Characterization of the oncornavirus particles in the plasma of guinea pigs with L2C leukemia.

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Characterization

of the Oncornavirus Particles in the

Plasma

of Guinea Pigs with L2C

Leukemia

R. MICHALIDES,l J. SCHLOM, J. PEARSON, K. PERK, AND J. DAHLBERG*

MeloyLaboratories, Inc.,Springfield, Virginia 22151,andNational CancerInstitute, Bethesda,

Maryland20014*

Received forpublication 28 November 1975

The inoculation of L2C guinea pig leukemia cells into strain 2 guinea pigs resultsinthe death of the animals within12to 15days.Deathisprecededbythe simultaneous appearance in the plasma of (i) elevated leukocyte levels, (ii) extracellularvirusparticles, and (iii)aparticle-associatedRNA-directed DNA

polymerase.This enzymeactivityhasacationpreferenceidenticaltothat of the

type B bromodeoxyuridine-induced guinea pigvirus, i.e., anMg2+ optimumat 20mMandnoactivityusing Mn2 . Competitive molecularhybridizationstudies also revealed that theplasmaofleukemicguineapigscontainedapproximately2 x 109 genome equivalents permlof anRNA that is homologous to the RNA of

the bromodeoxyuridine-induced guinea pig virus. Morphological observations

indicatethatmost,butnotall, of the extracellularparticles observedin leuke-miaplasmaarederived from the intracisternal particlesseeninthe L2Ctumor cells.Thepossibilities thateithertwoviralpopulationsarepresent orthat thein vivomorphogenesis of the typeBbromodeoxyuridine-inducibleguinea pigvirus ismarkedly differentfromits in vitro morphogenesis arediscussed.

The L2C strain of a transplantable guinea pigleukemia arose spontaneouslyin 1954 (3)in the strain2inbredline of guinea pigs andhas since been serially transferred in this strain. Intracellular and extracellular virus-like parti-cles were later observed in these tumor cells

(21, 23). The intracellularparticlesareformed bybudding through the rough endoplasmic re-ticulum membrane into intracisternal spaces (7). They differ from murine intracisternal A

particlesby being larger, having aclearly

de-fined intermediatelayer (2), and appearing less frequently connected to the cisternal mem-branes (25; Dahlberg and Perk, unpublished

data). Similar intracisternal particles have

been observedinguinea pigfetal germ cells (2),

spleengerminal centers of normal guinea pigs

(14), and in a chemically induced guinea pig hepatoma (6). Extracellular virus particles have been reported in the tissues of leukemic guinea pigs in the absence of any apparent budding from the plasma membrane (7), as well as intheblood plasma of the guinea pigs carry-ing the transplantable leukemia (23). Feldman andGross (7)have hypothesized thataprocess of reverse pinocytosis could account for the release of intracisternal virus from leukemic cellsinlight of the absenceofobserved budding ofvirus from theplasma membrane. Itisalso

IPresent address: Dutch Cancer Institute,

Sarphatis-traat108,Amsterdam,The Netherlands.

possible thatthe extracellular particles in leu-kemicguinea pigs are derived from the intra-cisternal particles after necrobiosis of the leu-kemic cells (Dahlberg and Perk, manuscriptin preparation).

Oncornavirus particles can be induced by halogenated deoxypyrimidines from any nor-mal guinea pigcell (11, 20, 22, 24, 27). These particles sharemost of the morphological fea-tures of the type B mouse mammary tumor virus (MMTV 4). Severalbiochemical parame-ters ofthis induced guinea pig virus (GPV), such as buoyant density in sucrose and CsCl and the cation preference of its RNA-directed DNA polymerase, are identical to those of MMTV (17) and are quite distinct from the comparable properties ofamammalian typeC virus, murineleukemia virus (MuLV).

In this study, the biochemical and morpho-logical features of the extracellular virus parti-clespresent inthe plasmaofguinea pigs carry-ing the transplantable L2C guinea pig leuke-miaarecharacterized and compared with those ofthevirusthatisinducedfrom normal guinea pig cells by treatment with 5-bromo-2'-deoxyu-ridine (BUdR).

MATERIALS AND METHODS

Animals. Strain 2 guinea pigs were obtained

through the Frederick Cancer Research Animal

Farm, Frederick, Md. The animals were kept in

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stainless steel cages and were fed Wayne guinea pig chow and cabbage daily with water ad libitum. Ani-mals of 300 to 500 g were used.

Plasm from leukemic animals. The L2C strain of guinea pig leukemia was passaged by subcuta-neousinoculation of 0.2 ml of a 10% (wt/vol) tumor suspension into strain2 guinea pigs. Under these conditionsasmallnodule forms at the site of inoc-ulation, and a rapidly progressing lymphoblastic leukemia kills the animal within 12 to 15 days. Plasma from leukemic animals with an elevated leukocyte count was obtained by heart puncture. All plasma samples were clarified by centrifugation at 8,000xgfor 10 min and stored at -76 C.

