Black Beetle Virus: Propagation in Drosophila Line 1 Cells and an Infection-Resistant Subline Carrying Endogenous Black Beetle Virus-Related Particles

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Black

Beetle Virus: Propagation in Drosophila Line 1

Cells

and an

Infection-Resistant Subline Carrying Endogenous

Black

Beetle

Virus-Related Particles

PAUL FRIESEN,' PAUL SCOTTI,2 JOHN LONGWORTH,2ANDROLAND RUECKERTI*

BiophysicsLaboratoryofthe Graduate School and Biochemistry Department of the College of Agricultural andLifeSciences, University ofWisconsin-Madison, Madison, Wisconsin53706,'andDepartment of

Scientific and Industrial Research, Entomology Division, Auckland, New

Zealand2

Black beetle virus (BBV), oneofarecently discovered class of viruses with a

bipartite genome, multiplied readily in Schneider's line 1 of Drosophila cells.

Virusyields, onthe orderof100mg perliterofculture,wereunusually high and

represented some20% of the total cell protein within3 days after infection. A

derivative subline of these Drosophilacells wasfoundto beresistant to infection

by BBV. Theseresistant cells werealsofound to carrysmallamounts of

BBV-relatedparticles, possiblyamaturation-defectiveformof BBV.

Blackbeetle virus (BBV), originally isolated

(7) from the New Zealand black beetle

(Heter-onychus arator),ischaracterizedbyanisometric

137S virion (30nmindiameter) whichcontains

a

single major

coat

protein (molecular

weight

[mol

wt],40,000)andtwoRNA molecules ofmol

wt 1.0x 106 (RNA 1) and0.5x106(RNA 2) (8).

These

properties

establish BBVas amemberof

a

recently

discovered group of small

divided-genome riboviruses whose prototype is

Noda-muravirus (10).

Theunusuallysmallgenomeof viruses of this

group,onlyhalfaslargeasthat of the

picorna-virusesorretroviruses, isespecially favorable for

molecular analysis. With only two RNAs, this

family

represents the

simplest

class ofviruses witha

segmented

genome, and itsmembersare

therefore

potentially

useful models for

studying

geneinteraction.

Mostofour

knowledge

of themolecular

biol-ogyof thisgroupofviruseshas beengained from

studiesonNodamura virus. Translationin

cell-free extracts has shown that both RNAs from

Nodamura virus are active messengers (11);

RNA1codesfora

105,000-mol-wt protein

(p105)

which may mediate

synthesis

of viral RNA,

whereas RNA2codesforacoat-related

protein

of mol wt 43,000 (p43) which is

probably

a

precursorofmature coatprotein (vp40).

Progress in understanding the molecular

bi-ology of members of this interesting group of

virusesrequirescellculturesystemssuitablefor

propagatingand

manipulating

virusunder

con-trolled conditions. We describe here a system

involvingBBVandDrosophila line1cellswhich

allows suchstudies to be made. Thissystem has

led directly to the recognition of a subline of

Drosophila line 1 which carries endogenous

BBV-related particlesand is resistant to

infec-tionwithBBV.

MATERIALS AND METHODS

Cells. Two strains ofDrosophilaline 1 cells (15) wereused. The WR strain, kindlyprovided by Imo-gene Schneider (Department ofEntomology, Walter Reed Army InstituteofResearch,Washington,D.C.),

waspropagatedinSchneider's complete growth me-dium (14)containing5mgofbacteriologicalpeptone (Difco Laboratories, Detroit, Mich.) per ml and sup-plemented with 15% fetal bovineserum.The NZstrain was a subline of Schneider's WR strain, obtained through T. C. Grace (Canberra, Australia)and main-tainedfor several yearsatAuckland,using the above growth medium but withalowerserumconcentration (10%).Cellswereroutinely grownat26°C in stationary tissue culture flasks (Falcon Plastics, Oxnard, Calif.;

3024) using 15 ml of medium per flask. To passage

cells,confluentmonolayers(5x 107cells)weregently

dislodged by flushingwith a pipetteand diluted 20-fold intofresh medium.

