Spontaneous regression of Friend virus-induced erythroleukemia. VI. Structural and antigenic differences between the regressing and conventional strains of virus.

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Vol. 33, No. 3 JOURNAL OFVIROLOGY, Mar.1980,p.1083-1096

0022-538X/80/03-1083/14$02.00/0

Spontaneous

Regression

of Friend Virus-Induced

Erythroleukemia

VI. Structural and Antigenic Differences Between the Regressing and

Conventional Strains of Virus

PHILIP FURMANSKI,* CLIFFORD LONGLEY, CHRISTOPHER S. BOLLES, DAVID L. HINES,AND

MICHAEL DIETZ

Departmentof Biology, Michigan Cancer Foundation, Detroit, Michigan 48201

The regressing and conventional strains of Friend virus were compared by

neutralizationassays, sodiumdodecylsulfate-polyacrylamidegelelectrophoresis, andtryptic peptide mapping of the individual viralcomponents. Neutralization rates of the two viruses differed in the presence of monospecific anti-gp70 antiserumandserafromregressedorimmunizedmice. Neutralization of regress-ing Friend virus, butnotconventional Friend virus,occurred when the viruses were incubated with anti-pl5(E) and complement. Human serum inactivated conventional Friend virusmorerapidly than regressing Friend virus, probablyas aresult ofvirolysis induced by the reaction of viral p15(E) with human comple-mentcomponent Cl. Structural differences between the viruses were detectedin theirgp7O viralglycoproteinsandp15(E) andp12proteins. Analysis of different stocksand clonalisolatesof theviruses showedthatthedifferences betweenthe gp70 and p15(E), butnotthe p12proteins, were associatedwith the regressing phenotype of theregressing strainof Friend virus.

In contrast to the progressive, chronic, and lethal erythroleukemia caused in miceby con-ventional strains of Friend virus (CFV), the re-gressing strain of Friend virus (RFV) inducesan

erythroleukemia that spontaneously regresses (28, 30). Both viruses induceaninitially identical disease characterizedby massive splenomegaly

and viremia. However, at 30 to 60 days post-virusinoculation, when the mice inoculated with CFVbegintodieduetotheirdisease,the

eryth-roleukemia in the mice inoculated with RFV

spontaneously regresses and, in about half the leukemic mice, the animals return to a

histo-pathologicallyandvirologicallynormalor near-normal state. This characteristic of the RFV strain of Friend virus isstable through routine

sequentialviruspassageandtoviruscloning.

Our previous studies have established that immunological reactivity isanessential compo-nent ofthe process ofregression and that re-gressed mice developpotentantivirus and anti-leukemia cell immunologicalactivity (5, 17, 29; C.S.Johnson, S.Fouchey,and P.Furmanski,J. Natl. CancerInst.,in press). Bothhumoraland cell-mediatedspecificimmunereactivitycan be detected in regressed mice. We have also sug-gestedthat thedifferencebetween the RFV and conventional strains of virus which results in disease regressionis that RFV or its structural components are morehighly immunogenicthan thoseof CFV(14).

These data have ledus tocomparethe struc-turalcomponents of RFV andCFV,both phys-ico-chemically and immunologically. We report here that in neutralization and neutralization kinetics assays RFV and CFV are distinguish-able on the basis ofreactivity with sera from regressed mice, from immunized mice, and with monospecific antisera againstgp70 andp15(E).

Furthermore,theviralgp70 and p15(E) compo-nentshave differenttryptic peptidemaps.

MATERIALS AND METHODS

Viruses. RFVwasmaintainedbyserial passage in

random-bredSwiss/ICRmice. Virusstockswere

pre-pared as20%cell-free spleen homogenatesfrom

leu-kemic mice and stored at -70°C. Unless otherwise

indicated,the CFV used in theseexperimentswasthe

N-tropic,nonregressingFriendvirus,PEN,originally

obtained from Robert J. Eckner,Boston University

School of Medicine, and maintained, as described

above,byserial passage inNIH/PLCRmice.

For use in polyacrylamide gel electrophoresis (PAGE) and tryptic peptide mapping experiments,

virus waspurifiedfromculturefluids of infectedNIH/

3T3 cells. Spleen homogenates were diluted 20-fold

withDulbecco modifiedEaglemedium(DMEM),

fil-tered, and inoculatedontocultures ofNIH/3T3cells

in 75-cm2 plastic tissue culture flasks.The cellshad

beenpretreated for1 hwith 25ug of DEAE-dextran

per ml and then washed with serum-free medium.

Afterinfection for 1 h at 37°C, 20ml of DMEM + 15%

calfserum (K-CBiologicals)was added to eachflask.

Three days after infection, thecellsof each flaskwere

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trypsinized andtransferred to a 490-cm2 plasticroller

bottle(Corning) in 60 ml of DMEM + 15% calf serum.

Thefollowing day, the medium was replaced with 60

ml of DMEM + 5%heat-inactivated fetal calf serum

(K-C Biologicals), and on day 7 postinfection, the

bottles were fedagain with the same medium.

Super-natants for viruspurification was harvested on days

10, 12, and 14 postinfection. The fluids were

centri-fuged at10,000 x g for 10min and then layered over

apadof 20% sucrose in TNE (0.01 M Tris, 0.15 M

NaCl, 0.002 M EDTA, pH 7.4) in an SW 27 tube and

centrifuged at 25,000 rpm for 60min. The pellets were

suspended in TNE buffer, layered onto a 20 to 50%

sucrose gradient in TNE in an SW 50.1 tube and

centrifuged at48,000 rpm for 60min. Virus bands were

removed, diluted with TNE buffer, and rebanded in a

20 to 50% sucrose gradient. The virus bands were

diluted with TNE buffer andpelleted in an SW 50.1

rotor at 48,000 rpm for 60 min. The pellets were

drained, and the tubes were sealed and stored at -700C.

