Ev 2, a genetic locus containing structural genes for endogenous virus, codes for Rous-associated virus type 0 produced by line 72 chickens.

Vol.33,No.1 JOURNALOFVIROLOGY, Jan. 1980,p.250-255

0022-538X/80/01-0250/06$02.00/0

ev

2,

a

Genetic Locus

Containing Structural Genes for

Endogenous Virus, Codes for Rous-Associated Virus Type

0

Produced by Line

72

Chickenst

S. M.ASTRIN,'* L. B.CRITTENDEN,2 ANDE. G. BUSS3

The InstituteforCancerResearch,Philadelphia, Pennsylvania 19111,1 U.S. Department of Agriculture

Science andEducationAdministrationRegional Poultry Research Laboratory, East Lansing, Michigan

48823,2andDepartment of Poultry Science, PennsylvaniaState University, University Park,Pennsylvania

16802

ev2 isoneofseven recentlydescribed geneticloci ofchickens which contain

structural genes for endogenous virus. ev 2 is present exclusively in line 72

chickens,aninbred strain of whiteLeghorns which is homozygousfor thecapacity

to produce Rous-associated virus type 0 (RAV-0), a subgroup E virus. This

phenotype is knownasV+and hasbeen assignedagenetic allele designated

V-E7.The segregationofev2wasfollowed inageneticcrossin which the V-E7+

phenotype was also segregating. The progeny of the cross were analyzed for

endogenousviral lociby cleavage of embryo DNA with restriction endonuclease SstI, electrophoretic separation oftheresulting fragments, andidentificationof

bands containing viral sequencesby hybridization of the DNAto radiolabeled

viral RNA. Fourendogenousviralloci,ev 1,ev2,ev4, andev5,wereidentified

intheprogenyof thecross.One of theprogenycontainednodetectable

endoge-nousviralsequences. ev1,ev4,andev5werepresent inprogenyof boththe

V-E7+andV-E7-phenotypes. ev2waspresentexclusivelyinprogenyof the V-E7+ phenotype,and allV-E7+progenycontainedev2.Inaddition,oneof the V-E7+

progeny containedonly ev2. Fromthese data,we concludethat ev2 codes for

RAV-0 virusproduced bythecellsof line72 chickens.

Line 72 is an inbred line ofwhite Leghorns

which is geneticallyhomozygous for the V-E7

allele, an allele controlling the production of Rous-associated virus type 0 (RAV-0), a sub-group E endogenous retrovirus (6, 8, 13, 19).

Recently, line 72 chickens have been shownto

containendogenousviralgeneticinformationat twospecific chromosomal sites of residence (1). The genes at these twosites have been

desig-natedendogenous viral loci1and2(abbreviated

ev 1 and ev2). ev 1 was found in all chickens

tested, butev2wasfoundexclusivelyinline72

chickens. This finding suggests that ev 2 may code for RAV-0 virusproduced by the cellsof

these chickens.

To testthishypothesis, wehavefollowedthe

segregation ofev 2 ingenetic crosses involving

the progeny of line 72 chickens. The progeny,

which wereheterozygousfor theproduction of

RAV-O, were mated with nonproducer (V-E-) chickens, and the resulting embryos were

as-sayedfor thecapacitytoproduceRAV-O(V-E7+ phenotype) and for the presence of ev 2. We found an exact correlation between the

segre-gation of the V-E7+ phenotype and the

segre-t Journal series no.5870 of the Pennsylvania Agricultural ExperimentStation.

gation ofev 2; in addition, one V-E7+ embryo containedonlyev2. Weinterpretthesefindings

toindicate thatev 2 codes forRAV-Oin line 72

chickens.

MATERIALS AND METHODS

Chickens. Line 72 isan inbred line of white

Leg-hornsof theV+ phenotype which is maintained at the

Regional PoultryResearchLaboratory, East Lansing,

Mich. (6). Hens of the V-E-phenotypewereobtained

fromSPAFAS, Inc., Norwich, Conn. V-E- henswere

negative for viralgroup-specific antigen(gs) and viral

envelopeprotein(chf).Individualmatingsweremade

between birds in individual cagesby artificial

insemi-nation.

Cells and cell DNAs. Chicken embryo cultures

were prepared from 11-day-old white Leghorn

em-bryosasdescribedbyRubin(16).Cellsweretypedfor

production ofRAV-0byinfectingamixture ofBryan

RSV-transformed quail cells and turkey cells with

culture supernatant.SubgroupE virusreleasedbythe

quail-turkey cell mixture wasassayed for focus

for-mationonsubgroupE-susceptible chickencells(5).