Propagationofvirusin waxmoth larvae. Wax moths (Galleria mellonella) wereraisedat 30°C as described (1). Fourth- and fifth-instar larvae(12to15 mmlong)wereinjectedwith 10,l ofafilter-sterilized BBV suspensioncontaining about 106waxmoth 50% lethal doses(LDro)/ml(seebelow). An ISCO model M

microinjectorwasused forthispurpose. After10days at

300C,

dead larvae (typically 90 to 100% of the

population)werecollectedand storedat-20°C. Stock virus wasprepared by homogenizing BBV-infected larvaeinice-cold0.05Msodiumphosphate, pH 7.2, usinga Sorvall Omnimixer. The 10% larval extract was clarifiedby centrifugationand sterilized witha0.45-,um membranefilter(Millipore Corp., Bed-ford, Mass.).Anequal volumeofglycerolwasadded, and viruswasstoredat-20°C. Infectivitywasassayed in waxmothlarvae asdescribed (1);yields averaged 109to 10"' LDr)operlarva.

Propagation ofvirus in Drosophila cell

cul-ture.Confluentcellmonolayerswereresuspendedand washedinSchneider's medium containing0.5%bovine serumalbumin (BSA). Cell viability (usually greater 741

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than 95%) was determined by staining with 0.05% Trypanblue,and thecellconcentrationwasadjusted to4 x107cells/ml.Cells(108)wereseededinto75-cm2 flasks and infectedwithamultiplicityof1waxmoth

LD)r,/cell,using thevirus stocks describedabove.The infectedcultureswereagitated gentlyfor 30min and then dilutedto107cells/mlwithSchneider's medium containing 10% fetal bovineserum.Cultureswere in-cubatedat26°Cand frozen 4days laterat-20°C.

Purification ofvirusfromDro8ophilacell cul-ture. Viruswasreleased from infectedcellswiththree freeze-thaw cycles. Cell debris wasremovedby cen-trifugation, and virus was pelleted through a2.0-ml cushion of 30%sucrose in buffer A (0.05 M sodium phosphate, pH 7.2, and 0.1% ,B-mercaptoethanol [18-ME]) containing 0.5% BSA. Centrifugation was at 25,000rpminaSpincoSW25.1rotor(60,000xg)for 4hat6°C.Viruspelletswereresuspendedin bufferA. Two-millilitersamplesweremade 1% withrespectto sodium dodecyl sulfate (SDS) and centrifuged at 25,000rpmintheSW25.1rotor(60,000xg)for4.5 h at11°Con28-mi 15to45%(wt/wt) sucrosegradients inbuffer A. Fractions(0.6 ml)werecollected fromthe topof eachgradient, usinganISCOdensity gradient fractionator equipped with aflow cell (0.2-cm path length) andamodel UA-5 absorbancemonitor. Frac-tionswere assayed for virus by spectrophotometric (absorbancyat260nm [As2])andradioactivity mea-surements. The virus-containing fractions were pooled,diluted with bufferA,andpelleted througha

30%sucrosecushioncontainingthesamebuffer.The

viruspelletswereresuspendedinbuffer Aandstored at-70°C.Virus concentrationswerecalculated from optical density measurements at 260 nm, assuming that 1mg/mlhasanabsorbanceof 4.15percmoflight path (8).

Preparation of radiolabeled virus.'Virus

prop-agatedinDrosophilacellculturewasradiolabeledby adding 1.0 mCiofL-[4,5-3H]leucine (AmershamCorp., ArlingtonHeights, Ill., TRK.170)or0.1mCi of L-[U-'4C]leucine (Amersham, CFB.67) to each 10 ml of culture 24 h afterinfection. Incubation with radiolabel wascontinued for 3days. Recovery of input radiolabel inpurifiedvirusrangedfrom0.7 to1.2%.

Viruspropagated inwaxmoth larvae was radiola-beledbyinjectinginfected larvae with80,uCi (40,uCi/ larvaondays3 and4postinfection) of L-[4,5-3H]leu-cine. Virus washarvested from dead larvae 10 days after infection. About 1% ofthe input radioactivity wasrecovered inpurifiedvirus.

Electrophoretic analysis.Methods for SDS-poly-acrylamide gel electrophoresishavebeendescribedby Medappa etal. (9). Gelscontaining9.8%acrylamide, 0.3% (vol/vol) ethylene diacrylate, 0.1% N,N,N',N'-tetramethylethylenediamine,0.1% SDS(Pierce Chem-icalCo.,Rockford, Ill., lot4102-5),and 1.0 Mureain 0.1 M sodiumphosphate, pH7.2, werecastin glass tubes(0.6by20cm).Polymerizationwascatalyzed by the additionofammoniumpersulfate toafinal

con-centration of 0.05%. The electrode buffer, 0.1 M

so-dium phosphate, pH 7.2, containing 0.1% SDS and 0.05 Mneutralized3-mercaptopropionic acid,was

cir-culated betweenupperand lower buffer vessels. Virussampleswerepreparedforelectrophoresis by

adjustingeachto1%SDS,0.5Murea,0.1%,8-ME,and

10 mMsodium phosphate, pH 7.2. Samples were made 20% insucrose and 0.01% in bromophenol blue and thenheated for5minat100°C before electrophoresis.

