Viral protein expression in producer and nonproducer clones of friend erythroleukemia cell lines.

0022-538X/81/020654-07$02.00/0

Viral Protein

Expression

in Producer and

Nonproducer

Clones

of Friend

Erythroleukemia

Cell Lines

RITA ANAND,1 FRANK LILLY,* AND SANDRA RUSCETTI2

DepartmentofGenetics,Albert EinsteinCollege of Medicine,Bronx,New York10461,1andLaboratory of

TumorVirusGenetics, National CancerInstitute, Bethesda, Maryland 202052

Erythroleukemia cell lines HFL/d andHFL/b, derived from tumors induced in vivo in BALB/c

(H-2d)

and congenic BALB.B

(H-2b)

mice, respectively, by a polycythemia-inducing strain of Friend virus, produced bothspleenfocus-forming virus(SFFV) and its nativeNB-tropic helper virus (Friend murine leukemia virus [FMuLV]) during

early-passage generations

in culture.

Eventually

each line ceased production of both infectious viruses but retained its

tumorigenic potential

in syngeneic hosts.

Virus-producer

and

-nonproducer

clones of these cell lines were examined for expression of proteins encoded

by

the SFFV or FMuLV genomes.Lysatesoflabeledcellsweretreated with various antiviralsera,and the precipitateswereexamined

by gel

electrophoresis. Expression

of the FMuLVenv gene-encodedprecursor

protein,

gPr85env,

wasobserved inall

producer

andmost nonproducer clones, but the FMuLV gag andpol gene products, Pr659'9 and

Pr2009a"'PI,

wereuniformly undetectable innonproducer clones.All HFL/d and HFL/b clones expressed

appreciable

amounts ofthe SFFV-encoded

envelope

protein, gp52, includingone

exceptional

clonewhich had ceasedto express any FMuLV-encodedproteins. The molecularweight of this SFFV-encoded

envelope

protein was consistently smaller in all HFL/b clones than in

HFL/d clones,

regardless of theirproducerornonproducerstatus. Thevirus-nonproducer phe-notype thus appears to be due to shutdown ofexpressionof the 5'portion of the FMuLV genome in twoindependent cell lines.

The Friend

erythroleukemia-inducing

virus complex (FV) consists ofthe

spleen

focus-form-ingvirus

(SFFV)

anda

helper

retrovirus

com-ponent,Friendmurineleukemiavirus

(FMuLV).

SFFVhas been showntobea

replication-defec-tive virus associated withspleen focus induction and therapid

development

of

splenomegaly

and erythroleukemia in susceptible mice (29). Al-though co-infectionwithahelpervirusis essen-tial forproduction of infectious SFFVparticles, helper virus is not needed for infection and expression of the SFFV genome in fibroblast cultures (3, 31) orpossiblyevenfor transforma-tionof erythroidprecursorcells (6).

Erythroleukemia cells from tumors induced byFVcan beadapted to growth inculture (15). Mice of theBALB/c

(H-2d)

andBALB.B

(H-2b)

strains, congenic with respect to the H-2 locus, have been used inourlaboratorytoestablish a series of H-2-congenic erythroleukemia cell lines, designated HFL/d and HFL/b, respec-tively(12). In these

cell lines,

theexpression of infectious virus and of some viralantigens

even-tually

ceased as eachcellline wascontinuously

passed in culture (13). Although virus-nonpro-ducercelllineswere

phenotypically

virus nega-tive, they still maintained the entire viral ge-nome, sincerenewedproduction of infectious FV

could be induced chemically. Recent studies fromourlaboratory (2)involving molecular hy-bridization ofcellular RNA with viral comple-mentary DNAprobessuggested that shutdown of Friend virus expressionin these erythroleu-kemiacell lineswasdueto amechanism involv-ing regulation oftranscription of proviral DNA. However, due to the unavailability of comple-mentary DNAprobestoindividual viralgenes, gag,pol,orenv,thepreciseandsequential role of thesegenes inthe shutdown of virus produc-tioncouldnotbeascertained.

