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Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

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CopyrightC)1977 American SocietyforMicrobiology Printed in U.S.A.

Purification of DNA

Complementary

to the env Gene of Avian

Sarcoma Virus and Analysis of Relationships Among the env

Genes of Avian Leukosis-Sarcoma Viruses

JACOV TAL, DONALD J. FUJITA, SADAAKI KAWAI,1 HAROLD E. VARMUS, AND J. MICHAEL BISHOP*

Department ofMicrobiology, University ofCalifornia, San Francisco, California 94143,* and The Rockefeller

University,NewYork,New York 10021 Received forpublication 30 August 1976

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that

determines the hostrangeandsurface antigenicity of virions. We have purified radioactiveDNA

(cDNAp)

complementary to at least a portion of the env gene for viral subgroupsAand C;

complementary

DNA was synthesized withpurified

virions of wild-type avian sarcoma virus, and RNA from a mutant with a

deletioninenv was used to select DNA specific to env by molecular

hybridiza-tion. The genetic complexity of cDNAg for subgroup A (ca. 2,000 nucleotides)

wassufficientto representthe entire deletion and most or all of the env cistron. The deletionsin env in twoindependently isolated strains of virus (Bryan and rdNY8SR) overlap, and

cDNAgp

represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletionsinthe adjacent viralgene src.Laboratorystocks ofviral subgroups

A,B, C, D, and E do not contain detectable amounts ofenvdeletions when tested

by molecular hybridization; hence, segregation of deletions in env is a less frequent event than the segregation of deletions in the viral transforming gene src (Vogt, 1971). Wefoundextensivehomology among the nucleotide sequences encoding theenvgenesofvirus strainsindigenous to chickens (subgroups A, B,

C, D, and E), although subgroups B, D and E appear to differ slightly from

subgroupsAandC attheenvlocus.By contrast, virusesobtained from pheasant cells (subgroupsFandG) have env genes with little or no relationship to env

genes ofchicken viruses. According to available data, virusesof subgroup F

aroseby recombination betweenanavian sarcoma virusandviral genes inthe

genomeof ring-necked pheasants, whereas subgroup Gviruses maybe entirely

endogenoustogolden pheasants.

The genome ofavian sarcoma virus (ASV) presence ofcomplete provirusforASV(20)and

contains at least four genes (1): onc or src, mayhave normalphenotypesdespitethe

pres-responsible for virus-induced neoplastic trans- ence of a competent viral transforming gene formation offibroblasts; env, encoding theen- (3).

velope glycoprotein of thevirion;pol, the gene To facilitate analysis of the origins and for RNA-directed DNA polymerase; andgag, expression ofviral genes, wehave

developed

a encoding a polyprotein that includes the four proceduretoprepareradioactiveDNA (cDNA)

polypeptides

foundintheinteriorof thevirion.

complementary

to

specific

segments of theASV Homologues of all fourgenesprobably exist in genome.The

procedure exploits

the existenceof

normal

chicken cells (9, 19), but the natural deletion mutants; cDNA

specific

for the dele-origins of both viral and homologous cellular tions isisolated

by

selectivemolecular

hybridi-genes are not known. Expressionof these genes zation. We previously isolated

cDNA6,,

com-ismodulated in both normal and virus-infected

plementary

toatleast part of the

transforming

cells:someuninfected chicken cells containone gene of ASV (18) and found nucleotide

se-ormoreviral geneproducts, and other chicken quences

homologous

to

cDNA..r,

inDNA from

cells do not (4); mammalian cells infected by uninfected cells ofa number of avian

species

ASV generally produce no virus despite the (19).

1Presentaddress: The Institute of MedicalScience,Uni- The gene product of env is a glycoprotein (or

versity of Tokyo, 4-61 Shiroganedai, Minato-ku, Tokyo, glycoproteins)thatdeterminesthe hostrange,

Japan. surface

antigenicity,

and pattern of

interfer-497

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(2)

498

enceof thevirus(9);onthe basisofthesepheno- the

following procedure.

Single-stranded

DNA

syn-typic properties, virusstrains canbearranged thesized with

detergent-activated

virions of Pr-C

intodistinct subgroups (9, 21). In thepresent ASV washybridizedto70S RNA of Pr-C ASV under

communication we describe the isolation of conditions that permitsaturationofthe RNA with

DNA complementary to nucleotide sequences DNA.Thehybridswerethenpurified by

chromatog-DNAcomplmtary

o

nucleoti

suences

raphy on

hydroxyapatite,

and the DNA was

re-encodng part or all of enV forbothsubgroupA covered by treatment with alkali followed by

ASV (cDNAgpA) and subgroup C ASV ethanol precipitation. DNA prepared in this manner

(cDNAgpc), and we report on relationships can saturate viral RNA whenhybridized at

DNA-amongnucleotide sequences encoding the env RNAratiosof<5 (unpublishedobservations ofthe

genes of different viral strains. Using tech- authors and C. T.

Deng);

hence, the entire viral

niquessimilartothosereported here,

Hayward

genome isrepresented in the DNA in a

relatively

andHanafusa haveisolated cDNAcomplemen- uniform manner (8). DNA complementary to the

tary to env ofsubgroup B avian leukosis-sar- genome of GPV (cDNAGpv) was synthesized with

comaviruses(13). detergent-disruptedGPV andpurified asdescribed

coma VirUSeS (13).

for

cDNAB77

(18).

