Cell-free synthesis of mouse mammary tumor virus Pr77 from virion and intracellular mRNA.

0022-538X/79/03-1131/11$02.00/0

Cell-Free Synthesis of

Mouse Mammary

Tumor

from

Virion

and Intracellular

mRNA

Virus Pr77

HANS-HENRIK M. DAHLt AND CLIVE DICKSON*

TranslationandViralOncology Laboratories, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, WC2A3PX, United

Kingdom

Received for publication10October 1978

Mousemammarytumorvirus (MuMTV)waspurifiedfromtwo celllines (GR

andMm5MT/cl), and the genomic RNAwasisolatedandtranslatedin vitro in

cell-freesystemsderived frommouseLcellsandrabbitreticulocytes.The major

translation product in both systems was a protein with the molecular weight

77,000. Several other products werealso detected,amongthema 110,000-dalton

and in minor amounts a 160,000-dalton protein. All three polypeptides were

specifically immunoprecipitated by antiserum raised against the major core

protein of MuMTV (p27), but theywere notprecipitated byantiserum against

the virionglycoprotein gp52. Analysis of the in vitro products by tryptic peptide

mapping established their relationship tothe virion non-glycosylated structural

proteins. The 77,000-dalton polypeptidewasfoundtobesimilar, ifnotidentic.al,

toananalogous precursorisolated from MuMTV-producing cells. Peptide

map-ping of the 110,000-dalton protein shows that it contains all of the

methionine-labeledpeptides found in the 77,000-dalton protein plussomeadditional peptides.

We conclude thatthe products synthesized in vitro from the genomic MuMTV

RNAarerelatedtothenon-glycosylated virion structural proteins. Polyadenylic

acid-containing RNA from MuMTV-producing cells also directed the synthesis

of the 77,000-dalton polypeptide in the L-cellsystem. If this RNA preparation

wasfirstfractionated bysucrosegradient centrifugation, the 77,000-dalton protein

appearedtobesynthesized from mRNA withasedimentationcoefficient between

25and35S.

Mousemammarytumorvirus (MuMTV) isa typeB oncovirus of thefamilyRetroviridae. The

typeB oncovirusesarecharacterizedby having

a subviral form called the intracytoplasmic

A-typeparticle,detectablebythin-section electron

microscopy, andbythemorphologyof the virion

particle which shows an eccentrically located

electron-dense nucleoid (3). In contrast to the

type C oncoviruses that are usuallyassociated

with leukemias andsarcomas, the MuMTV

in-duces mammary carcinomas in infected mice

(48). In cellcultures which shedMuMTV,

tran-scriptionof theintegratedviralgenomeappears

tobe underglucocorticoid hormone control(29,

38, 39, 43). The virions contain a 70S RNA

complex which is probably composed of two

subunitsofsingle-stranded genomicRNA which

sedimentatapproximately 35Sandanumber of

tRNA's of which the predominant species is

tRNALYS (9, 11, 53). The 70S RNA complex,

tPresent address: Section for Medical Enzymology and

Molecular Biology, University of Amsterdam, Amsterdam,

The Netherlands.

together with several non-glycosylated

low-mo-lecular-weight proteins,composesthecore

struc-tureof thevirion(5, 47).

The nondefective avian type C oncoviruses

have beenmore thoroughly characterized than

have thetype Boncoviruses and also shown to

containa70SRNAcomplex composed of similar

subunits. Type C oncoviruses contain the

ge-netic informationtocode forthe internal

struc-turalproteins (gag),theRNA-dependentDNA

polymerase (pol), the envelope glycoproteins

(env), andinthecaseof thesarcomaviruses the

src geneproduct. These genes are arranged in

the order5'-gag-pol-env-src-p(A)-3' (18, 19, 52).

Cell-freetranslation of the intactgenomicRNA

of severaltype C virusesprimarily gives rise to

apolyproteinthat is similartotheprecursorfor

thelow-molecular-weight proteinsthatcompose

thecorestructureof thevirion(gag) (13, 24-26, 33, 41, 50, 51). In some instances a protein of

180,000 daltons has beendetected, and this

ap-pearstocontain thegaggeneand thepolgene

products (25, 30, 35).Theenvelope proteinsand

1131

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the src gene product appear to be synthesized

onintracellular subgenomic mRNA's (4, 12, 15,

32, 46,54).