The products of the FMuLV genome are rel-atively well known (Fig. 1). The env gene, lo-cated in the3'portionofthe viralgenome, codes for a glycosylated precursor protein,

gPr85env,

which isprocessed togp7O,themajorviral sur-facemolecule, andp15(E) (9, 10, 20). At least threeprecursorproteinsappear to be generated fromthe 5'portion of the viral genome compris-ingthegag andpol genes:

Pr2009'91"'

isprobably a precursor to reverse transcriptase, whereas gPr80gagand Pr659ag have beenreported to be two independent primary translation products of the MuLVgag gene (7,9, 18, 21, 28).gPr80gg appears to be further glycosylated and trans-portedtothecellmembrane, whereas Pr659ag is processed to form the internal structural

pro-654

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Pr65P 9 virionstructuralproteins(p15,p12p3O,plO)

gPr800"oucellsurfaceantigens \ Pr200eDPo-0/.reversetronscriptose

.+ ~ *85""-wvralenvelopeglycoprotein (gp7O) 5, gog Po/ ,nv C 3' FMuLV genome

5 ---.- 3' SFFV genome

4

[image:2.485.44.235.71.173.2]

p45 gp52

FIG. 1. Diagrammatic summary of the relation-ship between the genetic maps of ecotropic MuLV and SFFV and the major protein precursors and products translatedfromthesegenomes. The dashed lines inSFFV represent the portion of this genome which is not shared withFMuLV, at least part of whichoriginates fromthe genome of anendogenous

xenotropic virus.

teins p30,p15,p12,and plO.

Several translational products of the SFFV genome have also been identified (Fig. 1). A relatively well characterized one is gp52 (also designatedgp55) (5, 24, 26,33).The gene encod-ing this protein isapparentlythe result of recom-binationbetween the env genes ofFMuLVand axenotropicMuLV (26, 30).The gp52 molecule shows marked immunological cross-reactivity with thegp70productof anumber of mink cell focus-inducingstrains ofMuLV,which also ap-pear to derive fromenvregionrecombinational eventsbetween

ecotropic

and

xenotropic

strains of MuLV (8, 11, 16). Certain strains of SFFV have also beenshowntocode foranother prod-uct,p45,which is encoded in the 5'regionof the virus and is

antigenically

relatedtocertain prod-ucts oftheFMuLVgag gene(27).

Thepresent

study

explores

the

expression

of theproteinsencodedbytheFMuLVand SFFV genomes in relation to the shutdown of infec-tious virusproductioninthe various

erythroleu-kemia cell clones.

Metabolically

labeled

trans-lationalproductsof FMuLVwere identifiedby immunoprecipitation and gel electrophoresis, using antisera to gag and env gene proteins. SFFVgene productswereidentified with anti-sera raised

by

immunizing

rats with

syngeneic

nonproducercellsinfected

only

with SFFV. MATERIALS AND

METHODS

Cell lines. Frienderythroleukemiacelllinesused inthese studies have beenpreviously described

(12-14). Theircharacteristicsareoutlined in Table1.

Controlcell linesincluded normalratkidney cells (NRK)orFisherratembryocells(FRE) infected with SFFValone,FMuLValone,both FMuLV andSFFV,

orneither. These lineswereprovided bythegenerosity

ofD. H. Troxler and E.M.Scolnick of theNational Cancer Institute.

Antisera. Rat anti-SFFVserum waspreparedby immunizing Fisher rats with syngeneic SFFV-FRE

cells.The rats were injected subcutaneously at multi-ple sites with a total of 107 cells each and developed solid tumors in 2 to 3 weeks after injection. Antisera from two rats were pooled and inactivated at 56°C for 30 min. Goat antisera toRauscher MuLV (RMuLV) gp7O and Pr65O" were provided by the Division of Cancer Cause and Prevention, National Cancer Insti-tute. Goat anti-Pr65O'' serum was prepared against the purified RMuLV gag precursor and reacts with all gagproteins.

Metaboliclabeling of cells.Subconfluent mono-layers offibroblast lines in 100-mm petri dishes or 6 x106 cells/ml from each mouse cell suspension culture were labeled with 100 MCi of [3H]leucine per ml in leucine-free medium containing 1% dialyzed fetal calf serum.Pulsingwascarried out for 30 min at 37°C.

