestability oftopcomponentis

probably

dueto

hydrophobic protein-protein

in-teractions. Virus

particles

might be lessstable

than top component atpH 1.45if the DNA was

partially

accessible to protons

and,

hence, if

some of the

negatively

charged

phosphate

groups onthe DNAwereneutralizedbyprotons.

ThepK of these

phosphate

groupsis about 1.0,

and thusthey arenot normally titrated.

How-ever, apH of1.45might be low

enough

forsome

of these

phosphate

groups to be neutralized.

Such neutralization

might

cause

disrupting

forces sufficient to overcome the

stabilizing

forcesand, hence, dissociation of virus

particles

under conditions in which top component is

stable.

Alternatively,

ionization ofsome of the

basesontheDNAcould make ita

destabilizing

influence.Theeffect of ionicstrengthagain

sug-gests that ionic forces are

partly

involved in

destabilizing

both virus

particles

and top com-ponent atthispH.

Continuous electrometric

titration.

Top

componentsafter exhaustive

dialysis

into 0.1 M

KCI hadapH of6.0.Itwasassumed that neither

K+ nor C1- ions bound

preferentially

to top components,andhencethis

pH

wastakentobe the

point

ofzero netproton

charge

for the pur-pose of

interpreting

the electrometric titration

curve. The titration curve of top components

obtained by

forward-titrating

samples

in 0.1 M

KCI with 0.1 NHCIand then

back-titrating

with 0.1NNaOHisshown in

Fig.

7.

Top

components

were

partially

precipitated

between

pH

4and6. Thetitrationcurves werereversiblebetween

pH

2.5and 6.0 and

partly

reversible,

butwith

time-dependent changes,in the

pH

range6.0 to 11.5.

The acid endpoint waswell defined at

pH 2.5,

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o 2 m

oOH

OH

m0

OH U+ O

U)

8

4

4

2 4 6 8 10

pH

FIG. 7. Forward electrometric titration ofDNVI

top componentsin 0.1 MKCI.

butthe alkaliendpoint hadnotbeenreachedby pH 11.5. Titration curvesofanequivalent vol-umeof 0.1MKC0 wereobtained, and the num-ber ofH+ ions bound by the proteinwasobtained in the usualway.

Apreliminary analysis of thegroupstitrated

was made as described by Tanford (32). Seven moles of H+ ions per 100 mol of amino acid residues were bound between pH 6.0 and 2.5. These may be assigned toside-chain carboxyl residues of aspartic acid and glutamic acid which have pK values of =4.0. The number of histidine, lysine, and arginine residues per 100 mol of aminoacid residues available for titrationabove pH 6.0 must therefore be seven, since at the isoionic pH (6.0)thenetnegativechargeequals thenet positive charge (32). Since 1 molofH+ ion per 100 mol of amino acid residues was dissociated betweenpH 6.0 and 7.3, presumably

from histidine, a total of 6 mol of lysine and

arginine residues per 100 mol of amino acid residueswas available fortitration. Amino acid analysis has shown that there is between 6 and 7mol oflysine and arginineper100 molof amino acidresidues inthe protein oftop components and virus particles (see Table 1 and reference 20); it islikely, therefore,thatallthese residues

arepositivelychargedatpH 6.0 andhence

avail-able for neutralizing negative charges on the DNA invirusparticles.

Between pH 7.3 and 11.5, between four and five H+ ions were dissociated per 100 mol of aminoacidresidues. The sidechains titrating in thisrangewouldhave been fromsome,butnot

necessarily all, of the cysteine, tyrosine, and lysine residues. The arginine residues wouldnot

beexpectedtotitratebelow pH12.Since only7 ofthe20aspartic acid andglutamic acid residues (20)per100molof aminoacid residues titrated,

13 of these residues in top component were

present in the amide form. Thus,all thelysine

and arginine residues in top componentare

ti-tratable andhencearepotentiallyavailable for

neutralizing negative charges on the DNA in virusparticles.