For thepreparation of radiolabeled virus, the

me-dium in theroller bottleswasreplacedwith 30 ml of

leucine-free DMEM containing 5% dialyzed,

heat-in-activatedfetal calfserum and either30uCi of

[3H]-leucine (Amersham Corp.) per ml or 4 MuCi of

["C]-leucine (Amersham) per ml. Supernatant fluids were

harvested and viruswaspurifiedasdescribed above.

Pseudotypes of the Kirsten sarcoma virus

(Ki-MuSV) werepreparedwith RFVand CFV forusein

theneutralization and neutralization kinetics assays.

Cultures ofnonproducerK-NIH cells(obtainedfrom

S. Aaronson, National Cancer Institute[1]) were

su-perinfected with about 500PFU of the appropriate

murineleukemia virus(MuLV). Supernatants of the

infected cultureswereharvestedat7 or 14days

post-inoculation and titrated forfocus-forming activityon

NIH/3T3 cells (see below).Pseudotypes ofMoloney

sarcomavirus(MoMuSV)werepreparedin thesame

way from cultures of a transformed nonproducer

MoMuSV-NIH/3T3 cell line isolated in our labora-tory.

Antisera.Monospecificgoat antiseratoRauscher

virus-derived gp7O, p30, p15, p12, and plOwere

ob-tainedthrough the Office ofProgramResources and

Logistics, Virus CancerProgram,National Cancer

In-stitute. Rabbit antiseratoRauscherp15(E) were

ob-tained from E. Fleissner, Memorial SloanKettering

Cancer Center, and S. Oroszlan, Frederick Cancer

Research Center.Regressedserawerepoolsfrom

in-dividualregressedmice. Immuneserum wasobtained

by inoculationof strain129/Jmice(Jackson

Labora-tories) with RFV; those mice that did not become

leukemicwere reinoculated with virus40dayslater

and then reinoculated again after another 40 days.

Fourteendaysafter the lastinoculation,theanimals

werebled, and theirserum wasseparatedand stored

at-70°C. Humanserum wasobtained from blood of

healthydonors,filtered,andstoredat-70°C.

PAGE. Virusproteinswereseparatedandanalyzed

by sodiumdodecylsulfate(SDS)-PAGEin15%

acryl-amide,15-cmgelsprepared bythe method ofLaemmli

(23). The gelswereelectrophoresed at1mApergel

for 16 h.Alternatively,thehighresolutiongelsystem

describedby Montelarowasusedexactlyasdescribed

earlier (24, 25), except for the equimolar substitution ofB-mercaptoethanolfor themercaptopropionic acid.

When the polypeptides were labeled with 125I (see

below), the gels were frozen, sliced on a Mickle gel

slicer (BrinkmannInstruments), and two 1-mm

seg-ments were counted in a Searle automatic gamma

counter.Gels of3H-or"4C-labeledvirus, or both, were

pulverized in2-mmsegments directly intoscintillation

vials with a Gilson automatic gel fractionator. The

radioactivity wasdetermined in a toluene-Triton

X-100scintillation cocktail with a Packardscintillation

spectrometer. Protein standards ("nI-labeled bovine

serum albumin, ovalbumin, chymotrypsinogen, and

RNase) were included with eachgel run for

determi-nationof molecular weights.

Bromelaindigestion. Intact, purified virus was

digested with purified bromelain as described earlier

(25, 26). Afterdigestionfor3hat37°C, the virus was

collectedbycentrifugationthroughadiscontinuous 20

to50%sucrosegradient andpelleted. Thepelletswere

dissolved in 1%SDS, 1%,B-mercaptoethanol, 0.01 M

phosphate buffer, pH 7.2, heated for 4 min at 100°C, andanalyzedby SDS-PAGE.

Trypticpeptide mapping.A

10-pl

amountof

pu-rifiedvirus (1 mg ofprotein/mlin water) was added to

40

pi

of TNE+0.2% Nonidet P-40(NP-40) and

incu-batedat4°Cfor30min.One mCi of'25I(Amersham)

wasthenadded,followedby20

pl

ofchloramine T (4

mg/ml). After incubationatroomtemperature for10

min, the reactionwasterminated with50

pl

of sodium

metabisulfite (5 mg/ml). The mixturewasdilutedwith

100

pl

of TNE containing 2% NP-40, 1% sodium

de-oxycholate, and 10 mM KI. Phenylmethyl sulfonyl

fluoridedissolved inisopropylalcoholwasthenadded

to a finalconcentration of100,uM. The labeled virus

was dialyzed against TNE containing 0.2% NP-40,

0.05% sodiumdeoxycholate, and 5mMKI. After

di-alysis,phenylmethyl sulfonylfluoridewasagainadded

to 100MM.

