Murine leukemia virus (T-8)-transformed cells: identification of a precursor polyprotein containing gag gene-coded proteins (p15 and p12) and a nonstructural component.

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0022-538X/78/0027-0809$02.00/0

Copyrighti 1978 AmericanSocietyforMicrobiology Printed inU.S.A.

Murine Leukemia Virus (T-8)-Transformed

Cells:

Identification of a Precursor Polyprotein Containing gag

Gene-Coded

Proteins

(p15

and

p12) and a Nonstructural

Component

THOMAS L. SACKS,' FREDH. REYNOLDS, JR.,2 DILEEPN.DEOBAGKAR,' ANDJOHN R. STEPHENSON'*

LaboratoryofRNA TumorViruses,NationalCancer Institute, Bethesda, Maryland20014,'and Viral Oncology Program,Frederick Cancer ResearchCenter,Frederick, Maryland217012

Receivedfor publication 11 May 1978

Minkcellsnonproductivelytransformed by the T-8strain of mink cell

focus-inducing virusexpress twotype C viral amino terminal gag gene-coded structural proteins, p15 and p12, in the form of a 90,000 to 110,000 molecular weight polyprotein that lacks detectable immunologicalreactivity with other known type Cvirus-coded translationalproducts.The observationconcurswiththeprevious

demonstration of similar high-molecular-weight precursor polyproteins in cell

lines nonproductively transformed byeither of two other mammalian sarcoma viruses also limited in virus-codedstructural protein expression to p15 andp12.

Replication-defectiveRNAtumorviruses

ex-hibitingboth in vitro and in vivo transforming

activity have been isolated from a number of

mammalian

species

including

the mouse

(13),

rat (9, 11), cat (7, 20) and woollymonkey (24).

Suchvirusesappear to representgenetic recom-binants between nondefective "leukemia" or

"helper" viruses and the host cell from which

thetransforming sequenceswerederived(6, 17,

26). The contribution of the former is limited

withonepossible

exception

(4) togenes

coding

fornonglycosylatedviral structural proteins

lo-cated at the 5' terminus of the helper virus genome (2, 3, 10, 14) andpossiblyanadditional, as yetundefined segment(s) from the 3' termi-nus. The host cell contribution tosuch recom-binant viruses are unique transforming se-quences known as onc or srcgenes (17). Non-defective mammalian leukemia virusesare char-acterizedbynonglycosylatedstructuralproteins with molecular weights of 30,000 (p30), 15,000

(p15), 12,000 (p12), and 10,000 (plO) coded for

byaviralgenedesignatedgag(1,2, 18,23,27).

Initial expression of thegaggenetranslational

products occursinthe form ofahigh (65,000)-molecular-weightprecursor in which the individ-ual proteinsare arrangedas follows: NH2

p15-p12-p30-plO COOH (2, 16). Cells

nonproduc-tivelytransformed by mammalianRNA tumor virusesexpress differentnumbers of gag gene-coded proteins in the absence of other viral proteins such as the envelope glycoprotein

(gp70) or RNA-dependent DNA polymerase.

Forinstance, gag geneexpression incells

non-productivelyinfected by the spleen

focus-form-ing viruscomponentof Frienderythroleukemia

virus is restricted to the amino terminal gag

geneprotein,p15 (3);the

Snyder-Theilen

strain

offelinesarcomavirus(FeSV)codes for

p15

and

p12(10),whereascells transformedbythe S+L-isolate of Moloneysarcomavirus(MSV)express

p15, p12,andp30 (2).

In view of evidence that nonproductively

transformed cells containgenetic material

cod-ing for viral structuralproteinsin additionto a gene(s) coding for putative

transforming

pro-tein(s), itwasreasoned that translation ofsuch

proteins might occur in the forn of common

high-molecular-weight (MW) precursor

poly-proteins that would be subject to subsequent

post-translational cleavage. Infact, analysis of

cellsnonproductivelytransformed

by

FeSV (10,

22), theAbelsonstrain of murine leukemia virus

(MuLV) (F. H. Reynolds, T. L. Sacks, D. N.

Deobagkar, and J. R. Stephenson, Proc. Natl.