Severalrecent studies havedemonstrated that

the internal structural proteinsofMuMTV are

synthesized in cells as part of a polyprotein

precursor of77,000to 75,000 daltons (Pr77/75)

(6, 36, 42). In this studywehave used in vitro

translationto investigate the genomic RNA of

MuMTV obtained from two mouse cell lines

continuously producing the virus. The products werecharacterizedbyimmunoprecipitation and tryptic peptide mapping. We found that the

major product synthesized was a 77,000-dalton

protein (p77) thatresembled the Pr77/75 found

in cultured cells. Inaddition, atranslation

prod-uctof110,000daltons (plIO) and aminor

prod-uct of 160,000 daltons (p160) were detected

which also have their counterparts in infected

cells (6). The in vitro-synthesized plO was

shown to contain all ofthe methionine-labeled

trypticpeptidesfound inthep77plus additional

peptides.

When cellular polyadenylic acid

[poly(A)]-containing mRNA from virus-producing cells

wasexamined,asimilarprotein of 77,000 daltons

could beimmunoprecipitated. This mRNA

giv-ing rise to p77appeared tosediment in a sucrose

densitygradientatabout 25 to35S.

MATERIALS AND METHODS

Cell cultures. Two cell lines producingMuMTV wereusedtoprepareviralgenomicRNA.One,the GR line, was grown in Eagle minimal essential medium withantibiotics, 10% horse serum, and 10jgof dexa-methasone per ml (37). The other cell line,

Mm5MT/cl,was aclone(10)fromamammarytumor

cell line derived from C3H mouse mammary tumor

(28). Thislatter cell line wasgrown ina similar

me-dium, butwith fetal calfserumsubstituted for horse serum.Theproductionof MuMTVbyboth celllines was routinelymonitored witha reverse-transcriptase

assayusingeitherMg2+orMn2'asthe divalent cation to distinguish between type B and type C viruses as

previously described (56).

Preparation of RNA. The cells were grown in roller cultures (480-cm2Corning flasks) and continu-ously labeled with 10 1iCi of [3H]uridine (Amersham/Searle) per ml. Culture mediumwas har-vestedat 5- or 16-hperiods andclarifiedby centrifu-gation at 12,000 x g for 10 min, and the virus was

pelletedby centrifugation at 140,000 xg for40 min. The viruswassuspendedin buffer(0.01M Tris-hydro-chloride, pH 7.2, 0.1 M NaCl, 0.001 M EDTA), dis-ruptedwith 0.5% sodiumdodecyl sulfate (SDS), and extracted three times with phenol-chloroform-iso-amyl alcohol at 24:24:1 and twice with chloro-form-isoamyl alcohol at 24:1. The RNAcontained in theaqueousphasewasprecipitated with 2.5 volumes of alcohol in 0.2 M sodiumacetate.The 70S RNA was fractionated on a 3 to 30%sucrose density gradient

containing0.05MTris-hydrochloride (pH7.5),0.05M EDTA, and 0.2%SDSbycentrifugationat280,000x g for 45min. The fractions composing the 70S peak werepooled, ethanolprecipitated,heatdissociatedat

100°C for 45 s, andrun on asimilargradient for100 min. The subunit RNAsedimenting ataround 28 to 35Swaspooled and ethanolprecipitated. Before

trans-lation, the RNAwasreprecipitatedto remove traces ofSDS, dissolved in water at 1 mg/ml, and stored at -700C.

Cytoplasmic RNA from MuMTV-producing cells was isolated, and poly(A)-containing RNA was se-lected on polyuridylic acid [poly(U)]-Sepharose 4B columns as previously described (32). 32P-labeled rRNAwaskindlysupplied byG. Peters.