Individual virusproteinswereimmunoprecipitated

from the labeled preparation and purified by

SDS-PAGE as follows: the virus suspension was diluted

with TNE containing 0.2% NP-40, 100MLM

phenyl-methyl sulfonyl fluoride, and 1 mg of bovineserum

albumin per ml. Normal goatserumwasadded(2

pi),

and the mixturewasincubatedfor1h at4°C, followed

by the addition of20

pl

ofa10%suspensionof

Staph-ylococcusaureus, Cowan strainI,prepared exactlyas

describedpreviously (21).After incubationat4°Cfor

30min, the bacteriawereremovedbycentrifugation

(2,000xg,20min),and thesupernatantwasusedfor

immunoprecipitation. A

2-pl

amountof the

appropri-atemonospecificantiserumwasaddedto200

pl

of the

labeled virus, and the mixture wasincubatedat4°C

for 1h. Staphylococcusorganismswereadded,

incu-bated, and removed asdescribed above. The pellet

containing the bacteria and antigen-antibody

com-plexeswasthensuspendedin0.5ml ofTNEcontaining

0.5% NP-40and 0.02% sodiumazide. Thesuspension

waslayeredover1mlofasolution of TNEcontaining

5%sucrose, 0.5% sodiumdeoxycholate,and 3%NP-40.

Aftercentrifugation (2,000xg,10min),thepelletwas

washed with TNE+ 0.5% NP-40and drained. The

complexes were dissociated with 0.01 M phosphate

buffer,pH7.4,2%SDS,and 2%

fB-mercaptoethanol

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STRUCTURE OF REGRESSING FRIEND VIRUS

100°C for3 min, and the bacteriawere removedby

centrifugation (2,000 x g, 10 min). The supernatant

wasthenremoved and electrophoresedon

high-reso-lutionSDS-PAGEgels,preparedasdescribed above.

After electrophoresis, the gels were frozen and

sliced, and the slices were counted in the gamma

counter. The slices containing the specific, purified,

immunoprecipitatedproteinwerepooled and washed

twice for30mineachat roomtemperaturewith 50%

methanol, 10% acetic acid inwater. The sliceswere

thendried ina vacuum ovenandrehydrated with0.5

mlof0.05M NH4HCO3, pH 8.0, containing 25 Lg of

L-(tosylamido 2-phenyl)-ethyl chloromethyl

ketone-trypsin (269U/mg;WorthingtonBiochemicalsCorp.).

The slices were incubated overnight at 37°C with

continuous gentle shaking. The supernatant was

re-moved andlyophilized.

The tryptic peptides were analyzed by

2-dimen-sional thin-layer electrophoresis/chromatography

(10). Thepeptidesweredissolved in acetic acid-formic

acid-water, 15:5:80, applied to a 0.1-mm

cellulose-coated thin-layerchromatography plate (EM

Labo-ratories), andelectrophoresed at900V for about 35

minat0°C. Theplatewasthen dried and

chromato-graphed in the second dimension in

butanol-pyridine-aceticacid-water,32.5:25:5:20.Theplatewasdried and

exposed to Kodak X-Omat R film.A single Cronex

Lightning PlusX-rayintensifyingscreen wasusedto

enhance theautoradiography(35).

Insomeinstances,sequential immunoprecipitations

werecarriedout onsingle labeled virus preparations.

Differences intryptic peptide mapswereconfirmedby

analysis of admixtures of the tryptic peptide prepara-tions.

Neutralization assays. The KiMuSV

pseudo-typeswerediluted with serum-free DMEMtoafinal

titerof about400focus-forming units per ml. Portions

of0.5 mlwere added to tubes containing0.5 ml of

serialdoubling dilutions of thetestantiserum made in

Dulbeccophosphate-bufferedsaline(GIBCO

Labora-tories).The mixtureswereincubated ina37°Cwater

bath for 1 h and chilled, and 0.1-ml portions were

inoculated onto NIH/3T3 cells in the individualwells

ofamultiwell dish (16-mmwells, COSTAR). Thewells

wereseeded the previous day with10"cells in DMEM

containing 15% calfserumand2,ugofpolybrene

(Ald-richChemical) per ml and washedoncewith DMEM

before inoculation. After the addition of the

virus-antibody mixture, thedisheswereincubatedfor 1 h at

37°Cin 10% CO2in air, andthen 0.4 mlof DMEM

with 15% calf serum was added to each well. The

following day andonday4post-virus inoculation, the

cultures were fed with DMEM containing 5% calf

serum and 1%dimethyl sulfoxide (22). On day 7

post-infection, the cells were fixed and stained with Giemsa.

Fociwerecountedmicroscopically.

For neutralization assays with complement, freshly

reconstituted,lyophilizedguinea pig serum (Colorado

Serum Co.) was addedto the virussuspension to a

final concentration of 5% before admixture with the antiserum.

All neutralization assays were carried out at least in duplicate.

Neutralization kinetics assays. The KiMuSV

pseudotypes of thetestviruseswere incubated with a

single dilution of serum, andatthe timesindicated,

portionswereremoved,diluted,chilled,and tested for

residual virus. The concentration ofserum used in

these assayswasgenerallytwo toeight timeshigher

than the concentration which caused 50%

neutraliza-tion ofthe moresensitivepseudotypeintheendpoint

assaysdescribed above. Portions of0.5ml of theserum

diluted in phosphate-buffered saline were added to

tubes. Anequal volume ofvirus,diluted in DMEMto

contain about 4,000focus-forming units per ml was

thenadded and mixed inaVortexmixer,and0.1 ml

wasimmediatelyremoved withanautomaticpipettor

(zero timesample).Theremainingvirus-antibody

mix-turewasincubated, with occasionalgentle shaking,in

a 37°C water bath. At 10, 20, 40, and 60 min of

incubation, 0.1-ml samples were removed.

