Attachment of avidin-coupled spheres to linear and circular forms of mengovirus double-stranded RNA.

0022-538X/81/070229-09$02.00/0 Vol. 39, No. 1

Attachment of Avidin-Coupled Spheres to Linear and Circular

Forms of Mengovirus Double-Stranded RNA

GEORGE B.THORNTON,' DONALD L.ROBBERSON,' AND RALPH B.ARLINGHAUS3* Department of Biology, Abilene Christian University, Abilene, Texas79699,1 and Department ofMolecular

Biology'

andDepartment of TumorVirology,3 The University of Texas System Cancer Center M. D.

Anderson Hospital and TumorInstitute, Houston, Texas 77030

Received23January 1981/Accepted1 April1981

Picomavirus-infected cells contain a double-stranded RNA (replicative forn

[RF] RNA), composed of viral genomic RNA hydrogen bonded to cRNA of

similarnucleotide length.Mengovirus RFRNA reacted with a succinimide ester

ofbiotinwasshownby electronmicroscopy to bind avidin-coupled

polymethac-rylate spheres. These binding sites are taken to indicate the presence of VPg

protein molecules by methodspreviouslyappliedto poliovirus RF RNA (Richards

etal., Proc. Natl. Acad.Sci. U.S.A. 76:676-680, 1979). One sphere was bound at

or very near one terminus of linear RF molecules while a second sphere was

boundat asitewhich was 1 to 4% of thegenome length from the other terminus.

Assignment of VPgpositions was limited bythe physicaldimension (ca. 60-nm

diameter) of the heavy metal-contrasted sphere observedbyelectronmicroscopy.

Athirdsite of sphere binding was detected at a lower frequency of occurrence at

asite whichwas 10 to20% of thegenomelengthfromone orthe other terminus.

Circular RF RNA molecules were also detected with two spheres attached at

juxtaposed sites. The termini of the linear RF RNA were located within the

circular structuresby sphere attachmentat sites whichwere very close to and

obscureda

nucleic

acid projection whichwehave described to occur on circular

mengovirus RF RNA (D. L. Robberson, M. V. Marshall, G. B. Thornton, and R.

B.Arlinghaus, manuscript submitted forpublication). CsCl gradient fractions of

RNA reacted with

avidin-coupled

sphereswere

highly

enriched incircular

struc-tures.

The

genomic

RNAof

picornaviruses

contains

asmall basicprotein

covalently

attachedto its

5' terminus(6-8, 10,14,16, 19,20).Thisprotein,

VPg, isalso attachedtothe 5'ends of

intracel-lular forms of viral RNA involvedinviral RNA

replication. Double-stranded

poliovirus

RNA

(replicative

form [RF] RNA) contains VPg at

the 5' end of the

plus

and minus strands(6, 13).

Onewaytodetect

protein

boundtosuch

struc-turesis

by

useof

electron-opaque

avidin-coupled

spheres (16). In such

experiments,

biotin is

chemically

linked to the

VPg-RNA

complex.

The high affinity of avidin for biotin is then

utilized in the attachment of the

electron-opaque avidin-coupled spheres to the protein bound to viral RNA.Richards etal. (16) showed

that avidin-coupled spheres were attached to

theplusstrand atonlyoneend ofpoliovirus RF thathadbeen reacted with biotin.

Ourstudies withmengovirusRF have shown

that purified preparations of RF contain both

circular and linear forms of viral double-stranded RNA (M. V. Marshall, R. B.

Arling-haus, and D. Robberson, J. Cell Biol. 70:248a,

1976;Robbersonetal., manuscript submittedfor

publication).

The circulardouble-strandedviral

RNAs are of genomic

length,

contain a short

projection, andarenoncovalent associations of

the linearforms. Similar circularstructures were

firstreported forRF RNAof

encephalomyocar-ditis virus

by Agol

andhiscolleagues (1,

2)

and

subsequently shown to be noncovalent

struc-tures (18). In this

study,

the

avidin-coupled

sphere

technique

(11)

(previously applied

to

poliovirusRNA) was usedtodemonstrate that

both ends of biotin-linked

mengovirus

RF can

bind

avidin-coupled spheres,

indicating

the pres-ence of

VPg-like

molecules at or near the

ter-mini,and toidentifythe locationof the termini

of linear RF within the circularstructures.