Cell-free protein-synthesizing systems. The rabbitreticulocyte lysate was prepared as described byPelham and Jackson (34),except the amino acids (minus methionine)were added to aconcentration of 0.2 mMeach, and2-aminopurinewasaddedto afinal concentration of6mM. The 25-1lreactions were ini-tiatedbythe addition of15 to251iCi of

[35S]methio-nine (Amersham/Searle; 600 to 1,100 Ci/mmol) and 0.1 to1

tig

ofexogenous RNA andincubatedfor 1.5h at34°C. To determine theefficiencyofincorporation of[35S]methionine,

2-pl

portionswerewithdrawn and treated with 10 Ml of 30%hydrogen peroxideand 100

Al

of 0.1 M KOHat370C for 15minbefore

precipita-tion with cold 10% trichloroacetic acid. The precipi-tates werecollectedon0.45-ammembrane filters (Mil-lipore Corp.), washed,andcounted inaliquid scintil-lation spectrophotometer. Samples for analysis on SDS-polyacrylamide gelswere prepared by mixing4

[Lof thefinal reaction mixture with 10Mlofwaterand 15 Mul of electrophoresis sample buffer, followed by heatingat100°Cfor 2 min.

TheL-cellS30wasprepared as previously described (31).To 1ml oftheL-cellS30were added all of the components necessary for protein synthesis, except methionine butincluding CaCl2(1.0 mMfinal concen-tration),togiveatotalvolumeof 1.6 ml. The S30 was then preheated to 20°C for 2 min, and micrococcal nuclease (P-LBiochemicals) was added to a concen-tration of 10Mg/ml. After a further 10min at 20°C, the mixturewascooledto0WC,and 200 mM ethylene-glycol-bis(,B-aminoethyl ether)-N,N-tetraacetic acid (pH7.0) wasaddedto afinal concentration of 2.0 mM. Portions of this nuclease-treated L-cell S30 were stored inliquidN2.Thereaction mixtureconsisted of 16

Al

ofthe nuclease-treated S30supplemented with 5 Mgof ascitestRNA,aminoacids(minus methionine)

at a concentration of 0.2 mM, 15 to 25 MCiof [s5S]-methionine, and 0.1 to 1 Mg ofexogenous RNA in a tinalvolume of 25Ml. The Mg" andKCl concentra-tions which gave optimum incorporation of

[35S]me-thionine,usingcellmRNA, were determined for each lysate preparation. Incubations were performed at 37°C for1 h. Portions of 2ML were taken for trichlo-roaceticacidprecipitation, and samples were prepared forelectrophoresisasdescribedpreviously (31).

Antisera. The preparation and characterization of antiserumtodisruptedMuMTVand to electrophoret-icallypurified p27have been described previously (6, 7). Theanti-gp52serum(lot8/10/25/77) was obtained

as a gift fromMeloy Laboratories Inc., through the

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Office of Program Resources and Logistics, National Cancer Institute.

Immunoprecipitation. Samples of the cell-free protein-synthesizing systems were immunoprecipi-tated as described by Paucha et al. (31). Briefly, 15 to 20 ,ul of the reactionmixture was diluted with 50AI of NET buffer (0.05 MTris-hydrochloride [pH 7.4], 0.15 MNaCl, 0.005 M EDTA, 0.05% Nonidet P-40, 1 mg of bovineserumalbumin per ml) and incubated with 5

Al

of antiserum at 22°C for 1 h. Immune complexes were absorbed to 15

p1

of a 10% Formalin-treated suspension of Staphylococcus aureus (22). The bac-terial suspension was washed twice with NET buffer (minus the bovine serum albumin), and the immune complexesweredissociated by the addition of electro-phoresis sample buffer followed by heating at 100°C for2min. The bacteria were removed by centrifuga-tion before application of the samples onto the poly-acrylamide gel.

Electrophoresis. The samples were separated on 15% discontinuous SDS-polyacrylamide gels as de-scribed previously (23). Marker proteins of known molecular weights were labeled with ['4C]iodoacetic

acid as described by Smith et al. (45) and run in a

parallel track. The proteins were ,B-galactosidase

(120,000), phosphorylase a (94,000), fructose-6-phos-phatase (81,000), catalase (60,000), glutamate dehy-drogenase (53,000), creatine kinase (40,000), and lyso-zyme (14,000).

Tryptic peptide mapping. The appropriate bands were cut outfrom dried gels, rehydrated with 75 ,ul of a 1-mg/ml solution ofbovine serum albumin, frag-mented, and extracted with 3 ml of 0.1% SDS. The eluted proteinswere concentratedby trichloroacetic acidprecipitation, washed with diethyl ether, oxidized, andtrypsinized aspreviously described (6). The sep-aration of the tryptic peptideswasperformedon cel-lulosethin-layer platesbyelectrophoresisatpH4.6in the first dimension andchromatographyinthe second

(7).