The forward titrationcurve of virusparticles

in 0.1 M KC0 is shown in Fig. 8. Since virus particles hadapH of 5.7 afterdialysis into0.1 MKCl,this wastaken asthepoint ofzeronet proton charge. From pH 2.5 to 5.5, virus was

partially precipitated. In contrast to top

com-ponents, the acid endpoint for virus particles wasnotreachedby pH 2.5. Between pH 3.0 and

5.7, 7 mol ofH+ ionper100mol of aminoacid

residues was dissociated. For top components

thecorresponding value between pH 2.5 and 6.0

was also 7 mol. Between pH 2.5 and 3.0, two

extra H+ ions were apparently dissociated per

100 mol of amino acid residues in the virus. Between pH 5.7 and3.0, therefore,thenumber oftitratablegroupsofvirusparticlesper100mol of amino acid residueswasthesame asthatof topcomponents, but below aboutpH 3.0 there

wereextratitratablegroups onvirusparticlesas

compared with top components. The alkali titra-tioncurveof viruswascarried outin mM KC1.

Fifteenorsixteen moles ofH+ ionper100 molof amino acid residues was dissociated for virus

particles between pH 5.7 and 11.5. The

corre-sponding value fortop componentswas 6 mol.

Clearly, thereareextratitratablegroupsinvirus particles as compared with top components in thispHrange.

Electrometric titrationswerecarried out un-der conditionswhich maintainedtop component

oUX

8uz

oQ.

OH

o H

Q

,o +

H

H

0

8

0

8

16

2 4 6 8 10

[image:9.510.69.261.49.234.2]

pH

FIG. 8. Forward electrometric titration of DNVI particles ( ), pH 2.5to5.7 in 1.0 MKCI and pH 5.7to11.5 in 0.001MKCI. Thedifference (---) is also shownbetweentheforward electrometric titrations of top components(Fig. 7) andvirusparticles.

I I

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[image:9.510.273.465.445.603.2]

and virus

particles

intact. Thiswas confirmed

by analyzing the titrated samples atboth high

andlowpHvalues inthe centrifuge. Inallcases

intact virus and top component wereobserved.

Therefore,anydifferencein the titrationcurves

betweentopcomponent and virus particles

can-not be due todissociation effects.

Thus, the electrometrictitrationexperiments

on top component (Fig. 7) and virus particles

(Fig.8)showed thatalargernumber ofhydrogen

ions per 100 mol of amino acid residues was

dissociated from intact virus than from intact

top componentinthe pHrange 2.5 to 11.0. The extratitratablegroupsof virus particles,as

com-pared with top component, weredissociated in

thepHrange 6.0 to 11.0andpossibly also inthe

pH range 2.5 to 3.0. No extra groups were

ti-trated from virus particles in the pH range 3.0

to6.0, ascompared withtop components.These

observations werenotduetothe differentionic

strength conditions used for the titrationsinthe

alkalinerange,since the effect ofincreasing ionic

strength would betodecrease the number ofH+

ions dissociatedathighpH (34).Thisis contrary

to the effect found. The difference in titration

curvefor virusparticles andtop component (see

Fig.8)suggested thatatpH greater than 6.0, the

twotypesof

particles

havedifferentcharges and

hence different electrophoretic mobilities. We

showedearlier that virusparticles hadamobility

about twice that oftop component atpH 9.0.

Themobility difference wasconsistent with

vi-rus

particles'

having a negative charge about

twicethat oftop componentatthispH. This is

confirmedby the titrationcurves.

The difference in titration behavior (Fig. 8)

for virus

particles

and top component may be

ascribed to titration of the DNA bases in the

virus. Thefour nucleotide bases ofDNAprovide

three titratable amino groups with pK'near 4

and two titratable

hydroxyl

groups with pK'

near 11. The fact that little or no virus DNA

titrated between pH 3.0 and 6.0 but that viral

DNAapparently titratedinthepHrange 6.0 to

11.0 means thatthe titrationcurve for the nu-cleic acid invirusparticlesisshiftedto a lower pH range compared with DNA in

moderate-ionic-strength solutionsand issimilartothatof

denatured, and hence essentially

single-stranded, DNA under conditions ofvery high

ionicstrength (4). Thus, the DNA in virus par-ticles appears from its titration curve to be in an environment equivalent to a very high ionic

strength,where the netchargeonthephosphate

groups isalmostcompletely screened, aswould

be the case if the phosphates were

efficiently

neutralized

bycounterions.