Immedi-ately afterremoval, each sample, includingthe zero

time sample,wasaddedto aprechilledtubecontaining

0.9mlof DMEM and mixed inaVortex mixer. The

dilutedsampleswereheldoniceuntil the serieswas

complete, and they werethen inoculated in

quadru-plicate ontomonolayers ofNIH/3T3 cells in 16-mm

multiwellplates for virus assayasdescribed above.

Virus clones. Cultures containing 105

DEAE-dex-tran-treated NIH/3T3 cells in 60-mm dishes were

infected with 100 XC PFU of virus (4). Thecells were

trypsinized and inoculated at 100cells perwell into

the individual wells ofa Microtest II plate (Falcon

Plastics).Whenthecellshad grown toconfluence, the

supernatantswereremoved and frozen, and thecells

in thewellswereUV irradiated and overlaid with XC

cells. Thesupernatants from XC-positive wells (-10

per96-wellplate)werethen usedtopreparestocks in

fresh NIH/3T3 cells (see above) and to prepare

pseu-dotypes in K-NIH cells,asdescribed above.

RESULTS

Immunological analysis of viral

compo-nents by neutralization. Differences in the antigenic properties of RFV and CFVwerefirst determinedby usingneutralizationassays.This approach, although limited to the analysis of external proteins or glycoproteins, relates di-rectly to the major humoral antiviral immune response detectable in regressed but not leu-kemic mice (17, 29; M. Dietz, D. Hines, M. A.

Rich,andP.Furmanski, Proc. Am. Assoc. Can-cerRes. 19:156, 1978) andmight, therefore, be relevantto theprocessofregression.

The effect of monospecific antisera against each of themajor viral polypeptideswastested inneutralizationassaysbothinthepresenceand absenceofcomplement. Aspreviouslyreported (13,32;M.Kende, S. Oroszlan,R.Donahue, and G.Kelloff,Proc. Am. Assoc.Cancer Res. 18:101, 1977), in the absence of complement, neutrali-zation wasobtained withanti-gp7O serum (Fig. 1B), but not with antiserum against p12 (Fig. 1A)orp30, p15,orplO (data not shown). In the absence of complement, anti-pl5(E) did not causeany greater degree of neutralization than did normal rabbit serum(Fig.

10).

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Theapparentneutralizingactivity of the anti-gp7Oserum wasincreasedtwo- tofourfold inthe presenceofcomplement (Fig. 1B). As expected, noneof theantisera against thegag gene prod-ucts (p30, p15, p12, plO) caused virus neutrali-zation in the presence ofcomplement. In con-trast, anti-pl5(E), which didnothave neutral-izingactivity against RFVorCFV in the absence ofcomplement, exhibitedavery strong neutral-izingeffect in itspresence,butonly against RFV. Atthe dilutions used in theseexperiments,

com-z A

0

-so

Geo

40-o 0

plementalone did not have any effect on either virus, nor did heat-inactivated (560C, 30 min) complement,either in the presence or absence of anti-pl5(E) (datanotshown).

[image:4.504.69.463.193.342.2]

To further assess immunological differences between the viruses, neutralizationassays were carriedout, usingserafromregressed mice and strain 129/J miceimmunizedwithRFV as test reagents. Bothserahad potentneutralizing ac-tivity against RFV and CFV which was en-hancedtwo- tofourfoldbycomplement(Fig. 2).

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DILUTION

FIG. 1. Neutralization ofRFVandCFV bymonospecificgoatantisera.(A)Neutralization by anti-p12and

normalgoatserum;(B) neutralization by anti-gp7O; (C) neutralizationby anti-pl5E and normalrabbitserum.

Neutralization ofRFVbyantiserum (0, 0); neutralization of CFVby antiserum (0, U); neutralization of

RFVbynormalserum(A, A);neutralizationofCFVbynormalserum(V,V). Neutralization in the absence

of complement,closedsymbols;neutralization inthepresenceofcomplement,opensymbols.

100

z 0

F:

Lu

n a

C-)

0

80

60

40

20

0

40 80 160 320 640 1280 40 80 160 320 640 1280

-I

DILUTION

FIG. 2. NeutralizationofRFV and CFVbymousesera.(A)Neutralizationby regressedmouse serumand

normalmouseserum; (B)neutralizationbyimmunemouse serum.NeutralizationofRFVbyantiserum (0,

0); neutralizationofCFVbyantiserum(0, U;neutralizationofRFVbynormalserum(E, A);neutralization

of CFV by normalserum(V, V).Neutralization in the absenceof complement,closedsymbols;neutralization

in thepresenceof complement,opensymbols.

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Asmallbut consistentdifference in theactivity of these sera against the two viruses was

ob-served.

Immunological analysis ofviral

compo-nentsby neutralizationkinetics. The results

obtained in the experiments described above

wereconfirmed and extended, using

neutraliza-tion kinetics. For these assays, antibody at a

single dilutionwasaddedtothe twoviruses,and

the rate of reduction in focus formation was

1 O.S

1.00

0.5

0.2

0

4-(9

z

0

-)

4 0.1

.05

determined. In this way, small differences in

antigenicityorreactivityintheendpointassays

could be magnified andevaluated.