RESULTS

In vitro translation of MuMTV virion

RNA. MuMTV virion RNA isolated from the

GR cellline asdescribed above sedimented on

sucrose gradientsas abroadpeak of 25to35S.

Harvesting atshorter intervals did not signifi-cantly change this size distribution. Since the

virion RNA containspoly(A) atits 3'terminus,

theRNA wasfurther purified by affinity

chro-matography on a

poly(U)-Sepharose

column. Theeluted

poly(A)-containing

RNA(1.5,Lg)was

fractionated on a 3to30%sucrose

density

gra-dientwith 32P-labeled28and18SrRNA assize markers (Fig. 1A). The RNA species still

sedi-ments as a broad peak of25 to 35S. An equal

portionof eachgradientfractionwastranslated

in themessenger-dependent rabbit

reticulocyte

lysate, and the products were analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 1B). The major translation

product

was a

pro-tein ofmolecularweight 77,000whichwas

effi-ciently translated off RNA sedimentingbetween 35and 25S.Aprotein of110,000daltonswasalso synthesized in the same fractions, and in frac-tions6 to 8 a product of160,000daltons (p160)

could be detected. Several otherpolypeptides of

lower molecular weightwerealso synthesized in

response to thefractionatedvirionRNA. When

these translationproductswere immunoprecip-itated withananti-p27serum, themajor product

p77andpllOandp160 werespecifically

precip-itated (Fig. 1C).

To comparethe efficacyof the cell-free

sys-tems torespond to 70S RNAcomplexorsubunit

RNA, MuMTVvirion RNA wasalso translated

in both the messenger-dependent mouse L-cell

S30 and rabbitreticulocyte systems.

MuMTV-relatedproducts synthesizedintheL-cellsystem appeared to be similar to those made in the rabbit reticulocyte lysate. When undissociated 70S virion RNAwasaddedtoeitherof the

cell-freesystems,thesamestimulationof aminoacid

incorporation andasimilarpolypeptide pattern were obtained aswith the subunit virionRNA

(Fig. 2),although the amount ofthe

lower-mo-lecular-weight productswasgreater with the 70S

RNA.Theseresultsdemonstratethatthe rabbit

reticulocyte lysatewas asefficientintranslating

MuMTV virion RNA as was the homologous

mouseL-cell system, and the products

synthe-sizedwereofsimilar molecularweight.

VirionRNA was also isolated from virus

pro-duced bythe Mm5MT/cl mouse cell line and

translated inbothcell-freesystems. This virion RNA,asexpected, coded for similar

products

to

the GR virion RNA; the main product was a polypeptide of about 77,000 daltons (Fig. 2). However, p77 and pllO from the Mm5MT/cl cells hadslightlylower molecularweights than the corresponding products from the GR cell

virus RNA. The ratio of pllO to p77 appeared

tobe similar with both strains of MuMTV.

Characterizationof theinvitroproducts using

specific

antisera. Theproteins

synthe-sized in response to the virion RNA werefurther

analyzed by

immunoprecipitation

and SDS-polyacrylamide

gel electrophoresis.

The results

of such an

immunoprecipitation

of theproducts

synthesized in the L-cell system are shown in

Fig. 3. The rabbit antiserum raised

against

the

majorvirionstructural

protein

p27

precipitated

p77 andp110,aswellasseveral

lower-molecular-weight proteins (Fig. 3, track 8). When goat

antiserum raisedagainstthevirion

glycoprotein

gp52 was used, no specific

polypeptides

were

immunoprecipitated

(Fig.

3, track

9), although

a small amount of p77 was detected. The

im-munoprecipitationofp77results fromalow anti-p27 activityin the anti-gp52 serum. We cannot

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1134 DAHL AND DICKSON

exclude the possibility that peptides related to

theglycoproteinweremade inthecell-free

sys-tem, but not

immunoprecipitated

withthe

anti-gp52 serum. For example, the antiserum may

not react with a

non-glycosylated

glycoprotein

precursorsynthesized in vitro. When the in vitro

products wereanalyzedwithantiserumraisedin

rabbits against whole detergent-disrupted

MuMTVvirions,thesame

polypeptides

and one

additional polypeptide of molecular weight

17,000 were

immunoprecipitated

as with the

anti-p27serum(Fig. 3,track10). Thisantiserum is known to recognize the virion

antigens

plO,

p14, and p27 and the

glycoproteins

gp36 and

gp52. The controland immunerabbitsera

non-specifically precipitate a 28,000-dalton poly-peptide seen in all tracks (Fig. 3, track 7). We conclude from the

immunoprecipitation

data

that the

majority

of the

products

synthesizedin

response to the MuMTVvirionRNAin the

cell-free systems are related to theinternalstructural

proteinsof the virion.