Acad.Sci.U.S.A.,inpress),ortheFriendspleen

focus-forming virus (Stephenson, in

prepara-tion) has led tothe identification ofprecursor

proteins containing

p15

and

p12

covalently

boundtononstructuralcomponentsof

approxi-mately 110,000to 130,000MW. Intwoof these threesystems, thedemonstration ofsignificant

amountsofthisprecursor

polyprotein

inan un-cleavedformwasprimarilypossible by analysis ofnonproductivelytransforned minkcell lines. Inviewofourpreliminary findingswith FeSV (10, 22) and Abelson leukemia virus (AbLV) (Reynoldset al., inpress),wehave undertaken

809

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810 NOTES

to systematically analyze mink cells

nonpro-ductively transformed by additional mammalian

sarcomaviruses for analogous high-MW

precur-sors.This communicationreportsonthe

identi-fication of another such protein in mink cells nonproductively transformed byoneofaseries

of recently described mink cell focus-inducing

viruses(8). These latter virusesarederived from

thymuses of preleukemic and leukemic AKR mice and appear to be genetic recombinants between different endogenoustypeC virusesof

mouse cells (5). Two such isolates have been

showntomorphologically transform minkcells,

and withoneof these anonproductively trans-formed minkcelllinehasbeenderived(21).

Cells weregrown in Dulbecco's modification of Eagle medium supplemented with 10% calf

serum. These included a fetal mink lung cell

line, CCL 64, obtained from the American Type Culture Collection, Rockville, Md., and sub-clones of CCL 64 nonproductively transformed by AbLV (Reynoldsetal.,inpress), the Moloney (M) strains of murinesarcomavirus(MSV) (25), theS+L-strain of MSV (15), and theT-8 virus isolate of Staaletal.(21) designated 64T8 in the present study. Other cell lines included rat

(NRK) cellsnonproductively transformed by M-MSV (M-NRK) orby the S+L-strain ofMSV

and a dog cell line transformed by the

S+L-strain of MSV (DoCl1). These various

nonpro-ductively transformedcelllines and their

desig-nationaresummarized in Table 1.

Competition immunoassays for MuLV gag

gene structural proteins of 30,000 (p30), 15,000

(p15), 12,000 (p12), and 10,000 (plO) MW, the pol-coded RNA-dependent DNA polymerase, and env-coded gp70 were performed as

previ-ously described (2; Reynolds et al., in press). Molecular size analysis of viral antigen

expres-sion in cellswasperformed byagarosegel

filtra-tion in thepresenceof 6Mguanidine-HCl (A-15 m) by methods described in detail previously (2). Immunoprecipitationof [35S]methionine-la-beledviralproteinsandsubsequent analysisof irnmunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were

per-formedby the method of Van Zaaneetal. (27). In initialstudies, mammaliancelllines

trans-formed by various replication-defective trans-forming virusesofmousecelloriginwereassayed

for type C viral protein expression by use of competition immunoassays for the known trans-lationalproducts of thegag,pol, andenv genes.

Assummarized in Table 1, mammalian cell lines nonproductively transformed by either T-8 or

AbLV were found to express the two amino terminal MuLV gag gene-coded proteins, p15

and p12,atreadilydetectable levels.Incontrast, neither the remaininggaggene-coded proteins, p30 andplO,theenvgeneproduct, gp7O,northe

reversetranscriptaseweredetectedatsignificant levels. Cells nonproductively transformed by S+L--MSV were limited in expression ofgag

gene-coded proteins to p15, p12, and p30, whereas M-MSV-transformedratorminkcells

lacked detectable levels of any of the known

typeC viral translational products. Of interest,

cells nonproductively transformed by

S+L--TABLE 1. TypeChelpervirusprotein expressionin mammalian cellsnonproductivelytransformed by various mouse-derivedtransforming viruses

Viralprotein expression (ng of viral protein per mg of cellular protein)"

Transform- Cellline Designationof

non-ingvirus producer clone gag pol env

p15 p12 p30 plO RDDP gp7O

T-8 Mink(CCL64) 64T8 1,600 1,300 <5 <5 <5 <20

AbLV Mink(CCL64) 64Ab2 2,600 1,800 <5 <5 <5 <20

S+L--MSV Mink(CCL64) MiCl, 2,200 2,400 3,500 <5 <5 800"

S+L--MSV Dog DoCl, 6,100 5,000 6,900 <5 <5 1,320b

S+L- MSV Rat(NRK) S+L--NRKC1, 5,000 4,400 7,100 <5 <5 950"

M-MSV Mink(CCL64) 64M1 <5 <5 <5 <5 <5 <20 M-MSV Rat(NRK) M-NRKC1, <5 <5 <5 <5 <5 <20

'Cellextracts,preparedbysonicoscillationfor 20s(BiosonicIIsonicoscillator)in 10 mMTris-hydrochloride (pH 7.8) buffercontaining100mMNaCl,0.5mMEDTA,10%glycerol,and0.5% TritonX-100,weretestedat

serial twofold dilutionsincompetitionimmunoassays usinggoatantiseraagainstdetergent-disruptedM-MuLV forprecipitation of'I51-labeledRauscher MuLVp15,p12,p30, plO, RNA-dependentDNApolymerase(RDDP), andgp7O.Resultsarebasedontheextentofdisplacementof'25I-labeledproteins bycellularextractsrelativeto

standards andrepresentmeanvalues from threeseparatedeterminations.Cellularproteinconcentrationswere

determinedby the method ofLowryetal.(12).

bAlthough thecompetition immunoassay titers for S+L--transformed cellextractsintheenvelope glycopro-tein(gp7O)assayswererelatively high,thefinal extentsofcompetitionwere nogreaterthan 50%evenatthe highestprotein concentration tested.