Drug-induced remission. At 11 days after inocula-tionof the L2C strain of guinea pig leukemia,three animals were treated with cyclophosphamide (Cy-toxan), 40 mg/kg, or MeCCNU (1-[2-chlorethyl]-3[trans-4-methylcyclohexyl]-1-nitrosourea), 10 mg/ kg, asdescribed previously (25). Under these condi-tions,cyclophosphamideinduced a remission of 3 to

4 weeks, followed by a reoccurrence of leukemia, whereas MeCCNU cured theanimals of the leuke-mia. Plasma samples (1 to 2 ml per animal) were obtainedbyorbital bleedingatvarious timesafter the administration of thedrugs.

Assays ofRNA-directedDNApolymerase. (i) Re-actions using the synthetic template poly(rA):-oligo(dT) and poly(dA):oligo(dT). Plasma samples

wereconcentrated by centrifugationthrough8mlof 20%sucrose in TNEbuffer (0.01 M

Tris-hydrochlo-ride, pH 8.3, 0.15 M NaCl, and 1 mM EDTA) at 95,000 x g for 1 h at4C inan SW41 rotor (Beck-man).Theresulting pellet was resuspended in 40u.l

of 0.01 MTris-hydrochloride (pH 8.3) and preincu-bated for10min at 0C in1mMdithiothreitol and 1% NP-40 (Shell). Thereafter, the following were addedtotherespectivefinalconcentrations, in 100 ,ul: poly(rA):oligo(dT) orpoly(dA):oligo(dT), 40 ug/ ml;Tris-hydrochloride (pH 8.3), 62.5 mM; KCl, 15

mM; MgCl2, 20mM; TTP, 32 uM; dATP, 200 ,uM;

and400

ACi

of[3H]TTPperml (50.1Ci/mmol,New

EnglandNuclearCorp.). The reaction mixturewas

placedat 37C,andaliquotswerewithdrawnat indi-cated intervals and assayed for trichloroacetic

acid-precipitable radioactivity. The

poly(rA):oligo-(dT)),2-18)

andpoly(dA):oligo(dT) 12-18)template

prim-ersusedwereequimolarmixturesof thepoly-and

oligonucleotide compoundandwerepurchasedfrom P-LBiochemicals, Inc.

(ii) Reactions using the synthetic template poly(rC):oligo(dG). Plasma samples were

concen-trated asdescribed aboveorbysucroseequilibrium centrifugationasindicated.Theplasmaconcentrate

wasresuspendedin 40,ulof0.01 M

Tris-hydrochlo-ride,pH 8.3, andpreincubatedfor 10minat 0Cin 0.1 Mdithiothreitol and2% NP-40. Thereafter, the followingwereaddedtotherespectivefinal

concen-trations, in 100 u.l: poly(rC):oligo(dG), 40 ,ug/ml;

actinomycin D, 50 ug/ml; Tris-hydrochloride (pH

8.3), 62.5 mM; KCl, 15 mM; MgCl2 or MnCl2 at indicated concentrations; and200 ACiof[3H]dGTP

per ml (5.5 mCi/mmol, New England Nuclear Corp.). The reactionmixtureswereplacedat37C,

andaliquotswerewithdrawnatindicatedintervals

and assayed fortrichloroaceticacid-precipitable ra-dioactivity. The

poly(rC):oligo(dG)(12 18)

template primerused was an equimolar mixture of the poly-and oligonucleotide compound and was purchased from P-LBiochemicals, Inc.

Viruspurification. Plasma samples were concen-trated as described above. The plasmaconcentrate, inTNE buffer, was layered over either 20 to 60% sucrose(wt/wt) gradients in TNE buffer or over 10 to 30% CsCl (wt/wt) gradients in 0.01 M Tris-hydro-chloride, pH 8.3, and centrifuged in an SW41 (Beck-man)rotor for 3 h(sucrose gradients) or 2 h (CsCl gradients) at 4 C at 100,000 x g. Fractions were collected from below, and the density of certain frac-tions wasdetermined from the refractive index. In-dicated fractions were pooled, diluted with 0.01 M Tris-hydrochloride, pH 8.3, and concentrated by centrifugation at 100,000 x g for 1 h at 4 C in an SW41 rotor.The resulting pellets were suspended in 40 ,l of 0.01 M Tris-hydrochloride, pH 8.3, and assayed for viral DNA polymerase as described above, using the synthetic template

poly(rC):-oligo(dG).

In mixingexperiments, [32P]phosphoricacid- and

[3H]uridine-labeled BUdR-inducedGPVwasmixed witheitherplasmafrom leukemicanimalsortissue culture medium for the indicatedperiodof time and temperatureand thensubjectedtoequilibrium

cen-trifugation as described above. Gradient fractions wereassayed for total radioactivity by countingin

Aquasol (NewEnglandNuclearCorp.). The proce-dures for the preparation of[32p]_ or

[3H]uridine-labeled BUdR-induced GPVaredescribed elsewhere

(17).