Tryptic peptide analysis of the MuMTV products.Tofurtherestablish the

relationship

of the invitro-synthesized productstothe virion

proteins, two-dimensional tryptic peptide maps were prepared. The in

vitro-synthesized

77,000-and110,000-daltonproteinswerecompared with thePr77/75 isolated fromGR cells (6) (Fig. 4).

From the maps we conclude that p77 made in

vitro and the Pr77/75 isolated from cells are

very similar,with only one spot (12A in Fig. 4) appearing in a

slightly

different position. Fur-thermore,the invitro-synthesized p110 contains

all of the methionine-labeled peptides foundin

p77 plus threeadditional peptides (spots 15, 16, and 17). The methionine-labeled peptides

con-tained in p77 can also be allocated to specific

viral structural proteins (Fig. 5). p27 contains peptides 3 and 9 to 14 as

previously

described

(6). In some peptide maps of p27, spot 14 is

absent and spot 13 doubles in

intensity;

the

reason for the variation in unknown. p14 and

plO of the MuMTVvirion are alsorepresented

by spot 2 and spots 1 and 6, respectively. A

mixingexperiment confirmed the relative

posi-tions of these methionine-labeledpeptides (Fig.

5). We therefore conclude that the internal

structuralproteins p27,p14,andplO of the virion

arecontained in the invitro-synthesizedp77 and

p110.

Translation of cellular mRNA from GR

cells. Total cell RNA was isolated from the

MuMTV-producing GR cell line and poly(A)-containing RNA selected by poly(U)-Sepharosse column chromatography. When

translated in the messenger-dependent L-cell

S30,

the mRNA gave rise to a

large

number of

polypeptides.

Immunoprecipitation yielded a

77,000-dalton protein

which could be

specifically

precipitated

with the antiserum against either p27or

disrupted

whole virions.To further

ana-lyze

the cellular RNA coding for the

77,000-dalton

protein,

thepoly(A)-containing RNAwas

fractionated on a 3to 30% sucrose density gra-dient.32P-labeled28and18S rRNA'swereadded

assize markers.ThemRNA'sfrom thegradient

fractions were translated in the L-cell system, immunoprecipitated with antiserum

against

MuMTV virions, and analyzed by

polyacryl-amide

gel

electrophoresis. As Fig. 6 shows, the

77,000-dalton

proteinwassynthesizedonmRNA sedimenting between25 and 35S relativeto the

28S rRNA marker.Thisisabroaderdistribution

than expected ifa single class of mRNA gives

riseto p77in the GR cells. However, the

sedi-mentationprofileofvirus-related mRNAis very

1

I

x

I1

10

0

I

n

x)

FRACTION NO

FIG. 1. Analysis of MuMTVvirion RNA by sedi-mentation on a sucrose density gradient, in vitro translation of thefractionatedRNA, and immuno-precipitation of the translation products. VirionRNA was extracted from [3H]uridine-labeled MuMTV shedbytheGR cell lineasdescribed inthetext. (A)

Profile ofthepoly(A)-containing virion RNA frac-tionatedon a 3 to30%osucrosedensity gradient. 32P-labeled28and 18Sribosomal markerswereincluded.

(B) Autoradiogram of the products synthesized in response tothefractionated RNA in the messenger-dependentrabbitreticulocyte lysate. Portions (4

[Ll)

wereanalyzed byelectrophoresison a15% SDS-poly-acrylamide gel. (C) Autoradiogram of the anti-p27 serumimmunoprecipitates separatedon a15% SDS-polyacrylamide gel. Tracks1 to 20referto the gra-dientfractions,track Mcontains"4C-labeledmarker proteins,and in track b no RNA wasaddedto the in vitroincubation mixture.

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[image:4.503.267.455.281.466.2]

similar to that of the genomic RNA shown in Fig. 1.

