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Cell Cycle-Dependent Activation of Rous Sarcoma Virus-Infected Stationary Chicken Cells: Avian Leukosis Virus Group-Specific Antigens and Ribonucleic Acid

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JUly 82-87

Copyright©1972 AmericanSociety forMicrobiology PrinitedinU.S.A.

Cell

Cycle-Dependent Activation of Rous

Sarcoma

Virus-Infected Stationary Chicken Cells: Avian

Leukosis

Virus

Group-Specific

Antigens

and

Ribonucleic Acid

ERIC H. HUMPHRIES AND HOWARD M. TEMIN

McArdle Laboratory, UniiversityofWisconsin, Madisoni, Wisconsil53706

Receivedforpublication 24 April 1972

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary

cells did not contain avian leukosis virus group-specific antigens or ribonucleic

acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV

endogenous RNA-directed DNA polymerase activity.

Roussarcoma virus (RSV) appears to replicate through a deoxyribonucleic acid

(DNA)

inter-mediate,

the DNA provirus

(14).

Initiation

of

RSV production requires two types of early DNA synthesis, one viral and one cellular (12). In stationarychicken cells exposed to RSV, viral DNA synthesis occurs, whereas cellular DNA

synthesis

does not.

Therefore,

there is no cell

division or virus

production. The

present

studies

were undertaken to determine whether

either

avian

leukosis

virus

group-specific (gs) antigens

or viral-specific

ribonucleic

acid

(RNA)

were present

in

stationary RSV-infected

chicken

cells.

MATERIALS AND METHODS

Generalexperimental procedureswerethesame as

thosepreviouslydescribed for thislaboratory(12,13). Cell culture. Primary cultures of fibroblasts were prepared from 12-day-old White Leghorn chicken embryos (Sunnyside Hatchery Co., Oregon, Wis.) and weregrown inmodifiedEaglemedium with 20% tryptose phosphate broth (ET medium) and 5% calf

or fetal bovine serum. Secondary andlattercultures ofchickencells wereprepared at 5.0 X 105cells per

60-mm plastic dish (FalconPlastics, Division of B-D

Laboratories, Inc., LosAngeles, Calif.) and at 2.0 X 106 cells per100-mmdish.Cultures of stationarycells

were preparedat 1.0 X 106cells per 60-mm plastic dish or at 4.0 X 106 cells per 100-mm dish in ET mediumandincubated for 3 days at 38 C in a humidi-fiedCO2incubator.

Viruses. Schmidt-Ruppin (SRV)and B77virus were previously described (1). A heat-resistant strain of

SRV (SRV-HR), at approximately 1 X 108 focus-forming units (FFU) per ml, was obtained from D. Boettiger (Ph.D. thesis, Univ. of Wisconsin, Madison,

1972). Viruswas concentrated by centrifugation and

purified by equilibrium density gradient

centrifuga-tion in 15 to 65% sucrose gradients in 0.01 M tris

(hydroxymethyl)aminomethane-0.001M

ethylenedia-minetetraacetic acid (pH 7.5) buffer.

Antisera. Two antisera were used. For immuno-fluorescence, rabbit immunoglobulin G

(lgG)

anti-serum prepared against Tween-ether-disrupted avian

myeloblastosis virus (AMV) was supplied by R. Nowinski. This serum had a fluorescence titer of

1:150onuninfected chicken cells andatiterof 1:2000

on SRV-infected chicken cells. Since the rabbit anti-AMV antiserum contained antibodies that reacted with both normal cellular components and virus-specific antigens, in vivo absorption was carriedout to

eliminate nonviral fluorescence (8). Absorption was

carried out in 2-week-old quail by intraperitoneal injection of 0.5ml of antiserum followed by

ex-sanguination6hrlater. Serawerecollected and stored

at -70C. This procedurereduced the titer of

fluo-rescence on normal chicken cells to 1:8, whereas

viral-specific fluorescence had a titer of 1:500. (Ab-sorption in 1-week-old chickens resulted in a large

reduction offluorescence on both uninfected and

in-fected chickencells.) Fortesting cell preparationsfor avian leukosis virus gs antigens, cellswere incubated with a 1:500 dilution of rabbit anti-AMV (purified

IgG) or with a 1:128 dilution of rabbit anti-AMV

(purifiedIgG) absorbed inquail.