Fortunicamycin inhibition experiments, cells were treated with 0.5,ugoftunicamycin per ml (E. Lilly & Co.,Indianapolis,Ind.) for1h and thenlabeled with 100uCi of[3H]leucineperml for 30 min.

Immunoprecipitation of pulse-labeled

ex-tracts.Labeledcellswerewashed in cold phosphate-buffered saline and then lysed in extraction buffer (0.01 Msodium phosphate buffer, pH 7.6, 0.1 M NaCl, 1%TritonX-100, 0.5%deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]). Extracts were then spun at 2,000 x g for 20 min and pretreated overnight by incubation withnormal ratserum(5

id/ml)

and 50ul

ofStaphylococcus aureus per ml (a 10% Formalin-fixedsuspension of Cowan strain I [17]). Extracts were clarified by centrifugation at2,000 x gfor 20min. Immunoprecipitationswere setupat4°Cby incubat-ing1 ml of eachextractwith10 1d ofratanti-SFFV serum per mlorwith5,ulof various goat anti-RMuLV

seraperml.After16hof incubationat4°C,S.aureus

wasadded (50pl/ml) and incubationwascontinued for2hmore.Reaction tubeswerethencentrifugedat

2,000 x gfor 15 min, and the pelletswere washed three times with extraction buffer andsuspendedin

samplebuffer (50 mMTris-hydrochloride,pH 6.8, 1%

SDS,1%,B-mercaptoethanol,10%glycerol,and 0.001%

bromophenolblue)inpreparationforpolyacrylamide gelelectrophoresis.

SDS-polyacrylamide gel electrophoresis. Seven percentpolyacrylamidegelswith3.5%

stacking

gelswere prepared, and electrophoresis wascarried

out as previously described (19).

125I-labeled

bovine

serumalbumin(68,000)and RMuLVp30(30,000)were

used as molecular weight markers. Gels were then

processedforfluorography(4)and

exposed

toKodak RoyalBlueX-Omat filmat-70°C.

RESULTS

HFL/d and HFL/b cell lines: virus pro-duction and

tumorigenicity

in vivo. The HFL/d and HFL/b cell lines and their clonal derivatives originated from the

spleens

of BALB/c and BALB.B mice,

respectively,

in-fected with FV of NB-tropic host range (14). Each cell line produced both infectious SFFV andFMuLV (titers,>102 infectiousunits/mlof

culturesupernatant)inearly passagesin culture. TheHFL/dline ceased

producing

both viruses very early, and no frozen stocks of this line

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656

TABLE 1. Tumorigenicityandexpressionof infectiousvirusbyclonesofratandmousecelllines

