Genome structure of avian sarcoma virus Y73 and unique sequence coding for polyprotein p90.

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Vol.38, No. 2 JOURNAL OFVIROLOGY, May1981,p.430-437

0022-538X/81/050430-08$02.00/0

Genome Structure

of

Avian Sarcoma

Virus Y73 and

Unique

Sequence

Coding for

Polyprotein

p90

MITSUAKI YOSHIDA,`* SADAAKI KAWAI,2 AND KUMAOTOYOSHIMA2

Departmentof Viral Oncology,CancerInstitute, Toshima-ku,' andDepartmentof Oncology,Instituteof

Medical Science, University of Tokyo, Minato-ku,2 Tokyo, Japan

Received 7November1980/Accepted 23 January 1981

Thegenomestructureofanewly isolatedsarcomavirus,Y73,wasstudied. Y73

isadefective,potentsarcomagenic virus and contains 4.8-kilobase (kb) RNAas

itsgenome;incontrast,helper virusassociated with Y73 had 8.5-kb RNA, similar

toother avianleukemia viruses. Fingerprinting analysis of these RNAs

demon-strated that the 4.8-kb RNA containsaspecific RNAsequence of 2.5kb, which

representsthetransforminggene(yas) of Y73.Thisspecificsequence wasmapped

in the middle of the genome and had at both ends 1- to 1.5-kb sequences in

commonwith Y73-associated virus RNA. Thisstructureisverysimilartothose

of avian and mammalian leukemia viruses. Invitro translation of the 4.8-kb RNA

and theimmunospecificity of the products directly demonstrated that polyprotein

p90,containing p19, isaproduct translated from capped 4.8-kb RNA and that the

specific peptide portion iscoded by theyas sequence.Protein 90,whichwasalso

found in cells transformed withY73,wassuggestedtobeatransformingprotein.

Amongavianretroviruses,Rous sarcoma virus

(RSV) has been most thoroughly studied

be-cause of itscompetencyinreplication and

sar-comagenic transformation. The sarcoma gene

(src)in RSV codesaprotein,p6OBrc (4, 21),which

has protein kinase activity

phosphorylating

ty-rosine residues in target proteins (5, 10).

Re-cently, we (27) reported that a newly isolated

avian sarcoma virus, Y73 (11), contains a

sar-comagenic transforminggene (yas) showingno

homology with the src sequence ofRSV. Fur-thermore, aspecific protein, p90, was found in

Y73-transformed cells and shown tohave

pro-tein kinase activity, which phosphorylated

ty-rosine residues in p90itself and in heavy-chain molecules ofimmunoglobulinGcomplexedwith

p90 (13). Fujinami sarcoma virus (7) was also recently reported to have a distinct class of transforminggene (8, 16).

Independent isolates of avian sarcoma viruses

RSVandB77 are competent inreplicationand

havevirtuallythe same genomestructure: 5' gag

polenvsrc 3' (26). On the otherhand,allavian

acuteleukemia viruses are defective in

replica-tion,deleting thepoland env genes and part of

thegag gene, instead of the region containing

the transforming gene in the middle of the

ge-nome(3, 6,9, 18,22).Since Y73 is a sarcomagenic

virus, but is defective in replication (11), it

seemed interesting to determine its genome structure.

Inthis work, wecharacterized the Y73 genome

by RNaseT1 fingerprintinganalysis and in vitro

translationof thegenome RNA. Results showed

that thestructural features of the Y73 genome are

essentially

thesame asthose of genomes of

otheracuteleukemia viruses and that polypro-tein p90 is a gene product of the specific

se-quence(yas),suggestingthatp90is a

transform-ingprotein.

MATERLALS AND METHODS

Cells and viruses. Chicken embryo fibroblasts

wereprepared from C/Oor C/BE chickenembryos

andculturedasdescribed before(28).Y73 viral stock

wasgrownasdescribed previously (13), and

Y73-as-sociated virus(YAV) wasisolatedbytwosuccessive

limiting dilutionsonchickenembryofibroblasts and

confirmedtohavenofocus-formingabilityunder the

standardagarlayer.

Viralpreparations were harvested every 12hand

purified byprecipitationwith ammoniumsulfate and

by successive discontinuous and continuous sucrose

gradientcentrifugations asdescribedpreviously (30).

Labelingof viral RNA with[32P]phosphatewascarried

outexactlyasdescribedpreviously (30).