Electrophoresiswasconducted at4V/cm until the

bromophenol blue marker migrated to the bottom of the gel (about15hfora20-cmgel). Gels were frac-tionated andanalyzedaspreviously described (3).

Preparation of antiserum. Virus used to raise antiserumwaspurifiedfrom BBV-infected wax moth larvae. To purify virus, frozen larvae were homoge-nized in ice-cold 0.05M sodium phosphate, pH 7.2, and carbontetrachloride (2:1 by volume). The heavier organic phasewasseparated by centrifugation at 2,600 xg,and thevirus-containing aqueous phase was clar-ifiedbycentrifugationat12,000xgfor 30 min. Virus was pelleted and sedimented on 15 to 45% sucrose densitygradientsasdescribed above. Afterrepelleting,

viruswassuspendedinbuffer A and storedat-70°C. Anti-BBVserum waspreparedbyimmunizing rab-bits withasingleinjectionof 0.5 mg ofpurifiedBBV

emulsifiedin Freundcomplete adjuvant.Serum was prepared5weeks later.

Immunoprecipitationassays.Serological assays wereconducted by thestaphylococcalprotein A-anti-body adsorbent method describedby Kessler (5). Ra-diolabeled virus inphosphate-bufferedsaline (2) con-taining 0.1% BSA(PBSA)wasincubated with appro-priate dilutions of rabbit anti-BBVseruminPBSA for 2.5hat roomtemperature. Staphylococcal

immuno-globulinG(IgG)adsorbent(IgGsorb;Enzyme Center, Inc.,Boston, Mass.) in NET buffer (150 mM NaCl, 5 mM EDTA, 50mM Tris, pH 7.4, and 0.02% sodium azide) containing 0.5% Nonidet P-40 and 0.1% BSA wasthenaddedto a final concentration of 1% (vol/

vol). After20minatroomtemperature,theIgG ad-sorbentwaspelletedwithanEppendorf microcentri-fuge model5412(12,000xg) and washed with NET, 0.5%NonidetP-40,and 0.1%BSA. The adsorbent was

resuspendedin0.1Msodiumphosphate,pH 7.2,

con-taining 1% SDS and heated for 5 min at 100°C to release radiolabeled virus. The inert adsorbent was removedbymicrocentrifugation,and the supernatant wasassayedforradioactivity.

Radioimmunecompetitionassays.Competition

assays were conducted by incubating increasing amountsofunlabeledantigenwithadilution ofserum

capable ofprecipitating 80%ofa radiolabeled virus probe (0.1 ,ug) for2.5hat roomtemperature. A stand-ardamountofprobe(0.1 ,tg)wasthenadded,and each sample was incubated foran additional 2.5 h. Virus complexedwithanti-BBVIgGwasprecipitated bythe addition ofstaphylococcal IgGadsorbent. The adsorb-ent waspelleted,washed, andassayedforradioactivity asdescribedabove.

Tryptic peptide analysis. Tryptic digestion of viralproteins and thechromatographyof theresulting peptideshas beendescribed(6).Differentiallylabeled virusparticlesweremixed andheatedinthepresence of 0.01% SDS for 5 min at 100°C. Samples were

adjusted to 0.1 M Tris-hydrochloride, pH 8.0, then reduced with 3.5 mg of dithiothreitol per ml, and alkylatedwith 9mg of iodoacetamideperml.Proteins were precipitated with 50% trichloroacetic acid and

suspendedin 0.1 M ammoniumbicarbonate,pH 8.0. After digestion with 0.05 mg of

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CULTURE OF BBV IN DROSOPHILA CELLS 743

nylchloromethylketone-treatedtrypsin (Worthington Biochemicals Corp.,Freehold, N.J.) perml, samples

were lyophilized and suspended in 0.2 M pyridine acetate,pH3.1.Digestswerefiltered to remove insol-ublematerial, and samples were chromatographed on a Chromabead type Pcation-exchange column. Frac-tions(1.5ml)werecollectedandassayed for radioac-tivity.