Cell line Symbol Tumora

SFFV

FMuLV

yin mor

~~~~~~(FFU/m-l)b

(PFU/rnl)

Rat

SFFV-FRE/FMuLV 8.5x102 2.5x105

FMuLV/NRK 0 6.25x102

SFFV/NRK 0 0

SFFV/FRE 0 0

NRK NT NT

Mouse

HFL/d-NB cl. A V+gp70+ + 15.8x 104 6.25x106

HFL/d cl.4 V-gp70+ + 0 0

HFL/d cl. A V-gp70- + 0 0

HFL/b cl. B2 V+gp70+ + 7.1x 102 2.5x103

HFL/b cl. C4 V-gp70+ + 0 0

a5x

l06 cells

were inoculatedsubcutaneously into young syngeneic recipients, and micewereobserved for

tumorgrowth.

bCulture

supernatants

weretestedbythespleenfocus assay in vivo.FFU, Focus-formingunits.

'Culture

supernatants

weretestedbythe XCplaqueassay.

before shutdown exist. TheHFL/bline ceased virusproduction much later in itspassage his-tory,andthis shutdown has occurredrepeatedly atapproximately thesame cumulativepassage level when stocks frozen before shutdownwere reestablishedinculture (22).

Characteristicsof the various clones of the cell linesused inthesestudies,representing different phenotypeswith respect tovirus

expression,

are summarizedinTable 1.Clonesrepresentingthe HFL/d cell line include: (i) a typical nonpro-ducerclone, HFL/d clone4, which expressesthe viralenvelope antigen (VEA) ofFV(12) andits majorcomponent,gp7O (14), calledV-gp70+; (ii) a unique nonproducer clone, HFL/d clone A, derived fromaV-gp70+

population

but

lacking

expression ofVEA orgp7O, calledV-gp7o-; and (iii)twovirus-producer clones obtained fromthe V-gp70- line by either treatment with 5-bro-modeoxyuridine (HFL/d-Bu) orsuperinfection withSFFV-freeNB-tropic FMuLV (HFL/d-NB clone A), bothcalledV+gp70+. Clones represent-ingtheHFL/b cell lineinclude: cloneB2,which producesbothSFFVandFMuLV and expresses VEAantigens(V+gp70+); and clone C4, which is avirusnonproducer butexpresses VEAantigen (V-gp70+).NoHFL/b clones which have ceased to express VEA antigen have as yet been ob-served.

Each of thesecloneswasstudied forthe pro-duction of infectious virus detectable in either the XC plaque assay for FMuLV (25) or the spleenfocus assay for SFFV (1).All lines,virus producers aswell asnonproducers, gave rise ta tumors in syngeneic mice 1 to 3 weeks after subcutaneous injectionof4 x 106 cells (Table 1).

Demonstration ofspecificity of antiviral sera.Various antisera to be used in

characteri-zation ofthe

erythroleukemia

cell clones were analyzed for their specificity, using rat fibro-blastsinfected withFMuLValone,SFFValone, or both FMuLV and SFFV. Anti-serum to Pr659' precipitatednotonly FMuLVgaggene products

(Pr200"ag'P,

Pr65V",

andp40[21];Fig. 2, lanes 11 and 12), but also the homologous SFFV gag protein (p45) (lane 13). Anti-gp7O serum precipitated the FMuLV env gene pre-cursor,

gPr85env

(lanes9 and10), aswellasthe analogous protein encoded by SFFV,gp52(lanes 7, 8, and 10). Rat antiserum prepared against SFFV-nonproducer cells precipitated FMuLV gag gene products (lanes 3 and 4) but not FMuLVenvgeneproducts.The rat serumalso precipitated the SFFV-encoded gp52 (lanes 2 and4).

The above antiserawere thenusedbelow to characterize the FMuLV and SFFV proteins expressedinthe variousmouseerythroleukemia cellclones.

Expression ofFMuLV-coded proteins in mouse tumorcellclones.

Suspension

cultures of clones ofthe HFL/d and HFL/b cell lines werepulse-labeled with

[3H]leucine

for 30min,

and

lysates

of the cellswere treatedwith

anti-viralsera.Electrophoresisoftheproteins precip-itated with anti-RMuLV gp7O serum revealed thatgPr85envwas detectable inall clones, irre-spectiveof theirvirus-producer or -nonproducer status, with the exception of HFL/d clone A (V-gp70-) (Fig. 3, lane 4). Thegp7Oproduct of gPr85env was detected only at very low levels aftera30-min pulse, but a 2-h chase after the

labeling

period ledto amarkedenhancementof

gp7O

detection (datanotshown) andpermitted confirmationthat its presenceorabsence corre-latedwiththatofgPr85enU.

Similarly prepared cell lysates were also

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[image:3.485.49.449.80.217.2]