Molecular hybridization. DNA was extracted fromspleensofhealthy guinea pigs,NIHstrain,and

processedusingaslightmodificationasdescribedby

Goodman etal. (10). Cellpelletsweredissolved in

five volumes of5% sucrose in TNE, disrupted by

homogenization, andcentrifugedfor10minat4 Cat

3,000 xg. Theresulting pelletwasresuspended in

20volumes of 1% sodium dodecyl sulfateand 1 M sodiumperchlorate inTNE. The mixtureswas

ex-tracted twice withphenol-cresol-chloroform (PCC) and four times with chloroform containing 1% iso-amyl alcohol. One volume of cold ethanolwasadded

totheaqueous phase, and the DNA was thenspooled

outand dissolved in 3 mM EDTA. The DNA was

thenadjustedto 0.4NNaOH andincubatedat 37C for 2 h. Nitrogen was bubbled through the DNA solution, and then the DNAwasfragmented (6-8S asmeasured byalkaline sucrosegradient centrifu-gation)by sonic treatment. The DNA solution was

adjusted to 0.1 M Tris-hydrochloride (pH 8.3) and neutralized using HCl. The DNA was then

reex-tractedwithPCCandprecipitatedwithtwovolumes of cold ethanol. After storage at -20 C, the DNA

was pelleted at 17,000 x g for 30 min at -20 C and then dissolved in 3 mM EDTA and stored at

-70 C until used. RNA from BUdR-induced GPV

and from Rauscher MuLV, released by

MuLV-infected JLSV-9 cells, as well as RNA from the plasma of healthy and leukemic strain2guinea pigs wasisolated. Virusandplasmasampleswere puri-fied by sedimentation and equilibrium

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tion as described above. The fractions from the sucrose gradients corresponding to the density of the viruses (fractions of 1.16 to 1.20 g/ml for the guinea pig plasma samples, 1.14 to 1.18 g/ml for MuLV) were used for RNA extraction by the sodium dodecyl sulfate-Pronase digestion method as previ-ously described (17).

Competitive molecularhybridizations were

per-formed,usingamodification of the method usedby

Wright and Neiman (29). The hybridization mix-tures in 50 or 100 gl contained 200 or 400 ,ug of cellular DNA fragments from guinea pig spleens,

1,000 counts/minof3H-labeled 60-70S RNA from the BUdR-induced GPV, 4x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate, 3 ,ug of yeast RNA, and varying

amounts of unlabeled RNA. The samples were

boiled for 3 min and incubated at 70C to a Cot

(corrected for saltconcentration) of 40,000. The

hy-bridization mixturewasthen transferredto 2mlof 2x SSC, and RNase A(Sigma,previouslyheated at 80Cfor 15 min todestroy DNase activity) andT, were added to 50,ug/ml and100U/ml, respectively. The mixtures were incubated at37C for 45 min, after which timetwovolumes of coldtrichloroacetic acidmix (100% acid-saturated

pyrophosphate-satu-ratedorthophosphate, 1:1:1) were added. The

sam-ples werekept in ice foratleast45min, after which time the nucleic acidswerecollected on nitrocellu-lose filters (Gelman, 0.45 ,um). The nitrocellulose filters were extensively washed with 5% cold trichlo-roaceticacid and dried, and theradioactivity of each

samplewascountedto2% accuracywithaBeckman

liquidscintillation counter.

Electron microscopy. Plasma pellets were pre-pared by centrifuging 4.5 ml of plasma samples

through 0.5 ml of 20% sucrose (wt/wt) in0.1M Tris-hydrochloride, pH 8.3, at 30,000 rpm in an SW50L rotor(Beckman). Pellets were fixedfor2h with3%

glutaraldehyde in Millonig buffer (18). The pellets

werethen washed in severalchanges of buffer for2

h, postfixed for 1 h in Dalton chrome osmium (5), washed for1to2h indistilled water, stained en bloc with 2% uranyl acetate in 50% ethanol for 16 h,

dehydrated in ethanol and propylene oxide, and embedded in Epon-Araldite (19). Thin sectionswere

stained with uranyl acetate and lead citrate and examined inaSiemens101electronmicroscope fit-ted with a50-,um objective aperture. For negative staining, virus was absorbed to the grid, and the gridwaspartially blotted, fixed for 30 sin3% glu-taraldehyde, blotted, and stained with 0.5%

phos-photungstic acid, pH 7.0.

RESULTS

Detection of RNA-directed DNA polymer-aseactivity intheplasmaofleukemic guinea pigs. Subcutaneous inoculation ofL2C tumor cells into strain 2 guinea pigs resulted in the formation ofanoduleatthe siteof inoculation and aproliferation of lymphoblasts, with death occurringatday 13 postinoculation. Leukocyte counts showed a sharp increase at approxi-mately day 12 (Fig. 1). The elevated leukocyte

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TIME AFTER INOCULATION(days) FIG. 1. Detection of DNA polymerase activity in the blood of leukemic guinea pigs. Three adult guinea pigs were inoculated subcutaneously on day 0 with0.2mlof a 10%(wtlvol)L2C tumor cell suspen-sion.On the indicateddays, 2-mlblood samples were withdrawn by orbital bleeding in the presence of heparin, and the total leukocyte (WBC) count of an

aliquotwasdetermined (A). A 0.5-ml plasma aliquot

ofeach blood sample was concentrated and assayed for the presence of DNA polymerase, using the tem-platepoly(rC):oligo(dG)asdescribed in the text. The counts per minute of[3H]dGMP (specific activity, 3,000counts/min per pmol) incorporated into acid-precipitable counts in a40-minassay are given (0).