Molecularhybridization. Standard conditionsfor MATERIALS AND METHODS hybridization were68°Cand 0.6 M NaCl (containing Cells and viruses. We have previouslydescribed 0.02 M

Tris-hydrochloride,

pH

7.4,

and

0.01

M

our procedures for propagation andpurification of

EDTA);

reaction

mixtures

were

contained

either in

virus,extraction of viralRNA, andfractionationof capillaries (volumes of 20

,ul)

or in conical tubes

viralRN by rate-zonal centrifugation '2 Vis under mineral oil (volumes of 5 to 50

,ul).

Each

virals

.rN

obyatezoas

centifug

Pato

(,18).

Viru

reaction mixture contained

10,ug ofyeast RNA and

ASVt

wubgroup

C (Pr-C

ASV)f

as a clone from p. 10 ug ofeithercalf thymus or salmon sperm DNA.

AVo

sub

goup

C

(Pr-CaSV),

ausp

alone

from P. Hybridization ofDNA wasmeasuredbyresistance

Vogtandasconcentratedsuspensionsfromuniver- hyrlsswt51ncee(6)adybiz-sity Laboratories, Inc., Highland Park, N.J. to

hydrolysf

s

with

Sl nuclease

(16) andhybridiza

(throughtheauspicesof the Offbice ofProgramRe A (5

gmA

y y

yNase

sources and Logistics, National Cancer Institute A (50 ug/m

0.3

M

NaCl-0.03

Msodiumcitrate

the Bratislava strain ofASV, subgroup C (B77-C

(370C,

45mm).

Nucleic acids

wereprecipitated with

ASV,fromR.Friis theShmidtRuppns n 5% trichloroacetic acid and filtered through

glass-ASV,subgroupA(SR-A

ASV)c

andtRous-apisciated

fiber filters. Results of hybridizations were

ex-As

,subgroupA

(SRAV-A

A),

and

R. Ru- iathe

pressedas afunction of

Cot

for DNAandCrtor

Vot

virus 2, sUbgrOUP B (RAV-2), from H. Rubin; the fo RNA (17)

Bryan strain of ASV [Br-ASV or RSV(-)] growing or

pc(1

C)o

s

in transformed quail embryo fibroblasts and the acids o

roxypuie

wma tographyof

nucledc

Schmidt-Ruppin strainofASV, subgroup D (SR-D

acids

on

hydroxyapatite

wascarriedoutasdescribed ASV), fromP.Vogt; andthesubgroupE virus RAV-

previously

(18), using sodium phosphate, pH 6.8,

0,growinginembryonicfibroblasts fromline 100 x

supplemented

withNaCl asindicated. Nucleicacids

7chickens,from L. Crittenden. RAV-60(12), RAV- wereprecipitated out ofphosphatebuffers with

ce-61 (10), ring-necked pheasant virus (RNPV) (7), tyltrimethylammonium bromide(18).Proteinswere

golden pheasant virus (GPV) (7, 11) and trans- removed from nucleic acids

by

treatment with

so-formed chicken fibroblastsproducingthedefective dium dodecyl sulfate (0.5%, wt/vol) andPronase (500

virum

rdNY8SR (NY8) (15) were preparedfias de- g/ml) at 370C for 1 h, followed by extraction with

virus

prdNY8s

( ( phenol

(2/3

volume)andchloroform

(i/3

volume).

scribed previously.

Preparation of virus-specific single-stranded

DNA.RadioactiveDNA was synthesized with puri- RESULTS

fiedASV, using 0.2% NonidetP-40,actinomycin D Preparation of

cDNA,,p.

DNA specific for env

(125,g/ml), [3H]TTP(49 Ci/mmol) at3 mCi/ml, and can be prepared by transcribing the genome of

the otherdeoxynucleosidetriphosphatesat 10-4 M in wild-type

ASV

with RNA-directed DNA polym-a standard polymerase reaction mixture as de- e

scribedpreviously (18).The course ofDNA synthesis erase andthen selecting theDNAthatcannot wasfollowed by withdrawingsamples for acid pre- hybridizeWithRNAfromamutantwitha

dele-cipitation;thereaction was terminatedwhen DNA tion in enV. In this procedure it is advisable

synthesis ceased. Enzymatic productwas extracted that the wild-type ASV and the deletion

mu-and fractionated into single- and double-stranded tant be congenic; otherwise, DNA sequences

DNAasdescribedpreviously (18). Thepurified sin- other than those specific for the deletion may

gle-strandedDNAcould be hybridizedcompletely to fail to hybridize with the deleted RNA. Two

anexcess ofhomologous70S RNA. strains of ASV with deletions in env are availa-The preparation of cDNAB77 (radioactive DNA

ble:

Br-ASV [or RSV(-)] (5) and the recently complementary to thegenome ofB77-C ASV) and

ile

NY8

ASV

(5) and the

ASVentl

cDNAsarc

(complementarytothetransforminggene isolated NY8

ASV

derivedfromSR-AASV (5, ofASV) have beendescribed (18).

cDNA,P

wassin- 15). SR-A ASV and NY8 ASV are congenic

gle-strandedDNAcomplementarytomost or all of strains (15), whereas the nondefective parent

the Pr-C ASVgenome. The bulk ofreiteratednu- for Br-ASV is not known.