1

2 3

4

m

Pr

20gogpoL

5

6 7

8 910

11121314

Pr209

68K-

_a.

am

p40-

*N

30K-r

Anti SF.V

Anti

SFFV

4I041

a..

-_ _p

I I

Anti

gp70

-gPr 85

env

-Pr

659°9

pS2

p45

I I

Anti

Pr659°9

FIG. 2. Identification of viral proteins in clones of rat cells infected with SFFV, FMuLV, orboth. Cells

were metabolically labeled with [3H]leucine for 30 min, 'and lysates were treated with various antisera. Antigen-antibody complexes were extracted with a 10% Formalin-fixed suspension of S. aureus and analyzed by electrophoresis on a 7% polyacrylamide gel containing SDS. Rat anti-SFFV antiserum (lanes 1-4), goat

anti-RMuLVgp7Oserum(lanes6-10),orgoatanti-RMuLVPr659' serum (lanes 11-14)was used to precipitate uninfected NRK (lanes 1, 6, and 14), SFFV-FRE (lane 7),SFFV-NRK (lanes 2, 8, and 13), FMuLV/NRK (lanes 3, 9, and 12),orSFFV-FRE/FMuLV(lnes 4, 10, and 11) cells. Lane 5 represents'"I-labeledmolecular weight markers bovine serum albumin (68,000) and RMuLVp3O (30,000).

1

2 3

4

5

6

_ W 0

\_-Pr8~Pr

5@n

[image:4.485.45.441.55.228.2]

ff:n_

~gp

48

FIG. 3. Immunoprecipitationofenvgeneproducts from mouse erythroleukemia cell lines. Clones of HFL/d and HFL/b cell lines were labeled with

[3Hlleucinefor30min.Celllysateswereprecipitated bygoatanti-RMuLVgp70serum.Different

virus-pro-ducer and-nonproducercelllinesusedwere HFL/d-Bu, V+gp70 (lane1); HFL/d-NBclone A Vgp70+ (ane2); HFL/d clone 4, V-gp70+ (lane 3); HFL/d

clone A, V-gp70-(lane4); HFL/b cloneB2,V+gp70'

(lane5);andHFL/b cloneC4, V-gp70 (lane 6).

treated indifferentexperiments withgoat

anti-serum prepared against the RMuLVgag gene precursor,Pr659ag(Fig. 4). Incontrasttotheenv gene product of FMuLV, the expression of

Pr65"" and gPr80gag correlated with the

virus-producer phenotypein thevariousclones(lanes

1 and 5). Virus-nonproducer clones showed no detectablelevels of this protein (lanes 2-4). This samecorrelationwith virus production was seen withrespect toexpression of the high-molecular-weight precursor,

Pr200"9`"',

a finding con-firmed in separate studies using goat anti-RMuLV reverse transcriptase antiserum (data notshown).

Theseexperimentsdemonstrated that the vi-rus-nonproducer phenotype among the eryth-roleukemiaclonesexamined correlated withthe absence ofgene productsof the 5' portion but notwith thoseof the 3'portion of the FMuLV genome.

Expression of SFFV-coded proteins in mouse tumor cell clones. The SFFV gene product, gp52, was precipitated by antibodies presentinratanti-SFFVandgoat anti-RMuLV gp70 antisera. When these sera were used to precipitate radiolabeled

lysates

ofthe

panel

of

erythroleukemia

cell lines

(Fig.

3 and

5),

gp52

couldbedetectedineach of theHFL/d clones whetheror nottheyproduced virusorexpressed FMuLV env gene products (Fig. 3, lanes 1-4; Fig.5,lanes1,2,5, and6).Thiswasalsotrueof the virus-producer and -nonproducer HFL/b clones,but the sizeof themajorprotein precip-itated with anti-gp70 and anti-SFFV sera was always slightly smaller,

approximately

48,000 daltons (Fig. 3, lanes5and6; Fig. 5,lanes3, 4, 7, and8).Thesmallerproteinwasalsoa

glyco-protein, since it, like gp52,

incorporated

[3H]-mannose

during

a 1-h

pulse

(data

not

shown).

Smaller amounts of a

52,000-dalton

protein

couldalso be detected in these cells.

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[image:4.485.46.238.313.484.2]

658 ANAND, LILLY, AND RUSCETTI

1

2

3

4

Pr6

:- f SQ

qPr80990

00W0;0<:4iWt

irom

FIG-.4f-.

Imuopei:tto

of

ga gen-oe

-nonproducerclonesoftheHFL/d and HFL/b cell lineswerepulse-labeledwith[3H]leucine for30min. Celllysateswereprecipitatedwithgoatanti-RMuL V

Pr65'g'serumand-separatedon a7%polyacrylamide

gel containingSDS. Clones analyzedwere HFL/d-NB clone A, V+,gp70+ (lane 1); HFL/d clone 4,

V-gp70+(lane 2);HFL/dcloneA, V-gp77- (lane 3);

HFL/b cloneC4,V-gp70+(lane 4);andHFL/b clone

B2, V~gp70+(lane 5).

Toascertain whether the difference in