RESULTS

Preliminary studies on the propagation

of

BBV in cell culture. Studiesonthe

multi-.plication

of the smalldivided-genome RNA

vi-ruses have beenhinderedby the lack of a system

inwhich to propagate and adequately radiolabel

virus incell culture.

Although

Nodamuravirus

grows in hamster (BHK-21) and mosquito

(Aedes albopictus) cell culture (1),

multiplica-tion

apparently

occurs at such low levels that

virus-specific

protein synthesis has not yet been

detected

(11). Since

the

ability

to radiolabel

virus and virus-specific proteins in culture is

crucial to studies on the molecular

biology

of

any

virus,

the search for such a cell culture

system hascontinued.

Because no

plaque

assay is yet available for

BBV, and because the

infectivity

assay based on

ability

to killwax moth larvae iscumbersome,

the searchfora

susceptible

line of hostcellswas

carriedout

by measuring

incorporation

of

radio-labeled uridine into virus-like particles. A

num-ber of insect and mammalian cell lines were

examined. Tothis

end,

virus-inoculatedcultures

were incubated for 5 days in the presence of

[3H]uridine

(50

,ICi/ml).

Virus, released from

cells

by

three freeze-thaw

cycles,

was

pelleted

and sedimented on sucrose

density gradients

essentially

asdescribed in Materials and

Meth-ods. Virus

multiplication

wasthen monitoredby

examining

these

gradients

forthe presence ofa

137S

peak

of

radioactivity.

No evidence of

multiplication

was found in

twomammaliancell lines

(baby

hamster

kidney

ormouseL-cells) norinseveral insectcell

lines,

including

those of the

mosquito (A. albopictus

andA.

aegypti),

the

cabbage

looper

(Trichoplu-sia

ni),

the fall armyworm

(Spodoptera

frugi-perda),

orline GM1of thefruit

fly (Drosophila

melanogaster).

The virus

did, however,

multiply

well in Schneider's

Drosophila

line 1;

thus,

10

days afterinfection, 10-ml cultures seeded with

108 cells

and

108

LD50

(about 10 ,ug) of virus

yielded4to 8

A20

units,representing1 to 2mg

ofvirus.Despitethishigh yield of virus,roughly

25 to 50

times

that

typically

obtained from

pi-cornavirus-infected

cultures, therewaslittle

cy-topathic effect and virus remainedlargely

cell

associated.

Low yields of virus from a subline of

Drosophila line

1. Preliminary attempts in

Madison togrow BBV inDrosophila line 1

(DL-1)cells obtained from New Zealand (NZ subline)

yielded only a few percent of the expected

amountsof virus. The lowyield was ultimately

tracedtotheidentity ofthecells used to

prop-agate the virus. Thus, low yields were obtained

with the NZsubline, whereas high yields of BBV

were produced by DL-1 cells obtained directly

fromImogene Schneider's

laboratory

(WRline).

This is illustrated by an experiment in which

equal

numbersof cells from the WR line and the

NZsubline of WR cells were infected in parallel,

incubated at

26°C,

and

radiolabeled

by exposing

the culturesto

[3H]leucine

(100

,uCi/ml),

begin-ning 1 day after infection. On the 4th day after

infection, virus was released by freezing and

thawing

and

analyzed

on sucrose

density

gra-dientsasdescribed above.

The parental WR cells yielded a prominent

peak of

radioactivity

which coincided with the

137S peak of optical density (Fig. 1A). As

ex-pected,

nosuch 137S

peak

wasobserved in the

gradient from mock-infected WR cellscarried in

parallel

(Fig.

1C). Incontrast, the BBV-infected

4,-DROSOPHILA (DL-1) CELLS Parental (WR) T Subilne (NZ)

II 20 30 40 10 20 30 40

FRACTION NUMBER

*0.2 o m

r-o0.4 32

*0.2

FIG. 1. Sucrosedensity gradient profiles showing

yieldsofBBV-likeparticlesfrom parental

Drosoph-ila line1cells and its NZ subline. Parental WR cells were (A) infected with BBV and(C) mock infected. NZ subline cellswere(B)infectedwith BBVand(D) mock infected. Cellmonolayerscontaining 108 cells wereexposedto0.025mlof5%extractsfrom BBV-infected (4 x 109LDr,o/ml) oruninfected waxmoth larvaeand incubatedfor4daysat26°C.