The antiserum used in complement fixation was

preparedagainst avianleukosis virus gs antigens ina

rabbit by using ether-Nonidet P-40 (Shell Chemical Co., New York, N.Y.)-disrupted SRV. The

anti-serum was absorbed twice withsheep redblood cells

andinactivatedat56Cfor30min. The

complement-fixing titer ofthe antiserum was 1:4 on 4 units of

antigenprepared from uninfectedquail,rat,ormouse

cells,and 1:16on4units ofantigenfrom uninfected

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VOL. 10, 1972 ACTIVATION OF VIRUS-INFECTED STATIONARY CELLS

chicken cells. Thetiteron4unitsof antigen prepared from chicken cells infected with SRV or from chicken cells found to release avianleukosis virus "spontan-eously" was 1:128. For testing antigen preparations, the antiserum was usedat 1:64 and 1: 128 dilutions. Complement fixation. Complement fixation was carried out by the microtechnique of Sarma (11). Fresh serumobtained from guinea pigs was absorbed twice with 0.3 ml of packed sheep red blood cells per1 ml ofcomplement and was frozen at -70 C in 0.2-ml fractions. Rabbitanti-sheep hemolysin (Miles Laboratories, Kanakee, Ill.) was inactivated at 56 C for 30 min. Antigens were prepared after two washes withVeronal buffer. Cells, 2 X 107 per ml, were frozen and thawed three times, extracted with an equal volume of ether, andcentrifuged for 1 hrat20,000 X g. Indirect immunofluorescence. Indirect immuno-fluorescence wasusedtotestcell preparationsforviral antigens by the methodofHilgersetal. (8).

Immunofluorescence absorption test. Cellswerealso

examined for the presence ofviral antigens bytheir

ability to absorb the positive fluorescence from a

known standard system by the method of Hilgers

etal. (8). Antigens were prepared from cells as de-scribed above for complement fixation. Antiserum

wasusedat afourfold dilutionbelowtheendpointof viral-specific fluorescence on standard positive cells.

Fifty

1liters

of antisera were absorbed overnight at

4Cwithvariousamountsoflyophilized antigen pro-tein. Antiserum without antigen was included as a

positive control. These antisera preparations were

then used in indirect fluorescence tests on standard

positivecells.

Polymerase reaction. The standard polymerase

re-action of Temin and Mizutani (16) was used to

produce large amounts of radioactive virus DNA product.

Reactions were carried out in a total volume of 2.50 to 6.50 ml of reaction mixture containing 160

pg ofpurified virus per ml and 20,Ci each of

3H-thymidine-5'-triphosphate (13.4 Ci/mmole; Schwarz BioResearch, Inc.) and

3H-deoxycytidine-5'-triphos-phate (22.5 Ci/mmole; Schwarz BioResearch, Inc.)

perml. Thereaction mixturewasdistributed in 0.5-ml samples andincubated at40 Cfor 1 hr, after which the reaction mixtures were pooled for nucleic acid extraction. Approximately 6,000 counts/min of

tri-chloroacetic

acid-precipitable

product

corresponded

to 1ngofDNA.

Nucleic acids and hybridizations. The procedures

usedforextraction of nucleic acids from

polymerase

reactions, purified virus, and cells by using

diethyl-pyrocarbonate (Baycovin; Bayer, Leverkusen, Germany) andsodium

dodecyl

sulfate andfor RNA-DNA hybridizations have been described by Coffin and Temin (3, 4). Control hybridizations between uninfected chicken cell RNA and B77 virus product

DNA contained a background of 3 to 5% of the

countsbandingat a density

equal

to orgreaterthan

1.54 g/ml. Percent hybridization with virus-infected chicken cell RNA was defined as the fraction of

countsbanding at adensity

equal

to orgreaterthan

1.54g/mlminus the

background

of

parallel

hybridi-zationswithuninfected chicken cellRNA.The

density

of 1.54 g/ml gave a sligbtly lower background com-pared with the density of 1.53 g/ml used earlier