Consequently,

we cleotide sequences was removedfrom the DNA by used SR-A ASV and NY8 ASV for the

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TABLE 1. Preparation ofcDNA,A

Elution fromhydroxyapatite (cpm) Recovery of

Purification step DNAin step

Single stranda Double strandb ) (A) Single-stranded DNA (95 x 106 cpm;4.8 jig) was hy- 35 x 106 34 x 106 73

bridized to 8 jigof SR-A ASV RNA; final Crt = 100 mol-s/liter

(B) DNA from thehybridscin step (A) was hybridized to 24 13.2 x 106 7.8 x 106 62 ,ugof NY8 RNA; final Crt= 162 mol *s/liter

(C) DNA eluted in step (B) fractions 3 and 4, Fig. la (2.5 x 1.6 x 106 6.6 x 105 90 106cpm), was denatured and hybridized with 15 ,ug of

NY8RNA; final

Crt

= 108 mol * s/liter aEluted by 0.1 to 0.16 M sodium phosphate. bEluted by 0.4Msodium phosphate.

cDNAwasrecovered from hybrids by hydrolysis with pancreatic RNase A (100 jig/ml,0.003 M EDTA, 37°C, 1 h) followed by treatment with sodium dodecyl sulfate, Pronase, and phenol-chloroform as described inMaterials and Methods.

tion of

cDNA.PA

as outlined in Table 1; this (iii) Step C: second selection of DNA

com-procedure is asimplified version of thestrategy plementarytothedeletioninNY8ASV.DNA

employed previously to isolate DNA comple- elutingassingle strands instepB (fractions3

mentary tonucleotide sequences in thetrans- and 4, Fig. la) was denatured by boiling,

hy-forminggeneofASV (18). bridizedagain with an excess of 70S RNA from

(i) Step A: elimination of highlyreiterated NY8 ASV, andfractionatedonhydroxyapatite

DNA.Single-stranded, virus-specificDNA pre- (Fig. lb). The DNA eluting as single strands

pared with SR-AASV asdescribed abovewas was now fully sensitive to hydrolysis by S1 hybridized to alimited excess (less than two- nuclease (Table 2)and could not anneal appre-fold) of SR-A ASV70S RNA, and the hybrids ciably with 70S RNA from NY8 (see below). We were isolated bychromatography onhydroxy- designated thisDNAcDNAIPA.

apatite. The conditions of hybridization re- Specificity of cDNAWA. The specificity of

stricted the amount of DNA hybridized (ca.

cDNA&I,A

was tested by hybridization with

50%) and served to eliminate much of the RNAsfrom the deletionmutantand itsparent

highly reiterated DNAthatis amajor compo- (Fig. 2); differentially labeled

cDNAB77

was

nentof DNA synthesized with ASV (8). Elimi- used as an internal standard. Both cDNAB77

nation ofreiterated DNA simplified the logis- and cDNAgpA reacted completely and with

vir-tics ofsubsequent steps in the purification of tually identical kinetics with the parental RNA

cDNAgPA.

(Fig. 2B), whereas only cDNAB77 hybridized

(ii) Step B: selection forDNAcomplemen- with RNA from the deletion mutant(Fig. 2A); tary to the deletion in NY8ASV. DNAfrom the kineticsof the reactions conformed to pre-thehybridsisolatedinstep A washybridizedto viousresultswithASV70S RNA (18). We

con-anexcessof70S RNA from NY8 ASVandthen cluded that cDNAgpA isspecific for nucleotide

fractionatedonhydroxyapatite(Fig. la). A sur- sequencesdeletedfrom the ASV genome in the prisinglylarge fraction of DNA (ca. 63%) eluted genesisof NY8 ASV.

inthegradient of0.10to 0.16 Msodium phos- Br-ASV is alsoadeletionmutantinenv (5); phate. From20to 80%of this DNA was resist- RNAfrom thisvirusdidnothybridize

cDNA&PA

ant tohydrolysis byS1nuclease (Table2);the (seeTable4).Hence,the deletionsinNY8ASV

resistance toS1nuclease waseliminated when and Br-ASV overlap, and cDNAgpA represents

a sample from the phosphate gradient was nucleotide sequencescommon tobothdeletions. treated with either alkaliorRNaseatlow ionic Size and genetic complexity of

cDNAIPA.

strength (Table 2). Moreover, after removal of Thebulkof

cDNA.pA

hadasedimentation

coef-RNA, at least 30% of theDNAinsamples from ficient of 3-5S when analyzed by rate-zonal

thephosphategradient (fractions3and 4, Fig. centrifugation in 0.1 M NaCl; unfractionated la)couldhybridize to 70S RNA from NY8virus cDNA had similar sedimentation properties (datanotshown).Theseobservations indicated (Fig. 3). These observations substantiate our

that the cDNAselectedinthis stepwassignifi- previousconclusion that theprocedurefor

iso-cantly contaminated with hybrids between lation of cDNA

specific

for deletions does not cDNAandRNAfromNY8ASV; consequently, introduce a bias with respect to size (18); fur-weperformed a second selection with the de- thermore,the DNAcanbe used without

correc-leted RNA. tion for ratedifferences due to length in

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500 ET AL. J. 8000 -(a) TABLE 2. Hydrolysisof chromatographic fractions

withSI nuclease

6000 Resistance to hydrolysis bySi

Chromatographic nuclease(%)