[3H]leucine

wasadded1dayafterinfection. Viruswasisolated,

purified, and centrifuged on sucrose density gra-dientsasdescribed inMaterialsand Methods. Sedi-mentationisfrom lefttoright.

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NZsubline yielded only3 to 10% as many137S

particles asthe WRline (compare Fig. 1B and

1A). From optical density measurements, the

totalyield of virus particles from theNZsubline

culturewasestimatedtobe about50,ug,whereas

the yield from the WR parental culture was

approximately 550

,tg.

Therecovery of

radioac-tivity inpurified viruswas106 dpm from theNZ

culture, compared with 30 x

106

dpm from the

WRculture.

Unexpectedly, virus-like particles (137S)were

also found in themock-infected NZ cell cultures;

moreover,theywerepresentinamountssimilar

tothose obtainedfrom BBV-inoculated NZ

cul-tures(compareFig. lB and 1D). The possibility

that these particles arose from an undetected

viralagent,inadvertently introduced by the

ex-tractsof

healthy

larvaeused in mock

infection,

was ruled out in other experiments which

showed thesame 137S particles in NZcellsnot

previously exposedtolarvalextracts.Thus, the

NZsubline ofDrosophila cells carries

endoge-nous particles (DL-1 particles) whichsediment

likeBBVbut whichare notfound in the parental

WRcell line.

Electrophoretic differences in the coat

protein of BBV and DL-1 particles. The

sim-ilarityinsedimentation velocitiessuggested that

DL-1 particles were related to BBV. This was

confirmed byelectrophoretic analysis on

SDS-polyacrylamide gels. Each of the 137S peaks

from thesucrosegradients shown in Fig. 1

dis-playeda single major bandinthe region of the

40,000-dalton

coatprotein ofBBV(Fig. 2). That

I

0

C5

90

60

30

6

4

2

A

50 100 1S0

.5 10.1.

so 1oo 1 so

DL-1 particles are not, however, identical to

BBVwasshownby differences in their electro-phoretic properties.

BBV wasfoundtocontaintwocoatproteins,

amajoronewith anapparentmolwtof40,000

(vp4O),andaminoroneof molwt44,000(vp44)

(Fig. 2); this agrees with the report of

Long-worth andCarey (8).vp44 may represent a

pre-cursor form of mature coat protein (vp4O) as

previously proposed for Nodamura virus (11);

indeed, the tryptic

profiles

of these two BBV

proteinsare virtually identical (P. Friesen,

un-published data). We have observed that the

relative proportion ofvp44varies fromonevirus

preparation to another; however, it is not yet

clear whether this variation reflects different

contentsofprecursorproteinvp44inindividual

particles or amixture oftwo different kinds of

particles.DL-1 particles, unlike BBV, exhibited

noevidenceof a minorprotein (Fig. 2C).

A second difference in the proteins ofDL-1

particles

and BBVwasdemonstrated

by

coelec-trophoresis of differentially labeled particles

(Fig. 2D). Theelectrophoretic mobility of DL-1

particlecoatprotein (molwt42,000)wasslightly

lower than that of the major BBVcoatprotein

(vp4O)buthigher than that ofvp44.

Finally,

electrophoretic

studies on the intact

particles revealedadifference innetcharge

be-tween BBV andDL-1 particles. AtpH 8.5 (1%

agarose in35 mM Trisbuffer), BBV migrated

towardthe

cathode,

whereas DL-1

particles

mi-grated in the opposite direction (data not

shown).

4.

2

6

-4.'

2

B

50 100 150

.1

D

D

50 100 150

3 o

0_a

2 K

*1

n

DISTANCE MIGRATED (mm)

FIG. 2. Electrophoreticprofiles ofcoatproteinof the radioleucine-labeled virus-likeparticlesshown in Fig. 1. (A) FromFig. IA, BBV-infectedWRcells; (B)from Fig.

lB,

BBV-infectedNZcells; and(C)fromFig.

ID,mock-infectedNZcells. (D)[3H]leucine-labeledDL-1particlesfrom Fig. IDversus

['4C]leucine-labeled

BBV. Virussamples weredisrupted and subjectedtoelectrophoresis onSDS-polyacrylamide tubegelsas described in Materialsand Methods.