MATERIALS AND METHODS

Cells and virus. BHK-21clone 13 cellsweregrown andpropagatedinroller bottlecultures,asdescribed

(24). Mengovirus was plaque purified by

Royce

Z. Lockhart, Jr. (Du Pont Laboratories,

Wilmington,

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230 THORNTON, ROBBERSON, AND ARLINGHAUS

Del.); theinitial stock of viruswasobtained from his laboratory. Inatypicalexperiment,tworollerbottles, having attained75 to90%confluencyof5x 108cells, wereinfected with 10 to50PFUpercell. Fresh me-dium containing 5,ug ofactinomycin D per ml was added2.5h afterinfection,and theisotope (1 mCi-of

[3H]uridine in25ml ofmedium)wasaddedat3h after infection. At5 to 6hafterinfection,the virus-infected cells were harvested by scraping and collected by

centrifugation.

Isolation andpurificationofmengovirusRF. Virus-infectedcellswereresuspended inahypotonic

buffer solution(0.02 M Tris[pH7.5],0.0036MCaCl2, and0.0014MMgCl2)andallowedtoswellfor5minat

00C.Thecellswerethen mixed withanequalvolume of hypertonic buffer (1.0% Triton X-100, 0.0036 M CaCl2, 0.01 MMgCl2, 0.5 M sucrose,0.25MKCI,0.04 MTris, pH 7.5)and homogenized withaloose-fitting pestle10times. Thebroken cellsuspensionwas cen-trifugedat5,000 rpmin aSorvall SS-34rotorfor 10 min, and the supernatant fluid wasdecanted. Cyto-plasmic RNAwasextracted from thesupernatant fluid by sodiumdodecyl sulfate(SDS)-chloroform-phenol,

asdescribed(15). The RNAwasmade0.2M inlithium acetate(pH 5.1) andprecipitatedbytheaddition of2 volumes of ethanolat-20°Covernight.

Mengovirus RFwasseparated fromsingle-stranded

virus-specific and other cellular RNAs based onits differential solubilityinhigh-saltsolutions (3, 4, 22). Thecytoplasmic RNAwasdissolved in1mlof TLE (10 mMTris-hydrochloride [pH7.5], 0.10MLiCl, 1 mM EDTA), andanequal volume of4MLiClwas addedtothat solution. Theresulting2MLiClsolution wasstoredovernightat4°C.Theprecipitate, contain-ingsingle-stranded RNAs, wasremovedby centrifu-gationat3,000xgfor45min. The2MLiCl-soluble

supernatant fluid,containing thedouble-stranded RF, DNA, and small RNAs,wasmade 1 M inLiClby the addition ofanequalamountof sterile deionizedwater and ethanol precipitated overnight at -20°C. The ethanol precipitate wassuspended in TLE-0.5% so-diumdodecylsulfate, made 2 MinLiCl, and kept at 4°C overnight. The precipitate, if any,wasremoved bycentrifugation,and thesoluble RNA was precipi-tated withethanol and suspended once again in TLE thatwasthen made2M inLiCl. After the third LiCl precipitation, the sample was ethanol precipitated and

suspendedinTLE-0.1% SDS and stored at -70°C. The2MLiCl-soluble RNA (in 0.5 ml of TLE-0.1%

SDS)wasmixed withanequal volume of 5% sucrose-NLE-SDS (10 mM sodium acetate [pH5.5], 100 mM LiCl, 1mMEDTA, and 0.1% SDS) and layered onto a5 to25%sucrose-NLE-SDS gradient. Water (3.5 ml) waslayeredontop of the sample layer and the gradient was placed in a vertical rotor (TVA-865; Du Pont,

Wilmington,Del.) and centrifuged for 75 min at 50,000 rpmat40C.The gradient was collected in 1-ml frac-tionsandassayed forradioactivity, and the peak frac-tions, which sedimented at 20S, were collected and precipitated by ethanol.