(3, 4). Approximately45 to 50% oft'hetotal B77 virus product DNA used in these studies was shown to hybridize with a large excess of viral RNA. However, in the experiments reported here withcell RNA, lower levels of hybridization were achieved because there were lower concentrations of viral-specific RNA. Recoveryof labeled DNA from gradients was always greater than 65 to 70%. Previous reports (3, 6, 7) have established that hybrids between RSV product DNA andviral-specific RNA band in the RNA region ofCs2SO4 gradients after equilibrium centrifugation. DNAcomplementary to the RSV RNA, "minus" DNA, was prepared aspreviously described by Coffin andTemin(5), byhybridization of total virus product DNAwith excess viral RNA and alkali hydrolysis of the fractionof DNA banding with a density greater than 1.65 g/ml after Cs2SO4 equilibrium centrifuga-tion.

RESUTLTS

Absence of avian leukosis virus gs antigens in RSV-infected stationary chicken cells.

RSV-infected

stationary chicken cells contain the

genome of RSV, but do not release infectious virus

(12).

These cells were tested by using three

immunological

techniques, indirect

immuno-fluorescence,

complement fixation, and

immuno-absorption fluorescence, to determine whether they contained avian leukosis virus gs antigens.

Stationary chicken embryo fibroblast cells

were

exposed

to SRV-HR, either ET medium or ET

medium with serum was added, and various times

after infection

the percentage of

cells

containing

gs

antigens

was

determined

by using indirect

immunofluorescence (Fig.

1). Whereas more

than

80% of

the cells

in theserum-containing cultures

became

positive for gs antigen within 48 hr,

cultures

of

stationary

cells were nevermorethan

10%

positive

even

after

130 hr. At 84 hr

after

infection, serum was added to cultures of sta-tionary cells, and the cells were examined for gs

antigens.

These

cells

were

50%

positive for gs

antigen within

48 hr

after addition of

serum.

The

low level

of positive cells in the cultures of

stationary

cells and the low level

of

virus

pro-duced

in

these

cultures are most

probably the

result

of division occurring inasmall

proportion

of

the

population (15). This interpretation is

sup-ported

by

the observation that autoradiography

of cultures of stationary cells

labeled

with

3H-thymidine

for 24 hr showedapproximately

10%

ofthe nuclei were labeled (data not

shown).

Infected

stationary

cells

werealsotested for gs

antigens by complement

fixation

(Fig.

2).

Again

nogs

antigens

werefound.

To demonstrate the specificity of the

immuno-logical

tests described above,

immunoabsorption

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HUMPHRIES AND TEMIN

A

80/ / /(106)

40 I /

| /(104) ~~(le4) X (104)/

1

o5(lo2)

~-g

--- x--x

0o 20 40 60 80 loo 120 140 160 180 HOURSAFTER INFECTION

I

C,)

w oI

0-'fo

8~--x _

8)2

~ ~ ~ ~ ~ ~

I L L

20 40 60 EC 100 120 140 160 180

HOURS AFTER INFECTION

FIG. 1. Absenice of avian leukosis virus

group-specificantigensfrom RSV-inifectedstationzarychicken cells. A,Stationarychickenembryofibroblasts, 1.0 X

106 cells per 60-mm culture dish, were exposed to

SR V-HR at a multiplicity of I FFU/cell, anid the inoculum was replaced with ET medium (X) or ET

medium with

4%lo

fetalbovine serum (0). At various

timesafter infection,cellswereharvested and examined

for gsantigensby usinig indirectfluorescence. Rabbit

anti-AMV(purified IgG) wasused ata1:500dilutiont.