Em

fractionive

Alkali- RNase-&Q 4000 A / \ fraction Native" treatedb treatedc

*A|

\, 3Fig. la

2000- Beforechromatography 61

Fractions 3-4 20

Fraction 7 53 2 5.3

__ Fraction 10 79

*

~~~~~~~~~Fig.

lb

250 -(b) Beforechromatography 35

Fraction 3 2.6

200 Fraction4 7.2

Fraction 5 16

laDNA (1,000 cpm) wastestedfor resistance to hydroly-E 150 sisby

Si

nucleaseasdescribedpreviously(16).Asampleof X. differentiallylabeledsingle-strandedDNAwasincluded in each analysistodocument theabsence of inhibitorsofSi

100 _ nuclease.

bDNA (3,000 cpm) wastreated with NaOH (0.3 N, 37°C, pI* 18h) in the presence of 50ggof calf thymus DNA and then 50- |

*\/

tested for resistance to hydrolysis by S1 nuclease as

de-scribed in footnotea.

cA sample (3,000 cpm) was treated with pancreatic

_* 8_ 1 RNase A(100

i.g/ml,

0.003MEDTA,pH7,37°C,1h)inthe 5 10 15 presence of 20 ,g of calf thymus DNA and then tested for FRACTION NUMBER resistance to hydrolysis by Si nuclease as described in FIG. 1. Fractionationofhybridizednucleic acids footnote a.

by chromatography on hydroxyapatite. (a) Single-

10

stranded 3H-labeledcDNA (2.4 x 107 cpm, 12 jg) A. was hybridized to rdNY8SR RNA as described in NYSRNA Table1, step A.Thehybridizationmixturewasthen 93P2 DNA

877

dilutedto 1 ml with 10 mM sodiumphosphatecon- 03H-cDNAp/

taining0.6 MNaClandloadedon ahydroxyapatite _ / column (3-mlpacked volume) at60°C. The column 0.5

was washed with thesame solution, followed bya gradientof 0.1 to 0.16 Msodiumcontaining 0.6M NaCl(fractions2to12)and afinalwash with 0.4 M sodium phosphate.Fractions (1ml)werecollected at

a rate ofabout 20 ml/h. A sample (1 p1) of each I °o4 1-3lo02 10 l0° fractionwasassayed for acid-insoluble radioactivity. < C,1(mole sec/liter)

a

Samples

from fractions3, 4, 7, and 10 were also l-o

B-assayed forresistance to hydrolysis by S1 nuclease z SR-AASVRNA

(Table 2).(b)DNAelutinginfractions3and 4(a) ,

was recovered by precipitation with cetyltrimethyl- e _ 2P-cDNAB77

ammoniumbromide, denatured by boiling for5min 0"H-cDNAgp/

in0.003 MEDTA,hybridizedto15pg ofNY8 RNA 0.5 9

(final Cot = 108 mol-slliter), and fractionated by chromatography on hydroxyapatite as in (a). Sam-ples(4p)weretakenfromeachfraction for

determi-nation ofacid-precipitable radioactivity. Lo

*0

10-4 lo-, lo- lo1-, lo0

parativehybridizationswith unselected cDNA. Cri(mole-sec/liter)

The genetic complexity of

cDNABpA

waseval- FIG. 2. HybridizationofcDNAUPA with the RNAs

uated by hybridization with radioactive 70S of SR-A ASV and rdNY8SR ASV. 3H-labeled

RNA of ASV (Fig. 4). As a convenience, we

cDNAUPA

and

32P-labeled

cDNAB77 (1,000 cpm of ued RNA from Pr-C ASV withan envgenethat each)werehybridizedto

viral

RNA involumesof

20

cross-reacts Rompletely withcDNAPA gene

Tna-pl

for14 hat68°C; the extentof hybridization was

cross-reacts completely withCDNASPA (see Ta- measuredbyhydrolysiswith

S1

nuclease.Symbols:

ble 4). The data fromtwoexperimentsindicated 0, 3H-labeled

cDNAgpA;

*,32P-labeled

cDNA877.

(A)

that cDNASPA represented at least 20% of the Hybridization with rdNY8SR RNA; (B)

hybridiza-ASV genome (Fig. 4). The stoichiometric re- tionwith SR-A ASV RNA.

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I500

2000 1 urements carried out at 10-fold excesses of

*145*oo_ e

cDNA

gave similar results (16 and20%; Table

,0 s X <, 3). In parallel experiments, we tested a recent

e 300- rS< Zu preparationof

cDNA.,r,

and found that it was

w100o complementary to between 16 and 20% ofthe

viralgenome(Fig.4and Table3);theseresults

100

conform

to our previousestimates ofthe

com-plexity of

cDNA.r,,

(18).

. We evaluated the combined complexities of

10 20 30

FRACTIONNUMBER

cDNA..r,

and

cDNA.PA

by hybridizing mixtures

FIG. 3. Rate-zonal centrifugation ofcDNA,,IA.3H- of the two cDNA's to ASV RNA; each cDNA

labeled cDNA

OVA

and unfractionated 3H-labeled was in 10-fold excess of itscomplementary RNA

cDNAfromSR-A ASV were centrifuged in separate (Table 3). The amounts of RNA hybridized in

gradients of 15 to 30% sucrose containing 0.1 M two experiments (29 and 33%) wereslightly less

NaCl-0.001 M EDTA-0.02 M Tris-hydrochloride, than the total hybridization obtainedwith the

pH 7.4. Purified tRNA labeled with32Pwas included cDNA's in separate reactions (32 and39%).We

in both analyses to serve as a reference.

Centrifuga-tion was carriedoutinanSW65rotor at65,000rpm

conc

ta the

nucleotide

sequences repre-for16 h at4°C.The resultsfromthe two analyses are sentedi cDNAsar and CDNAgpA are distict, at

superimposed in thefigure. Symbols: 0, cDNAOVA; least inlarge part, butourdata cannotexclude

0,CDNASR-A someoverlap (see below).

Preparation of

cDNA,Vc.

We have also

pre-pared cDNAgc by usingPr-C ASVto