DNase Itreatmentof RF.RFobtainedby velocity sedimentation centrifugation asdescribed above was suspended in 0.5mlof NTM (100 mM NaCl, 5 mM

MgCl2, and 10 mM Tris[pH7.8]). DNaseI,obtained from Calbiochem-Hoescht, Behring, La Jolla, Calif.,

was added to a final concentration of50 U/ml and incubated for 30to60minat250C. The reactionwas terminatedby the addition of 50,ul of 0.1 M EDTA (pH 7.0). An equal volume of 5% sucrose-NLE-SDS wasaddedtothe terminated reactionmixture, and the resulting solutionwaslayeredonto a5to25% sucrose-NLE-SDS gradient and subjected to velocity sedi-mentation in a vertical rotor for 75 minat50,000 rpm (4°C),asdescribed above. Thegradientwas fraction-ated, and the peak fractions, which sedimented at20S, werepooled andprecipitated with ethanol.

Bondingofavidin-coupledspherestoRF. Av-idin-coupled sphereswereboundtoRF moleculesas describedby Richardsetal.(16). Briefly, RF prepared asdescribedwassuspendedin0.1Msodium bicarbon-ate toyieldafinal concentration of 25,ug/ml. A

100-pl

sampleof the RFsolutionwasmixed with 10,lIof dimethyl formamidecontaining 100Mug of the

N-hy-droxysuccinimidylesterof biotin(kindly provided by JerryManning, University ofCalifornia, Irvine) and incubated at 4°C for 3 h. This mixture was then

dialyzedovernight against 1 MNaCl-0.1 M HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid) (pH 7.5). To the dialysatewasadded30,ul of the

avidin-coupled spheres (20mg/ml), and the suspen-sionwasincubatedat40C for24h. Thepreparationof avidin-coupled spheres boundtoRFwasthenlayered onto aCsCl stepgradient (2.45 ml of CsCl in0.1 M HEPES [pH7.5],p = 1.52; 2.45ml ofCsCl in0.1 M HEPES[pH7.5],p =1.23) andsubjectedto centrifu-gation inanSW50.1rotor at45,000 rpmfor22 hat

40C.Theavidin-coupled spheresandspheres bound tothe RFmolecules formed an opalescentband ap-proximately one-third of the distance from the top of thegradient. Theopalescent band contained 77% of theuridine-labeled RFRNA; theremaining 23%was found in the pellet. The band wasremoved with a syringe, dialyzedagainstTNEbuffer (10 mM Tris[pH

7], 100mMNaCl, and1 mMEDTA), and examined byelectronmicroscopy.

Electronmicroscopy.Samplesfrom the CsCl gra-dient fractions containing RF reacted with poly-methacrylate sphereswerediluted in0.1MNaCl-0.1 MTris[pH8.0]-0.01 M EDTAtoafinal RNA con-centration of 0.5 Mug/ml and prepared for electron microscopy by usingthe aqueous basic protein

tech-nique of Kleinschmidt as described previously by Davisetal.(5). Gridswerestained with uranyl acetate asdescribed(17) and rotary shadowed with Pt:Pd (80: 20) after which theywereexamined in a Philips 300 electron microscope. Photographswere taken on 35-mmflrm,andlengths of projectedimagesof the mol-eculesweredetermined withaNumonics 1224 digitizer interfaced withaHewlett-Packard 9825A computer.

RESULTS

Purification ofmengovirus RF.

Mengovi-rusdouble-stranded RNA, termed RF, was

iso-latedfrom infected BHK-21 clone 13 cells and

purified essentially as described previously by

Spector

andBaltimore (22).Theisolationof the RF is basedonitssolubility in2MLiCl.

Men-govirus RF, subjected to three LiCl

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andthelatterwas8entrifugedinaparallelgradient.(B)The peak(A)was

d+

DNase

I

centrifuge0 on

ti

TREATMENT

(LIt

i 0

4 It II

10 I

I

6-0

4~~~~14

0

0

20

0 10 20

0o

10 20 30

FRACTION NUMBER

FIG. 1. Sucrose density gradient centrifugation of mengovirus-specific RF RNA. (A) ViralspecificRNA waslabeled withf8HJuridinein thepresenceofactinomycinD,and 2 MLiCi-soluble RNAwasappliedtoa

5to25%linear-sucrosegradient. Theviral RNAtracing(@)wascoplottedwithacellular RNAmarker(0),

and the latterwascentrifuigedinaparallel gradient. (B) The208peak(A)waspooled,DNaseI-treated, and

centrifugedon a5to25% linearsucrosegradient.

tions, sedimented as a

homogenous

peak at a

rateof 20S

(Fig.