Eachpointrepresents300 cells. At 84 anid 130 hrafter

infection, serum-containing medium was added to

cultures without serum

(I

.). Culturefluids were nlot

changed during theexperiment. Virus titers (indicated

inparentheses)representFFU per mlofculturefluidat

the timeofcellharvest. Uninfectedcellswereincubated in medium with 4% fetal bovine serumfor48 hr and

lusedas a negative control (*). B, Cell niumbersper

culture dish were determinedJbr the infectedcultures

with ETmedium (X), for the infectedcultures with

ETmedium with4%.fetalbovineserum (0), atdfor

the uninfectedcultures withETmedium with4%fetal

bovine serum (U) described int A.

fluorescence was carried out with the antigen preparations from the experiment described in

Fig.2. Theantigenswereusedexactlyasprepared forcomplementfixation. Theimmunoabsorption

wascarriedoutby using overnightabsorption of z

O

g, 60~

o

Z0

X

--20

0 z

8 oo

0

A

--;L___~~~~~~~~~~a-

-24 48 72 96 120 144 168

HOURSAFTER INFECTION

| ~~~~~~~~~B

24j

o20L 16-'0

Cr 122-//

Z 8- //

U 4s/ ---J-.

0 24 48 72 96 120 144 168

[image:3.493.59.253.58.373.2]

HOURS AFTER INFECTION

FIG. 2.Absetnce ofaviatn leukosis viruisgroup-specific

antigenis from RSV-inifected stationary cells. A,

Stationiary cells, 4.0 X 106 cells/100-mmculturedish,

wereexposedto SR V-HRatamultiplicity of1FFU/

cell, and the inoculum was replaced with ETmedium

(X) or ETmedium containinig 4%O fetal bovineserum

(0). At 24-hr intervals, cells were harvested and processedforcomplementfixationi.Anztigen preparations

werenormalizedto30 ug of proteiniper25ulitersand

assayed with complement fixation forgs antigenis by

using rabbit anti-SR Vata1:64dilutionz. Culture fluids

were changed on all cultures 72 hr after inifectionz.

Serum was added to ciultures with ETmedium (i)

120hrafter infection. B, Cell numbersperculture dish

weredeterminedfor the cultures with ETmedium (X)

and ET medium with 4%, fetal bovinie serum (0)

describedinA.

rabbit anti-AMV at4 Cwith variousamountsof

lyophilized protein from different antigen

prepara-tions(Table 1). Onlydividingcells at 72 and 120 hrafter infectioncontained enoughgsantigensto absorb specifically the capacity of the antiserum

to react with the positive controls. The fact that

equivalentamounts offetal bovineserumand of

84

J)

-J

-i

> 0

0.

z I)

LU

J. VIROL.

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VOL. 10, 1972 ACTIVATION OF VIRUS-INFECTED STATIONARY CELLS

TABLE 1. Detectioni ofavian leukosis virus group-specific anitigensbyusingimmunoabsorption

fluorescence

Antigenpreparationa

RSV-infected cells Stationary Dividing Stationary Dividing Stationary Dividing Fetal bovine serum

Uninfected chicken cells

HAIb

24 24 72 72 120 120

Proteinc (mg)

500 500 500 50 500 50 500 500

Pres-ence of fluo- res-cenced

aAntigens prepared from cell extracts as

de-scribed in Fig. 2 for complement fixation. RSV,

Roussarcoma virus.

I Hoursafter infection.

cMilligrams of protein usedtoabsorb50 ,tliters

of 1:500 rabbit anti-avian myeloblastosis virus

(purified immunoglobulin G).

dObservedpresenceoffluorescenceonstandard

slides usingantisera afterabsorption.

ePresence ofgroup + specific (gs) antigens in

protein used for absorption (indicated bythe

ab-sence offluorescence).

uninfected chickencellprotein didnot absorbthe fluorescence illustrates the specificity of this test.

The results of these experiments demonstrate thatgsantigensarepresentonlyin low amounts in

culturesofRSV-infectedstationarycells,whereas

theyarepresent inlargeamountsindividingcells.

Absence of viral-specific RNA in stationary RSV-infected cells. To determine whether

viral-specific RNA was present in RSV-infected

stationary chicken cells, RNA-DNA hybridiza-tions were carried out with RNA from infected

stationary cells and DNA from the B77 virus

endogenous RNA-directed DNA polymerase

re-action. Sample gradients show the analysis of

hybridizations between total B77 virus product

DNA and RNA from stationary and dividing chickencells 120 hr after infection with SRV-HR

(Fig. 3). A controlhybridization withuninfected chicken cellRNAis also included.