synthe-0.2- size single-stranded DNA and the RNA of NY8

n ,___- ASVtoselect the specific cDNA.Thisselection

-N

- -- isjustified by the close homology between the

envgenesof

subgroups

AandC ASV(seeTable

4).The specificity of

cDNA.,c

for the deletion in

0.1

1'

env was

documented

as described above for

cDNAOPA (unpublished data of theauthors), but

o & we have not tested the genetic complexity of

cDNAOVc.

0 8 9 1 Kinetics of hybridization of cDNAOPA and

Ro (cDNA RNA) cDNA,,c with RNA from different viral

sub-FIG. 4. Geneticcomplexity of

cDNAO)

A. Constant

groups.

The kinetics of hybridization of

amounts of 3H-labeled

cDNAgOVA

or 3H-labeled cDNAgpA and

cDNAisC

with representative

cDNA,0,,

(6,000 cpm, 0.3 ng) were mixed with var- viral RNAs are illustrated in Fig. 5.

cDNAs77

ious amounts of 32P-labeled 70S RNA from cloned was included in each reaction to serve as an

Pr-C ASV (2 x 107 cpm/ug). The amounts of RNA internal

standard;

irrespective of the final

ex-werechosensothattheratiobetweenthecDNAand

itscomplementarynucleotide sequences in the RNA TABLE 3. Geneticcomplexities of

cDNAUPl

and would range from 10-2 to 10; computation ofthe cDNAaarca

ratios wasbasedonthepreviously measured sizesof

the deletions inthegenomes ofNY8 (ca. 20%; see

Exvt

no. DNA added Fractionof reference5) andPr-C tdASV (ca.16%;seereference hybridizedb 6).Hybridizations weredoneinvolumesof5Pifor

40h at68°C; the Cot ofeach reaction was thuskept 1 cDNAgPA 0.16 constant at0.15mol*slliter. The extentof hybridiza- cDNAsarc 0.16 tion was measured by hydrolysis with pancreatic cDNAgpAandcDNA., 0.29 RNaseA;all the data were correctedfor theintrinsic

resistance ofthe RNA tohydrolysis(3.7%). Symbols: 2 cDNAgpA 0.20 0,A,hybridizationwith

cDNAOgA;

*,hybridization cDNAsarc 0.19

withcDNAr,.,. cDNAgPA andcDNAsarc 0.33

aThe cDNA's were hybridized with 32P-labeled

quirements for hybridization indicated that 70S RNA (5,000 cpm) from cloned Pr-C ASV as

someportionsof env wereunder-representedin describedin the legendto Fig. 4. EachcDNAwas

cDNA.,pA;

thereactions reached approximately usedin10-foldexcessofcomplementaryRNA;

reac-CDNAXA; the reactions reachledapproximately tions were carriedto

Cot

= 0.15 mol*s/liter.

half of the apparent maximum when the ratio bHybridization was measured by testing resist-of cDNA tocomplementaryRNA was0.5,but a ance of RNA to hydrolysis by RNase as described in

10-fold excess of cDNA wasrequired to obtain Materials and Methods. The data have been

cor-maximumhybridization. Two additionalmeas- rected for the intrinsic resistance ofthe RNA(3.7%).

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502 TAL ET AL. J. VIROL.

1.0

A B

-Q101 - __O

~~0

a / 0

uj 0.5 L Je_

0/ /

10-410-i 10- 10'° 100 101 10-4 lo-, 1o-2 lo- 1 0 10 1lo2

Zn Crt Crt

LI 1.0

c

D

0

[image:6.501.107.399.58.294.2]

-0.5 *1

FIG. 5. Hybridization of

cDNA,,,

with RNAfromsubgroupsC, D,and E avian leukosis-sarcoma viruses. RNAwasextractedfromsedimented virus andeitherpurified byrate-zonalcentrifugation(A andB)orused

directly(CandD). Variousamountsofviral RNAwerehybridizedwith 800cpm(0.04ng)ofeither 3H-labeled

cDNAs,pAor3H-labeled

cDNA,,,c

and800 cpm(0.01 ng) of32P-labeledcDNABIIforappropriateperiodsat

68°C. (A)HybridizationofcDNA,,p,c (C) andcDNA,,,, (O) with RNAfromPr-BASV; (B)hybridization of

cDNAgpc

(C)andCDNAB77(O) with RNAfromPr-CASV; (C)hybridizationofcDNAs,PA(A) andcDNAB77(O) with RNA from SR-DASV; (D) hybridization ofcDNApA (A) andcDNAB77 (O) with RNAfromRAV-0

(subgroupE).

tent of

hybridization,

cDNAgp for either sub- 32P-labeled

cDNAB77

wasincluded in reaction

groupAor

subgroup

Cand

cDNAB77

reactedat mixtures as aninternal standard and

hybrid-identical rates with the various viral RNAs. ized

extensively

with RNA from all

subgroups

Apparently,

noneof the virus stocks contained

except

G. Wealso

analyzed

reactionsbetween

appreciable

numbers ofmutants

bearing

dele-

cDNArep

and viral RNA from several of the

tions in env;

otherwise,

the reaction with

subgroups;

these reactions

provided

amore

sat-cDNAgp

would have been slower than that with

isfactory

testof

homology

because

cDNArep

had

cDNAB77

(seebelow). beenselectedtobea

nearly

uniformcopyofthe

Homologies

amongtheenv genesof differ- ASV genome, whereas

cDNAB77

contained ent

subgroups

of avian leukosis-sarcoma vi-

highly

reiterated DNA ofverylow

genetic

com-ruses. We

analyzed homologies

amongtheenv

plexity

andasmallamountof DNAtranscribed genesfrom differentviral

subgroups by hybrid-

from the bulk of the ASV genome (8). The

izing

cDNAIpA

to70S RNAfrom all the known results with

cDNArep

conformed to those with

subgroups

of avian leukosis-sarcoma viruses

cDNAB77;

in

particular,

therewas

only

