Differential expression of helper viral structural polypeptides in cells transformed by clonal isolates of woolly monkey sarcoma virus.

Copyright 01975 AmericanSocietyforMicrobiology Printed inVol. 16,U.S.A.

Differential

Expression

of

Helper

Viral

Structural

Polypeptides

in

Cells Transformed

by

Clonal Isolates

of

Woolly

Monkey

Sarcoma Virus

STUART A. AARONSON,* JOHN R. STEPHENSON, SHIGEO HINO, AND STEVEN R. TRONICK ViralCarcinogenesis Branch, National Cancer Institute, Bethesda, Maryland 20014

Received forpublication9July 1975

Celllines transformed by woolly monkeysarcomavirus(WSV) in theabsence

of infectiousvirusproductionwereanalyzedfor theexpressionofwoolly monkey

helper viral p30, p12, and gp7O antigens. Several linesproduced high levels of

both p30 and p12, whereas gp7O was not detectable. One transformed clone

expressed only p12,and in another cellline,noneof thehelperviralantigenswere

detected. The properties of each sarcoma virus bred true upon transmission,

indicating that each variant represents a distinct genotype. The different cell

lineswereexamined with respecttoproperties characteristic ofthe transformed

state.The in vitrogrowth propertiesandoncogenicityof eachWSV-transformed

clone were indistinguishable, indicating that transformation by WSV occurs

independentlyoftheexpressionofatleast threehelperviralpolypeptides.

RNA-containing sarcoma viruses that have

beenisolated from anumberofspecies differin

their competence for replication. Many avian

sarcoma virus strains appear to replicate

inde-pendently (12, 14, 29), while infection by

mam-malian sarcoma viruses requires the presence of

a nontransforming typeC RNA helper virus (3,

5, 7, 15). Among known replication-defective

murine sarcoma viruses, there appears tobe a

range of abilities to express helper viral

anti-gens. Certain strains transform cells in the

absence ofdetectable

helper

ex-pression (3, 5, 15, 17). Another classof murine

sarcoma virus genome, termed S+L-

(6,

which codes for the mousehelper virus 30,000-mol wt major structural polypeptide (p30) (6,

30) and a 12,000-mol wtpolypeptide (p12) (30),

also produces low levels ofnoninfectious virus

(6).Finally, cells transformedbyathird murine

sarcoma virus variant have recently been

re-portedtoexpressa70,000-molwt mousehelper

virus glycoprotein (gp7O) in the absence of

detectable p30 orcomplete virus (10).

A sarcoma virus isolated from a New World

primate, the woolymonkey (16, 28, 32), has also

been shown to transform cells in the absence

ofinfectious virus production (2, 22). Radioim-munological techniques have been developed for

detectionofgp7O(15),p30(21, 26), andp12 (31)

of the woolly helper virus initially isolated in

associationwiththis sarcoma virus. In the

pres-ent studies, we have utilized competition

im-munoassays for these viral polypeptides to

in-vestigate the expression of woolly helper viral

antigens by cells transformed by individual

clones of woolly sarcoma virus. The results

provideevidence concerning the genetic

compo-sition of this sarcoma virus and preliminary

information concerning the ordering of some of

its genes.

MATERIALS AND METHODS

Media.Cellsweregrown inDulbecco modification ofEaglemediumsupplemented with 10% calf serum

(Colorado Serum Co.,Denver, Colo.) in 50-mm plas-ticpetridishes (Falcon Plastics, Los Angeles, Calif.).

Cells and viruses. Cells used included a line of normal ratkidney (NRK) cells (11). Marmoset cells,

chronically infected with woolly sarcoma virus

(WSV), were generously provided by F. Deinhardt,

RushPresbyterian St. Luke's Hospital, Chicago,Ill. Theviruswas passagedinastrain of normal human fibroblasts immediately prior to use in these studies. The isolation of the woolly type C helper virus associated with WSV has been reported (2, 32). A clone of Kirsten murineleukemiavirus(KiMuLV)(5) was also used.