Preparation ofviral RNA. Virion RNAwas

ex-tractedby proteinase treatment followed by phenol extraction (30). Virionsweredisrupted by adding0.1 volumeof10%sodiumdodecyl sulfateinthepresence of 100

,Lg

ofproteinaseKper ml. After incubation for

30minat37°C,RNAwasextracted three times with

phenol-chloroform (6:4) and then precipitatedwith 2

volumes of ethanol.RNAwasdissolvedin0.1%sodium

dodecyl sulfate and heated at 100°C for 1 min and

then fractionatedon a gradientof15 to30%sucrose

containing50mMTris-hydrochloride (pH 7.5),0.1M

NaCl, and 1 mM EDTA by centrifugationat 45,000

rpm for3h inaBeckmannSW50.1rotor at 15°C. In 430

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GENOME STRUCTURE OF Y73 431

[image:2.500.137.371.411.582.2]

someexperiments, RNA containingpolyadenylicacid

[poly(A)] wasseparated by passing the preparation

through acolumnof oligodeoxythymidylic

acid-cellu-lose as described before(31).

Fingerprinting analysis of[32PJRNA.

Two-di-mensional gelelectrophoresis was used for

fingerprint-ing RNase Ti-resistant oligonucleotides. The

proce-dure wasessentiallyas described by Lee and Wimmer

(17).Oligonucleotides separated on gels were extracted

with 0.5 ml of water, lyophilized, and digested with

RNase A (0.1mg/ml). The digests were analyzed by

electrophoresis at pH 3.5 onDEAE-cellulose paper as

described by Barrell (1).

Invitro translation of viral RNA.

mRNA-de-pendentreticulocyte lysate was prepared as described

by Pelham and Jackson (20) and used to translate Y73

RNA asdescribed previously (29). Each reaction

mix-ture(12

pl)

contained 0.2

jlg

of viral RNA and 20

ACi

of [3S]methionine in addition to the standard

re-agents. Twomicroliters of the reaction mixture was

usedforgel electrophoresisin thebuffer described by Laemnmli (15), and the gels were dried for

autoradiog-raphy orfluorography. Cells were labeled with

[3S]-methionine as described previously (31), and immu-noprecipitation was carried out according to Kessler (14).

RESULTS

RNA of Y73. Since the original Y73 stock

containedalarge excessofYAVofsubgroupA ashelper (11), the transformed cellswerecloned

in softagarmedium. Virusesreleased from the selected producer cellswerelabeled with

[32P]-phosphate, and the RNAwasanalyzed by aga-rose gel electrophoresis. As shown in Fig. la,

a b c

I

8.5kb-28S

-

48kb-0

x

E

a

18S-most of the RNA containing poly(A) migrated

as high-molecular-weight aggregates, probably

50-70S dimers. After heat treatment at 1000C

for 1 min, the aggregates were dissociated into

two RNA species: 4.8-kilobase (kb) and 8.5-kb

RNA components (Fig. lb). The 4.8-kb RNA

extracted from the gel sedimented as 26S on

sucrosegradient centrifugation (Fig.le),and the

RNAwassmaller thanthose ofknown defective

acute leukemia viruses (3, 6, 12, 18). On the

otherhand, 8.5-kb RNA sedimentedas35S,and its mobility in the gel was the same as that of

RNA of transformation-defective RSV. From these results, and from the findings that

non-transformingYAV isolated bylimiting dilution contained only 8.5-kb RNA and that the4.8-kb

componenthasaspecificsequence, asshownin

thispaper, we concluded that the 4.8-kb RNA

species isthe genome ofY73 carrying the

sar-comagenic transforminggene.

Analysis of Y73 RNA byfingerprinting. The relationship between 4.8-kb and 8.5-kb

RNAs wasanalyzed by

fingerprinting

RNase

T1-resistantoligonucleotides. 32P-labeled 4.8-kband

8.5-kbRNAs wereseparated byagarosegel elec-trophoresis andselected forpoly(A)-containing

RNA on oligodeoxythymidylic acid-cellulose.

The fingerprint of the 8.5-kb RNA (Fig. 2D) showed that thehelper 8.5-kb RNAwas asingle

component, and asexpected, the fingerprintof

4.8-kb RNA contained thespecific

oligonucleo-tides thatwere missing in that of 8.5-kb RNA,

f ract'ion no.