limited (Table4).Allreactionswerecarriedtovalues of

hybridization

with RNA from GPV.

By

con-C,t

suffilcienttoensure a

plateau

of

hybridiza-

trast,

cDNAGpv

hybridized

extensively

(80%)

tion.

Hybridization

was

complete,

or almost with RNAfirom GPV butnotwith RNA from

complete,

for

subgroups A, C,

and D, whereas ASV(Table 4).Weconclude that there is

exten-reactionswere

incomplete

with RNA from sub- sive

homology

among the genoxnes of all the

groups B (80%), D (88%), and E (70%) and virusestestedexceptGPV,whichappears tobe

barely

detectable with RNA firom

subgroups

F

largely

unrelatedtotheotherviruses,asshown

(15%) and G(10%o).Therewas no

hybridization

previously

(11).

with viral RNAs

containing

deletions in env Amore limited setoftests was also carried

(NY8 and Br-ASV), whereas RNAs with dele- out with

cDNA,,,c

(Table 4). The results

mir-tions in the

adjoining

genesrc (B77-C tdASV rored those with

cDNA.PA. In

summary,all the

and Pr-C tdASV)

hybridized

cDNAIIPA

com- virus strains

indigenous

tochickens

(subgroups

pletely.

A,

B,

C,

DandE)share extensive

homology

at

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VOL. 21, 1977 ENVELOPE GENE OF AVL4N RNA TUMOR VIRUSES 503 TABLE 4.Homologies among the env genes of avian leukosis-sarcoma virusesa

Hybridization of DNA(%) Subgroup Viral RNA

cDNAgPA cDNA,pc cDNABr7 cDNA,,p dDNAGPV

A SR-A-ASV 100 100 95

B RAV-2 80 86

Pr-B ASV 80 85 4

C B77-C ASV 100 100 100 100

B77-C tdASV 100 100

Pr-C ASV 100 97 100 100 0

Pr-C tdASV 100 95

D SR-D ASV 88 90

E RAV-0 70 77 70 70

RAV-60 70 70

F RNPV 15 9 76 73

RAV-61 15 17 73 68

G GPV 10 12 9 15 80

rdNY8SR 0 0 85

Br-ASV 0 85

aRNA wasprepared from the indicated viruses and hybridized with the various radioactive cDNA's (1,000

cpm) asdescribed in Materials and Methods. All reactions contained RNA in at least 100-fold excess of DNA and were carried to values Of Crt in excess of 1 mol * s/liter. Differentially labeledcDNAB77was included in all reactionswiththe other cDNA's; listed are representative results forcDNAB77combined withcDNAgPA. The data have been corrected for the intrinsic nuclease resistance of the various cDNA's (3 to 5%).

theenvlocus, whereas theenvgenesofviruses sentthe portion ofenv present in Br-ASV but

from pheasants (subgroups F and G)arelargely deleted from NY8 ASV. (ii)The congenic

non-or completely different from the env genes in defective parent of Br-ASV is not available. chickenviruses. Consequently,the size of the deletion in the Br-DISCUSSION ASVgenome cannot be

properly

assessed and

could be larger than presently estimated. Complexity of cDNAgpA and the extent of Thegenes ofsrc andenvprobably adjoin on

deletions in env. Results with molecular hy- the linearmapof theASVgenome

(25),

and it bridization indicatethatcDNAgpArepresents at isconceivable that deletions affecting thetwo

least 20% of the ASVgenome,correspondingto genesmayoverlap.The combined

complexity

of

a genetic complexity ofca. 2,000 nucleotides.

cDNAsar

and

cDNASPA

isatleast3,000

nucleo-This is a minimum estimate; the amounts of tides (Table3);theanticipatedvalue would be

material available for analysis were limited 3,500 to 4,000, based on the sizes of the two andwemayhavefailedtoachievea

plateau

of deletions (5,

6, 18).

In

addition, cDNAgpA

hy-hybridization (see Fig. 4). However, the reac- bridizes completely with RNA isolated from tionmusthaveapproachedsaturationbecause transformation-defective strains of ASV that the deleted RNA (NY8 ASV) is only 21% have deletions in src

(Table

4), and

cDNNam

smaller than RNA from the nondefective pa-

hybridizes completely

with RNAs from Br-ASV rental strain ofASV (5), and the nucleotide andNY8ASV(18;

unpublished

observationsof sequences of

cDNA.PA

are all included within the

authors)

that have deletions inenv

(5).

We

the deletion (Fig. 2A). We conclude that conclude that there isnomajor overlapbetween

cDNAgpA

represents mostifnotallof the dele- deletionsaffectingsrc and thoseaffectingenv,

tion in NY8. If the deletion doesnotextendinto butourtechniquescannotexclude the existence

othergenes, it islarge

enough

toencodemost ofsome

overlap.

or all of the envelope glycoprotein; hence,

Frequency

of deletions in env. We have

cDNAgPA

mayrepresentmostofenv. shown previously that mutant viruses with

The env deletion in Br-ASV apparently af- deletions insrc canbe detectedinvirusstocks

fectsonly10 to 15%ofthe viralgenome (5), yet by hybridizing viral RNA withboth

cDNAsa,

there is no appreciable homology between and unselected viral cDNA; the presence of

cDNABpA

and RNA from Br-ASV (Table4).We deletions

specifically

retards therateof

hybrid-suggest twopossible explanations for this dis- ization with

cDNA,ar,,