DISCUSSION

Poly(A)-selected virion RNA from MuMTV sedimenting between 25 and 35S wasefficiently translated in the cell-free systems. The results

show that themajor product synthesizedwas a

77,000-dalton protein (Fig. 1 and2). The maxi-mal synthesis of viral proteinsseen inFig. 1 is detected at 28S. The 28S virion RNA could resultfromdegradationorpossibly bythe prod-uctofa defective MuMTV provirus. However,

whenvirionRNA sedimentingat28Sinsucrose

density gradients is subjectedtoelectrophoresis on methylmurcury agarose gels (1), the RNA migrates with a molecular weight of approxi-mately 3 x 106, in agreement with other

esti-matesof thegenomesize (D. Robertson and H.

Varmus,personal communication).

Immunopre-cipitation analysis demonstratedthat the

77,000-dalton protein has antigenic determinants in

common with the major core protein of

MuMTV, p27, and tryptic peptide mapping of the [35S]methionine-labeled proteins confirmed

thatp27 and p77sharecommontryptic peptides.

Theresults also suggest that p77 shares peptides

in common with virion proteins plO and pl4.

The structural protein p21 appears to contain

little methionine, and, consequently,wecannot concludefrom theseexperiments whether p21is containedwithin p77.However,inanotherseries of experiments using

[14C]lysine

and

["4C]argi-nine tolabelthecell-associated Pr77/75 and the

virion proteins, all four small virion proteins

werefoundtobe present in thePr77/75

(unpub-lished data). The data shown in Fig. 4

demon-stratethat cell-derivedPr77/75and thecell-free

synthesized p77 were very similar.The doublet

natureof Pr77/75appears toresult froma

phos-M b 1 2 3 4 5

120-- so

94-8

81--?

60-

$

5

3-40--

Al

6 7 8 9 10 11

7-_

_-4ALio

12 13 14 15 16 17 18 19 20M B

160

- ---110

- 77

S

Up

M b 1 2 3 4 5 6 7 8 910 11 12 1314 15 16 17 18 1920 M C

120--94

-

81-60-_

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DAHL AND DICKSON

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4Wv

FIG. 2. Autoradiogram of polyacrylamide gel analysis of products synthesized in vitro in response to MuMTVvirion RNA.MuMTVvirion RNA wastranslated ineither themessenger-dependentL-cellS30(A) or themessenger-dependent reticulocyte lysate (B)andanalyzedbyelectrophoresis on 15%

SDS-polyacryl-amidegelsasdescribed in thetext.The[3Slmethionine-labeledproductsweredetectedbyfluorography. The in vitro systemswereincubated with(1)noRNA, (2)0.5pgofMuMTV70SvirionRNAfromthe GR cellline, (3) 0.5 ,ugof 30S MuMTV virion RNAfromtheGR cellline,and(4)0.5jigof30S MuMTV virion RNAfromthe Mm5MT/cl cell line.Track M is"C-labeledmarkers.

s

-..,,p

-~

FIG. 3. Autoradiogram showing the

immunopre-cipitation analysis of the in vitro-synthesized

MuMTVproducts.Noadded RNA (tracks to5)or

0.5 pLgofMuMTVvirion RNA (tracks 6 to 10) was translated in the messenger-dependentL-cellS30as described in the text. A 4-jil portion was taken for

directanalysis(tracks1and6). Theremainderofthe reactionmixturewasdivided intofour equal portions

and immunoprecipitated, as described in the text, withcontrol rabbit serum (tracks2and 7), anti-p27

serum(tracks3and8),anti-gp52serum (tracks4and

9),oranti-MuMTVserum(tracks5and10). TrackM is '4C-labeled markerproteins. The products were

analyzed by electrophoresis on 15%

SDS-polyacryl-amide slabgels, and labeledproducts weredetected

byfluorography.

phorylation ofp77 (R. Nusse, F. A. M.

Assel-bergs, M. H. L. Salden, R. J. A. M. Michalides,

andH. Bloemendal, Virology,inpress).

The MuMTV genomic RNA is in many

re-spects analogous to the type C virion RNAs

from Roussarcomavirusorthemurineleukemia

viruses insynthesizinga polyprotein that gives

rise tothe smallnon-glycosylated structural

pro-teins oftheir respective virions (20, 25, 26, 33,

49-51). We are not aware of any examples of

eucaryotic mRNA's which have more than one

active initiation site and, although the mRNA

might potentially possess internal initiation

sites,these appeartobenonfunctional (44).