Virus assays.Thefocus-forming assay forsarcoma virus was performed as previously described (5). An assayfordetection ofvirion-associated reverse tran-scriptaseactivityin tissueculture fluidutilizing the synthetic template poly(rA)-oligo(dT) has also been reported (27).

Isolation of WSV transformants.

WSV-trans-formed lines were isolated as previously described (2). Briefly, exponentially growing NRK cultures were 1117

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exposed to limiting dilutions of WSV (1 to 2 focus-forming units [FFUlplate) in 0.5ml of medium for4 hat 37C.Cultures werethen overlaid withmedium

containing 0.3% agar to minimize infectious virus spread.At around7days, theagar wasaspirated and

transformed fociwereselectedby the cloning cylinder

technique (3). Each focus-derived line was recloned

by the microtiter procedure (27) priortoanalysis. Analysis of biological propertiesof WSV-trans-formed cells. Cellswereinoculatedonto50-mmpetri dishes at a density of 5 x 109 cells/cm2, and the

mediumwaschangedevery3days. Cells from

dupli-cate plates were counted with a hemocytometer at 2-day intervals. The doubling time was determined

during growth in exponential phase. Thesaturation densitywastakenasthe valuewherethreesuccessive

cell counts showed no increase in cellnumber. Cell

colony formation on confluent monolayers of NRK

cells and in soft agar was determined according to methods previously reported (4, 19).

Tumorigenicity studies. Athymic (nude) mice (6 to8weeksold)were inoculatedsubcutaneously in the

intrascapular region with washed cells suspended in 0.2mlofphosphate-buffered saline. The animalswere

examined at weekly intervals for the appearance of tumors.

Radioimmunoassays. Competition immunoassays for woolly viral gp7O (14a), p30 (21, 26), and p12 (31) have been described in detail. Reaction mix-tures contained 0.01 M Tris, 0.3 M NaCl, 0.15 M EDTA, 0.4% Triton X-100, and 1% bovine serUim aIbumin, pH 7.8,inavolume of 0.7 ml. Antiserawere

assayed atserialtwofold dilutions for theirabilityto precipitate approximately 10,000 counts/minof 20I-labeled antigen.Unlabeled viral antigensweretested

atserialtwofold dilutions for theirabilitytocompete with "25I-labeledgp7O,p30,orp12forbinding limiting

amounts of antiserum. Antiserum and unlabeled competing antigen were incubated at 37 C for 1 h;

1211-labeled antigenwasthenadded, and the

incuba-tionwascontinued for 3 hat37 C andafurther 18 hat

4C. After addition of 0.025 ml of undiluted swine anti-goat immunoglobulin G to eachtube, the mix-turewasincubated3hat4C and centrifugedat2,500

rpm for 15 min, and the resulting precipitate was

measured for radioactivity. The antisera used were

generously provided by R. Wilsnack through the Office of Resources and Logistics, National Cancer Institute. Protein concentrationsweredeterminedby the method ofLowryetal. (18).

RESULTS

Sarcoma virus-release by cells infected at

limiting dilution with WSV. Cells transformed

at limiting sarcoma virus dilution by WSV

associated with woolly helper viruswere grown

up asfocus-derived linesforanalysis of virus re-lease. Twenty of 27 tranformants tested

re-leased high levels of virion-associated reverse

transcriptase activity and focus-forming virus (Table 1). With each of these lines, it was

possible to isolate woolly helper virus beyond

TABLE 1. Virological properties ofindividual WSV-transformed lines

Virusreleasea No. of Virion-associatedreverse

lines transcriptase activity WSV titer (pmol/ml of tissue (FFU/ml)

culturefluid)

20 103- 104 JOS.8_104-4

1 100.0 10°

6 <1O-l° <10-l °

aTissue culture fluids of exponentially growing

cultures of each transformed line were assayed for virion-associated reverse transcriptase activity and for focus-forming activity on NRK assay cells as described in the text.

the end point for focus formation. Thus, they werepresumed tohave arisen from co-infection by both WSV and helper virus. One

transform-ant (Table 1) released a very low level of

focus-forming virus (100°0 FFU/ml), and it was

possible to detect a low level of reverse

tran-scriptase activity in tissue culture fluids of this line. The remaining six transformants showed

noevidence of virus release, but contained WSV rescuable by superinfection with either woolly helper virusorKiMuLV.