The kinetics of neutralization of RFV and

CFV pseudotypes of KiMuSV differed when

tested withserafromregressed mice,immunized

strain 129/J mice, or monospecific goat

anti-gp70 (Fig. 30). Since neutralization with the

latter serum gave results whichwere the con-verseof the results obtainedwith theregressed

A

_ B

D

.02

.01

1.0

0.5

E F

0.2

0.1

.05

.02

F

.01 I , I

0 10 20 40 60 0 10 20 40 60

MINUTES

FIG. 3. NeutralizationkineticsassaysofRFV and CFV.(A)Neutralization of RFV in the absence ofserum

(0),orin thepresenceof normalmouse(O)ornormalgoatsera(A);(B) neutralization of CFV in the absence

ofserum(0), orin thepresenceof normalmouse (U)ornormalgoat(A); (C) neutralization ofRFV (open symbols) and CFV (closed symbols) in thepresenceof regressedmouse serum(0, 0), immunemouse serum

(El, ),oranti-gp7Oserum(A, A); (D)neutralization ofMoMuSVpseudotypes ofRFV andCFV by regressed, immune,oranti-gp70sera(symbolsarethesame asinC);(E)neutralization ofRFV-B23 andCFV-FES-7by regressed, immune,oranti-gp70sera(symbolsarethesame asinC);(F) neutralization of RFV (0,O,A)and CFV(0, Ui A) by three different pools ofserafrom regressedmice (0,O,A).

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orimmune sera,it isunlikelythat thedifferences in ratesof neutralizationweredueto aninherent susceptibility of RFV to neutralization or an excessofcompetingantigenin oneof the virus stocks.

Toeliminatepotential artifacts duetotheuse of KiMuSV focus-forming pseudotypes in the

assaymethod, similar determinationsweremade

usingpseudotypes ofMoMuSV, withthe same results (Fig.3D). Neutralization kineticsassays withthe intact Friend viruscomplexes,using in vivospleen focus fornation for virus titration, also showedasubstantial difference in therate ofreaction of RFV andCFV with anti-gp7O and serafromregressed mice (M. Dietz and P. Fur-manski,unpublisheddata).

Thesamedifferential sensitivitiestothe neu-tralizing antiserawereobtainedwhentwo

inde-pendently derived regressing and conventional virus stocks were compared by kinetics assay (Fig. 3E), and the distinction between RFV and CFV was also obtained when the assays were carriedoutusing three otherpoolsofserafrom regressed mice (Fig. 3F).Finally,KiMuSV pseu-dotypespreparedusingotherindependently de-rivedRFV and CFV stocks andaseriesof clonal isolates of theMuLV's of RFV andaCFV (Fig.

4) also showed thesamedifference in neutrali-zationrates asthe

parental

virusstocks.

Neutralization ofvirusbyhumansera.A major difference was detectedin the endpoint neutralizationassays of RFV and CFV with

an-tiserumtop15(E)andcomplement. Neutraliza-tionof MuLV's byhumanserum has been

re-ported and has been showntobe dueto adirect and specific reaction between human

comple-ment component Cl and viral p15(E) in the absence of antibody (2). Complement is acti-vatedby thisreaction,resultinginvirolysis.We therefore used normalhumanserum asanother

reagent tofurther establish differences between the pl5(E)'s of RFV and CFV.

Individualhuman sera were titrated for activ-ity against RFV and CFV. A typical result is shown inFig. 5A.Thekinetics of neutralization of the viruses by this same human serum is shown in Fig. 5B. A clear distinction was ob-served between theratesofreaction of RFV and CFV with the humanserum. Nosignificant neu-tralizationwasdetected ifthe human serum was heat inactivated

(560C,

30min) before use, or if guinea pig complement, which itself does not neutralize MuLV's,wasadded to the heat-inac-tivated humanserum. Thus, theneutralization

of the viruses observed in theseexperiments was likely duetocomplement and its reaction with

anti-p15(E)

asreported(2),andnot to antibody or complement-dependent antibody in the hu-manserum.

Thesignificanceof this reactivity with normal human serum lies in the difference it demon-strates in the p15(E)'s of RFV and CFV.

Fur-thermore, since the resultswith human serum are the converse of the results obtained with neutralization of RFV and CFV byrabbit anti-serum to MuLV p15(E) plus complement, the difference inreactivity is probablynotdueto an inherent difference in susceptibilitytovirolysis, differences in virus concentration or the pres-enceofcompeting proteins, anti-complementary

activity, orother artifacts inthe virus prepara-tions. However, the exact role in regression of this difference in the pl5(E)'s remains to be determined.

PAGE analysis of viral proteins. The structural basis for the immunological

differ-1.0

0.5

100

Zs 0

u

0

a

w

In

0 $0

0

N1- 0.2 _

0 10 20 40 60

DILUTION' MIN.JTES MINUTES

FIG. 4. Neutralization (A) andneutralization ki- FIG. 5. Neutralization kineticsofclonal isolates netics(B) ofRFV(0)andCFV(0)inthepresenceof oftheMuLVofRFV(0)andCFV(0)inthepresence

normalhumanserum. ofimmune129/Jmouse serum.

0.1

0.05

lI i, .

0 10 20 40 60

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encesdetected in the neutralizationassays was firstinvestigated by comparing thepolypeptide

composition ofarepresentativeRFVandCFV,

using SDS-PAGE in 15% gels preparedby the method of Laemmli (23). We found no major differences between the viruses; both prepara-tions contained all of the resolvable viral pro-teins and glycoproteins in approximately the same proportions. However, analysis on the same gel of admixtures of these two viruses labeled with different isotopes (Fig.6A)revealed

thattherewas asmall, reproducibledifference inthe mobilities ofone of the smnll viral pro-teins,identifiedasp12onthe basis ofmigration

and coincidence withimmunoprecipitated viral p12 (datanotshown). Reciprocal labeling(Fig. 6B)andelectrophoresis of mixtures of thesame virustypelabeled with eachisotope(Fig. 6C and D) established that the difference observed in the p12 was notduetolabelingor

electropho-reticartifacts. The difference inelectrophoretic

mobility of the p12 proteins of thetwoviruses

24 7

-20

6-16 5

4.