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Resistance of NZ cells to productive in-fection with BBV. Comparison of Fig. lBand 1D shows that inoculation of the NZ subline with BBV didnotsignificantly increasethe yield

of 137S material. Thecoat protein of particles from BBV-inoculated NZcells wasthat of DL-1 particles, not BBV (Fig. 2B). The sharpnessof

the peak and thepresenceof onlyaslight shoul-der in the region ofvp4O from BBV indicates that therewaslittle ifanyBBV protein in the

137S peak fromBBV-inoculated NZ cells. The absence of any detectable BBV in even this

smallpeak of virus-like material implies that NZ cellsareeffectively resistanttoproductive infec-tion by BBV.

Serological relationship between BBV and DL-1particles. Both BBV (derived from the WRline) and DL-1 particles (derived from the NZsubline)wereprecipitated by antiserum raised against BBV (Fig. 3A). The capacity of this antiserumtoprecipitateaconstantnumber ofparticles was 12-foldgreater for BBV; thus, the precipitin titerwas90,000for BBV but only 7,000 for DL-1particles.

Further evidence for serological relatedness was provided by a radioimmune competition assay which measured the ability of unlabeled BBVtocompetewitharadiolabeled DL-1

par-ticle probe for a limited number of antibodies (Fig. 3B). Theassaydemonstrated that the

pre-cipitation of the DL-1 particle probe could be reduced to background levels when anti-BBV serum waspreincubated withexcessBBV. Thus, antibodies directed against BBV were also ca-pable of precipitating DL-1 particles.

Gel diffusion tests using antiserum raised against DL-1 particles independently confirmed that BBV and DL-1 particles have common surfaceantigens (datanotshown). The precipi-tation arcs of these tests indicated that both particleswereserologically related butnot

iden-tical. Thisnonidentityissupported bythe sub-stantialdifference inprecipitation titersof anti-BBVserumtowardeachparticle (Fig. 3A).

Similaritiesinthetrypticfingerprints of the coat proteins of BBV andDL-1 parti-cles. Comparison ofthe tryptic profiles of the coatprotein of BBV andDL-1 particles,

differ-entially labeledwithradioleucine,revealed sev-eral identical tryptic fragments along with a coupleof differences atfraction114(Fig. 4).The

smallnumber oftryptic peaks exhibited by both particlesmaybeduein parttothe insolubility ofafraction(20%) of the tryptic digest. Similar behavior has beenpreviously observed with No-damuravirus (11) andmaybe characteristicof

thecoatproteinof thisclass of viruses.Although

the degree of difference cannot be accurately

assessed because ofthesmallnumber of tryptic

0.U

!

0 S? .2!?

(

W.W* Kr" W^, 0.0 0.10 100

Anti-BBV Serum(ILI) Unbbeled BBV Added(/Lg)

FIG. 3. (A) Titration curves for BBV and DL-1 particles, usingantiserum directedagainstBBV. A fixed amount (0.1 pg) ofradioleucine-labeledprobe antigen (BBV, -5,000 dpm; DL-1 particles, -1,000

dpm) in PBSA was incubated with the indicated volume ofserum under standard assay conditions (2.5hatroom temperaturein atotal volumeof100

,ld). Radioactivityin the immune

complex

was

deter-minedafterprecipitationwithstaphylcoccal IgG ad-sorbentasdescribed inMaterials and Methods. The precipitation titer, defined as thereciprocal ofthe volume(microliters) ofserumrequiredto half-precip-itate1 ngofvirus understandardassayconditions, was9x 104 forBBVand7x 103forDL-1 particles.

(B) Radioimmune competition assay ([3H]leucine

DL-1 particle probe versus BBV). The indicated amountsofunlabeled BBV in PBSA were preincu-batedfor2.5 hatroomtemperaturewithadilutionof

serumcapable ofprecipitating80%oftheradioactive

DL-1particle probe.A standard amount(0.1 pg) of DL-1particle probe(-1,000 dpm)in PBSAwasthen added, and incubationwascontinuedunder stand-ard conditionsforanadditional2.5 h.Radioactivity in thestaphylococcal IgGadsorbentprecipitatewas determinedasdescribed.

peaks observed, the similarities among those tryptic fragments displayed again suggest that

bothparticlesarerelated butnotidentical. DISCUSSION

We have shown that BBV multiplies well in

Schneider'sDrosophilaline1.However,the in-fection isnotverycytolytic and the infectedcells

remain intactforperiods of4or moredaysafter infection.Thus, arelatively long period of

syn-thesis probably accounts in part for the high yields of virus which rangefrom 1 to 2mg per

108cells(40mgequivalentwetweight) within3

days of infection. By the 3rd day postinfection, the cellmasshasincreasedtoabout 70mg(wet

weight). Assuming that protein constitutes 10%

of the cellular wetweight, 1.5 mg of virus cor-respondstoabout20%of thetotalcell-associated protein,a yield 100-foldgreaterthan that typi-callyobtained frompicornavirus-infected cells.