1A). Treatment of

RF,

which

had been isolated fromasucrosegradient, with

DNase I hadnoeffectonitssedimentationrate

(Fig.

1B).

DNase-treated mengovirus RF molecules

were isolatedasdescribed above and examined

by

electron

microscopy.

Several formswere

ob-served

(Fig. 2):

(i)linear RF molecules

(Fig. 2A,

B, and

C), (ii)

circular molecules

possessing

a

shortprojection

(Fig.

2A,

arrow),

and

(iii)

linear

molecules with their ends

opposed

(Fig. 2B,

ar-row, and

C,

upper

part).

Although

some

mole-cules smaller than

genomic

length

werepresent

(Fig.

2C, arrows),

the

preparation

of RF

ap-peared to be rather

homogeneous

in

length

in

each of the three

forns.

Before

sphere

attach-ment and

CsCl

gradient

fractionation

(see

be-low),

the RF

preparation

contained 7.1% circular

duplexes

witha

projection,

6.0% linear

duplexes

with

opposed

ends,

and

only

1.0%circular

du-plexes withoutadetectable

projection;

atotal of

298moleculeswasexamined

by

electron

micros-copy.

Binding

of

avidin-coupled spheres

toRF

molecules. The

genome-linked

protein,

VPg,

has been found to be

covalently

linked to the

virus-specific RNAs of

poliovirus (6,

7, 10, 16,

25), the genomicRNAs of foot-and-mouth

dis-easevirus(9,20),

encephalomyocarditis

virus

(8,

10), mengovirus

(14),

and calicivirus

(21).

In

these

studies,

bothbiochemical andelectron

mi-croscopictechniqueshave beenemployed. Using

the avidin-coupled sphere method ofRichards

etal. (16), weinitiallysought to locate VPgon

mengoviruslinearRFmoleculesandcircular RF

molecules. Since VPg is expectedtobe found at

or near the termini, its presence would then allowustoidentifytheterminiof themolecules

in thecircularforms,a majorobjectivein these

studies.

An excess of theN-hydroxysuccinimidylester

ofbiotin was reacted with mengovirus RF by

meansoftheprotein VPg. Themengovirus

RF-VPg-biotin complex was thenboundto

avidin-coupledpolymethacrylate spheres,asdescribed

above.Electron microscopic examinationofthe

RF-bound spheres revealed several classes of

linear molecules: (i) full length RF molecules

possessing spheresonbothtermini (Fig. 3A and

B), (ii)molecules possessingasinglesphere on

oneendandmultiple spheresontheother(Fig.

3C), (iii) molecules possessing multiple spheres

on both ends (Fig. 3D), (iv) molecules ofless

than genomiclength possessingasingle sphere

(Fig. 3E), and (v) infrequently occurring

mole-culespossessing intemallyboundspheres (Fig.

3F).Itshouldbeemphasized that when multiple

sphere attachment was involved, only one

sphere of theaggregate wasfoundtobein direct

physical contact with the RF RNA molecule.

Furthernore, in linear RF molecules, it was

frequently observed thataspherewasattached

at oneterminuswhileasecondsphere attached

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232 THORNTON, ROBBERSON, AND ARLINGHAUS

FIG. 2. ElectronmicrographsofmengovirusRFRNA. Both linear and circular RF RNA moleculeswere detected. The circular molecules typically contained ashortprojection (indicated by the arrow inA). In

addition, linearmolecules withopposedendswerealsodetected(indicated bythearrowinB;upperpart

of

C).Double-strandedmolecules much shorter than genomelengthareindicatedbythearrowsin C.Bar, 1,m.

verynearbutnotpreciselyatthe other terminus, i.e.,oneterminuswasseentoextendbeyond the site of sphere attachment (Fig. 3A and F,

ar-rows). This terminus extends approximately

1.0% of thegenomelengthbeyond the boundary of the sphere. Furthermore, a second site of

sphere attachmentappearedatadistance of10 to 20% of the genome length from one of the

termini(Fig. 3Datright and Fig. 3F) in

approx-imately 6% of the linear duplexes and13% of the

circular duplexes with one or more attached

spheres, which may reflect the presence ofan

additionalVPg-like moleculeor someother

pro-teinboundtotheRF RNA.