FIG. 1. Analysis ofY73 RNA byagarosegelelectrophoresis (a, b, c)andsucrose

gradient

centrifugation

(d, e).[32P]RNAofY73 virionscontainingpoly(A) before (a)andafter (b)heattreatment at1X0°C for1 min

and[32PJRNAoftransformation-defective PragueRSVwere

subjected

to

electrophoresis

in 2% agarose

gel

in40mMTris-hydrochloride (pH 8.0),20mM sodium acetate, and1mM EDTA.After

electrophoresis,

RNAs

wereextractedfromthe bandscorrespondingto4.8-kb and 8.5-kb RNA and

centrifuged through

15to30%

sucrosegradientscontaining20mMTris-hydrochloride(pH 7.5),0.1MNaCi, 1mMEDTA,and 0.2% sodium

dodecyl sulfatein anSW50.1 rotorat45,000rpmand15°C

for

180min. (d) 8.5-kbRNA; (e)4.8-kb RNA.

Dotted linesare[3H]rRNAaddedasinternal markers..

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432 YOSHIDA, KAWAI, AND TOYOSHIMA

00:.-fE-;S.-Sffi-.

:0

0'tV,0000000..00.:00077.?

A 00 ;f}0 -u:0- 0-0 SS00: :00 000

:E. :fX

FIG. 2. RNaseT, fingerprinting analysis ofY73 RNA.[32PJRNA of Y73wasfractionatedinto26S(A) and

35S(D)fractions,and eachfractionwastreated with weak alkalitointroduceoneortwo cutsin the RNA

molecule,asdescribedpreviously(27). Then RNA fragments containing poly(A)wereseparatedon

oligodeox-ythymidylicacid-cellulose andfractionatedintosixfractionsbysucrosegradient centrifugation. Eachfraction

wasrecentrifuged, and thepeak fractionwasdigestedwith RNaseT,andfingerprinted by two-dimensional

gelelectrophoresis. (A) 26S RNA;(B) 18-20S RNAfrom26SRNA; (C) 5-14S RNA fromt 26S RNA; (D) 35S

RNA; (E)19-25S RNAfrom35SRNA;(F) 5-14SRNAfrom 35S RNA.

indicating that 4.8-kb RNA contained the

spe-cific nucleotide sequences (Fig. 2A). All ofthe

oligonucleotidespotsfound in the fingerprintof

8.5-kb RNA were detected in that of 4.8-kb

RNA, butmostofthemwereminorcomponents.

These minorspotswereprobablycontamination

ofthe 4.8-kb RNA fraction with fragments of

8.5-kb RNA. To verify this explanation, each

spotwascut outfrom thegelsand quantitated

by counting the radioactivity of32P. Since the

accuratelength of eacholigonucleotidewasnot

known, the ratio of the yield of each

oligonu-cleotide from the4.8-kbRNA fraction to thatof

the corresponding spot from 8.5-kb RNA was

used to estimate the apparent molar

yield

of

eacholigonucleotide, assumingthatpure8.5-kb

RNA

gaveeach

oligonucleotide

inamolarratio.

Inthe case ofspotsspecificto4.8-kbRNA, the

yield ofoligonucleotides of 8.5-kb RNA which

were similar in size was used to calculate the

ratio. TheresultsaresummarizedinTable1.

All

spots wereclassifiedinto three groupsby their

yield ratio,thatis,less than0.5, between0.8and

1.2,andmorethan1.4.

Oligonucleotides

showing

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VOL. 38,1981

TABLE 1. Yi

oligonucleotidesfS

Spots in 26S RNA fraction No. Yield (cpm) 1 775 2 736 3 295 4 985 5 291 6 126 7 978 8 230 9 201 10 745 11 168 12 494 13 779 14 556 15 192 16 139 17 353 18 320 19 185 23a+b 867 24 389 25 131 27 201 28 216 29 806 30 164 31 616 32 142 38 564 39 575 40 101 41 227 42 568 43 607 44 320 45 500 46 164 47 177

'ields ofRNaseTi-resistant

rom26S and 35S RNAfractions

Spots in 35S RNA

fraction Ratio 26S/

No. Yield 35S (cpm)

(1.03)a (0.98)

51 753 0.39

52 643 1.50

53 779 0.37

(0.18)

54 702 1.39

55 502 0.46

56 491 0.41

57 553 1.35

58 441 0.38

(1.12)

59 562 1.38

(1.19)

60 466 0.41

61 379 0.36

(0.91) (0.82)

62 388 0.47

65 466 1.86

(1.08)

66 358 0.36

68 536 0.37

69 524 0.41

70 542 1.49

71 486 0.34

72 367 1.67

73 486 0.29

80 393 1.43

81 355 1.61

82 234 0.43

(0.97)