.92

t8

C

l0 20 30 40 50 60 780 4.

2+

'4

1 .

20

7-B

64

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54.

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2-.4 Il

'0 -30i 0i-0 6 8

lb 2-0

30 40 50 60 70 '

12

10

-8

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6 _

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0

2 -,

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0.~

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2

0

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FRACTION

FIG. 6. SDS-PAGE analysis of RFVandCFV.Symbols:(A)3H-labeledRFV(0), '4C-labeledCFV (0):(B) 'H-labeled CFV(0), 14C-labeledRFV(0);(C)3H-labeledRFV (0),14C-labeledRFV (0): (D)3H-labeledCFV

(0), 4C-labeked CFV(0). Arrow inAindicatesthe positionof viralp12.Migrationofthemolecular weight

markers: b,bovineserumalbumin;o,ovalbumin;c,chymotrypsinogen;r,RNase.

12

10o

8

A

I

N I 0

x

z

0 cr

N.L.

CL

6 4 2Z

20

16+

12+

8+

4+

W--. a

4

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corresponded to an apparent difference in mo-lecular weight of about 600 to 800, as calculated from the relative migration of standard proteins. The high-resolutionPAGE system of Monte-laro et al. (24, 25) was employed to further analyzethepolypeptidecomposition of RFV and CFV. In this gel system the two viruses were

N

0

x

z

0

0 LL

Um.

0

6

5

4

3

2

composed of thesame polypeptides in approxi-mately the same proportions, with one of the smallproteins,identified as p12 (25), differing in electrophoreticmobility (Fig. 7A).

Montelaro et al. have previously demon-strated that the proteolytic enzyme bromelain will remove the major external glycoprotein

;70

°

C

r

A

60

-50

.40

_30

O

-20

z

10 0

w6

.6 s%

B

_a.)

4 (m

3

2

[image:8.504.88.445.150.626.2]

lb

20 30 40 50 60 70 80 90 i00

FRACTION

FIG. 7. High-resolutionSDS-PAGEofintact(A)andbromelain-treated(B)RFV and CFV. Thearrowin

A indicates theposition ofviralp12.Symbols: 3H-labeledRFV(0), 14C-labeledCFV(0).Migrationofthe

molecularweightmarkers:b,bovineserumalbumin;o,ovalbumin;c,chymotrypsinogen, r,RNase.

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gp7Oand thep15(E) from intact virus particles without affecting the internal (inaccessible)

polypeptides p30, p15, p12,andplO (25).When

RFV andCFVweretreated withbromelain,the gp7O and p15(E) peaks of both viruses were

eliminatedorreduced(Fig. 7B). The other com-ponents, including the peak which showed a

difference inelectrophoreticmobility,the

(inter-nal) p12,remained intact.

Trypticpeptidemappingof viral compo-nents.Tofurtheranalyze the structural differ-ences between RFV and CFV, tryptic peptide

maps werepreparedof eachof the virion

com-ponents, isolated, and purified by

immunopre-cipitation withmonospecificantisera and SDS-PAGE. The tryptic peptide maps for the gag geneproductsareshown inFig.8.

Amongthegaggeneproducts, the p12protein

was significantly different in RFV and CFV, confirming the differences observed in electro-phoreticmobility.Thep30'sandp10'swere iden-tical. Thep15 showedtwoadditional minor pep-tidesintheprotein isolated fromCFV,but be-causetherewerenoreciprocaldifferencesinthe map ofp15 from RFV andno apparent differ-encein molecularweights of theproteins (Fig.

7), these addedpeptidesmayhave been dueto contamination.

Both env gene products, gp70 and p15(E), were different in RFV and CFV (Fig. 9). For gp70, the differencewasdueto ashiftin asingle majorpeptide.Incontrast,thedifferencesinthe p15(E) weremuchmorecomplex, and onlyone ofthe 12distinguishablepeptides could be con-sideredcommon tothetwoviruses.

To determinewhether these structural differ-enceswererelated totheregressingphenotype

of the viruses, tryptic peptide maps were pre-pared from theproteins isolated fromtwo

inde-pendently derived stocks of RFV and CFV (RFV-B-23andCFV-FES-7) andclonal isolates of the MuLV's ofRFVand CFV. Thedifferences observed inthe gp70's ofRFV and CFV were also observedinthe other stocksandclones(Fig. 10). Similar results were obtained with the

p15(E)'s (notshown). Incontrast, thep12 pro-teins of each of these viruses gave identical

tryptic peptide maps, andhence differences in thep12 donotappear tobe relatedtoregression.

DISCUSSION

We havecompared thestructural components of two strains of Friend virus: a conventional strain, CFV, which induces a progressive and lethal erythroleukemia, and the RFV strain, whichinduces an erythroleukemiathat sponta-neously regresses. Two approaches have been used to make these comparisons: physico-chem-ical, based on electrophoretic mobility and

tryp-ticpeptidemapping, andimmunological, based onneutralizationassays.

SDS-PAGE analysis revealed that RFV and CFV both consisted of all of thegenerally ob-servedmajor viral proteins inapproximatelythe same proportions. One polypeptide, p12, ex-hibitedasmall, consistent difference in molecu-lar weight(mobility) in thesetwoviruses.