Figure 4 summarizes the results of hybridiza-tions of RNA extracted 36 hr after infection from SRV-HR-infected stationary chicken

em-bryo fibroblasts with whole B77 virus product DNA (Fig. 4A) and with the "minus" DNA

isolated from whole B77 virus product DNA

(Fig. 4B). In both sets ofhybridizations, 150 ,g

of RNA from infectedstationarycellsshowedno

morethan

0.5%c,

hybridization above background values of 4.5 and 2.5%o hybridization with unin-fected chicken cell RNA. There was approx-imately a 100- to 150-fold difference between the amount of viral-specific RNA in RSV-infected dividing and RSV-infected stationary cells.

Figure 5 shows the results ofhybridizations of B77 virus product DNA and various amounts of RNA extracted 72 and 120 hr after infection fromstationary and dividing cells. RNA prepara-tions from dividing cells 72 and 120 hr after

infection

gaveapproximately 21 to

22%

hybridi-zation at saturation. A

150-,ug

amount of RNA from parallel stationary cells gave only 3 to

4%

hybridization. Hybridization with uninfected chicken cell RNA gave a background value of 4%. There was at these times approximately 20-fold more RNA hybridizable with viral product DNA in RSV-infected dividing cells than in RSV-infected stationary cells.

The results of these experiments demonstrate

that

viral-specific

RNA is present only in low

amounts in cultures of RSV-infected stationary cells,whereas RSV-infected dividing cells contain 20-to 150-foldmoreviral-specificRNA.

DISCUSSION

Provirus formation occurs in stationary cells of RSV-infected chicken embryo fibroblasts, whereas cell cycle-dependent activation and release of

infectious

virus does not (12). The results

pre-sented above demonstrate that cultures of

stationary

cells

contain

only small amounts of

avian leukosis

virus gs

antigens

and

viral-specific

RNA. Although

90%

ofRSV-infected dividing cells contained gs

antigens

48 hr after

infection,

only

10%

of the cells in cultures without serum

contained gsantigens6 daysafterinfection. Since

a small proportion of cells in cultures without

serum

proceed through

cell

division,

it is

likely

thatthe cells in cultures withoutserumthat were positive for gs antigens had proceeded through mitosis. In

parallel

experiments,

RSV-infected

stationary

cells contained

only

small amounts of

viral-specific RNA compared to the amounts

found in

parallel

dividing

cells. At 36 hr after

infection,

dividing

cells contained 100-fold more

viral-specific RNA than found in

stationary

cells. Three and four

days

after

infection,

this ratio

decreasedto a 15to20-fold difference. It is

likely

that this decrease is the result ofcell division in

the cultureswithoutserum.Itappearsthat neither

viral-specific

RNA nor avian leukosis viral gs

antigens are

produced

in

stationary

cells and

that, until cell

cycle-dependent

activation,

trans-cription of the

provirus

does not occur.

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UNINFECTED CHICKEN A INFECTED STATIONARY CHICKEN INFECTED DIVIDING CHICKEN

- 17 7 17

Q16Qb,v8 8- 1:

662 20 2 4 6 2 4 6

Q. ~~~~~~~~~~~~0.0.

2- 2~ 2

4 8 12 14 20 24 4 8 12 16 20 24 4 8 12 16 20 24

FRACTION FRACTION FRACTION

FIG. 3. Hybridization withB77 virusproduct DNA and RNA extracted 120 hrafter infection fromculturesof

stationaryanddividingSRV-HR-infectedchickencells.Stationarychickenembryo fibroblasts,4.0X 106 per

100-mmdish, wereexposedto SR V-HR at amultiplicityof100FFU/cell, andthe inoculum was

replaced

withET mediumorET medium containing4%fetal calfserum.At 120 hrafterinfection,cellswereharvested,andthe RNA

wasextracted. RNAwashybridizedwith6,000counts/minofB77virusendogenous

3H-labeled

DNA

product

for

5hr at68 C,andthe mixturewasanalyzed by Cs2SO4 equilibrium density gradientcentrifugation.The

hybridizations

werecarried outwith RNAasfollows:A, 150,sg of uninfectedchickencellRNA;B, 150 ,&gof infected stationary

chicken cellRNA, 120 hrafterinfection;C, 75pg ofinfected dividingchicken cellRNA,120hr

after infection,

plus

75ugofuninfected chicken cell RNA. Thearrowsindicatethedensityof1.54g/ml.