Severalunusualfeaturesrequirefurther com-ment.Therewere numerousunattachedas well

asattachedspheres which possessedsmall

pro-tuberancesthatweredistinguishedfrom the

pro-jections on circular structures. A small

protu-berance is evident on the single sphere in the

lowerrightcornerof Fig.3C.Thesourceof these

protuberances is unknown, but it should be

noted thatvery smallRNAs havebeenobserved in

preparations

of

mengovirus

andmayaccount

forthese

protuberances

(manuscript

submitted

for

publication).

These

protuberances

werenot

observedin the

poliovirus

studies described

by

Richards et al.

(J.

Manning,

personal

commu-nication). A second unusual

phenomenon

was

the

clumping

of

avidin-coupled spheres.

The

clumps of

avidin-coupled

spheres

were

rarely

foundexcept when boundto

mengovirus

RF. A

random sampling of 212 spheres in the

back-groundof theCsCl

gradient

fraction

containing

RFrevealedthat 73%occurredas

single

nonag-gregated spheres, 17% as aggregates of two

spheres, 6% as aggregatesofthree

spheres,

2%

asaggregates of four spheres,and 2% as aggre-gates of five spheres. The significance of the

clumps ofspheres boundtooneend of the RF

remainstobe determined.However, the

clump-ing of spherical aggregates of antibodies was

reported by Wuet al. (25) in antibody-treated

2,4-dinitrophenol derivatives of

poliovirus

RF

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VOL. 39,1981

(seebelow).

Having demonstrated thatbothtermini,

bear-ing VPg, could be labeledwith the

avidin-cou-pled spheres, itwasofinterest todeterminethe

location of thetermini in circular forms of

men-govirus RF. As mentioned above, mengovirus

circular RF molecules possessed a single short

projection per circle. In 73% of the molecules

scored (sphere-bound circles)theprojection was

obscured. The termini of the RF

molecules,

therefore, are at or in close proximity to the

projection.Figure 4A presents a typical circular

RFmolecule withtwospheresattached in close

proximity but lying on the circular contour of

MENGOVIRUS RF RNA 233

the RFmolecule. Most circularformshad two

spheres attached (Fig. 4A and B). There were

also circular formspossessing a single attached

sphere (Fig. 4D and E), multiple attached

spheres (Fig. 4E), and

internally

boundspheres

(Fig. 4F).Inthose circular structures possessing

twoor more spheres which indicated the

posi-tions of the termini of linear RF within the

circularstructures,itwasfound that the termini

were physicallyseparated by only a small

un-measurable distance on the circular contour

(Fig. 4A, B, C,and F). This would indicate that

in the circularforms of RF, the VPg molecules

at the termini of the linear molecules are not

FIG. 3. Electronmicrographs ofmengoviruslinear RF RNA molecules with attachedpolymethacrylate spheres. (AandB)A single sphereis attachedneareach terminusof the linear molecules. Thearrow (A) depictsoneterminus whichprotrudes slightly beyondtheboundary ofthesphere. (C) An aggregate of four spheres is attached to one terminus and a single sphere is attached to the otherterminus of the linear molecule.(D) Twospheresareattachedatoneterminusandanaggregateof six spheres is attachednearbut not atthe otherterminus,whereasingle sphereis attached. (E)Asphereis attached atonlyoneterminus. (F) Onesphere isattachedneareach terminusaswellas asinglesite locatedonthe linear molecule. The arrow(F)depictstheterminusofthe molecule whichprotrudesbeyondtheboundary of the sphere. Bar,1,tm.

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234 THORNTON, ROBBERSON, AND ARLINGHAUS

FIG.4. Electronmicrographs of mengoviruscircular RF RNA molecules with attachedelectron-opaque polymethacrylate spheres. Samples wereprepared for electron microscopy by the aqueous technique of Kleinschmidtdescribedpreviously byDavisetal.(5)and contrastedbyrotaryshadowingwith Pt:Pd.(Aand B) Two spheres are attached at apparently adjacent sites indicated by arrows on each ofthe circular molecules. (C)Twospheresjoinedin tandem appearattachedat asinglesiteonthe circular molecules. (D

andE[atleft])A single sphereis attachedtothe circular molecules. (E [at right])Anaggregateofseven

spheres is attachedat asinglesiteofthe circular molecule. (F)An aggregateof four spheresis attachedat onesite, andasingle sphereisattachedat asecond siteonthesamecircularmolecule.Bar, I,um.

juxtaposedbutdo lieinclose proximity toone

another.