Woolly helper viralantigen expression by

non-virus-releasing WSV transformants.

Each non-virus-releasing transformant was

tested inimmunoassaysforwoolly viral p12 and p30. Goat antiserum prepared against the

woolly helpervirus wasusedtoprecipiateeither

"2'I-labeled woolly viral p12 or p30 in the presence of increasing quantities of unlabeled WSV-transformed cell extracts. Immunoassays

forwoolly viral p12 and p30detected high levels

ofboth viral polypeptides inNRK cells

chroni-callyinfected withwoollyhelper virus (Fig. 1A).

In contrast, uninfected NRK cells (Fig. 1B)

showed only slight reactivity at very high

pro-tein concentration. This was considered to be

nonspecific because at high concentrations the

same extractsalso showed similar lowdegreesof

reactivityinimmunoassays for unrelatedtypeC

viral polypeptides (data not shown).

Threedistinct patterns ofimmunological

re-activity were observed among the

non-virus-releasing WSV transformants examined. One

clone demonstrated no greater reactivity than

uninfected NRK cells in the immunoassay for

woollyviral p30and only slightly greater

reac-tivity in the assay for viral p12 (Fig. 2A). In

contrast, another WSV-transformed line (Fig.

2B) was strongly positive in the immunoassay

for woolly viral p12 but lacked detectable p30

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1119

-1 0 1 2 3 -1 0 1 2 3

COMPETING PROTEIN(log1o,LgImI)

FIG. 1. Analysis ofviralantigen expressionin cellextractsprepared from(A) NRKchronically infectedwith woolly monkey virus and(B) control NRK. Extracts were prepared by sonication and clarified by low-speed cen-trifugation aspreviously described (26). Protein determinationswere performed by the method of Lowry et al. (18). Assays included an immunoassay for woolly viral p12 utilizing antiserum to woolly monkey virus to precipitate '25I-labeled woolly monkey virus p12 (0), and an immunoassay forp30 utilizing antiserum to woolly monkey virus to precipitate '25I-labeledwoollymonkey virusp30 (A).

-o Ia- a A

-Ob

\4\b

B ~~~~~~~C

AA A.AAAA

A1A

-V

b0 _'A

H--1 0 1 2 3 -1 0 1 2

COMPETINGPROTEIN(Iog1gopg/mlI

FIG. 2. Viralantigenexpressionincell extractsprepared from (A) WSVCl9,(B) WSVCl2,and(C) WSVCl 11.Extractswereassayed for woolly monkeyvirusp12 (0)andp30 (A)immunological reactivityasdescribedin thelegendtoFig.1.

expression. Finally, high levels of both woolly viral p12 and p30 weredetected in each of the

remaining non-virus-releasing WSV

transform-antsasrepresentedby results withonesuch line

showninFig.2C.ItcanbeseeninFigure2that, where reactivity of WSV-transformed lines was

detected in either assay, it closely

approx-imated the amount of viral polypeptide ob-served with cells chronically infected with

woolly helper virus.

Recently, immunoassays that specifically

de-tect gp7O of the woolly helper virus have been developed (14a). Itwasofinteresttoinvestigate theexpression of thisviral antigen by

represen-tative non-virus-releasing WSV transformants. NRK cells productively infected with woolly helper viruswerehighly positive for woolly viral

gp7O(Fig. 3). Incontrast,the reactivities of the threephenotypic variants of WSV-transformed cells were no different than that of uninfected

NRK cells.

Evidence that the differences in woolly

100

60

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0-

I-z C:

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. AA

B A~~~~~~N

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20

100

z8

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00

80

a

z

3

0

260

a. u 0.

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w40

w

a.