"MINUS STRAND"

WHOLE VIRUSPRODUCT DNA VIRUS PRODUCT DNA

8 A '

20I20 B

Al~~~~~~~~~~~~~~A

Al

2~~~~~~~~~~~~~~~~2

2

z DIVIDING CELLm IIIGCL

2

w z

n

~~~~~~~~~~ID

Zlo

_

<

10_

/

z

8I-/ STATIONARY CELL ~~~~~~~z /

CL STATIONARY CELL

a.

I0 I0 IO Oc

&)50 100 150 0 50 100 150

jxg RNA 9ugRNA

FIG.4. Hybridizationbetween B77 virusproductDNAandRNAextracted 36 hrafter infectionfromculturesof

stationaryanddividing SRV-HR-infectedchickencells.StationarycellswereinfectedasdescribedinFig.3and

wereharvested36 hrafter infection. A, VaryingamountsofRNAfrom infected stationarycells(X)andinfected

dividing cells ( )werehybridized with 3,000counts/minofendogenous3H-labeledB77 virusDNAproduct for5hr

at68Candanalyzed by

Cs2S04

equilibrium density gradientcentrifugation. Varyingamountsofuninfected chicken

cellRNA were addedsothat allhybridizationswerecarried out in thepresenceof150

pg

ofchickencellRNA. A

150-pg

amountof uninfectedchickencell RNAwasalsohybridized without addedinfected cell RNA

(U).

B,

Dupli-catehybridizations werecarriedoutas in Aexceptthat 3,000counts/minof"minus"strand B77 virusproduct DNA

wasusedineachhybridization.

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VOL. 10, 1972 ACTIVATION OF VIRUS-INFECTED STATIONARY CELLS

10, 17). Itispossible that activationof transcrip-tion isinvolved inallthesesystems.

ACKNOWLEDGMENTS

We thank R.Chen, R.Grimstad,V.Kassner,and A.Kato for technicalassistance.

DIVIDING CELL This investigation was supported by Public Health Service researchgrantCA-07175 from the National Cancer Institute atsd grant VC-7 from the American CancerSociety. E. Humphries wassupported by traininggrantTOI-CA-5002from the National CancerInstitute. H. M.Temin holds Research Career

Develop-mentAward1OK3-CA-8182 from the National Cancer Institute.

STATIONARY CELL

,ug RNA

FIG. 5. Hybridization between B77 virus product

DNAand RNAextracted72and 120hrafter infection

from SRV-HR-infected chicken cells, dividing and stationary. Cells were infected, and RNA was

ex-tracted and hybridized with whole B77 virusproduct

DNA as described in Fig. 3. Under these conditions,

1.5 ugofB77 virusRNAgaveapproximately25to27%

hybridization. The RNA species in the hybridization

mixtures were: uninfected chicken cell RNA (-);

infectedstationary (A) anddividing (A) chicken cell RNAat 72hrafterinfection; infectedstationary (a)

anddividing (0) chicken cell RNA at 120 hr after infection.

The RNA-DNA hybridizations were carried

out with an endogenous B77 viral DNA

polym-eraseproduct capableofdetecting approximately 20% ofthe viral RNA (5). Although the large

amounts ofviral-specific RNA found individing

cells were not present in stationary cells, it is

possible that some viral-specific RNA was in

stationary cells which was not detected by the DNAusedin thishybridizationprocedure.

Leong et al. (9) have reported that therate of

productionof RSV-RNAvarieswiththestageof

thecellcycleoftheinfectedcells.Thiswould bea second control of viral transcription once the

transcription was activated by the initial cell

division.

Cell cycle-dependent activation of differentia-tion hasbeenreportedinseveral othersystems(2,

LIERATURE CITED

1. Altaner,C., and H. M. Temin. 1970.Carcinogenesisby RNA sarcomaviruses. XII. A quantitative study of infection of

rat cells in vitro by avian sarcoma virus. Virology 40: 113-134.