Linearandcircular dimersofmengovirusRF RNAhave alsobeendetectedatalow frequency

ofoccurrence (D. Robberson, M. Marshall, G.

Thornton, and R. Arlinghaus, manuscript

sub-mitted for publication). These species also re-actedwithavidin-coupled spherestogive struc-tures presented in Fig. 5. The circular dimers frequently containedtwoor moresites of sphere

attachment (Fig. 5A), whereas linear diners

most frequently were seen to contain sites of

sphere attachment ator nearthe termini (Fig.

5C)aswellasatasiteinternally locatedonthe molecule (Fig. 5D). A circular monomer was

occasionally seenattachedtothe terminus ofa

linear moleculethroughone ormore

avidin-cou-pledspheres(Fig.5B). In such cases, the

projec-tionon thecircular RFmoleculewasobscured

bysphere attachment and, byinference, must

belocatednearthesite ofsphere attachment.

Table 1summarizes thefrequenciesof

differ-ent structures observed in the avidin-coupled

sphere boundRFpreparation.Ofsignificanceis

the fact that 63% of the linear RF molecules

possessed spheres on both ends, indicating at

least oneVPgmolecule is at or neareach

ter-minus.However,98% of the linear RFmolecules

had one or more spheres attached. Of the

cir-cular forms, 98% of the molecules contained

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MENGOVIRUS RF RNA 235

FIG. 5. Electron micrographs of mengovirus circular and linear oligomeric RF RNA molecules with attachedpolymethacrylate spheres. (A)The circular dimerformhastwospheres attached near the same site

(indicatedby arrows)andasingle sphereattachedat aremotesite. A linearmonomerwithasphere at each terminus is shownatthetopofthemicrograph. (B)Acircularmonomerisjoinedto alinear monomerthrough anaggregateof fourspheres in theregion indicated byarrow. (C) A linear dimer has one sphere attached near oneterminus andtwospheres, close to each other, attached near the other terminus. (D) One sphere is attachedateach terminus andonesphere is attachedat aninternal site indicated by thearrow on the linear dimer molecule.Bar,1

tum.

spheres, 43% contained one sphere, and 50%

containedtwospheres.Itshould beemphasized

that

sphere

attachmentnearly always occurred

at asite thatobscured the projection. Also, the

frequency

of circular forms in these preparations

was

significantly

higher than in preparationsnot

reacted with biotin-bound avidin-coupled

spheres. One possible explanation for this

occur-rence is that the reaction withthe

avidin-cou-pled spheresmayhave inducedagreater

stabil-ityin circular forms, preventing their

dissocia-tionuponspreadingfor theelectronmicroscopy.

DISCUSSION

We have been ableto demonstratethe

pres-enceofprotein, presumably VPg, onboth

ter-mini ofmengovirus RF by the avidin-coupled

sphere technique of Manning et al. (11).

Pre-vious studieswith this method ofpoliovirusRF

(16) revealedapreferentiallabelingofonlyone

end of the RFmoleculewith theavidin-coupled

spheres, the endpossessing the 5' terminus of

thepositivestrand. Wu etal. (25),

however, by

using adifferent method

(that

of

reacting

anti-2,4-dinitrophenol antibodies with the

2,4-dini-trophenol-derivatized VPg of RF molecules),

wereabletolabel both ends of

poliovirus

RF. It

was suggested by Richardset al. (16) that the

inabilitytolabel the 5' end of the negative strand

of the poliovirus RF might be due to one of

several

factors,

themost

probable

of whichwere

(i) that thepolyadenylic acidsequence onthe 3'

end of the positive strand is longer than the

polyuridylic acid sequenceon the 5'endofthe

negativestrand, whichmight maskVPgatthe

5' end of thenegativestrand, preventing

esteri-ficationwithbiotin,and (ii) that there are two

different VPg

molecules,

one capable of

being

esterified tobiotinmore

readily.