molec-ular weight of HFL/d gp52 and HFL/b gp48

resided in the protein or carbohydrate portion

of the molecule, the virus-producer clones of

both lines were treated with tunicamycin, an

inhibitor ofglycosylation, before labeling with

[3H]leucine.Smallergp52-related proteinswere

expressedinbothclones aftertunicamycin

treat-mentof thecels(Fig.6,lanes2and3),and the

difference in molecular weight between these

unglycosylated proteins appeared to be about

thesame asthat between gp52andgp48.

Con-version of FMuLV gPr85en to Pr66" in the

presence of tunicamycin (lanes 2 and 3)

con-firmned that glycosylation was indeed inhibited in this experiment. It thus appears that the

difference between the HFL/d gp52 and the

HFL/bgp48resides in theirproteinmoieties. In experiments not shown, spleen cells from bothBALB/cand BALB.B mice infected9days

earlier with FVexpressedagp52butnot agp48. Thus, the expression of gp48 in clones of the HFL/bcell lineis most

likely

anidiosyncracy of this line which isnotrelatedtoits

H-2b

type.

AlthoughtheSFFV-encoded

gag-related

pro-tein,p45,could be

consistently

immunoprecipi-tated withgoat

anti-Pr65Vqg

andratanti-SFFV sera from SFFV-infected rat celllines

(Fig. 2),

its presence in any ofthe mousecell lineswas

notreadily detected, suggestingthat itwas ex-pressed only atverylow

levels,

ifatall. Ithas previously been shown that certain strains of SFFV donotexpressp45(27), andthestrainof SFFVin thelines studied here may be sucha p45-negative strain. Rat anti-SFFV sera

did,

however, precipitate

Pr65Vag

in all producer clones (Fig. 5, lanes 5 and 8), but not in any nonproducer clones (Fig. 5,lanes6and7), con-finning thedata obtained with goatanti-Pr65ag serum.

DISCUSSION

Two

FV-inducederythroleukemia cell lines of independent origin, HFL/d and HFL/b, have given risetoclones representing both the virus-producer and virus-nonvirus-producer phenotypes. Presentstudiesdemonstrate that virus nonprod-uction is correlated with the absence of gene products encodednearthe 5'end of the helper FMuLV component of the FV complex. Gene products encodedin the 3' envregion of FMuLV continuedtobe detectableinnonproducer

cells,

1 2 3 4 5 6 7 8

env

gPr85

X4

gp52- Pr65909

5

-p48

Virus +-- +

gp7O + - + + + - + +

l~l

Serum anti gp7O antiSFFV

FIG. 5. Comparativeanalysis of viral proteins of

mousecell lines by immunoprecipitation with goat anti-RMuLVgp70serumandratanti-SFFVserum.

HFL/dandHFL/bvirus-producerand-nonproducer cloneswerelabeled with

[3H]leucine

for30min.CeU

lysatesweredividedintotwoportionsand immuno-precipitated separatelywithgoatanti-RMuLVgp70

serum(lanes1-4) andratanti-SFFVserum(lanes

5-8). Various cell linesused wereHFL/d-NB clone A,

V+gp70+ (lanes1 and5); HFL/d clone A, V-gp70-(lanes2 and6); HFL/b clone C4, V-gp70+ (lanes3 and 7); andHFL/b cloneB2, V+gp70+ (lanes4and

8).

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1 2 3 4 5

68K-1

30K-HFL clone

gp7O

Tunicomycin

-gPr

85&nv

-p66env

-gp52 gp48

-p46

p42

d b d b

+ + + +

_-FIG. 6. Viralgeneproducts in tunicamycin-treated

cells. HFL/d-NBcloneA, V+gp70+ (lanes2and4) and HFL/b cloneB2, V+gp70+ (lanes3and 5)were

incubated either in thepresence(lanes2 and 3) or

absence(lanes4and5) oftunicamycin for1h, labeled

with[3H]leucine for30min, and then

immunoprecip-itated with goat anti-RMuLVgp7O serum. Lane 1

represents'I-labeledmolecular weight markers.

althoughoneofthenonproducer lines (HFL/d,

V-gp70+) eventuallygaverisetoadifferent

var-iant (HFL/d, V-gp70-) whichexpressedneither 5'nor3'genesofFMuLV. Incontrast,allclones,

producer and nonproducer alike, clearly

ex-pressed the gp52 product of the 3' end of the SFFVgenome.

Our present resultsare consistentwith other

recentstudies inthislaboratory which involved analysis bynucleic acidhybridization techniques of theexpression ofFMuLV-specificand SFFV-specific RNA sequences in the cytoplasm and