Values plotted are average for the three animals.

level atdays 12and 13largelyrepresents lym-phoblasts (tumorcells), whicharepresumably released from metastatically involved tissues (25). A high-speed pellet from 0.5 ml ofeach plasma samplewas assayed for RNA-directed DNApolymerase activity, using the synthetic

templatepoly(rC):oligo(dG)asdescribedabove.

The particles containing the RNA-directed

DNA polymerase activity were concentrated from plasma thatwasfirst clarifiedat8,000 xg for 10 min at4 C. The supernatant was then diluted 10-fold with TNE buffer containing 3 mM EDTA and sedimented through 20% su-crosefor 1 h at 100,000 x g at 4C. The RNA-directed DNA polymerase activity measured increased proportionally with the increase of leukocytecount ineachblood sample(Fig. 1).

Theextracellularvirus-like particles, as ob-served inultrathin sections ofhigh-speed pel-lets, are present in the plasma at maximal levelsjustbefore the animal dies of leukemia and at the same time that peripheral

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cyteshave reached their highest level. No ex-tracellular particlesorRNA-directed DNA po-lymerase activity has been observed in su-crose densityfractions of 1.13 to 1.19g/ml or in high-speed pellets from the plasma of healthy guinea pigs (seebelow). There appears, there-fore, to be a correlation between the RNA-di-rected DNA polymerase activity observed in the plasma of leukemic animals and the pres-ence of extracellular virus particles in the plasma.

Treatment of the guinea pigs, inoculated 11 days before with an L2C tumor suspension, with the antitumor drugs cyclophosphamide

and MeCCNU (25; seeabove)resultedina pre-cipitous decline in less than 24hofthe leuko-cytecount tobelow-normallevels. Atthe same time,the RNA-directed DNApolymerase activ-ity dropped to base line levels. Cyclophospha-mide-induced remission was followed by re-lapse, with the animalsdyingin35 to 37 days

due to meningeal leukemia with slight

in-creases in leukocyte number and in

particle-associated RNA-directed DNA polymerase ac-tivityinplasma.Thetreatmentwith MeCCNU resulted in apermanent chemotherapeutically induced remission,asdescribedpreviously(25). RNA-directed DNA polymerase in the plasma of leukemic animals. Attempts to ob-tain anendogenous RNA-directed DNA

polym-eraseactivityortoisolateradioactivelylabeled

60-70S RNA fromequilibrium density gradient

fractions from the plasmaof leukemic guinea

pigswere unsuccessful;oneof several possible

explanations is the presence ofhigh levels of

RNase in this plasma, as tested by the

rapid

degradation of3H-labeled rRNA when mixed

with plasma from leukemic guinea pigs. To

characterize the RNA-directed DNA

polymer-aseinparticlesfromplasmaof leukemicguinea

pigs, its abilityto incorporate [3H]TMP into a

dT polymer, using the synthetic

template

poly(rA):oligo(dT),wascomparedwith that

us-ing the template poly(dA):oligo(dT). Particles

from 20 ml ofplasma from leukemic animals

wereconcentratedbysedimentationand

equi-libriumdensitycentrifugation.Thefractions of the sucrose gradient corresponding to densi-tiesbetween 1.13 and 1.19 g/ml were

concen-trated and used inthe poly(rA):oligo(dT)- and

poly(dA):oligo(dT)-templated DNApolymerase

assays, asdescribed above. As seenin Fig. 2,

the DNA-synthesizing activity

preferred

the

template poly(rA):oligo(dT) above poly(dA): oligo(dT); this is one characteristic ofa viral RNA-directed DNApolymerase (12).

Asshown previously (17), the RNA-directed DNApolymerase activity of the BUdR-induced

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REACTION TIME (MINUTES)

FIG. 2. Characterization of the DNA polymerase activity inthe plasma of leukemic guinea pigs. Parti-cles from 20 ml ofplasma from leukemic guinea pigs

were concentrated afterpooling sucrose equilibrium fractions with densities between 1.13 and1.19 g/ml. The plasma concentrate was then added to DNA polymerase reaction mixtures containing either the synthetic template poly(rA):oligo(dT) (-) or

poly(dA):oligo(dT) (0) asdescribed in the text. Ali-quots(10pi)of a100-plreaction mixture were with-drawn at the indicated times and assayed for trichlo-roacetic acid-precipitable radioactivity. The pico-moles of [3H]TMP (specific activity, 7,000 countsl

min per pmol) incorporated in a trichloroacetic acid-precipitable polymer are given versus reaction time.

GPV preferred Mg2+ over Mn2+ in a

poly(rC):oligo(dG)-templated assay. The

opti-malMgCl2concentration was 20 mM, whereas

no incorporation of [3H]dGMP into an rC:dG

polymer occurred atany of the MnCl2

concen-trationstested(from0.1 to 200mM). Theuseof the synthetic template poly(rC):oligo(dG) was shownby others (1, 13)tobe another character-isticofaviral RNA-directed DNA polymerase. The cation preference of the RNA-directed DNA polymerase activity from the plasma of leukemic guinea pigswas determined by con-centrating 50 ml of leukemic plasma by sedi-mentation andequilibrium density

centrifuga-tion. The fractions with densitiesbetween 1.13

and 1.19 g/ml were pooled, concentrated, and

used in poly(rC):oligo(dG)-templated DNA po-lymerase assays, as described above, with the

indicatedMgCl2 orMnCl2 concentrations (Fig.