201

_1 0 2 3

COMPETING PROTEIN (Iogj,oug/ml)

FIG. 3. Analysis of gp70 expression in cellextracts prepared fromWSVCl9(A), WSVCl2(O), WSVCl 11 (0), and NRK cells chronically infected with woolly monkey virus (0). Extracts, preparedas

de-scribed in the legend to Fig. 1, were tested in the

woolly viralgp70immunoassay in whichantiserumto woolly monkey virus was used to precipitate 1251_ labeledwoolly monkey virusgp70.

helper viral antigen expression by WSV

transformants are virus coded. The

varia-tions in expression ofwoolly type C viral

anti-gens among differentnon-virus-releasing WSV

transformants could reflect virus-coded

differ-encesorcell-specificeffectsontheexpressionof

an identical viral genome. To examine this

question, WSV was rescued by KiMuLV from

cells of eachtransformedphenotype and

trans-mitted to new NRK cells. The expression of

woolly viral antigenswasthenanalyzedin these

cultures. As with the parental line, cells

in-fected with sarcoma virus rescued from the

WSV Cl 9 transformant failed to demonstrate detectable woolly viral p12 cross-reactive anti-gen(Table 2), despite the factthatthe cellswere

morphologically transformedand released high-titered WSV (102 FFU/ml). Similarly, like

their respective parental WSV transformants, NRK cultures infected with sarcoma virus

res-cued from WSV Cl 2 and Cl 11 transformants

expressed high levels of woolly viral p12 (410

and 320 ng/mg of cell protein, respectively).

Due to cross-reactivity of KiMuLV in the

im-munoassay forwoolly viralp30, it was difficult

to quantitate the expression of woolly viral p30

in these cells. These results argue that the

differences in expression of p12 cross-reactive

antigensby eachWSVvariant are viruscoded.

The genetic stability of viral p30 expression

by WSV present in awoolly viral

p30-contain-ing transformant, WSV Cl 11, was next

exam-ined. Cells transformed by progeny sarcoma

virus ofthis linein theabsence of helper virus

co-infectionwereisolated and analyzed for viral

polypeptide expression. Six separate clonal

transformants induced by thefirstcycleof virus

from WSV Cl 11-transformed cells each

con-tainedboth woolly viral p30 and p12

cross-reac-tive antigens at levels similar to those of the

parentaltransformant(Table3). A second cycle

ofsarcoma virus transmission to new cells was performed after its rescue by KiMuLV from a

first-generation transformant. Again, each of

fourclonal transformantsexpressed both woolly

viral polypeptidesatlevelscomparabletothose

detected in the original parental line. These

findings strongly indicate thatwoolly viralp30,

in addition to p12, is coded by this WSV

genome and provide further evidencethat each of theimmunologically distinguishable

replica-tion-defective WSV variants represents a

dis-tinct genotype.

TABLE 2. Woolly type C viralp12expressionby cells infected withKiMuLVpseudotypesof

differentWSVvariants

Level of viralantigen WSV associated with Cellsinfected (KiMuLV) infectedcells with KiMuLV release (ng/mgofcell pseudotypeof: (FFU/ml)a protein)'

Woolly virus MuLVc

p12 p12

WSVCl2 104.8 410 440

WSVCl9 105.2 <2 380

WSV Cl11 104.5 320 430

aNRKcultures were infected with WSV rescued by KiMuLV from each WSV-transformed clone and thenpassagedintissueculturefor 3weeks. Thetiter of WSV in tissue culture fluids of each infected culturewasassayedasdescribed inthetext.

Extracts were prepared and immunoassays were performed as described in the legend to Fig. 1. The results, expressed asnanograms ofp12permilligram ofcell protein, werecalculated from standard calibra-tion curves (30, 31) and represent mean values of three separate determinations.

IMuLV, Murine leukemia virus.

& ~~~* A

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CLONAL ISOLATES OF WOOLLY MONKEY SARCOMA VIRUS 1121

TABLE 3. Genetic stability of woolly viral p12 andp30expression by progeny of one WSV variant

Level ofcell-associated woolly helper viral WSVtransformanta No. ofclones polypeptide(ng/mgofcellprotein)

tested

p12 p30

Parental (WSVC1 1) 380 850

1stviruscycle 6 200-450 500-900

2nd viruscycle 4 210-460 610-920

aWSV was rescued from WSV-transformed NRK cells by superinfection with KiMuLV and transmitted at

limiting dilution to NRK assay cells. Transformed foci were isolated as described in the text and those transformants that failed to release high-titered WSV were tested immunologically. Transformed foci, the product of a second cycle of WSV release, were inducedby.WSVrescued from a transformant of the first cycle of virus transmission. Each line was assayed for reactivity in woolly viral p12 andp30immunoassays as described inthe text.