Con-sequently, from an analogy with the type C

viruses, we might expect that the gene coding

for p77 is located near the 5' terminus of the

MuMTV genomic RNA.

A comparison ofthemajor translation

prod-ucts synthesized in vitro, using viral RNA

ob-tained from both GR and Mm5MT/cl cells,

showed that the77,000-dalton andthe

110,000-dalton proteins obtained with the latter virus

areslightly smallerand seem torepresent avirus

straindifference.

The cell-free translations also gave rise to

products of 110,000 and 160,000 daltons, the

latter inverysmall amounts. Both of these

pro-teins are immunoprecipitable with anti-p27

se-rum(Fig. 1C), and, furthermore,pl 10 was shown

to contain the methionine-labeled peptides of

p77 as a subset. It is not clear at present what

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CELL-FREE 1137

.% 12

w13 *, 11

0

12A

13

+ 0

12A

13 11

17

+ 0

FIG. 4. Autoradiogram

sionalseparation ofthe/'

ticpeptides obtained wit}

p110,p77,andPr77/75ob

latterwaspreviouslypubl

electrophoresis was carrie

origin (0) and is shown

Chromatographywasfrom

additional information

ofsimilarmolecularwei,

inGRcellculturesbyirr

PR77/75 the

anti-p27

serum

(6).

High-molecular-weight

polypeptides containing the gag-pol gene

prod-ucthavebeenfound in Rous sarcoma virus- and

MuMLV-infected cells and in

cell-free

transla-10 tionsofthese virionRNAs

(17,

20,

25, 27, 30,

35).

9 5 MuMTV p160 might represent a

similar

poly-4 protein. A

protein equivalent

to

pllO

has not 8 4w3 been described for the translation products of

d3

2 other nondefective oncoviruses. This protein

7 may be synthesized on the same mRNA as p77,

6 and a low level of suppression of the p77

termi-nation site would lead to a read-through product. Alternatively,initiation at a site to the 5' side of the p77 initiation site and direct read-through to the normal termination codon would give a sim-ilar product. However, this would imply from

the molecular weightestimatesthat this second

p77 initiation site is approximately 1,000 bases to the

5' side of the p77 initiation site. Synthesis on a

separate species of RNA present in minor

10 amounts in the virion preparations is also a

9 4 possibility. This minor species of RNA might

8 have the termination

condon(s) spliced

out or

403 deleted, as seems to be the case for several of

7 2 the defective oncoviruses (2, 14, 55). The

110,000-dalton polypeptide could also be the

6 normal gene product of another MuMTV (16,

56). However, the observation that the

molecu-lar weights of pllO and p77 are both slightly

lower in the Mm5MT/cl viral RNA translates

suggests that the messenger(s) coding for

pllO

and p77 is derived from the same provirus. The reticulocyte lysate and the messenger-P110 dependent L-cell

S30

produce several proteins of lower molecular weight that by

immunopre-cipitation arerelated to p77. The origin of these

lower-molecular-weight proteins is unclear,

al-10 5 though premature termination, internal

initia-4 16 tion, and proteolytic cleavage of p77 are

plausi-8 ble

explanations.

Authentic

proteolytic

process-- 3 ing does not seem to occur, since the size of these

15 ₇ 2 proteinsdoes not agree with those of the known

intracellular

intermediates detected in

virus-6 producingcells(6).

The 70Svirion RNA complex from MuMTV

was as efficient a messenger as the heat-dena-tured virion RNA in the cell-free systems tested, similartothe findingof Naso et al. (26), for type

showing the two-dimen- C 70S viralRNA.

Nevertheless,

thiswas

unex-S]methionine-labeledtryp- pected

since

the

subunit

RNA is probably the

i the in vitro-synthesized naturalmessengerin vivo. Wewerenot able to

5tained from GR cells (the determine whether the 70S RNA

complex

was

'ishedinreference 6). The dissociated during the incubation period or

sd out at pH 4.6 from an whether the complex itself functioned as a

mes-in the horizontal plane. senger. If the latter case applies, it might mean

bottomtotop. that the initiation codon and probably the

ribo-some-binding site are positioned to the 3' side

pllO contains. Proteins with respect to the primer tRNA-binding site.

ghts have been detected The70SRNA of MuMTV has also been shown

imunoprecipitation

with to act as an efficient messenger when

microin-VOL. 29,

4HIlI

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[image:7.503.50.236.72.559.2]

1138 DAHL AND

DICKSONJ.VO.