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NOTES 811 MSVcompeted partially (around50%), although

at high titer, in an MuLV gp7O competition immunoassay.

ExtractsofT-8-transformed minkcellswere subjectedtomolecular sizeanalysisby gel filtra-tion in thepresenceof6Mguanidine-HCl, and

individual column fractions were tested for

MuLV p15 andp12. Over 90% of bothp12 (Fig.

1A) and p15 (data not shown) reactivities

co-chromatographedat anMW of around100,000,

withasmall amountofreactivityfor both pro-teinsappearingat25,000MW. Similar analysis ofAbLV-transformed mink cells revealeda ma-jor p12 (Fig. 1B)- and p15 (data not

shown)-containingprecursor at a somewhathigher MW (110,000to 130,000) (Fig.1B).Incontrast, in the

caseofS'L--transformedmink (Fig. 10),rat, or

human cells (data notshown), thehighest-MW protein containing antigenic cross-reactivity with MuLVgagproteins occurredat anMW of around 57,000 and contained p15, p12, and p30 antigenic determinants.

Furthercharacterization ofvirus-coded anti-gen expression in T-8 and AbLV nonproduc-tively transforned mink cellswasperformedby immunoprecipitation and subsequent sodium dodecyl sulfate-polyacrylamide gel

electropho-resis analysis of [3S]methionine-labeled pro-teins. The results (Fig. 2) indicate the presence

of virus-codedpolyproteins ofaround 90,000 to

110,000and110,000 to 130,000 MWinT-8- and AbLV-transforned mink cells, respectively. In both cases, these proteins were efficiently

pre-cipitated byseraprepared against

detergent-dis-ruptedRauscherMuLVorM-MuLV,butnotby

controlgoat sera orby high-titeredsera directed against MuLV gp7O. Althoughin some experi-ments the AbLV-codedprecursorwas also pre-cipitated by antibody toMuLV p30, theextent of suchprecipitation wasextremelylimited, in-dicating that the precursor contained at most

onlyasmallportionofMuLVp30.Moreover,in

pulse-chase experiments,we have not, to date,

detectedappreciable levels ofananti-p30

precip-itable protein lacking p15 and p12 in either AbLV-orT-8-transformed minkcells.Thus, the

possibilitythat the lowlevels of

immunoprecip-itation of the AbLV precursors by anti-MuLV

p30maybedue tolow titers ofanti-p15orp12

in theanti-p30serumcannot beruledout. The difference ofabout 20,000 in MWbetween the T-8-andAbLV-codedprecursorsmay be due, at least in part, to differences in MW of their nonstructural components since the contribu-tion of viralstructuralproteins other thanp15

andp12appearstobeminimal.

These present findings increase to three the number ofexamples where minkcells nonpro-ductively transformed by replication-defective

oL

g

<U. 3

0

15-210

wU 5

z

c §

§§

> 9 ci

10

5

20 40 60 80 100

FRACTIONNUMBER

FIG. 1. Molecular sizeanalysis ofMuL Vgaggene-codedproteinsexpressedin thefetalmink lungcell lineCCL64nonproductivelytransformed by (A) T-8, (B) AbLV, or (C)SL--MSV. Cell extracts (20 mg)

weresubjectedto agarosegel filtration(A-15 m) in the presenceof6Mguanidine-HCl(16),and individ-ualfractions weretested in aheterologous competi-tionimmunoassay in which antibody directedagainst detergent-disrupted Rauscher MuLVwas used for precipitationofM-MuLVp12. The total recoveryof p12antigenic reactivity followingagarosegel filtra-tionwas around 50%. Resultsareexpressedas the percentage of antigenic reactivity in each column fraction andarebasedonthedegreeof displacement of competitioncurvesrelativetostandards. Molecu-larweightstandards includedtracer amountsof 2'I-labeled bovine serum albumin (69,000), Rauscher

MuLVp30(30,000),andRauscher MuLVpl2(12,000). mammalian sarcoma viruses have been shown toexpress thetwoaminoterminalgagproteins, p15 and p12, in the form of a high (90,000 to 130,000)-MW precursor. These include FeSV (10, 22), AbLV (Reynolds etal., inpress), and, as shown in the present study, the T-8-trans-formingvirus isolate. In each case, the sarcoma 27,1978

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812 NOTES

A B C

0

E

F

G

H

I

Std

Wp .