2. Bischoff, R., and H. Holtzer. 1969. Mitosis and theprocesses ofdifferentiation of myogenic cells invitro. J. Cell. Biol. 41:188-200.

3. Coffin,J. M.,and H. M. Temin. 1971.Comparison of Rous sarcoma virus-specific deoxyribonucleic acid polymerases invirions of Roussarcomavirus andRoussarcoma virus-infected chicken cells. J. Virol. 7:625-634.

4. Coffin, J. M., and H.M.Temin.1971.Ribonuclease-sensitive

deoxyribonucleic acid polymerase activity in uninfected ratcells andratcellsinfected with Roussarcomavirus. J. Virol. 8:630-642.

5. Coffin, J. M.,andH.M. Temin.1972.Hybridization of Rous sarcoma virus deoxyribonucleic acid polymerase product and ribonucleic acids from chicken andratcellsinfected with Roussarcomavirus. J.Virol. 9:766-784.

6. DuLesberg, P. H., and E. Canaani. 1970. Complementarity between Rous sarcoma virus (RSV) RNA and the in

vitrosynthesized DNA of thevirus-associated DNA poly-merase. Virology 42:783-788.

7. Fanshier, L., A.-C. Garapin, J. McDonnell, A. Faras, W. Levinson, andJ. M. Bishop. 1971. Deoxyribonucleic acid polymerase associatedwith avian tumorviruses:secondary

structureof the deoxyribonucleic acid product. J. Virol. 7:77-86.

8.Hilgers, J., R. C. Nowinski, G. Geering, and W. Hardy. 1972. Detection of avian and mammalian oncogenic RNA viruses (oncornaviruses) by immunofluorescence. Cancer Res. 32:98-106.

9. Leong, J. A., W.Levinson, andJ.M.Bishop. 1972.

Synchroni-zation of Roussarcomavirusproductioninchickembryo

cells.Virology 47:133-141.

10.Pietsch,P., andS. B.McCallister. 1965.Replication and the activation of muscle differentiation. Nature (London)

208:1170-1173.

11. Sarma,P. S., H. C. Turner, and R. J. Huebner. 1964. An

avianleukosisgroup-specific complement-fixation reaction: applicationforthe detectionandassayof non-cytopatho-genic leukosisviruses.Virology23:313-321.

12. Temin. H. M. 1967. Studies on carcinogenesis by avian sarcomaviruses. V. RequirementfornewDNAsynthesis and forcell division. J. CellPhysiol. 69:53-63.

13. Temin, H. M. 1968. Studies on carcinogenesis by avian sarcoma viruses. VIII. Glycolysisand cellmultiplication. Int. J. Cancer 3:273-282.

14. Temin, H. M. 1971. Mechanism of cell transformation by RNAtumor viruses. Annu. Rev. Microbiol. 25:609-648. 15. Temin, H. M. 1971. Stimulation byserumofmultiplication ofstationarychicken cells. J.Cell Physiol. 78:161-170.

16.Temin, H. M., and S. Mizutani. 1970. RNA-dependent DNA polymerase invirions of Roussarcomavirus.Nature (London) 226:1211-1213.

17. Yamada,T.1966. Control of tissuespecificity:thepatternof cellularsyntheticacdvities in tissuetransformation. Amer.

Zool. 6:21-31. Q

0

Al

z m z

w H z

N C,,

z

0

0 z w

w

a-87

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[image:6.493.51.244.76.347.2]

Figure

FIG.specific
TABLE 1. Detectioni of avian leukosis virus group-specific anitigens by using immunoabsorption
FIG. 3.chickenstationarymediummmwerewas575 hr ug Hybridization with B77 virus product DNA and RNA extracted 120 hr after infection from cultures of and dividing SR V-HR-infected chicken cells
FIG.5.fromDNADNAandstationary.RNAhybridization.mixturesinfectedtracted1.5 Hybridization between B77 virus product and RNA extracted 72 and 120 hr after infection SR V-HR-infected chickencells,dividing andCellswere infected, and RNAwasex- and hybridized wit

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