The sires, dams, and progeny embryos were not

susceptibletoinfectionby subgroupEvirus.DNA was

prepared from cultured cells or erythrocytes as

de-scribedbyVarmusetal.(18).Erythrocyteswere

iso-lated from heparinized whole blood by differential

centrifugation. 250

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Enzymes, gels, andtransfer of DNA to

nitro-cellulose filters. RestrictionendonucleaseSstI was

purchased from Bethesda Research Laboratories.

DNAs at aconcentrationof250,ug/mlweredigested

by using 250 U of endonuclease perml at 37°C for 4 h.

Digestion was monitoredby the additionof SV40 or

A DNA to a portion of the digestion mixture and

visualization of the products, after digestion and elec-trophoresis, by staining with ethidium bromide. Elec-trophoresis of digested DNAs was carried out in 1%

agarose (Sea Kem) slab gelsasdescribed by Ketner

andKelly (10). A 25-,tg amountof DNA was run in

eachwell. DNAwastransferredtonitrocellulose filters

(HAWPOO010; Millipore Corp.) by the method of

Southern (17) as modified by Ketner andKelly (10).

Hybridization and autoradiography.

3P-la-beled RAV-2 RNAwasusedas aprobe for endogenous

viral sequences. RAV-2 RNA has greater than 80%

sequencehomology withthe RNA of RAV-0 (9, 11).

RAV-2 RNA was chosenfor these studies since

RAV-2grows to high titer whichfacilitates the preparation

of the hybridization probe. RAV-2-infected cell

cul-tures werelabeled with 32P as described by Parsons et

al. (12), using 1.5 mCi of[32P]phosphate (carrier-free,

NewEngland Nuclear Corp.) per ml of culture fluid.

Virus wascollectedandpelletedat 24-hintervals, and

70S RNA was purified by pronase-sodium dodecyl

sulfate (SDS)treatmentfollowedbysucrosegradient

sedimentation asdescribed by Robinson et al. (15).

Purified 70S RNA washybridized at 70°C to DNA

immobilized onnitrocellulose filtersin amixture

con-taining4xSSC(lx SSC is0.15MNaClplus 0.015 M

sodiumcitrate,pH 7.4),500tigof carrier wheatembryo

RNA perml,0.1%SDS, and 32P-labeled 70S RAV-2

RNA(specificactivity,1 x 107to2 x 107cpm/,ug) at 0.3,ug/ml.The salt and temperature conditions chosen

forhybridizationarethosedescribedbyBaludaetal.

(3). The filters wereincubated in hybridization

niix-turesfor10 to 20h,washed with2xSSC,treatedwith

pancreaticRNAase (20pg/mn in2xSSC for0.5h at

37°C), washed with4x SSC and 0.1% SDS at70°C,

and dried.Hybrid bandsweredetectedby

autoradiog-raphy at -70°C by using Kodak XR-5 film and a

DupontQuanta IIintensifyingscreen.

RESULTS

Endogenousviralloci in line 72chickens.

A

B

C

D

E

F

G

H

DNA was extracted from erythrocytes of 26 birds of line 72 and cleavedwith restriction

en-donuclease SstI. This enzyme was chosen be-cause it produces a single major fragment for each site of residence ofendogenous viral genes in the chicken genome(1). DNA fragmentswere

fractionated by gelelectrophoresis, denatured in situ, and transferredtonitrocellulose filters(17).

Endogenous viral genes were detected by hy-bridization with 32P-labeled70SRNA extracted

from virions of RAV-2. Bands containing viral sequences were visualized by autoradiography. All 26 DNAs produced identical patterns with fragments of 5.8 x 106, 3.7 x 106,and 0.9x 106 daltons. Of the 26patterns, 12 are shown inFig.

1, lanes A through L. After a long

autoradi-ographic exposure, a third band at 0.9 x 106

daltonscould be seen in every well (inset, Fig.

1). The band at 5.8x 106daltons has previously

been identified as representing a single site of residence of endogenous viral genes and has beendesignated ev1.Thebandsat3.7 x106 and 0.9 X 106 daltons represent a second site of residence, designated as ev 2 (1). Digestion of the DNAs withHindIIIwasusedtoconfurm the presence of ev 1 and ev 2 in these six DNAs. HindIII cleavage producedbands at 8.5 x 106,

3.2 x 106, 2.4 x 106, 1.9 x 106, and 1.0 x 106 daltons (data notshown). The 3.2 x 106, 1.9 x

106, and 1.0x 106 bandsrepresent ev 1,and the 8.5and 2.4 bands arecharacteristic ofev2 (1).