nucleus of thesesameproducerandnonproducer clones (2). These studies indicated that both

typesof clones expressed essentiallyall SFFV-specificRNAsequencesand all RNAsequences

shared by both SFFV and FMuLV. However, nonproducercloneswerepartiallyortotally

de-ficient in expression of FMuLV-specific RNA

sequences. The presentstudies havepermitted

ustoidentifytheportionof theFMuLVgenome

whose expression is deficientinthesecell lines.

Clones found earlier to express only 75% of

FMuLV-specific RNA sequences are those

which express gene products of the 3' but not

the 5' end of FMuLV, and the unique clone

found earliertoexpressno detectable

FMuLV-specific RNA sequences also fails to express

products of either endof the viralgenome.

Thenonproducererythroleukemiacellclones analyzed in this study are apparently different fromthe single nonproducer clone analyzed by Racevskis and Koch (23), who concluded that the nonproducer status ofcellsthat they were studying was due to a defect in the processing of

Pr6Y5g

rather than to its failure to be tran-scribed. Thus, more than one mechanism may beresponsible for the nonproducer phenotype of a givenerythroleukemia clone.

The SFFV-encoded gp52 molecule was de-tected inallclones of both the HFL/d andHFL/ b cell lines. However, in HFL/b clones, itwas routinelyfainter in theautoradiographs, and a newband, absent inHFL/d clones, was always present at a position corresponding to about 48,000 daltons. This new protein showed the samepattern of reactivity withthe various an-tiseraasgp52. Treatmentof the cell lineswith tunicamycin to inhibit glycosylation produced

parallel

effects on the electrophoresis patterns

ofgp52 in HFL/d cells and gp48 in HFL/b cells, theapoprotein of gp52 being about 46,000 dal-tonsand thatof gp48beingabout 42,000daltons. Preliminary observations of electrophoretic pat-terns of productsofpartial enzymatic digestion of gp52 and gp48 also show them to be very similar (datanotshown). Thepresenceofgp48 inHFL/bcellsappears to be an idiosyncracy of

this

particular

line, since onlygp52andnotgp48

could be detected in cells from the enlarged spleens of BALB.B mice infected in vivo with FV. Apossible explanation for the phenomenon is thatgp48originates fromavarient SFFVenv gene; since gp52isalso detected in these

cells,

it seemspossible that the HFL/b line originated fromaprogenitorcell containingmorethan one SFFV proviral genome, one of which bears a mutation in the gp52 region. Consistent with thisideaistheobservationthat somestrains of SFFV code for

slightly

smaller gp52 molecules (27,32).

Each of the clones examinedinthese studies has retained its

capacity

to form tumors upon reintroductioninto

syngeneic

mice.The

finding

that one ofthese

clones, HFL/d

clone

A,

failsto

express anyFMuLVgeneproducts while

contin-uing

toexpressthe SFFVgp52 lendssupportto the ideathatSFFV and notFMuLV is respon-sible for theoncogenic

potential

of the FV

com-plex. Also, the observation that the SFFV-en-codedgp52 isexpressed ineach of the

erythro-leukemiacellclones,whereas theSFFV-encoded p45 is not, is consistent with thehypothesisthat the oncogenic

potential

of SFFV resides in its env gene. However, additional

V-gp70-

clones of FV-induced

erythroleukemia

cell

lines,

tu-morigenic as well as

nontumorigenic,

must be analyzedinordertoestablishafirm correlation

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[image:6.485.46.238.59.274.2]

between SFFV gp52 expression and leukemic transfornation.

ACKNOWLEDGMENTS

The progressof these studies has been abettedby discus-sionswith Alan S.Berkower, Ruy Soeiro,and Richard A. Steeves. We thank IdaShapirofor expert secretarial assist-anceand KennethBlumbergforhelpwith virus assays.

Supportfortheworkwasfrom the National Cancer Insti-tuteunder Public Health Service grant09173.R.A.was sup-ported by Public Health Servicetraininggrant 09060from the National Cancer Institute.

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