(1.06) (1.13) (0.83)

77 383 1.30

78 415 0.39

79 397 0.44

'Theratio ofoligonucleotides specificto the 26S RNA fractionwascalculated by using the yield (counts

per minute; cpm) of oligonucleotides of 358 RNA,

whicharesimilar insizetothespecificone;this ratio

isshown inparentheses.

aratio of 0.8 to 1.2 were all specific to 4.8-kb

RNA, and all otheroligonucleotides showinga

ratio of less than 0.5 or more than 1.4 were

confirmed by RNase A digestion to have the

samenucleotidecompositionasthe

correspond-ingspots obtained from 8.5-kb RNA (data not

shown). From these findings it was concluded

that 22 oligonucleotides produced ataratio of

morethan 1.4(10 spots)andbetween0.8 and 1.2

(12 spots) originatedfrom 4.8-kb RNA andthat

the restofthe minor spotsproducedataratio

of less than 0.5 represented 8.5-kb RNA

frag-mentscontaminatingthe 4.8-kb RNAfraction.

Spot

no.6 showedavery lowyield

although

itwas

specific

tothe 4.8-kbRNAfraction. This

spot

might

beexplainedbya

microheterogeneity

in the sequence of 4.8-kb RNA,but thereason

isunknown.

Thus,

spotno.6wasremovedfrom the

following

considerations.

Analysis

ofthe nucleotide composition of 22

oligonucleotides

originating

from 4.8-kb RNA

suggested

that 11

oligonucleotides

were

specific

and

produced

inaratio of 0.8to1.2andthat 10

oligonucleotides were shared with YAV 8.5-kb

RNAandthuswereproducedat aratio ofmore

than 1.4

(Table 2). Assuming

a random distri-butionofthe

oligonucleotides

alongthe genome,

the results suggest that about half the 4.8-kb

sequenceis

specific

to4.8-kbRNA.

For determination ofthe location of the

oli-gonucleotides

specificto4.8-kbRNA within the

genome, RNA

fragments

containing

poly(A)

were fractionated and purified bysucrose

gra-dient centrifugation, followed by

recentrifuga-tion of eachfractionandfingerprinting (Fig. 2B

and

C).

Althoughthe 4.8-kb RNA fraction was

contaminated with fragments of 8.5-kb

RNA,

only

22spots, whichwere concludedtobe

pro-duced from 4.8-kb RNA (Table 2), were

fol-lowed,

and the otherspotswereignored.

Thus,

atentativeoligonucleotidemap wasconstructed

(Fig. 3).

YAV 8.5-kb RNAwasanalyzed

similarly

(Fig.

2E and F), and the two oligonucleotide

[image:4.500.54.245.98.497.2]

mapswerecompared. Tenoligonucleotideswere commontoboth

RNAs,

and 4 ofthese 10were

TABLE 2. Qualitativeanalysis ofRNaseT,

oligonucleotides of Y73 26S RNA and YA V 35S RNA

Sptn.Spot

no.

ofY73

oSf

YA RNase A digestion products 26S RNA RNA

1 U,C,G

2 U,C, AC, G

4 52 U,C, AC, AU, AAG 6 U,C, AC, AU, AAAU,G

7 54 U,C,AC,AU,AAC,AAG

10 57 U, C, AAG, AAAU 12 U, C,AC, AG, AAAU 13 59 U,C,AU, AG,AAC, AAAC

14 U,C,AC,AU,AAC,G

17 U, C, AC,AU,AAC, AAU, G 18 C, AC, AU, AAC, AAU, AAAC, G 23 a + b 65? U,C, AC, AU, AG, AAC, AAAG

24 C,AC, AG, AAAC

29 70 U,C,AC,AU, G 31 72 U,C,AC,AU,AAC,G 38 80 U, C,AU,AAU,G 39 81 U, C,AU, G

41 U, C,AU, AG

42 U, C, AC,AU, AAU, AAAN,a G 43 U, C,AC,AU, AG,AAC,AAU

44 U, C,AC, AU, AG 45 77 C, AU, AAU,AAAC, G

aN, Not identified.

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mapped within about 1kb adjacent topoly(A)

atthe 3'end;the other6 were detected

only

in

the

poly(A)-containing

RNA fragments larger

than20SfortheY73and 30S for

YAV,

andwere

thusmappedwithin about 1 to 1.5kb in the 5'

regionsof the genomes. The

remaining

oligonu-cleotides specificto4.8-kbRNAwere

mapped

in

the middleportionof the 4.8-kb genome RNA.