The 26 line 72 chickens that weanalyzedwere

specifically chosen to be representative of the

gene poolpresent in thebreeding stocks of line

72.Wecan,therefore,conclude fromourresults

that line 72 chickens contain onlytwo loci for endogenous viral genes, loci ev 1andev 2.Two roosters (Fig. 1, lanes A and B) were chosen

from the group of 26birdsanalyzed tobe used

assires in themating experiments.

Segregation ofgenotype andphenotype

in backcross matings. Two line 72 roosters wereeachmated withtwodifferent hens of the

I

J

K

ip

L

5.8-_%W 00

4_0

am_ 00

0,*W.

-

-w.-3.7-b

W o l".,%W

Iwo

iw

_0. -w _.N

LINE

72

CHICKENS

_-5 8

_ II

- 9

-O g

FIG. 1. SstIdigestionof line 72chicken DNAs andidentification of endogenousviralsequences. A marker

ofEcoRI-digested 32P-labeledADNAwasusedtodeterminemolecularweights(whichareshown x10').The

insertattherightshowsalongautoradiographicexposureofthesamplein lane A.

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[image:2.504.104.391.513.622.2]

252 ASTRIN, CRITTENDEN, AND BUSS

V-E-phenotype.Progeny from each of the

mat-ingsweregrown tomaturity,andonemale from eachwasselected foruse as asire inabackcross. Eachof these four F-1 roosters wasmatedto a

V-E-gs-chf-hen, and10to 12progenyembryos fromeachmatingwereanalyzedforproduction

of RAV-0 and for endogenous viral locias de-scribed above.

TheSstI patterns for thesire, dam,and prog-enyfor each of the fourmatings areshown in Fig. 2 through 5. Various combinations of four bands (12 x 106, 5.8 x 106, 5.4x 106,and3.7 x

106 daltons) canbe seenin the patterns. After longautoradiographicexposures, afifth bandat 0.9 X 106 daltons could beseen always

accom-panying the 3.7 x 106 molecular weight band (data not shown). As described above, the 5.8

X 106 daltonband represents ev 1; the 3.7 and 0.9molecularweightbandsrepresentev2.The bandsat5.4x106and12x 106daltonsrepresent twoother distinct sites of residence of viralgenes

(1) that have been designated ev 4 and ev 5,

respectively. ev4 and ev5 have been observed

tosegregateinaMendelianmanner(2).

Appro-priate DNAs have been cleaved with BamHIto

confimnthepresenceofev4andev5(seelegends

toFig.2and4).

Inmating1 (Fig.2), the sire carriedev 1and

ev 2 (Fig. 2, laneA) and the dam carried ev 1

and ev 5 (Fig. 2, laneB). Of the 11progeny, 9

inheritedev1 (Fig.2, lanesC, D, E, F, G, H, I,

K, and L), fourprogeny inherited ev5 (Fig. 2,

lanes F, G, L, andM), andfour inherited ev2

(Fig.2,lanesD,F, L,andM).One of theprogeny

(Fig. 2, laneJ)appearstolack

endogenous

viral loci. To ensure that therewasindeed DNA in

wellJ, thegelswerestained withethidium bro-mide before transfer of the DNAto

nitrocellu-lose filters and the DNAwasvisualized with a

UV lamp.Well J

appeared

tocontainanornal

amount ofDNA.Inaddition,this DNA

sample

was analyzed asecond time and again showed nohybridization. The frequencies ofoccurrence

of individual loci in these eleven progeny are

consistent with the dam beingheterozygous for

ev1andev5andthe sirebeing heterozygous for

ev 1 andev2.Inthiscase, 1/16oftheprogeny

wouldbe expectedtolack endogenousviralloci. All theprogenythatinheritedev 2(Fig.2,lanes

D, F, L, and M)were of the V-E7+phenotype.

The remaining progeny were of theV-E- phe-notype. Thus, ev 2 segregated with theV-E7+

phenotype ineveryinstanceinthismating.