Characterization of a low level

virus-re-leasing WSV transformant. The properties

of individual transformed foci induced by the

low level virus-releasing WSV-transformed line

WSV Cl 4 were next investigated. Each of five

separate transformants isolated after infection

with tissue culture fluids of this line

demon-strated levels of infectious sarcoma virusrelease

and reverse transcriptase activity similar to

that of the parental line. In contrast, cells

infected beyond the end point for focus

forma-tion and even nontransformed cells isolated

from the same petri dishes were virus negative

by these assays. Analysis ofwoolly viral

anti-gens expressed bytheparental line and several

ofitsdaughtertransformants revealed that each

containedhighlevels ofwoollyviralp12andp30

in the absence of detectable gp7O (data not

shown).

Biological properties of different WSV

gen-otypes. The

biological

transform-ants containing representative WSV variants

were nextcompared. Themorphological

charac-teristics of cells transformed by each were

indistinguishable. The transformants all grew

to similar saturation densities (1.8 x 101to 2.3

x 10'

cells/cm'),

each line resembled that of uninfected NRK

cells (18 to 22 h). Properties of cells in tissue

culture that are known to correlate with their

malignant potential in vivo include the

ability

toformcoloniesonmonolayersof

contact-inhib-ited cells (4) and to grow in suspension in

semisolid medium (19). Each WSV

transform-anttested, incontrast touninfectedNRKcells,

possessed the capacity to form colonies under

both selective conditions (Table 4). The

tu-morigenicity ofeach cell line was also

investi-gated. Representative WSV-transformed lines

of each genotype produced solid tumors at a

dose of 5 x 106cells inapproximately40to50%

of athymic mice at the site of subcutaneous

inoculation; control NRK cells, similarly tested,

were nontumorigenic. Tumors that formed

were histopathologically diagnosed as

fibrosar-comas. The lack of detectable differences in the transformed properties of the different WSV

variants both in vitro and in vivo argues that

the differences in their capacities to induce

helper viral antigens have little, if any,

re-lationship with those viral functions involving transformation.

DISCUSSION

Analysis of the immunological properties of

cells transformed by clonal sarcoma viruses

obtained from a natural isolate of WSV has

revealed the existence of several

immunologi-cally distinguishable variants. One isolate

ap-pearsanalogoustopreviouslydescribed

nonpro-ducer transforming sarcoma viruses of murine

origin (3, 5, 15, 17). Cell extracts of this

WSV variant lacked detectable reactivity in

immunoassays forwoolly helper viralp12, p30,

orgp7Oand didnotreleasefocus-formingvirus.

Asecond variantinduced theexpressionofhigh

levels of both woolly viral p12 and p30 in the

absenceofdetectablegp7Oorvirusrelease. This

WSV variant resembles the S+L- genome of

MSV, whichhasbeen shownto induce

produc-tion of mouse type C viral p30 and p12 in

infected cells (7, 30). However, unlike

S+L--transformed mouse or rat cells which also

pro-ducealow level of noninfectious virus (6), cells

transformed bythis WSV variantdid notshow

evidence of virus release. Cells transformed

by

third WSV variant expressed woolly viral p12

cross-reactiveantigen inthe absenceof

detecta-blep30, gp7O, orinfectiousvirus.Findingsthat

the properties of each variant bred true upon

passageto newcellsargue that thevirusesdiffer

genetically.

In addition to several non-virus-releasing

WSV transformants, one clone, which released

VOL.16,1975

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TABLE 4. Invitro and in vivobiological properties of WSV-transformed cells

In vitroacolony-forming efficiency (%) Invivo'tumor-forming NRK cell line _________________________abilityinathymicmice

(no.oftumors/no.