These results suggest that the 4.8-kb specific

RNAsequence iscomposedofa continuous

se-quence of about 2.5 kb of RNA and is flanked at

the two endswith 1- to 1.5-kb RNAsequences

commonwith those ofhelperYAV RNA.

Invitro translationof Y73RNA. To

study

YAV / O X 0t1-0 J o 1f CNi

YAV ( Ln UINr), U" 0 00 i.0 D P.,1 r-...N- to LODto

the gene

products

coded

by

the specific

se-quence, poly(A)-containing RNA isolatedfrom

Y73 virions was fractionated by size (Fig.

4A)

andtranslated in an mRNA-dependent

reticu-locyte lysate (20). Polyacrylamide gel analysis

(Fig. 4B)

of thetranslationproducts showedthat

Pr76wasthe mainproduct fromRNAfractions

larger

than 28S, as found in previous

experi-ments on translation of avian erythroblastosis

virus RNA (29). This result was also expected,

becausethehigher-molecular-weightfraction of

RNA contained mainly helper YAV RNA in

which the gag gene is located near the 5'

ter-minus and theprotein-synthesizingsystemfrom

L1 1. _-. He_CANDoiox .1. U i

S

s

to r-I

s

P. toW

s

to A

)>

5' I I I I C0_Ci

8.5kb 8 7 6 5 4 3 2 1 0

4.8kb

Y73

CaPE2ZE=

I I 71;7yrovKzj :!

(..oow-.o.oo~~~~~~~~~~~~~cNc%JXN-o0.0-.0~~~~~~~~~~~~~-r!.X- C4__ ____

( 4-tn X--_-CN M - xW "- -); a

.5

o90 Protein r

9 + p

-pl9+? specific polypeptide

FIG. 3. Physicalmapsofoligonucleotides and polypeptide codedby Y73.Spotnumberswithasingleline

areoligonucleotidesshared with4.8-kband8.5-kbRNAs, and those withadoublelinearespecificto4.8-kb

RNA. The ordersof theoligonucleotideswithin the respectiveparenthesesareuncertain.

A

(I) /) (I)

04

CI N oo

B b 3 4 5 b 7 8

2<

90-

Pr76-fraction no.

FIG. 4. Polyacrylamidegelelectrophoresisof thetranslationproducts of Y73 RNA and helper YA V RNA.

Y73 RNAcontaining poly(A) was fractionatedon asucrose gradient(A) asdescribedinFig. 1, and about 0.2

pgofRNAineachfractionwastranslatedin anmRNA-dependentrabbitreticulocytelysate, containing20

pCiof[35S]methionine, andanalyzedon a 7 to

15%o

polyacrylamide gradientgel(B). Track b had noadded

RNA;othertracks arelabeledwithnumberscorrespondingtothefractionsofRNAfromthesucrose gradient

in (A). TrackYA V is 0.5

pg

ofheat-treated70S RNAof helper YA V.

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VOL. 38, 1981

eucaryotic cells translates only one gene near

the 5'end of polycistronic viral mRNA (19, 21,

25).

IncontrasttoPr76,a productwithamolecular weight of about 90,000 (p90) was synthesized mostefficientlyfrom slightlysmallerRNAthan

28S (Fig. 4b). Since fractions of similar sized

RNAofYAV didnot support detectable

synthe-sis ofp90, the results indicatethatp90wascoded

by the 4.8-kb RNA of Y73. The effect of

7-methyl-GTP, a cap analog, onthe synthesis of

p90wastestedtoknowwhetherp90was

synthe-sizedby intact cappedRNA orfragmentedRNA

(2). As seen in Fig.5, thesyntheses ofp90and

Pr76were bothstrongly inhibited by the

addi-tion of7-methyl-GTP. On the other hand, the

amount of smaller minorproducts was not

re-ducedby addition of 7-methyl-GTP.These

find-ings strongly support the idea thatp90 is

syn-thesizedby intact 4.8-kb RNAwhich has a cap

structure atthe5'terminusandalsothatsmaller

productsaresynthesizedbyfragmentedRNA.

The in vitro product p90 was found to be

immunoprecipitated with serum against

dis-ruptedvirions ofB77 (Fig. 5).Sincethis antise-rummainly containedantibody to gag proteins,

p90 was suggested to contain amino acid

se-quences related togagproteins. Thisanti-B77

antiserum precipitated p90 from an extract of

cellstransformed with Y73,and, as seen in Fig.