an effect which canbe crepancy. (i) Some of the nucleotide sequences

reproducibly

detected whendeletions constitute

deleted from NY8 ASV are very scarce in more than 30%of the virus stock

(18;

D.

Ste-cDNAg,A (Fig. 4); thesesequencescouldrepre- helin et

al.,

submitted for

publication).

Since

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[image:7.501.48.442.76.257.2]
(8)

504 TAL ET AL. J. VIROL.

manystrainsof ASV segregate deletions in src leased isolates.) It is possible that the env genes

when propagated by serial passage (22), we in all strains of chicken leukosis-sarcoma

vi-found that deletions ofsrc werereadily detecta- ruses (subgroups A, B, C, D, and E) have a

ble by molecular hybridization in uncloned common ancestry but have evolved into the

stocks of most strains of ASV (Stehelin et al., different subgroups during horizontal and/or

submitted for publication). By contrast, dele- vertical transmission innature;recombination

tionof theenv gene is a rareevent. Wecannot among leukosis-sarcoma viruses is frequent

detect this deletion by molecular hybridization (14, 23) and could havefacilitated divergence at

inhigh-passage stocks ofanyofthesubgroups env.

of avian leukosis-sarcoma viruses (Fig.2and 5) We havepreviously proposed that the

nucleo-and the frequency of env deletions in stocks of tide sequences in src were derived from the

SR-ASV was very lowwhen measured by bio- normal genome of the chicken or a closely

re-logical means (15). lated bird (19). By analogy, env for subgroup E

Homologies among the env genes ofavian could be theprogenitorofenvforsubgroups A,

leukosis-sarcoma viruses. Our data indicate B, C and D, and genes related to the env of

that there is extensive homology among the env subgroup E should be present in avian species

genes of all the subgroups of avian leukosis- otherthan chickens. The results withpheasant

sarcoma viruses indigenous to chickens (i.e., viruses suggest that such genes do exist, but

subgroups A, B, C, D, and E). Subgroups B, D, are extensively diverged from the env found in

and E appearto differ slightly from subgroups normal chickens. We are examining this issue

Aand C at the env locus (Table 4). In the case of further by using cDNAgp to test DNA from

subgroup E, the difference is apparent with different species for homology withenv.

both RAV-0, an endogenous virus of chickens

(24), and RAV-60, a recombinant between

RAV-1 (subgroup A) and viral genes in the WethankH.Hanafusa for advice andsupport,L. Crit-genomeof chickens (12); these dataconformto tenden for materials, J. Jackson for technical assistance,

and B. Cookforstenographicalassistance. This workwas

the view that theenv gene ofRAV-60 wasde- supported by Public Health Service grants CA12705,

rivedfromthe chickengenome (9, 12). We are CA19287, CA14935, and 1T32 CA09043 from the National

analyzing divergence inenv further by testing CancerInstitute, grantVC-70 from the American Cancer the thermal stability of hybridsformed between Society, and Public Health Service contract no. N01 CP 33293 within the Virus CancerProgramof the National cDNAgp and viral RNAs (workinprogress). Cancer Institute. J. T. received supportfrom theCalifornia The virusesof subgroupsF(RNPV andRAV- Divisionof the American Cancer Society. D. J. F. wasa

61) and G (GPV)wereobtained from pheasant Fellowand S. K.aSpecial Fellowof theLeukemia Society cells after infection by Br-ASV (7, 10, 11) and ofResearch CareerAmerica. H. E. V.Developmentis a recipientAward CA70193 from theof PublicHealth Service wereoriginally considered tobe recombinants National Cancer Institute.

between the infecting virus and viral genes

endogenous topheasants. This may betruefor LITERATURE CITED

subgroupFviruses:wefound that cDNAB77and 1. Baltimore, D. 1974.Tumor viruses. Cold Spring Harbor

cDNArephybridized extensively with RNAfrom Symp. Quant.Biol.39:1187-1200.

both RNPV andRAV-61, yettheenv genes of 2. Bishop, J. M., W.E. Levinson, D. Sullivan, L.

Fan-RNPVandRAV-6arenotcloselyrlatedto shier,N. Quintrell, and J. Jackson. 1970. The low RNPV and RAV-61 are not closely related to molecular weight RNAs of Rous sarcomavirus. II.

envof subgroupA(Table 4) and are presumably The 7S RNA. Virology42:927-937.

derived from the ring-necked pheasant ge- 3. Boettiger, D. 1974.Virogenicnontransformed cells iso-nome. By contrast, GPVappears tobe an en- lated following infection ofnormal rat kidneycells

dogenous virus ofgoldenpheasants

l11)

havig.withB77 strain Rous sarcoma virus. Cell 3:7-15.

dogenous virus Of golden pheasants (11) having 4. Chen, J. H., W. S. Hayward, and H. Hanafusa. 1974.

littlehomologywiththegenomesof other avian Avian tumor virus proteinsand RNA in uninfected