Although BBV multiplies well in one strain (WR line) of Schneider's line 1, it multiplies poorly, ifat all, in aderiVative strain (NZ sub-line). The observation that the resistant NZ

subline carries BBV-relatedparticles (DL-1 par-0-A

i° BBV* /

'5

._{_4}.-_{,^f ,,}2 ,^f}

lOO B

75

so

\,/)L~~~-I p*

25

.

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50 100 150

FRACTION NUMBER

500

'

300

-100 :

200 250

FIG. 4. Comparison of differentially radioleucine-labeled proteinsofBBVand DL-1particles by tryptic fingerprinting. A mixture of[14C]leucine-labeled BBV and [3Hlleucine-labeled DL-1 particles was heat denatured, digested with trypsin, and chromatographed on a cation-exchange column as described in Materials and Methods.

ticles)whereas thesusceptible WR line doesnot

suggeststhat resistance toinfectionby BBV is mediated by the DL-1 particle. However, further evidence is required to rule out the possibility that such resistance is an independent trait of cellscarrying DL-1 particles.

Recent studies (P. Scotti, unpublished data) show that DL-1particlescanmultiply in cells of Drosophilaline 2 and GM1. Thisinfectivity of

DL-1particlesnotonly establishes theagentas a virus (DLV), but may also open the way to clarifying the role of DLV in BBV resistance by determining its abilitytoalter thesusceptibility of DLV-infected cellstosuperinfection by BBV. Theproperties of DLV indicate that itbelongs

tothesamefamilyasBBV andNodamuravirus.

Each virus sedimentsat135to137S andcontains one majorcoatprotein in the molecularweight

range of40,000. Moreover, recent studies

indi-cate thatDLV, like BBV and Nodamura, con-tains two RNAs sedimenting at 15 and 22S, respectively.

Thediscovery of DLV raisesaquestionabout itsorigin. In light ofreportsof virus-likeparticles in the nucleus and cytoplasm of cultured Dro-sophilacells,including Schneider's line 1 (4, 12, 16), it is possible that DLVwas acontaminant of theoriginal tissue explant of the cell line. It could then have been eitherlost from the WR line ormaintained ina suppressedstate. Alter-natively, the virus could have arisen in the NZ

subline because of derepression at some later

stage ofsubcultivation.None of these explana-tions, however, accountsfortheremarkable co-incidence that the immuneDrosophilacellline carriesanagentclosely relatedtoBBV, avirus

originallyisolated fromblackbeetles.

A more likely way ofexplaining this coinci-dence is that DLV arose from an inadvertent infection of line 1 with BBV itself. Such an infection might easily have been overlooked since BBV does notcause extensive cytopathic effects inDrosophila cells. Continued subculti-vation of the infected cells may then have

se-lected formutantsmorecompatible with contin-ued cell growth, thus leadingto the

establish-mentofapersistent infection.

The rather small differences in the tryptic profiles of thecoatproteins (Fig. 4)are

consist-entwith thehypothesis that DLV isamutantof BBV. These tryptic similarities and the minor shift inelectrophoretic mobilityoftheDLV coat

proteinsuggestthatrelatively fewchanges have occurred. The apparent increase in molecular

weight of the DLV protein might be accounted forbya changeassmallas asingle amino acid residue since it has been demonstrated that re-placement of such a residue can significantly alter SDSbindingand thus the relativemobility of certainpolypeptidesonSDS-gels (see discus-sion in reference6).

On the other hand, our electrophoretic and serological studies suggest that BBV and DLV differsubstantiallymorethan wouldbeexpected

for asimpleselection ofviralmutants. For

ex-ample, DLV appears to lack the minor coat

found in BBV. Moreover, the particles have opposite chargesat pH 8.5 and have a 12-fold difference in immune reactivity, both implying substantial differences in the surfaces of BBV

and DLV.