Inmating2 (Fig. 3), the sire carried ev 1 and ev 2 (Fig. 3, lane A) and the dam carriedev 1

and ev 5 (Fig. 3, lane B). Of the 10 progeny,8

inherited ev 1 (Fig. 3, lanes C,D, E, F, I, J, K,

andL),7progenyinheritedev 2(Fig.3,lanesD,

E,G, H,I,K, and L), and threeprogenyinherited

ev 5 (Fig. 3, lanesH, I, and J).As inmating 1,

these frequencies are consistent with the dam

being heterozygous forev 1andev 5 and the sire

being heterozygous for ev 1 and ev 2. All the progenythatinherited ev 2(Fig. 3,lanes D,E,

G,H, I, K, and L)wereofthe V-E7+ phenotype, whereas the remaining progenywere ofthe

V-E-phenotype. In the case of the V-E7+ embryo

whosepatternis shown inFig. 3laneG, ev 2is

the only locus present. Therefore, the RAV-0

produced by the cells of this embryo mustbe

codedfor byev 2.Thesegregation ofev 2with theV-E7+phenotype in the otherprogeny sup-portsthis conclusion.

In matings 3 and 4 (Fig. 4 and 5), the sires

carried ev 1 and ev 2 (A lanes) and the dams carriedev 1andev 4(Blanes). All of theprogeny

of bothmatings inherited ev 1. Four of the 11 progenyofmating3inheritedev 4(Fig.4,lanes E, F,K, andM), asdid 3ofthe 12 progeny of

mating4(Fig.4,lanesE,I, and K). Nineprogeny

of mating 3 and seven progeny of mating 4

inheritedev 2(Fig.4,lanesC, D,E, F, G, I, J, L,

A B C D E F G H u K

LM

A N

_hi _

4-S-_

_to

_{_1}

*

PROG E NY

-FIG. 2. SstIdigestionof DNAsfrom the sire, damandprogenyotmating no. 1.Molecularweignts(xiu)

areshown. AmarkerofEcoRI-cleaved32P-labeledA DNA isshown inLane N. +,Embryo wasof the V'

phenotype;-, VWphenotypeembryo. Assays forRAV-Oproduction wereperformed as described in the text.

BamHIdigestionofthe DNA shown in lane Byielded bandsat 7.8 x 106,3.2 x106, 1.0 x 106,and 0.7 x106

daltons(datanotshown). This is the expectedpatternforaDNA containing only ev 1 and ev 5 (1). The sire

(laneA)isanF-I betweenamalehomozygous forev1andev2and afemale heterozygousfor ev 4.

J. VIROL.

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[image:3.504.120.409.497.599.2]

VOL. 33,1980

A B C D E F G H

_;~~~~~~~~*,O*

J K L M

}13 7t

ev 5)

12-ev 1) 5.8- _ i_ ii ev 2) 3.7-to

t~~~~~~~~~~~~~~~~

we i4Iiit-33

-21

v + + -+4 ±

--1-1

+

PROGENY

FIG. 3. SstIdigestion ofthe DNAsfromthesire, dam, and progenyof matingno.2. Molecularweights

(x106) are shown. A markerof EcoRI-cleaved32P-labeledA DNA is shown in lane M. +,Embryowasofthe

V+ phenotype; -, V-phenotype. The sire(lane A)isanF-I betweenamalehomozygous forevI andev2 and

afemale heterozygous forev 4.

A

B

C D E

F G

H

I

J

K

L

M

ev 1

-ev 4

%a -ev 2

+ t t -4- - + - + +

[image:4.504.96.384.72.178.2]

d

-o

PROGENY

FIG. 4. SstIdigestionofDNAsfromthesire, dam, and progenyof matingno.3.Molecularweights(x106)

wereestimatedby usingamarkerofEcoRI-cleaved32P-labeled A DNA (not shown). +,EmbryowasoftheV+

phenotype; -, V-phenotype. BamHIdigestionofthe DNA shown in lane Byielded bandsat4.5x106 3.2x

106, 1.0x106,and0.7x106 daltons(datanotshown).This is theexpectedpatternforaDNAcontaining only

ev 1 andev4(1). The sire(lane A) isanF-i between amale homozygous forev 1 andev2andafemale

homozygousforev 1.

A B C D E F G H I J K L M N

5.8_- u_ "

5.4-3.7

-ev 1

- I..

VW. to*

-oo$

-111

ev_ev 4₄

1.-,: -ev 2

Ce""

- + -

-f

+ + + - t -

+

t --

-PROGENY

FIG. 5. SstIdigestionofDNAsfromthesire, dam,and progenyofmatingno. 4.Molecularweights(x106)

wereestimatedby usingamarkerof EcoRI-cleaved32P-labeledX DNA (not shown). +, Embryo was of the V+

phenotype; -, V-phenotype. The sire(lane A) isanF-I betweenamalehomozygous forev Iandev 2anda

female homozygousforev1andheterozygous forev4andev 5.

andM;Fig.5,lanesE,G,H,I,J, L, and N). All the progeny thatinherited ev 2 wereof the V+

phenotype; the remaining progeny were of the V- phenotype. Thus in all 45 progeny, ev 2

segregated with theV+phenotype. In addition, in oneof theV+progeny, ev 2 wasthe only locus present. We conclude that ev 2 codes for the virusproduced bycells ofline 72 chickens.