3). No significant incorporation of [3H]dGTP

into an rC:dGpolymer occurred at any MnCl2 concentrationtested, whereasMgCl2 provedto

be essentialtothereaction mixture. The

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1124 MICHALIDES ET AL.

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CATION CONCENTRATION (mM)

FIG. 3. Divalent cation titrationofthe DNApolymerase activityinplasma fromleukemicguinea pigsina

poly(rC):oligo(dG)-templated assay. Particles from200 ml ofbloodplasma ofleukemicguinea pigs were

concentratedandpurified bysucroseequilibrium density centrifugationasdescribedinthetext.Theplasma concentrates were then added to anRNA-directed DNA polymerase assay, using the synthetic template

poly(rC):oligo(dG)(j2.18) with the indicated cation concentrations (see text). Thepicomoles of [3H]dGMP (specific activity, 3,000 counts/min perpmol) incorporated in a trichloroacetic acid-precipitable rC:dG

polymerina40-minreactionaregivenversusthe concentrationofMgCl2orMnCl2inthe reaction mixture.

mal incorporation occurredataMgCl2 concen-tration of 20 mM (Fig. 3). This divalent cation preference is identical to that of the RNA-di-rected DNA polymerase of the BUdR-induced GPV (17). An analogous plasma concentrate

from 30 ml ofplasma from normal guinea pigs didnot showany [3H]dGMPincorporation into anrC:dG polymer ina poly(rC):oligo(dG)-tem-plated assayusing 20 mM MgCl2 (Fig. 4, open

circles). In thesameexperiment,aconcentrate of 15 ml ofplasma from leukemic guinea pigs was added to a reaction mixture containing4

ggofoligo(dT)(l_l8) (Fig. 4, open squares), in-stead of4 ,ug ofpoly(rC):oligo(dG)(l2_18) (Fig. 4, closed circles). The absence of any incorpora-tionof[3H]dGMP, using only oligo(dT) primer, reduced the possibility that, under thereaction

conditionsused, the measured activitywasdue

to an endogenous cellular terminal deoxyribo-nucleotidyl transferase (15).

Density of particles containing the RNA-directed DNA polymerase activity in the leu-kemic guinea pigs. The buoyant density of the particles present in the plasma of leukemic guinea pigs that contain RNA-directed DNA

polymerase was determined by assaying

frac-tions ofanequilibrium density gradientfor the

presence of the enzyme. Samples (100 ml) of

plasma from leukemic guinea pigswere concen-trated andsubjectedtoequilibrium centrifuga-tion insucroseorCsCl,asdescribed above. The fractions with the indicated densities (Fig. 5)

were pooled and concentrated by

centrifuga-tion, and the resulting pelletswereassayed for DNA polymerase activity, using the synthetic template poly(rC):oligo(dG) in the presence of 20 mM MgCl2or0.4 mMMnCl2. The principal RNA-directed DNA polymerase activity was observed in sucrose gradient fractions with a buoyant density of approximately 1.15to1.16g/ ml (Fig. 5A) and in CsCl gradient fractions withabuoyant density of approximately 1.17to

1.18 g/ml (Fig. 5B).

Because of thepresenceof RNase anda con-siderableamountof cellular debris(see below) in the plasma of leukemic guinea pigs, the density of the particles containing the RNA-directed DNA polymerase activity could have been alteredbyvariousproteolyticorother deg-radative enzymes.Totestthis, [32P]phosphoric

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REACTION TIME (MINUTES)

FIG. 4. Assay for DNA polymerase activities in

the plasma of normaland leukemic guinea pigs. A 15-mlsample ofplasma from leukemic guinea pigs anda30-ml plasma sample from normal guinea pigs wereconcentrated andpurified by equilibrium

cen-trifugation insucrose asdescribed in the text. The

plasma concentrates were then added to a

poly(rC):oligo(dG)-templated reaction mixture. Symbols: *, plasma from leukemic guinea pigs (the

assaycontains20mMMgCl); 0,plasma from

nor-mal guinea pigs (theassaycontains 20 mMMgCl2); El, plasma from leukemic guinea pigs (the assay

contains4 ug ofoligo(dT)(1_18), insteadof4 pgof

poly(rC):oligo(dG), and 20mMMgC12).Atindicated times 10-pl aliquots from100-pl reaction mixtures werewithdrawn and assayed for trichloroacetic

acid-precipitable radioactivity. The picomoles of [3H]dGMP (specific activity, 3,000 counts/minper

pmol) incorporated inatrichloroacetic

acid-precipi-tablepolymeraregiven versusreactiontime.