Apossible resolutiontotheseapparently con-tradictory findings is suggested by an analogy with poliovirus. The surface antigenicity of poliovirus is radicallyalteredbyalarge confor-mational change which occursduringthe

mat-urationstepin whichthe virionacquires infec-tivity (reviewedinreference13);moreover,

mat-urationisaccompanied by cleavageofeachpro-.

tein subunit in theprecursorparticle (provirion). AnanalogousprocessinBBV,wherebythe

pro-tein subunits(vp44) of the provirionarecleaved

toformthemajorcoatprotein(vp4O)of mature

virions, could also be responsible formajor an-tigenic differences between the BBV provirion and the mature virus particle. This raises the

possibility, then, that DL-1 particles represent uncleaved provirions ofa maturation-defective

a-,

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747

mutant of BBV and that the single peak of

protein in DL-1 particles (Fig. 2C) represents

the precursorprotein, not the mature coat

pro-tein, ofthe defectiveforn of BBV. It should be

possible to test this hypothesis by comparing

DL-1 particle coat protein with the cell-free

translation product directed by its mRNA. ACKNOWLEDGMENTS

Thisresearch was supported in part by grantMV-33I from theAmerican Cancer Society and by Public Health Service grant CA-08662 from theNational Cancer Institute. P.F. was supported by Public Health Service training grant 5-T32-GM07215 from the National Institute of General Medical Sciences.

Wethank Harry Coppel, Department of Entomology, Uni-versity ofWisconsin-Madison, for the gift of wax moths, for instruction on propagationand injection of larvae, and for useful discussions. We also thank DanOmilianowskifor chro-matography of tryptic digests.

LITERATURE CITED

1. Bailey, L,J.F.E.Newman, andJ. S.Porterfield. 1975.Themultiplicationof Nodamura virus in insect andmammaliancellcultures.J. Gen.Virol.26:15-20. 2. Dulbecco,R.,andM.Vogt.1954.Plaqueformation and

isolationofpure lines withpoliomyelitisviruses. J.Exp. Med. 99:167-182.

3. Gilson,W.,R.Gilson,and R. R.Rueckert 1972. An automatic highprecision acrylamide gel fractionator. Anal. Biochem. 47:321-328.

4.Hirumi,H. 1976.Viral, microbial,and extrinsic cell

con-tamination of insect cellcultures, p. 233-268. In K. Maramorosch (ed.), Invertebrate tissue culture: re-search applications. AcademicPress, Inc.,New York andLondon.

5. Kessler, S. W. 1976. Cell membrane antigen isolation with the Staphylococcal protein A-antibody. J. Immu-nol. 117:1482-1490.

6.Kew, 0. M., M. A.Pallansch, D.R.Omilianowski, and R.R.Rueckert. 1980. Changes in three of four coat proteinsof oral poliovaccine derived from type 1 poliovirus. J. Virol. 33:256-263.

7. Longworth, J.F., and R. D. Archibald. 1975. A virus ofblack beetle, Heteronychus arator (F) (Coleoptera: Scarabaeidae).N.Z. J. Zool. 2:233-236.

8. Longworth,J.F.,andG. P.Carey.1976.Asmall RNA virus with adivided genome from Heteronychus arator (F)[Coleoptera:Scarabaeidae].J.Gen. Virol. 33:3140. 9.Medappa, K. C.,C. McLean, and R. R. Rueckert. 1971. Onthe structureofrhinovirus 1A. Virology 44:259-270. 10. Newman, J. F.E.,and F.Brown. 1973. Evidence for a divided genome in Nodamuravirus, anarthropod-borne picornavirus. J. Gen. Virol. 21:371-384.

11.Newman,J. F.E.,T.Matthews,D. R.Omilianowski, T.Salerno,P.Kaesberg, and R. Rueckert. 1978. In vitrotranslation of the two RNAs of Nodamura virus, anovel mammalian virus with a divided genome. J. Virol.25:78-85.

12.Plus, N. 1978.Endogenous viruses ofDrosophila mela-nogastercelllines: theirfrequency, identification,and origin.In Vitro 14:1015-1021.

13.Rueckert,R.R. 1976. Onthestructureand morphogen-esis ofpicornaviruses,p. 131-213. In H.Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology. PlenumPublishing Corp.,New York.

14.Schneider,L.1964.Differentiation of larvalDrosophila eye-antennal discs in vitro. J.Exp.Zool. 156:91-104. 15. Schneider, L.1972.Celllines derived fromlateembryonic

stages ofDrosophilamelanogaster. J.Embryol. Exp. Morphol.27:353-365.

16. Williamson,D.L,andR. P.Kernaghan.1972. Virus-like particles in Schneider'sDrosophila cell lines.Dros. Infect. Serv. 48:58-59.

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