+ 0

12

13 11 10

9

3

2

6

+ 0

FIG. 5. Autoradiogram showing the f0Slmethionine-labeled trypticpeptide mapsofthe isolated virion

proteins p27,p14,andp10 and the mixturep27plusp14plusp10.

jected intoXenopus oocytes. Under these

con-ditions Pr77 isprocessed to yieldseveral of the virionproteins (Nusseetal.,inpress).

The poly(A)-selected mRNA from GR cells

also contained mRNAspecies whichtranslated

togive p77and sedimentedat35 to25S. This is consistent with the observations for both the avian and themurinetypeC viruses thatappear to produce a genomicsize mRNA in cells, and

this yields the analogous viral structural poly-proteinupontranslation in vitro. Both thevirion

and thecellular RNAs whichgiverisetop'77are

ofsimilar size, but more heterogeneous in size thanexpected whencomparedwith the

equiva-lenttypeC virussystems (unpublisheddata; 13, 32). At higher sedimentation values more pll0

is produced compared with p77, whereas at a

sedimentationvalue well belowgenomicsize the

p77alone isefficiently produced,indicatingthat p110couldbesynthesizedoffaseparatemRNA. Theseresultsarecomparabletothefindingthat

35Sandnot30Savianmyeloblastosisvirus RNA

hasthecapacitytosynthesize gag-pol(30). The

heterogeneityof RNAsizemightbedueto deg-radation, splicing ofRNAs, the products of dif-ferent proviruses, or an artifact of the sucrose

gradient centrifugation (seeabove).

In the experiments with poly(A)-selected RNA, an anti-MuMTV serum was used to

im-munoprecipitate the virus-specific proteins. However, the presence of a candidate for the

non-glycosylatedglycoproteinprecursorwasnot detected. The results obtained with the avian

type C virus mRNA's would indicate that this

polyproteinprecursorismoredifficulttodetect

(32).Alternatively, thenon-glycosylatedformof theprecursor mayhaveno, oramuch reduced,

specif'icity for the antibodies which wereraised

against the fully glycosylated proteins. An an-swerto this problem could rely upon the addi-tion ofa microsomal fraction, which results in

glycosylation in vitro (40), or the use of the

oocytes to translate a putative glycoprotein

mRNA.

p27

12

13 10

9 14

-~3

*2

p10

27+14+

IC:

6

+ 0

J. VIROL.

I

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[image:8.503.62.456.67.406.2]

285 I8S

i-o

34-A

-8

-1CN

-6 °

x

0E

u .4

0E

N

-2

5 10 15

FRACTION NO.

1 2 3 4 5 6 7 8 9 10 11 12 13 .4 15 16 B

---110

- _ +--- ~~~~~~77

'W,,

FIG. 6. Autoradiogram of polyacrylamide gel analysis of immunoprecipitated MuMTV products synthesized in the micrococcal nuclease-treated L-cellS30in responsetofractionated cellularmRNA. Cytoplasmic RNAs from virus-producing GR cells

werepoly(A) selectedon apoly(U)-Sepharose column,

and 25,ug was fractionated on a 3to 30%Q sucrose

gradient. mRNA in the 16 fractions collected was

concentratedbyethanolprecipitationinthepresence

of 10,ug of calf liver tRNA, and one-fourth of the

RNA in each fraction was added to the cell-free

system. After the incubation, the products were

im-munoprecipitatedasdescribed in thetextwith4,ul of anti-MuMTV. The immunoprecipitates were

ana-lyzed by electrophoresison 15%SDS-polyacrylamide

gels, andlabeledpeptidesweredetected by

fluorog-raphy. Tracks 1 to 16 are the immunoprecipitated gradient fractions.

ACKNOWLEDGMENTS

We thank M.A. Atterwill for excellent technicalassistance,

M. J.Hayman,H. F.Lodish,G.G.Peters,A. E.Smith,andR.

A. Weiss forhelpful discussions and critical reading of the manuscript, and A. Horwich for running the methylmercury gels.

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