Furtheranalysis of the structuralcomponents of the viruses was achieved by using tryptic peptide mapping of the isolated viral proteins and glycoproteins. The difference in the p12 detected by electrophoresis was confirmed by this method. In addition, we found that the primarystructureofthe twoenvelope constitu-ents,gp70 and p15(E),differedinRFV andCFV. The p30's and p10's of the two viruses gave identical tryptic peptide maps, and, with the exception oftwo additional minor peptides in theprotein fromCFV, the p15'swerealso iden-tical.

The p12of the MuLV's isaninternal, acidic, RNA-bindingphosphoprotein (33).Itexhibitsa very high degree of type specificity, both in terms ofantigenic structure (34) and function-ally,in termsof its interaction with viral RNAs (31). In additiontodifferences in primary amino acidstructure,changesinthepatternordegree ofphosphorylation might also beassociated with differences observed betweentwoviruses.Itis, therefore, perhaps notsurprising that compari-sonofanRFVandaCFVmight reveal different p12's. However, when additional virus stocks and clonal isolateswereexamined, the difference in p12was not aconsistentfinding,andtherefore isnotrequired for induction ofaleukemia that spontaneouslyregresses.

Both env gene products, pg7O and p15(E), yielded different tryptic peptide maps for the two viruses. The differences observed between the p15(E)'s were extensive, whereasa change in onlyone tyrosine-containing tryptic peptide distinguished thegp7O's. Elderetal. have made adetailedanalysis ofthegp7O's of MuLV'sand havecategorized the virusesonthebasis of the peptidemaps of theirgp7O's (9).Recombinant viruses have alsobeenidentified by analysis of the tryptic peptidemapsoftheirgp7O's (8, 11). The differenceswehaveobserved between RFV and CFV might be the result ofa recombina-tional event which occurred during the genesis of RFV.

It is of interestthat thep30'sofRFV and CFV gave identical tryptic peptide maps in view of reports from severalinvestigators on differences betweenp30'sofcloselyrelatedMuLV's. Buch-hagenetal. reporteddifferencesintryptic pep-tide patterns forp30's isolatedfrom N- and B-tropic viruses derived from a single BALB/c 1091

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B

d.

0 .:

K

J

0

c

li~~~~i

'I~~~~~~~~~~~~~~~

1")~~~~~~~~~~~~~~~~~~~~~~

M.4t~~~~~~~~~~~~~~~~~~~~~~

.II

0*OR

L

*1,

FIG. 8. Tryptic peptideanalysis ofgag geneproducts of RFV and CFV. (A) RFV p3O; (B) CFV p3O; (C) composite ofp30maps;(D) RFVpl5; (E) CFVpl5; (F) composite ofp15maps;(G) RFVpl2; (H) CFVp12;(I) composite ofp12maps;(J)RFVplO; (K) CFVplO; (L) composite ofplOmaps.Electrophoresiswascarriedout from righttoleft; thin-layerchromatography in the second dimensionwasascending from the bottom. In the composites, peptides uniquetoRFVareindicated inwhite,peptides uniquetoCFVareindicated inblack, andcommonpeptidesarestriped.

A

D

E

.4.

I

.S

S*

G

H

A a

U.

S' 0

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STRUCTURE OF REGRESSING FRIEND VIRUS 1093

A

B

I

D

E

C

9 F

FIG. 9. Trypticpeptide analysis of env gene products of RFV and CFV.(A)RFVgp7O;(B)CFVgp7O; (C)

composite ofgp7Omaps;(D)RFVpl5(E);(E) CFVpl5(E); (F) composite ofpl5(E) maps. Symbolsarethe same

asinFig.8.

A * B C D

*

e

04

0

e_s

F G

Ab,-"W.,,:.At

. _'

.-.

H

9

.._ m

a _*._*.

FIG. 10. Tryptic peptideanalysisof thegp7O(A-D) andp12 (E-H)ofRFV-B-23 (A,E),CFV-FES-7 (B, F)

andclonalisolates of theMuLVof RFV (C, G) and CFV (D, H).

mouse(3).Hopkinsetal. showed that

p30's

from NB-tropic MuLV's haddifferentelectrophoretic mobilities than the parent B-tropic virus (18), and Gautsch et al. have found subtledifferences inp30tryptic peptide maps that areassociated with virus tropism (15). We have previously

shown that RFV and CFV differ in tropism

(RFV is NB-tropic, whereas CFV is N-tropic)

andthatthisdifference

apparently

relatestothe regressingphenotype of the virus (4). Itmight

thusbeexpectedthat thep30'sofRFVandCFV would also differ. However, Hopkins et al.

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ported that there was little to no difference in thep30'sof N and NB viruses, incontrast to the clear differencebetween NB and B isolates (18), and themajor differences observed by Gautsch et al. among the p30's ofalarge series of eco-tropic MuLV's was between N- and B-tropic viruses (15). Itistherefore possible thata change in p30, although relatedtotropism,is not nec-essarily associated with theN to NBconversion, or that RFV and CFVareexceptional with re-spect to their identical p30's. It is also possible that subtle differences in the p30's would not havebeendetected byourmethods.