Mh

-135,000

-

69,000

-s--130,000

--

12,000

FIG. 2. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of viralproteins expressed in thefetalmink cell line CCL 64nonproductively transformed by either T-8 (A through D)orAbL V(F through I) labeled with[:'S]methioninefora2-h pulse.Nontransformed control CCL

64cells(E)areincludedas anegative control. Antisera included goat Rauscher MuLV (A, E, F), anti-RauscherMuLVp30 (B, G), anti-Rauscher MuLVgp70 (C, H), anti-M-MuLV (D), andnormalgoat serum (I). Molecularweight standards included '25I-labeled/8-galactosidase(135,000), bovine serum albumin (69,000), Rauscher MuLVp30 (30,000),andRauscher MuLVpl2 (12,000).

virus-codedpolyproteinuponpost-translational cleavage gives riseto a25,000-MWcomponent containing p15 andp12andasecondcomponent ofaround60,000to95,000MWlacking detecta-bleimmunological reactivitywith known virion structural components. In contrast, mink cells

transformedby the S+L-strainof MSVexpress threegagproteins, p15, p12,p30, andpossiblya portion oftheenvelope glycoprotein (gp7O),but lack evidence of a precursor containing non-structural components. Similarly, analysisofa

rat (NRK) cell clone nonproductively trans-formedby thewoolly monkeysarcomavirus and expressingpi5,p12,andp30hasfailedtoreveal aprecursorofgreater than55,000MW (unpub-lisheddata).Thus,todate,identificationof

high-MW virus-coded precursor polyproteins con-taining structural and nonstructural components

has been limitedtothose transforming viruses coding for only thetwoaminoterminal gag gene proteins, p15 and p12. To determinethe

gener-alityof thisphenomenon,itwill be of interestto

search foranalogousprecursors in mammalian cells nonproductively transformed by sarcoma virusesthatexpressvariable numbersof the gag geneproducts.

The natureofthe nonstructural components of the various sarcoma virus-coded precursor polyproteins has not been conclusively estab-lished. Forinstance, suchproteinscould repre-sentsrcgenetranslationalproductsinvolved in malignant transfornationorcould be codedby

nontransforming cellulargenesacquired by sar-coma virus isolates along with the src genes.

Altematively, these polyproteins may be the

productofin-phase deletions encompassing both J. VIROL.

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VOL. 27, 1978

p30 and p10 andresulting in a read-through of the carboxy terminal portions of pol or env.

Althoughthe fact that neither pol norenvgene

products were detected by competition immu-noassay would tend to argue against such a

model, the possibility that themajor

inmuno-logical determinants measured in such assays

arelocated inpartially deleted amino terminal

regions of theseproteins cannot be ruled out.A final consideration is that of a frameshift muta-tion within the gag gene resulting insynthesis

ofa newprotein(s). The major argumentagainst

thispossibilityis the lowprobability of a

frame-shiftresulting in such extensive translation with-outencountering a termination codon. The fact that in the feline system the 60,000-MW com-ponent of the FeSV-coded precursor is immu-nologically reactive with certain antisera di-rected against the tumor-specific feline oncor-navirus cell membrane antigen (22) favors the

possibility that these newly described proteins

aretransformationspecific.

Inprevious studies both the FeSV (19; A. S.

Khan,D. N.Deobagkar,and J. R.Stephenson,

inpress)- and AbLV (Reynolds et al., in

press)-coded110,000-to130,000-MW polyproteins have

been showntobeincorporatedinto certain

pseu-dotypevirions in an uncleaved forn. A similar

product is beingsought in various helper virus-rescued T-8 pseudotype virions. Such a finding

would provide an effective means of obtaining

thepurified proteininrelatively high yield. By

utilizingsuchpurified proteins,it should be

pos-sible to analyze these three independent

sar-coma virus-coded putative transforming pro-teins forimmunologically shareddeterminants and alsototestforexpressionof cross-reactive antigens in spontaneous and inducedtumorsin

variousmodel systems.

WethankS. P.Staal,J. W.Hartley,and W. P. Rowe for generously providingthe T-8-transformed nonproducer mink cellline usedinthestudyand C. A. Hanson and E. Hershey for excellent technical assistance.

Thiswork wassupported by Public Health Service Con-tractNo.NOI-CO-25423 of the Virus CancerProgramof the NationalCancerInstitute.

ADDENDUMINPROOF

Biochemical and immunological analysis of thegp7O

reactivity expressedinS+L- MSV-transformed mink

cellsindicated it to contain mink cellfocus-inducing virusdeterminantsandtorepresentthe translational

product ofarecombinantenLvgenecontained within theS+L- genome (F.H.Reynolds,U.R.Rapp,G.J. Todaro, and J. R.Stephenson, inpreparation).

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