acid- and [3H]uridine-labeled BUdR-induced GPV (17)was addedto plasma from leukemic guinea pigs and incubated at37 Cfor 1 and 24 h. The buoyant density of the labeled BUdR-induced GPV wasthen determined after equi-librium centrifugation in sucrose for 3 h as described above. Control [32p]_ or [3H]uridine-labeled BUdR-induced GPV, held in cell

cul-turegrowth media at4 C for 24h, bands ata density of1.19 g/ml in a sucrose equilibrium gradient. Incubation of the labeled virus for1h

at 37 C in plasma from leukemic guinea pigs ledtoaloss ofradioactivity in the region of 1.19 g/ml,with ashifttoboththe1.17g/ml and top regions of the gradient, indicating considerable degradation. By24 hthe shift from the 1.19 g/

ml region to the 1.17 g/ml density regionwas

essentially complete, withmostof the radioac-tivityreleasedtothetopofthe gradient. When labeled GPVwasincubated at 37Cfor24hin growth medium, theprofile was similar to that of virus incubated for 1h at 37 C in leukemic plasma. This indicated that the virus is rela-tively thermolabile, since murine mammary tumor virus, in an identical experiment, showed no degradation whatsoever, but the more extensive degradation after 24 h in leu-kemic plasma implies that degradative en-zymes may play an additional role in deter-mining thedensity of any virusparticlepresent in the plasma of leukemic guinea pigs. This experiment illustrates that the density ofthe particle containing the RNA-directed DNA

polymerase activity in the plasma from

leu-kemic guinea pigs could represent a partially degraded oncornavirus with a true density higher than1.16g/ml.

Competitivemolecular hybridization.

Com-petitive molecular hybridization studies were

performedtodetermine whether therewasany

nucleic acid sequence homology between the RNA of the virus particles observed in the plasma of leukemicguinea pigs andthe RNA of the BUdR-inducible GPV. The induction proce-dure, the 3H labeling, and extractionof the

60-70SRNAof the BUdR-induced GPV have been described (17). The competitive hybridization assay wascarriedoutasfollows: 3H-labeled

60-70S RNA from BUdR-induced GPV was an-nealedtoDNAfrom normalguinea pigspleen cells. Unlabeled RNA from BUdR-induced GPV (as a positive control) as well as RNA from other sources was addedto this mixture, and thepercentage of competition of theannealing

reactionbetween the 3H-labeled GPV RNA and guinea pig spleen DNAwasmeasuredby the amount of the [3H]BUdR-induced GPV RNA that is rendered RNase sensitive. Virus parti-cles from 200 ml of plasma from leukemic guineapigswerepurifiedby sedimentation and

equilibrium density gradient centrifugation.

The fractions with densities between 1.13 and 1.19g/ml wereconcentrated and used for RNA isolation using the sodiumdodecyl

sulfate-Pro-nasepredigestionmethod (17). A

65-p,g

amount

of RNA was obtained from 200 ml ofplasma

fromleukemicguinea pigs. Less than0.5,ugof RNAwasobtained from200ml ofplasmafrom normalcontrol strain2guinea pigs obtainedin the same manner.

The addition ofincreasing amountsofRNA isolatedfrom the BUdR-induced GPV (Fig. 6,

opencircles)orthe plasmaof leukemicguinea

pigs(Fig.6,closedcircles)displacedthehybrid

formation between 3H-labeled 60-70S RNA of BUdR-induced GPV and guinea pig spleen

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FIG. 5. Equilibrium density centrifugation analysis ofparticlesinplasma from leukemic guinea pigs,as

assayed byRNA-directedDNApolymeraseactivity. Plasma samples (100 ml) from leukemic guineapigswere

concentratedandsubjectedtoequilibrium centrifugationinsucrose(A)orCsCI(B)asdescribed in thetext. Fractions were pooled and concentrated by ultracentrifugation. The resulting pellets were used in a

poly(rC):oligo(dG)-templated assay using 20 mM MgCI2 (-) or 0.4 mM MnCl2 (E). The picomoles of [3H]dGMPincorporatedin atrichloroaceticacid-precipitable polymerina40-min assay are given versus the averagedensity valueof thepooled fractions.

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20

-40

-60

_-80

-6 5 4 3 2

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LOG(CELLULARDNA:ADDEDRNA) FIG. 6.Displacement ofthemolecularhyb

tion betweenpurified3H-labeled60-70SRN, BUdR-inducedGPV and DNAfragments of?

guinea pig spleens by the addition ofcom RNA.CompetitorRNAsaddedwere asfollot labeled RNA from BUdR-induced GPV(0) extracted fromparticlesfrom the 1.13 to1.1

fraction of plasma from leukemia guineapih

andRNAofRauscherMuLV (A). RNAswere

attheindicated levelstocompetewith thehyb

tionof radioactive RNA.Thehybridswereinc toaCatvalueof 40,000and thenassayed for

RNA by at least 90%. This indicated that, in

theplasmaof leukemicguinea pigs, RNA

mole-cules arepresentwith anucleic acid sequence

at least 90% homologous to the RNA of the

BUdR-induced GPV. No displacement (less

than2.5%)of hybrid formationwasobservedby the addition of the small amount of RNA

ob-tainable from 200 ml ofplasma from

normal

guinea pigs. The addition of RNA isolated from Rauscher MuLV also didnotdisplacethe radio-activelabel inthishybridformation. Further-more, neither the RNA of the BUdR-induced

GPVnorthe RNA from the "viral" fraction of

plasma from leukemic guinea pigs competed

with thehybrid formation between 3H-labeled

MMMTV

6070S

RNA and mouse mammary tu-morDNA; thisindicated littleornonucleic acid sequencehomology between MMTV RNA and

'ridiza-Afrom normal Lpetitor ws:

un->,RNA

'9gIml gs (0), ?added

~ridiza--ubated RNase

resistanceasdescribed in thetext. Thepercent hy-bridization ofradioactively labeled BUdR-induced

GPV60-70SRNAtoguineaspleenDNA inthe ab-senceofanycompetitor RNA(60%)wasnormalized to100%aftersubtractionof theamounthybridizedto calf thymus DNA under identical conditions

(11.4%). Thepercentdisplacement of the 3H-labeled

BUdR-induced GPV 60-70S RNA in the hybrids

formed withguineapigspleen DNA is givenversus

the ratio ofthe logof the cellularDNA to added

competitorRNA.