5, no detectabledifference wasobserved inthe

molecular sizes of thein vivo and in vitro

prod-ucts. This stronglysuggeststhatthese twop90

proteinsareidentical. Whenanotherantiserum that had noantibody activity against p19 was

used, Pr76 andp27 wereprecipitated from

trans-formed

cells,

but

p90

was not(Fig. 5). This result demonstrated that

p90

contained the amino acid

sequence ofp19, butnotthat ofp27.However,

this

experiment

does not exclude the possible

presenceofapartialsequenceofp27 in the

p90

molecule.

DISCUSSION

In this paper, we characterized the genome

structureof

Y73,

a

newly

isolatedaviansarcoma

virus

which, like RSV, induces fibrosarcomain

chickens (11). The

transforming

gene

(yas)

of Y73was

previously

characterizedas a newtype

of sarcomagene with no

homology

withsrcof

RSVor

fps

ofFujinamisarcomavirus (27). In

thepresentstudy, the yassequence, whichwas

characterized by

specific

oligonucleotides,

was

mapped in the middle

portion

of the genome, with portions of 1 to 1.5 kb common to the 3'

and 5' ends ofhelperYAV RNAatthetwoends.

Thisstructural feature of the genome makes it

very similar tothe genomes of avianacute

leu-GENOME STRUCTURE OF Y73 435

Bh

[image:6.500.260.450.79.347.2]

-p9O

~Pr76

FIG. 5. Comparison of in vitro and in vivo

trans-lationproductp90. (A)Effect of 7-methyl-GTP on in

vitrosynthesisofp90; 35S RNA (a, b)and26S RNA

(c, d) were translatedas described inFig. 4 in the

absence (a, c)orpresence(b, d) of 200 mM

7-methyl-GTP. The translationproducts in the absence of

7-methyl-GTP were immunoprecipitated with rabbit

antiserumagainst disruptedvirions,andprecipitates

were analyzedon a9%gel; (e) and

(t)

are

immuno-precipitated translation productsfrom 35S RNA and

26SRNA,respectively. (B)Extractsofcellsinfected

with YA V(g)orwith Y73(h,i)weremixed with

anti-gagserum, which hadanti-p19activity (g, h) or no

anti-p19activity (i), and theprecipitateswere

ana-lyzedon thegel.

kemiaviruses such asavian

erythroblastosis

vi-rus(3) or MC29 (18),tothose of

Fujinami

sar-comavirus (8, 16), andtothose of mammalian

sarcoma-acute leukemia viruses

(23, 24).

Be-causeof theclose

similarity

of thegenome

struc-ture of Y73 to that of avian acute leukemia viruses,wehavetested its

leukemogenic

capac-ity in chickens. One-week-old chickenswere

in-oculated

intravenously

with Y73(1.5x104

focus-forming units) or with avian

erythroblastosis

virus pseudotyped with Rous-associated virus

type 1 (104 focus-forming

units)

as control. All

chickensinoculated with avian

erythroblastosis

virusdeveloped erythroblastosiswithin 8

days;

however, no sign of leukosis was detected in

chickensinoculatedwith Y73

by

examinationof

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peripheral blood at 3- to 5-day intervals up to 1

month.Most of these birds developed

subcuta-neous sarcoma at the site of intravenous

inoc-ulation within 2 to 3 weeks. Eight surviving

chickens were autopsied at 1 month; six had

sarcomas in one or multiple sites or internal

organs suchasspleen, lung, liver, ormuscle, in

additionto subcutaneous tumors.These

obser-vations unequivocally confirmed Y73 as a

defec-tive sarcoma virus. Thus, the

replication-com-petentavian sarcoma virusesRSV and B77 are

exceptionalin genome structure, and generally

the features of sarcoma-acute leukemia viral

genomes are defective, with the transforming

genelocatedin the middle of the genome.

Invitrotranslation of 4.8-kb RNAhasdirectly

shownthat apolyproteinof90,000daltons (p90)

containing p19 butnotp27 is aspecific

transla-tion product. The p90 was translated from a

gene near the 5'end of theintact genomeRNA

containing thecap structure. The presenceofan

approximately 1.5-kb sequence homologouswith the 5' region ofYAV RNA suggested thatp19 was the N-terminal polypeptide componentin

p90,because evenifalong untranslated region

suchas 0.5kbwerepresentin the5' region, the

RNA sequence coding for p19, an N-terminal

peptide in Pr76, could be included in the 5' region homologous with YAV RNA. Thus, a

polypeptide of about 71,000 daltons should be

codedbyaspecific nucleotidesequenceofabout

2.5kb andtherefore would beaspecific

polypep-tide.Thisconclusionwassupported by the

find-ings that anti-gagserum that had no anti-p19

activity did notprecipitate p90 andthatrabbit

antiserumagainst

p6Oarc

codedby Schmidt-Rup-pin strain RSV didnotcross-reactwithp90(13).