The nearequivalenceof the isoionicpHof the virus and topcomponent leadstotheimportant

conclusion that the net charge on the protein

doesnot change when it is combined with the

nucleic acid. This means that the negatively

charged phosphate

groups

(pK

1)

ontheDNA

must be balanced by an equivalent number of

positivecounterionsin thevirus particlewhich

do not come from the viral protein. In other words, the majority, if not all, of the positive

chargeson the viralproteinare notinvolvedin

neutralizing DNA phosphates. These must be

neutralized, therefore, by small counterions

present within the virus particle. The

electro-static interaction between chargesontheprotein

and DNAappears tobeminiimal.

Acomparison of the titration curvesof virus

andtopcomponentshows that theprotein

side-chain

carboxyl

groupshaveverysimilar pK

val-uesin bothcomplexes. They areprobably

situ-atedonthe surface of theprotein capsid andare

probably responsible for the solubility of the

virus in aqueous solution. Similar conclusions

mayalso be drawnconcerningthearginine,

his-tidine, and lysine side chains. These, too, are

freely accessible to H+ ionsin both complexes

and may therefore be situated on the outer

surface of theprotein capsid.

Circular dichroism studiesonDNV2

par-ticles andtop components. The circular

di-chroicspectraofDNV1, DNV2, and the

respec-tivetopcomponents intherange 230to320nm

have already been reported (11). The circular

dichroicspectra inthe195- to250-nm

region

of

virusparticles andtop components areshown in Fig.9. The spectra were

significantly

different.

Therewere decreases in negative dichroismof

the top component between 230 and 215 nm

when theproteinwasincorporated into the virus

capsid

and increasesintherange 215to198nm

which might at first sight be

interpreted

as

changesin the secondary structure of the

pro-tein.However,itis also

possible

that the

changes

arosefrom the alteredenvironment of side-chain

aromatic chromophores, such as

tyrosine

or

tryptophan, which might have interacted with

the DNA in the virus, or fromaltered

confor-mation of thebases inthe DNAitself.Itisnot

possible to distinguish between these various

possibilitiesonthe basis ofthese spectraalone.

It can be concluded, however, that there is a

definite interactionbetweenviralDNAand

pro-tein whichgives risetoalteredoptical properties.

DISCUSSION

DNV1and DNV2 are

similar,

butnot

identi-cal,virusesas

judged by

biophysical,

biochemi-cal, and

serological properties

of the

protein

coat. This basic conclusion

complements

the demonstration that the viruses possess

closely

homologousDNAsofsimilarstructure

(13).

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Cl)

l,

rz

075 .

190 200 210 220 230 240 250 WAVELENGrH (NM)

FIG. 9. Circular dichroism spectra at room tem-peratureof DNV2 virus particles(-) andtop com-ponents( ).

Thetwoviruses show typical parvovirus

mor-phologyand size(9),althoughnodetailed

struc-ture could be determined. We were unable to confinn thededuction of Kurstak and Cot6 (18) that thevirus contains 42capsomeres.

Thepolypeptide compositions ofagiven DNV and itscorresponding top componentare iden-tical, and both containatleastfourpolypeptides.

The sum (265,000) ofthe molecularweights of

thepolypeptidesexceedsthecodingcapacity of

agenomewithnooverlappingorsplicedgenes the molecular weight of the DNV genome is

approximately 1.95 x 106, and so itpotentially

codes for about 2 x 105daltonsofprotein.

Pre-liminary peptide mapping experiments show

that thepolypeptides ofDNVsareinterrelated (N. F. Moore and D. C.Kelly, unpublished data).