A B

SUCROSE CsCI

20

15-10

5

-~~~~~

I

,,

ol

A 0

0

I

A

n

-0

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[image:7.503.68.260.370.544.2]

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the RNA in theparticles from guinea pigs and also indicated the absence of any RNase or other substance that would have interfered withthehybridization assay.

Although the extent of competition by the RNA from leukemic plasma RNA was com-plete, as shown inFig. 6, the amount of RNA needed to effect 50% competition, compared

with the control (competition with unlabeled 60-70S viral RNA from the BUdR-induced GPV), indicated thatonlyapproximately5%of the RNAisolatedfrom theplasmaofleukemic guinea pigs contained nucleic acid sequences homologous to the 60-70s RNA of the BUdR-induced GPV. Because ofthe high level of ne-crosis observed by electron microscopy in the tissuesof the leukemic guineapigsatthetime ofcollecting theplasma,ahighlevel of contam-inating cellular RNA could be expected. As shownabove, approximately5%of the65,gof RNA from 200 ml of plasma from leukemic guinea pigsis RNA thatishomologous tothe

BUdR-induced GPV 60-70S RNA. Assuming

that the60-70S genome ofanRNAtumorvirus

weighs approximately 7 x 10-12 ,ug (28), then the frequencyof virusparticlesidentical to the BUdR-induced GPV in the plasma of leukemic guinea pigs is approximately 2.5 x 109 parti-cles/ml. This titer should be taken as a mini-mum valuebecause it assumes 100% recovery of the viral-related RNA.

Electron microscopy. Electron microscopyof selected areas of high-speedpellets of leukemic plasma revealed ahigh concentration of virus

particles (Fig. 7A and B). Although most of

theseparticles contained condensed nucleoids, alow percentage was immature and exhibited thedouble-shell structure of the nucleoid typi-cally observed inbuddingor detached but im-mature particles. In most cases(Fig. 7A, small arrows), the nucleoids were not completely

formed and the inner layer was moreelectron dense than the intermediate layer. Occasion-ally, the nucleoidappearedtobecompleteand,

in afraction of these nucleoids, the two layers

appearedtobeofapproximately equalelectron

density (Fig. 7B,large arrow). The latter parti-cle issimilar in appearance to immature forms

/'''

'

':'

it t.;

$~~~~~~~~15i

6,<

t

4A

i<4er

Is.,Fwte

*m

~

S~-4k14

IL~~~~~~~If

FIG. 7. Electron microscopyofvirusparticlespresent in theplasma ofleukemicguineapigs. (A andB)

Thin sectionofaselectedareaofa high-speed pellet.Mostparticleshave condensed nucleoids, butseveral (arrows) donot.(CandD)Typicalnegativelystainedparticlespresentintheplasma fromleukemicguinea

pigs.(E and F)Lessfrequentlyobservedparticleswithspikes10nminlength.

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[image:8.503.49.450.327.619.2]

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ofthe BUdR-induced GPV. Note that many of the matureparticlesappearpartiallydegraded, with the outer membrane broken or partially missing.

Negativelystainedplasmasamplesfrom leu-kemic animalsappeared to contain two types of particles. Themajorityof theparticles (Fig.7C and D) were devoid ofspikes. The core could frequently be observed under these staining conditions (Fig. 7D). Occasionally, particles with spikes could also be observed in plasma preparations (Fig. 7E and F). The spikes are approximately 10 nm in length, and the particles appear similar to negatively stained MMTV and BUdR-induced GPV (4). Electron microscope analysis of plasma samples from leukemic guinea pigs, therefore, indicatesthat twopopulationsofviruses appear to be present. The relative levels cannot be estimated with any accuracy; the majority ofparticles appear to be derived from the intracisternal particles ofthe L2C tumor cells, and a smaller but still significant level of particles appears to be simi-lar to the BUdR-induced GPV obtained from supernatantfluidsofculturesofnormal guinea pigcells (4).