Sincemostofthespecificsequence in the

4.8-kbgenomewouldbeusedforp90, p90seems to

bethe only polypeptide coded by the Y73

ge-nome. This possibility is consistent with the

finding that 4.8-kb RNA is the only species

detectable with specific complementary DNA

(cDNAya,)

incellstransformed with Y73. These

considerations strongly suggest that p90 is

in-volved inthe mechanism ofsarcomagenic

trans-formation of infected cells. This prediction is

supported by the finding thatp90 detected in

cells

transformed

with Y73 had protein kinase

activity for phosphorylation of tyrosineresidues

in

p90

itselfand inheavy-chain immunoglobulin

Gcomplexedwithp90(13). However, wedid not

exclude the possibleformation of smaller

prod-uctscodedby the Y73 and undetectable by our

methods.

ACKNOWLEDGMENTS

WethankR.Ishizaki forproviding rabbit antiserum, and

Y.Hirayama for excellent technical assistance.

This work was partly supported by a Grant-in-Aid for

CancerResearch from theMinistry of Education, Sciences andCulture of Japan.

LITERATURE CITED

1.Barrell, B. G. 1971. Fractionation and sequence analysis ofradioactive nucleotides. Proc. Nucleic Acid Res. 2: 751-795.

2. Beemon,K., and T.Hunter. 1977.In vitrotranslation yieldsa possible RSV-src geneproduct. Proc. Natl. Acad. Sci.U.S.A. 74:3302-3306.

3. Bister, K., and P. H.Duesberg. 1979. Structureand specific sequences of avianerythroblastosisvirusRNA: evidence for multiple classes of transforming genes among avian tumor viruses. Proc. Natl. Acad. Sci. U.S.A. 76:5023-5027.

4. Brugge, J.S., and R.L.Erikson.1977.Identification of

atransformation-specific antigeninducedbyanavian sarcoma virus.Nature(London)269:346-348. 5. Collet, M.S.,and R.L.Erikson. 1978. Protein kinase

activity associated with the aviansarcomavirus src

geneproduct. Proc. Natl. Acad. Sci. U.S.A. 75:2021-2024.

6. Duesberg,P.H.,K.Bister,and P. K.Vogt.1977.The RNA of avianacuteleukemia virus MC29. Proc. Natl. Acad.Sci. U.S.A.74:4320-4324.

7. Fujinami,A.,and K. Inamoto.1914.UeberGeschwulste

bei japanischenHaushuhnerninsbesondere uber einen transplantablenTumor.Z.Krebsforsch.14:94-119.

8. Hanafusa, T., L.-H. Wang, S. M. Anderson, R. E. Karess, W. S. Hayward, and H. Hanafusa. 1980. Characterization of thetransforminggeneofFujinami sarcoma virus. Proc.Natl. Acad.Sci. U.S.A.

77:3009-3013.

9. Hayman,M.J.,B.Royer-Pokora,and T. Graf. 1979. Defectiveness of avianerythroblastosisvirus:synthesis of a 75Kgag-related protein.Virology92:31-45.

10. Hunter,T.,and B. M. Sefton.1980.Thetransforming gene product of Rous sarcoma virus phosphorylates tyrosine.Proc.Natl.Acad.Sci. U.S.A.77:1311-1315.

11. Itohara, S.,K.Hirata,M.Inoue, M.Hatsuoka,and K.Sato.1978.Isolation ofsarcomavirusfrom sponta-neous chickentumor.Gann 69:825-830.

12.Kamahora,T.,H.Sugiyama,A.Nomoto,M.Yoshida, and K.Toyoshima.1979.RNAspecificfor the trans-forming component of avian erythroblastosis virus

strain R.Virology 96:291-294.

13. Kawai,S.,M.Yoshida,K.Segawa,H.Sugiyama,R.

Ishizaki,and K.Toyoshima.1980.Characterization

ofY73,anewly isolatedaviansarcomavirus:aunique transforminggeneand itsproduct,a phosphopolypro-tein withproteinkinaseactivity.Proc.Natl.Acad.Sci. U.S.A. 77:6199-6203.