Both of these

possibilities may, in

fact,

be

responsible

for

at-tachment of onlya single sphere topoliovirus

RF. Infact, the studies described in thispaper

wereinitiallyundertaken,inpart, because

men-govirus has been shown topossess a short

se-quence of

polyadenylic

acid on its 3' terminus (12,23).Withthis in

mind,

wefelt thatwecould label bothtermini of the

mengovirus

RF without

havingtocontend witha

long

polyadenylic

acid

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236 THORNTON, ROBBERSON, AND ARLINGHAUS

TABLE 1. Frequencies of mengovirus RFRNAs with attachedavidin-coupledpolynethacrylate

spheresa

No.of %Of

to-RF RNA mole-

-cules

Genomic-lengthmolecules

Linearforms 43 45

Circular forms 44 46

Opposedends 8 9

Genomic-lengthlinears

Nospheres 1 2

Sphereson oneendonly 15 35 Spheres on both ends 27 63

Genomic-lengthcircles

One sphere,projection absent 19 43 Twoadjacent spheres, 13 30

projection absent

Two adjacent spheres, 9 20 projection absent (loops)

Spherespresentonprojection 2 5

Nospheres 1 2

aThe frequencies of

internal

sphere attachment mentioned in the text have been omitted from this tabulation.

sequence similar to that found in poliovirus

RNA. Asreported here, 65% of the mengovirus

RF molecules werelabeled atboth

termini,

in

contrast tothe labeling of 13% of the RF

mole-cules ofpoliovirus (16). Althoughwewereable

tolabel both endsofapicornaviralRF

possess-ingashortpolyadenylicsequence,itcannotbe

ruledoutthat therearechemicallydifferent VPg

moleculespossessing different labeling affinities

(see

below).

In

fact,

Kingetal. (9) haverecently

demonstrated that thereare twochemically

dif-ferent VPg's in foot-and-mouth disease virus

RNApreparations.

Wu et al. (25) have described a class of RF

molecules possessingaproteinaggregate on one

end of poliovirus RF and have termed these

complex molecules.Mostoftheseprotein

aggre-gates could be removed by banding the RF in CsCl-guanidine hydrochloride gradients. These

additionalproteinmolecules were not,therefore,

covalentlybound.

We report here the occurrence ofcomplexRF

moleculesin preparations of mengovirusRF and

note that there appears to be a preferential

labeling of one or the other end of these

mole-cules with two or more spheres; however, the observation thatonly onesphereof anaggregate

lies in contact with the RF molecule suggests

thatonlyoneVPg molecule hasreactedwith a

sphereoraggregateofspheres at this site.Other

VPg molecules may infact be nearby but are

notdetectedas aresult of physicalexclusion by

the sizes ofspheresthat were utilized. Whether or not the VPg or protein molecules on both ends of the RF are the same or different remains

to be determined. It is also possible that one

typeofVPg molecule,orprotein, has agreater

affinityfor one end of the RF molecule than the

other.

A second major objective of this work was

locating the termini of linear RF molecules

within the mengovirus circular structures

pre-viously described (Marshall etal.,J. Cell Biol.

70:284a, 1976) (Fig. 2).We havepresented

evi-dence that the sphereswhich are boundto the

circularstructuresobscure a smallprojectionwe

have previously observed to occur on circular

molecules(Marshalletal.,J. Cell. Biol.70:284a,

1976). The observation that the spheres are

joinedtothe circularRF moleculesat two

dis-tinct butclosely separated pointsand that most circles possess two or more suchspheres lying

sideby sidesuggeststhe close relative position

of theVPg's but excludes the

possibility

that the

VPgmolecules are

juxtaposed

withinmost

cir-cularstructures.

Another observation which must be

ac-counted for is the increasedfrequencyof circular

RF RNAinthe

preparations

reacted with

avi-din-coupled spheres.Itappearedthat the

reac-tion increased thestability of the

circles,

possibly

by

cross-linking

the

VPg

moleculesor

effecting

a more stable structure which is ableto

with-stand the spreading forces during preparation

forelectronrnicroscopy.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grantsCA-25465, CA-16672 and CA-16527 from the National InstitutesofHealth and grantsG-429and G-841 from The Robert A.Welch Foundation. G.B.T.wassupported byfunds from the Research Council ofAbilene ChristianUniversity.

We thank Susan Berkley, CarolynPeterson, and James Syrewiczforexcellenttechnical assistance and Rebecca Bazer forassistance in manuscript preparation. We especially thank Jerry Manningforprovidingreagents withwhichtodo the biotincross-linking and the avidin-coupled sphere binding.

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