On monolayers In agar inoculated)

Uninfected <0.001 <0.001 0/10

Transformedby

WSV Cl 2 15 1.0 4/10

WSV Cl9 10 1.5 5/10

WSV Cl11 12 0.8 4/10

a Cellswereinoculatedatserial 10-folddilutions eitherontoconfluent NRKmonolayersorinsuspensionin medium containing0.3% agar. Colonieswerecountedat 12to 15daysandthecolony-forming efficiencywas expressed asthe percentageofcellsinoculated. The results representmeanvalues from twoexperiments.

'Athymic mice (6 to 8 weeks old) were inoculated with 5 x 106 cells suspended in 0.5 ml of phosphate-buffered saline. The site of subcutaneous inoculation was observed weekly for 12 weeks. Tumors reaching adiameter of 1 cm were scored as positive.

a verylowlevel of infectioussarcomavirus, was

isolated. This virus also appearedtobreedtrue

upon transmission to new cells, since each

daughter transformant possessed the parental

phenotype. These results could not be

ac-counted for by the presence of excess helper

virus,sinceattempts todetect suchaviruseven

at a titer comparable to that of the sarcoma

virus were unsuccessful. The transformed cells

werefoundtocontainhigh levels of woolly viral

p12and p30butnotdetectable gp7O, indicating

that if this WSV genome were competent for

replication it must be relatively restricted in

viral gp7O production. The present results are

also consistent with the possibility that this

sarcomaviruscomplements theendogenousrat type C virus present in NRK cells (1, 17),

causing activation and release of infectious

sarcoma virus in the envelope ofthis virus.

Murine and avian sarcoma viruses contain

genetic information both unrelated (8, 23, 25)

and related (8, 9)tothatofknowntypeChelper

viruses. Thus, one possibility is that these

transforming viruses have arisen as a result of

recombination between the helper virus and

some other cellular genetic information.

Analy-sis ofnucleic acid homologyof WSV expressed

in one WSV-transformed nonproducer clone

with woolly helper virus has indicated some

sequence relatedness between the two viruses (24). The presentfindings, byestablishing that

different amounts of helper viral information

are expressed by different WSV variants, and

that these properties breed true upon virus

passageto newcells, indicatethatwoollyhelper

virus genetic sequences canbestably associated

with the WSVgenome.

The present findings do not resolve whether

the WSV variants isolated here differ at levels

involving their transcription or translation or

result from specific deletions of helper viral

information. However, the pattern of woolly

viral antigens detected among the

transform-antstested in the presentstudies would suggest an ordering of genes coding for viral structural polypeptides relative to those coding for

trans-formation as follows: transformation

gene(s)-p12-p30-gp70. A recent report (10) of a murine sarcoma virus genome which codes for murine

leukemiavirus gp7O in the absence of p30 would

appear to be at variance with the above data.

However, when more information is available

concerning the origin of sarcoma virus and the mechanisms by which recombination with helper viruses occur, it may be possible to explain the apparentdifferencesbetween these twosystems.

The present evidence, as well as previous findings with murine and avian sarcoma virus, indicates that sarcoma viral genes coding for transformation may be expressed

independ-ently of those codingfor helper viralstructural components. Evidence is accumulating that

high-molecular-weight precursor polypeptides

are involved in thereplicationof typeCviruses (13, 20; J. R. Stephenson, S. R. Tronick, and S. A. Aaronson, Cell, in press). Ifthe sarcoma viralgenescoding fortransformationand helper viralpolypeptidesare present in the same mes-senger RNA, WSV genomes expressing woolly viral p12aloneor in combinationwithp30 may provide antigenic markers to monitor

purifica-tion ofthose proteins responsible for the

trans-formed phenotype. Studies of this nature are

currently in progress.

ACKNOWLEDGMENTS

WethankMae C.Wong, LindaK.Long,andVirginiaK.

Steiner for excellent technical assistance.

This work wassupportedbycontract no.NCI-E-73-3212of

the VirusCancerProgramofthe NationalCancerInstitute

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CLONAL ISOLATES OF WOOLLY MONKEY SARCOMA VIRUS 1123

and by a Public Health Service International Research Fellowship(FO5TW-2207-01) toS. Hino.

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