14.Kessler, S. W.1975.Rapidisolation ofantigensfromcells with aStaphylococcus proteinA-antibody adsorption: parameters of the interactionofantibody-antigen

com-plexeswithproteinA. J.Immunol. 115:1617-1624.

15.Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly oftheheadofbacteriophageT4.

Nature(London) 227:680-685.

16.Lee, W.-H.,K.Bister, A.Pawson,T.Robins, C. Mos-covici,andP. H.Duesberg. 1980.Fujinamisarcoma

virus: an avian RNA tumorviruswith aunique trans-forming gene. Proc. Natl. Acad. Sci. U.S.A. 77:2018-2022.

17.Lee,Y.F., andE.Wimmer.1976.Fingerprinting high

molecularweightRNAbytwodimensionalgel electro-phoresis: applicationtopolio virus RNA. Nucleic Acids

Res. 3:1647-1658.

18. Mellon,P., A. Pawson, K. Bister, G. S. Martin, and P.

H.Duesberg.1978.Specific RNA sequences and gene

products ofMC29avian acute leukemia virus. Proc. Natl. Acad. Sci.U.S.A.75:5874-5878.

19.Pawson,T., R.Harvey, and A. E. Smith. 1977.The J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

(8)

sizes of RSV mRNA active in cell free translation. Nature (London) 268:416-420.

20. Pelham,H.R.B., and R. J. Jackson.1976.Anefficient mRNA-dependenttranslation system from reticulocyte lysates. Eur. J. Biochem. 67:247-256.

21. Purchio, A.F., E.Erikson, and R. L Erikson. 1977. Translation of 35S and subgenomic regions of avian sarcomavirusRNA. Proc.Natl. Acad. Sci. U.S.A. 74:

4661-4665.

22. Rettenmier, C. W., S. M. Anderson, M. W. Riemen, and H. Hanafusa. 1979. gag-relatedpolypeptides

en-codedbyreplication-defectiveavianoncoviruses. J.

Vi-rol. 32:749-761.

23. Reynolds, F. H., Jr., T. L. Sacks, D. N. Deobagkar, and J. R. Stephenson. 1978. Cells nonproductively transformed byAbelson murine leukemia virus express

ahighmolecular weight polyprotein containing struc-tural and nonstrucstruc-tural components. Proc. Natl. Acad. Sci. U.S.A. 75:3974-3978.

24. Shields, A., S. Goff, M. Paskind, G. Otto, and D. Baltimore. 1979.Structure of Abelson murine leuke-mia virus genome.Cell18:955-962.

25. VonderHelm, K., and P. H. Duesberg. 1975. Trans-lation ofRSV RNA in a cell free system from ascites Kregs II cells. Proc. Natl. Acad. Sci. U.S.A. 72:614-618. 26. Wang, L-H., P. Duesberg, K. Beemon, and P. K.

Vogt. 1975.Mapping RNase Tl-resistant oligonucleo-tides of avian tumor virus RNA: sarcoma-specific oli-gonucleotides are near the poly(A) end and oligonucle-otides commontosarcomaand transformation-defec-tive viruses are atthe poly(A) end. J. Virol. 16:1051-1070.

27. Yoshida, M., S. Kawai, and K. Toyoshima. 1980. Un-infected aviancellscontainstructurally unrelated pro-genitors of aviansarcomagenes. Nature(London) 287: 653-654.

28. Yoshida, M., M. Owada, and K. Toyoshima. 1975. Strainspecificity of changesinadenylate cyclase activ-ity incellstransformed by avian sarcoma viruses. Vi-rology 63:68-76.

29. Yoshida, M., and K. Toyoshima.1980.In vitro

trans-lation of avianerythroblastosis virus RNA: identifica-tion of twomajorpolypeptides. Virology 100:484-487. 30. Yoshida, M., M.Yamashita, and A. Nomoto. 1979.

Transformation-defective mutants of Rous sarcoma vi-ruswithlonger sizesof genome RNA and their highly frequentoccurrences. J. Virol.30:453-461.

31. Yoshida, M., and H. Yoshikura.1980.Analysis of spleen focus-formingvirus-specific sequencescoding for spleen focus-formingvirus-specificglycoprotein witha molec-ularweightof 55,000 daltons(gp55). J. Virol.

33:587-596. VOL. 38, 1981