Calculations show that the relative amountsof the various polypeptides are not easily accom-modatedinvirusparticles, assuming that parti-cles containing plus and minus strands have identical polypeptide compositions. The virus contains about 60 polypeptide molecules, of which about half are the major polypeptide

(VP4).The relativeamountsofeachpolypeptide

present differ considerably from the amounts calculatedby Tijssen etal. (33). This probably reflects theirunconventional method of calcu-lation. Under conditions which resolve three

polypeptidesforadeno-associatedvirusparticles

(30), four DNVpolypeptideswereresolved,and so itappearsthat thesevirusesareindeed dif-ferent despite similarities in the finestructures oftheirDNAs.Iodinationexperiments with both

J.

lactoperoxidaseandchloramine-T showedthat

only

the two high-molecular-weight

polypep-tides anda minor polypeptide were accessible

foriodination.Thisobservationagaincontrasts

with observations on the iodination of

adeno-associatedvirustype3capsid protein (22),where

allthreeadeno-associatedviruspolypeptidesare

iodinated.

However,

theobservationthat

chlor-amine-T failstoiodinatethe two

lower-molecu-lar-weight

polypeptides does not necessarily

mean that these are "core" polypeptides. It couldindicatemerelythat in the native

protein

state,theirtyrosine residuesareinaccessibledue

to tertiary folding of the polypeptide chain.

Omission ofa reducingagentfrom the sample

buffer failedto alter the polypeptide profileof

DNA polypeptides, confirmingthe observation

of

Tijssen

et al. (33), whichdemonstrates that

disulfide bondsarenotinvolvedin thecreation

of theremarkablystable coat of these viruses. Gel

electrophoresis

ofintact virusparticlesat

pH9.0 showedthat, in the case ofDNV1 (but

not DNV2, which failed to enter the gel), the

virusparticlescould beseparated fromtop

com-ponentandindicated that the virusparticleshad

a

higher

net negative charge than did the top

component; this isprobably attributableto the

negative chargeoftheDNAin thevirusarising

fromtitrationof theDNAbasesatpH9.0.

The conclusionthat most of theDNA

phos-phateisneutralized by cations other thanbasic

amino acids issupported by ourearlier obser-vation that the virusparticles,but not the top

component, are rich in polyamines, having a

sufficient amounttoneutralize 26% of the DNA

(15).

Thebiochemical,biophysical, and serological

tests described so far in this discussion have

failed to show that there is any difference

be-tweenthe top component

protein

and the

pro-teincoatin situ in the virusparticle foragiven

virus. Althoughwehavepresentedindirect evi-dence that there is little direct interaction be-tweenthe viralgenome and itsprotein,

never-theless it is probable that interaction occurs,

albeiton alimitedscale, and thattheinteraction

hasacertain

specificity.

Todetermine whether

aconformational differenceoccurredin thecoat

whenpresent in topcomponentorin part ofthe

virus particle, circular dichroism studies were

performed, and differencesweredetected.

Anal-ysis of theresultsbystandardmethodsfailedto

demonstrate that the virusproteinexistedina

predominant

secondary

structuralform (similar

observations have been made for other

single-stranded RNA viruses[11, 28,31].Interactionof

single-stranded nucleic acid with the protein

induced amarked change in optical properties

of theprotein,nucleicacid,orboth.

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[image:11.510.84.233.52.280.2]

235

Theanalyses of the titration data suggest that

theelectrostatic interactions between DNA and

protein in the virus are minimal. The negative

charge on the DNA appears to be completely

screened by small counterions, such as

polya-mines.Althoughnonpolar interaction cannot be

excluded, and indeed thedifferences in the

cir-cular dichroic spectra between top component

and virus would support such interaction, the

picture that emerges is one where the DNA is

physically

contained within the protein, with

little

direct interaction between them.

ACKNOWLEDGMENTS

Wethank C.F.Rivers,M. H.Bew, M. D. Ayres, M. K. Arnold,andT. Lescottfor technicalasaistanceand R. H. Pain and R.Burgessfor use of theirDichrographe HI.

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