DISCUSSION

Subcutaneous inoculation of L2C guinea pig leukemia cellsintostrain2guineapigsresults, atapproximately day12, in arapid increasein theleukocytecount,the appearanceof extracel-lular virus particles as observed by electron microscopy, and the appearance of particles with anRNA-directedDNApolymeraseinthe plasma of these animals (Fig. 1).Itisclear that the appearance ofthe enzyme activity in the plasma of leukemicguinea pigs (Fig. 1) occurs veryrapidly. This coincides with themaximum level of necrobiosis that would release cellular polymerases but that alsoappears tobe critical for the release of the intracisternal virusfrom the L2Ctumorcells (Dahlberg and Perk, man-uscriptinpreparation). It is, therefore, impor-tant toestablish whether the DNApolymerase activityobservedispresent inplasmaas a con-stituentof a virusor as aconstituentofa cellu-lar substructure. The following data are con-sistentwith theenzymeobserved beingan on-cornavirus RNA-directedDNApolymerase. (i) Thedensity ofthe particle in which the DNA polymerase activityispresent inthe plasma of leukemicguinea pigs isapproximately 1.16 g/ ml insucrose and 1.17 g/ml in CsCl (Fig. 5). In reconstruction experiments, radioactively la-beled BUdR-induced GPV, with a density of 1.19g/ml, underwent ashift in density to 1.17 g/ml when incubated in leukemic plasma at

37C for 24 h. This indicated that the original

densityoftheparticle containingtheDNA po-lymerase activity in the plasma could have

beenhigherthanthe observed values of 1.16g/

ml in sucrose and 1.17 g/ml in CsCl. (ii) The DNA polymerase activity in the plasma from leukemic guinea pigs preferred the synthetic

template poly(rA):oligo(dT) over

poly(dA):-oligo(dT) (Fig. 2). TheonlyDNApolymeraseof cellularoriginthusfarreported(8)thatprefers

the synthetic template poly(rA):oligo(dT) over

poly(dA):oligo(dT)doessowithacation

prefer-encefor Mn2+ (9, 16). TheDNA polymerase of the plasma from leukemic guinea pigs cannot use Mn2+ under any conditions tested (Fig. 2) and is therefore different from that cellular DNA polymerase. (iii) The DNA polymerase activity ofplasma from leukemic guinea pigs utilizes the synthetic template poly(rC):oligo-(dG) only in the presence of Mg2+, with an

optimum at20 mM. The Mg2+ titration result (Fig. 3) is identical to that of the reverse tran-scriptase activity of the BUdR-induced GPV

(17).

When a sample of leukemic plasma was

con-centrated and centrifuged to equilibrium on a

sucrosegradient, the RNA extracted from frac-tions containing maximum levels of RNA-di-rected DNA polymerase completely displaced the annealing reaction between radioactively :TA-labeled 60-70S RNA isolated from BUdR-induced GPV and DNA from normal guinea pig spleens (Fig. 6). No competition of this annealing reaction was observed by the addi-tion of unlabeled RNA from the plasma of normal guineapigs.Becauseofthe deteriorated appearance of mostof the virus particles (Fig. 7A and B) and the presence ofhigh levels of RNase in the plasma ofthe leukemic guinea pigs, ourinability to isolate pure 60-70S RNA from the plasma of leukemic guinea pigs ap-pears due to the RNAbeing partially degraded atthe timeofbloodcollection. Itispossiblethat the RNA extracted from leukemic plasma, which is identical to the RNA of the BUdR-inducedGPV, could represent either one of two ormorepopulationsof viral RNA or viral RNA among an excess of cellular RNA. It is impor-tant to note that negatively stained prepara-tions ofplasmafromleukemicguinea pigs con-tainparticleswithspikes10 nm inlength(Fig. 7E and F)similarinappearance tothe MMTV andtheBUdR-inducedGPV.Thedataobtained from these experiments and electron micro-scopeobservationspermit twoexplanations. (i) The observed population of virus particles in the plasma from leukemicguinea pigs is com-posed oftwodifferentvirusparticles,the

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cellular virus particles derived from intracis-temal virusparticlespresentintumorcells and virus particles identical to the BUdR-induced

GPV. (ii) The extracellularvirus particles de-rived from L2C tumor cells are related to or

identicaltotheBUdR-inducedGPV.The RNA-directed DNA polymerase activity and the RNAobservedinthemolecular competition hy-bridization (Fig. 6) would then be ascribed to

one type ofvirus, which exhibits a variety of

morphological states in electron micrographs.

Thehigh level ofRNA-directed DNA

polymer-ase activity observed in the plasma from

leu-kemic guinea pigs and the minimal estimate of

2.5 x 109particles/mlindicatethat theplasma

containsrelatively high levels of particles simi-lar or identical to the BUdR-induced GPV. Thus,itwouldbepossibletoarriveatthe work-inghypothesisthatonlyonevirusispresentin

the plasma and that itexhibits variable

mor-phology, depending onwhetherit isformedin

vivoorinvitro.

Definitive proofthattwodifferentvirusesare

present or only one virus is present in the

plasma ofleukemic pigshasnotbeenobtained fromthestudiesreportedhere.Theappearance

of high levels ofavirusrelatedto, oridentical

to, the type BBUdR-induced GPVduring the

terminal stages of the L2C leukemia is, how-ever, an unusual occurrence and warrants

furtherattention.

ACKNOWLEDGMENTS

WethankA.J.Dalton and D. Colcher formanyfruitful discussions andsuggestions, G.Schirmer and J.Torgersen forexcellenttechnical assistance,and D.Jonesfor photo-graphicservices.

Thisworkwassupported byPublicHealth Service

con-tracts WC1-CP-43223 and 3391 within the Virus Cancer Program of theNational Cancer Institute.

K.PerkisaVisiting Scientistfrom TheHebrew Univer-sityofJerusalem, Rehovot,Israel.

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