DISCUSSION

Recently, the identification of seven genetic

loci containingstructuralgenesforendogenous virus was reported (1). Two of these loci, ev2

andev7,arefoundexclusivelyinembryosofthe

V-E7+ or V-15+ phenotypes, respectively, ev 2

beingcharacteristic of line72 embryosandev7

5.8=ImF s _ %W

3.7-eV 2 253

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[image:4.504.99.387.235.354.2] [image:4.504.86.387.427.529.2]

254 ASTRIN, CRITTENDEN, AND BUSS

being characteristic of line 15B embryos. The virus produced by line 72cellsis a subgroup E

retrovirusdesignatedas RAV-0 (5, 19), whereas

line 15Bcellsproducenoninfectiousvirus

parti-cles of unknownsubgroup (14). We have recently documented the existence of three additional

loci inchickens, one of which, ev 10, is

charac-teristic ofV-E+ embryos oflines1514 and C (L.

B. Crittenden and S. M. Astrin, manuscript in

preparation). Lines 1514 and C produce a sub-group Evirussimilar butnotidenticalto RAV-0 (P. Tsichlis and J. Coffin, unpublished data). Thus there appear tobe atleastthree distinct types of virus produced by thesethree pheno-typesof V+ embryo, each of which is associated with the presence of a distinct genetic locus containingendogenous viralsequences. One in-terpretation of thisrelationship isthateachof these loci codes for the virus produced by the cells containing that locus.Alternatively,

corre-lation between locus andphenotype could sim-ply reflect the fact that the chickens inwhich they are observed are genetically related. In experiments reported herewepresentevidence thatev 2 codes for RAV-0produced by cells of line72embryos. Notonly didev 2segregatewith

the V-E7+ phenotype in everyinstance infour

matingexperiments, butoneof theV-E7+ prog-enycontainedonlyev 2.Thusev 2mustcontain the genetic information for the virus produced by the cells of thisembryo.

Virus produced by cells of line 72 embryos

grows to a high titeronly after induction with BUdRorafter co-cultivation with cells

suscep-tibletosubgroup Evirus (7, 13). Therefore, ev 2mustnormallybeexpressedatonlyaverylow level. Recentexperiments ofCooperand

Silver-man (4), using line 100 chickens (a subline of line 72), indicate thepresenceofregulatory

se-quences,adjacenttotheviralsequences, which

repress transcription. Their data showed that theDNAofsubgroupE-resistantV-E+ cellswas

infectious in a transfection assay only after shearing to the approximate size of the virus

genome. Since the results presented here indi-cate that ev2 codesfor the virus produced by

cellsofline72 chickens, thefindingsofCooper

and Silverman implythat ev 2transcription is

regulated by linkage to cis-acting control

ele-ments. Furtherworkwill benecessaryto

deter-mine the nature and mode of action of these

control elements.

One of the progeny in the experiments

re-ported here lacked detectableendogenousviral genes (Fig.2,laneJ).We haveanalyzed

endog-enousviralsequences inover500chickens, and

this is the firstexampleofanindividual

appar-entlylackingthese genes. We haverepeatedthe

J. VIROL.

mating which produced this embryo and have succeeded in hatching a rooster which has no

detectable endogenous viral sequence (la).

Theseresultsindicatethatthe endogenous viral genes perform no essential function in the

chicken, atleasttothe age of7months,which

isthe present age ofourrooster. Chickens lack-ing endogenous viralgenesshould prove useful

inassessing the role of thesegenesinontogeny,

oncogenesis, and disease.

ACKNOWLEDGMENTS

Wethank DebraKeefer,Joanna Fang, FrancesGulvas, and Deborah Eagen forexcellent technical assistance.

This work wassupportedby grant VC232 from the Amer-icanCancerSociety, grant PCM 76-09721 from the National ScienceFoundation,grants CA06927andRR05539 from the National Institutes ofHealth,Interagency agreement Y01-CP-40214 with the Division ofCancer Cause and Prevention, National CancerInstitute, and by an appropriation from the Commonwealth ofPennsylvania.

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