leukosis-sarcoma viruses (reference 11; Table chickenembryocells. J. Virol. 14:1419-1429. 4). (The strain of GPV used in the present 5. Duesberg, P. H., S. Kawai, L.-H. Wang, P. K. Vogt, H.

studieswas isolated after infection ofgolden M.Murphy, and H.Hanafusa. 1975. RNA of

replica-studiles was iSOlated after infection of goldeen tion-defective strains of Rous sarcoma virus. Proc.

pheasant cellswith Br-ASV [7] and could be a Natl. Acad. Sci.U.S.A. 72:1569-1573.

recombinant between ASV and viral genes in 6. Duesberg, P. H., and P. K. Vogt. 1970. Differences

thepheasant genome; this couldaccountfor the between the ribonucleic acidsoftransforming and

*ri h i o bewe .N and the '

,nontransforming

._ ... avian tumor viruses. Proc. Natl.

partial hybridization betweencDJNA,ep andlthe Acad. Sci. U.S.A.67:1673-1680.

genome of GPV [Table4]. Recently, strains of 7. Fujita,D. J., Y. C. Chen, R. R. Friis, and P. K.Vogt.

GPV have also been isolated from uninfected 1974. RNAtumorvirusesofpheasants:

characteriza-golden pheasant cells [personal communica- tion of avianleukosissubgroups F and G.Virology

tionsfrmH.Hanfusa andP. Vogt] but we 60:558-571.

tions from H. Hanafusa and P. Vogt], but we 8. Garapin, A. C., H. E. Varmus, A. J. Faras, W. E.

have yet to analyze these spontaneously re- Levinson, and J. M. Bishop. 1973. RNA-directed

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DNA synthesis by virions of Rous sarcoma virus: 17. Ringold,G., E. Y.Lasfargues,J. M. Bishop, and H. E. further characterizationof the templates and theex- Varmus.1975. Production of mouse mammary tumor tentof their transcription. Virology 52:264-274. virus by cultured cells inthe absence and presence of 9. Hanafusa, H.1975.Avian RNAtumor viruses, p. 49-90. hormones: assay by molecular hybridization.

Virol-InI.F. Becker (ed.), Cancer:acomprehensivetrea- ogy 65:135-147.

tise. Plenum Press,NewYork. 18. Stehelin, D., R. V. Guntaka, H. E.Varmus,and J. M. 10. Hanafusa,T.,and H. Hanafusa. 1973.Isolation of leu- Bishop. 1976. Purification of DNA complementary to kosis-type virus from pheasant embryo cells: possible nucleotidesequencesrequiredfor neoplastic transfor-presenceof viral genes in cells. Virology 51:247-251. mation of fibroblasts by avian sarcoma viruses. J. 11. Hanafusa, T.,H. Hanafusa, C. E. Metroka, S. Hay- Mol. Biol. 101:349-365.

ward, C. W. Rettenmier, R. C. Sawyer, R. M. Dough- 19. Stehelin, D., H. E. Varmus, J. M. Bishop, and P. K. erty,andH.S. DiStefano.1976.Pheasantvirus: new Vogt.1976.DNArelatedtothe transforminggene(s) class ofribodeoxyvirus. Proc.Natl.Acad. Sci. U.S.A. ofavian sarcoma viruses is present innormal avian

73:1333-1337. cells. Nature(London)260:170-173.

12. Hanafusa, T., H. Hanafusa, and T. Miyamoto. 1970. 20. Temin, H. M. 1971. Mechanism of cell transformation Recovery ofa new virus from apparently normal by RNA tumor viruses. Annu. Rev. Microbiol. chickcells by infection with avian tumor viruses. 25:6094A8.

Proc. Natl.Acad. Sci. U.S.A.67:1797-1803. 21. Vogt, P. K. 1965. A heterogeneity of Rous sarcoma 13. Hayward, W. S., andH. Hanafusa. 1976.Independent virusrevealed by selectively resistant chick embryo

regulationof endogenous andexogenous avianRNA cells. Virology25:237-247.

tumor virus genes. Proc. Natl. Acad. Sci. U.S.A. 22. Vogt, P.K. 1971.Spontaneoussegregationof nontrans-73:2259-2263. formingvirusesfrom clonedsarcomaviruses. Virol-14. Kawai, S., andH. Hanafusa. 1972.Geneticrecombina- ogy46:939-946.

tionwithavian tumor virus.Virology49:37-44. 23. Vogt, P. K. 1971. Genetically stablereassortment of 15. Kawai, S.,and H. Hanafusa.1973.Isolation of defective markers duringmixed infection withavian tumor

mutantof aviansarcomavirus(recombination/RNA- viruses.Virology46:947-952.

dependent DNA polymerase/glycoprotein/endoge- 24. Vogt,P.K., and R. R. Friis. 1971. An avianleukosis nous virus). Proc. Natl. Acad. Sci. U.S.A. 70:3493- virusrelatedtoRSV(0):propertiesand evidence for

3497. helper activity. Virology43:223-234.

16. Leong, J.A.,A.C.Garapin, N. Jackson, L. Fanshier, 25. Wang, L.-H.,P. H.Duesberg,S.Kawai,and H. Hana-W.E.Levinson, and J.M.Bishop. 1972.Virus spe- fusa.1976.Locationofenvelope-specificand sarcoma-cific ribonucleic acidincellsproducingRoussarcoma specific oligonucleotidesonRNA ofSchmidt-Ruppin virus:detection and characterization.J.Virol.9:891- Roussarcomavirus. Proc. Natl. Acad. Sci. U.S.A.

902. 73:447-451.

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Figure

TABLE 1. Preparation ofcDNA,A
FIG. chromatography
FIG. 3.pHgradientsNaCl-0.001forsuperimposedlabeledtionincDNA0, both CDNASR Rate-zonal centrifugation ofcDNA,,IA
FIG. 5.RNAcDNAs,pA68°C.cDNAgpcdirectly(subgroupwith Hybridization ofcDNA,,, with RNA from subgroups C, D, and E avian leukosis-sarcoma viruses
+2

References

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