Neutralization kineticsassays werechosento assessimmunological differences between RFV and CFV.Neutralizationassays,although inher-entlyrestrictedtoviral surfaceconstituents,can yieldinformative results independent of differ-ences in theprimary structure ofthe proteins tested. This is becauseadifference in the struc-tureofanexternalprotein oftwoviruses might notbereflected inadifference inneutralization. This could result if the changes were in an inaccessibleportion of the molecule when incor-porated into whole virusparticles and thus be irrelevanttotheprocessofinfectionor neutral-ization. Likewise, it is conceivable thattwo vi-ruseswhose externalproteinswereidentical in primary structure might differ in reactivity to

neutralizing antibodybecause of conformational or accessibility differences induced by juxta-posed internal or adjacent surface proteins

themselves having differentstructures. Invivo neutralization kineticsassayshave been success-fully usedto distinguishamongdifferent pseu-dotypes of Friendspleenfocus-formingvirus(7).

By using the neutralization assays, we de-tected differences inreactivityof RFV and CFV withanti-gp70 serum, serafrom regressed and immunizedmice,andsera (andhuman

comple-ment) reactiveagainst anti-p15(E).The neutral-izationof RFVbyanti-pl5(E) andcomplement

is likely duetovirolysis(27). The effect of hu-man serumontheviruses,which is dueto viro-lysis mediatedbydirectcomplementactivation by viralp15(E),shows thatCFV alsoexpresses p15(E)onits surfaceand issusceptibletolysis.

Thus, both RFVandCFVapparentlyhavetheir

p15(E)'saccessibleonthe viralsurface,but with distinctantigenicsitesexposed. Fischingeretal. reported that (conventional) F-MuLV was not

neutralizedbyanti-pl5(E)andcomplement (13).

Incontrast, axenotropicMuLVwassusceptible.

It is possible, therefore, that the p15(E) sites exposedonRFVarerelatedtoxenotropicvirus sequences acquired during the genesis of this strain.

Thestudiesreported here have been carried

outprimarily byusing the in vivo-derived Friend viruscomplex,consisting of spleen focus-forming virus andthehelperMuLV, as startingmaterial for preparation of virus stocks in cultures of mousefibroblasts in vitro.With someexceptions (6, 37),however, growth of spleenfocus-forming virus in short-term fibroblast cultures is re-stricted or atleastoccurs atmuch lower levels thanthe MuLV helper.Furthermore,theMuLV specifies the antigenicity of the complex (7). Also, data obtained withthevirus complex were confirned by using clonal isolatesof theMuLV's from both RFV and CFV. Therefore,ourresults on both physico-chemical and immunological differences between RFV and CFV reflect, pri-marilyorexclusively, differences in theirMuLV components. This isjustified by the results of previous studies which established that the re-gressing phenotype of RFV is entirelyafunction of its MuLVhelper (4).

Comparisonof thestructuresof RFV and CFV might be instructivewith regardto the origin of theregressing strain. Our previous studies have established that viruses that inducearegressing disease can be generated from conventional strainsby forcedpassagethroughaspecific sub-line of Fv-1 heterogeneous Swiss/ICRmice(4). In these circumstances, virus tropism changes from N to NB and the resultant virus stock inducesanerythroleukemiathatspontaneously regresses. Recombination between MuLV's has been reported in several systems, and these eventsand thederivative virus strainsare con-sideredto beimportant inthe pathogenesis of thevirus-inducedleukemias (12, 16). The SFFV componentof theFriendvirusleukemias is itself a recombinant between its ecotropic MuLV helper andamouseendogenous xenotropic virus (36), and non-replication-defective recombi-nantsbetween thehelper MuLV of Friend virus andxenotropicviruses have beenreportedafter passage of the isolated helper in NIH/Swiss mice(38).Thus,itistemptingtospeculatethat RFVarose byaspecificrecombinationalevent

involving the helper MuLV component of the

parental CFV and endogenousmouse viral

se-quences to yield a new virus strain with the regressingphenotype.Several ofourfindingsin this study,as noted above,areconsistentwith this hypothesis, although altemative

explana-tions havenotbeenexcluded.

The exact role of any of the structural or

antigenic differences between CFV and RFV in theprogressionorregressionofleukemiacannot

bedeterminedatpresent. This isespeciallytrue

ofthe structural differenceswhich may be

to-tally

unrelatedtothe

biological

behavior ofthe viruses. It is ofinterest,however,thatthe viral

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STRUCTURE OF REGRESSING FRIEND VIRUS 1095

components that exhibit significantdifferences are related toimmunological reactivity against

the virus and leukemia cells which develops duringregression. The two env gene products, gp7O and p15(E), are clearly involved in neu-tralization of viralinfectivity.The enhanced

sus-ceptibility of RFVtoneutralizationby regressed serumandanti-p15(E) antiserum couldhelpto limitdiseaseprogression by preventing reinfec-tion oferythroblastic stemcells. Furthermore,

these sameviralcomponentsarepresentonthe surfaces of infected cells andserve astargetsfor cytotoxic reactions mediatedbytheappropriate

antisera andcomplement. Thus,if the structural and antigenic differences found in the compo-nents of RFV and CFV alter their in vivo

im-munogenicity or immunosensitivity, profound

changes in the dynamics of the disease might

result, including its regression. Further studies willberequiredtoestablish theexact relation-ship betweenthese changesand the biological

properties of the viruses.

ACKNOWLEDGMENTS

Wethank E. Fleissner and S. Oroszlan for theirgiftsof

anti-p15(E)antisera and S.Marcusfor his criticalreadingof themanuscript.

This studywas supported by grant CA-14100 from the National Cancer Institute andaninstitutional granttothe MichiganCancer Foundationfromthe United Foundationof Greater Detroit.

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