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 withpurifiedvirions 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 subgroupsA,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
tospecific
segments of theASV Homologues of all fourgenesprobably exist in genome.Theprocedure exploits
the existenceofnormal
chicken cells (9, 19), but the natural deletion mutants; cDNAspecific
for the dele-origins of both viral and homologous cellular tions isisolatedby
selectivemolecularhybridi-genes are not known. Expressionof these genes zation. We previously isolated
cDNA6,,
com-ismodulated in both normal and virus-infected
plementary
toatleast part of thetransforming
cells:someuninfected chicken cells containone gene of ASV (18) and found nucleotide
se-ormoreviral geneproducts, and other chicken quences
homologous
tocDNA..r,
inDNA fromcells 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 ofinterfer-497
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498
enceof thevirus(9);onthe basisofthesepheno- the
following procedure.
Single-stranded
DNAsyn-typic properties, virusstrains canbearranged thesized with
detergent-activated
virions of Pr-Cintodistinct 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
onucleoti
suences
raphy onhydroxyapatite,
and the DNA wasre-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 viralniquessimilartothosereported here,
Hayward
genome isrepresented in the DNA in arelatively
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,
and0.01
Mour procedures for propagation andpurification of
EDTA);
reaction
mixtures
werecontained
either invirus,extraction of viralRNA, andfractionationof capillaries (volumes of 20
,ul)
or in conical tubesviralRN by rate-zonal centrifugation '2 Vis under mineral oil (volumes of 5 to 50
,ul).
Eachvirals
.rN
obyatezoas
centifugPato
(,18).Viru
reaction mixture contained
10,ug ofyeast RNA andASVt
wubgroup
C (Pr-CASV)f
as a clone from p. 10 ug ofeithercalf thymus or salmon sperm DNA.AVo
subgoup
C(Pr-CaSV),
ausp
alone
from P. Hybridization ofDNA wasmeasuredbyresistanceVogtandasconcentratedsuspensionsfromuniver- hyrlsswt51ncee(6)adybiz-sity Laboratories, Inc., Highland Park, N.J. to
hydrolysf
swith
Sl nuclease
(16) andhybridiza(throughtheauspicesof the Offbice ofProgramRe A (5
gmA
y yyNase
sources and Logistics, National Cancer Institute A (50 ug/m
0.3
MNaCl-0.03
Msodiumcitratethe Bratislava strain ofASV, subgroup C (B77-C
(370C,
45mm).Nucleic acids
wereprecipitated withASV,fromR.Friis theShmidtRuppns n 5% trichloroacetic acid and filtered through
glass-ASV,subgroupA(SR-A
ASV)c
andtRous-apisciated
fiber filters. Results of hybridizations wereex-As
,subgroupA(SRAV-A
A),
andR. Ru- iathe
pressedas afunction ofCot
for DNAandCrtorVot
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
sin transformed quail embryo fibroblasts and the acids o
roxypuie
wma tographyof
nucledc
Schmidt-Ruppin strainofASV, subgroup D (SR-Dacids
onhydroxyapatite
wascarriedoutasdescribed ASV), fromP.Vogt; andthesubgroupE virus RAV-previously
(18), using sodium phosphate, pH 6.8,0,growinginembryonicfibroblasts fromline 100 x
supplemented
withNaCl asindicated. Nucleicacids7chickens,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 withso-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 withvirus
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- escribedpreviously (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) andile
NY8ASV
(5) and theASVentl
cDNAsarc
(complementarytothetransforminggene isolated NY8ASV
derivedfromSR-AASV (5, ofASV) have beendescribed (18).cDNA,P
wassin- 15). SR-A ASV and NY8 ASV are congenicgle-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 theon November 10, 2019 by guest
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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 DNAcom-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 with50%) and served to eliminate much of the RNAsfrom the deletionmutantand itsparent
highly reiterated DNAthatis amajor compo- (Fig. 2); differentially labeled
cDNAB77
wasnentof 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 deletionsinNY8ASVresistance 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 ThebulkofcDNA.pA
hadasedimentationcoef-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 withoutcorrec-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. la2000- Beforechromatography 61
Fractions 3-4 20
Fraction 7 53 2 5.3
__ Fraction 10 79
*
~~~~~~~~~Fig.
lb250 -(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 inhibitorsofSi100 _ 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 asde-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 DNA877
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-oB-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 RNAsuated by hybridization with radioactive 70S of SR-A ASV and rdNY8SR ASV. 3H-labeled
RNA of ASV (Fig. 4). As a convenience, we
cDNAUPA
and32P-labeled
cDNAB77 (1,000 cpm of ued RNA from Pr-C ASV withan envgenethat each)werehybridizedtoviral
RNA involumesof20
cross-reacts Rompletely withcDNAPA gene
Tna-pl
for14 hat68°C; the extentof hybridization wascross-reacts completely withCDNASPA (see Ta- measuredbyhydrolysiswith
S1
nuclease.Symbols:ble 4). The data fromtwoexperimentsindicated 0, 3H-labeled
cDNAgpA;
*,32P-labeledcDNA877.
(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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[image:4.501.77.224.51.336.2] [image:4.501.259.450.76.232.2] [image:4.501.261.450.353.580.2]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 wasw100o complementary to between 16 and 20% ofthe
viralgenome(Fig.4and Table3);theseresults
100
conform
to our previousestimates ofthecom-plexity of
cDNA.r,,
(18).. We evaluated the combined complexities of
10 20 30
FRACTIONNUMBER
cDNA..r,
andcDNA.PA
by hybridizing mixturesFIG. 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 RNAcDNAfromSR-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 thenucleotide
sequences repre-for16 h at4°C.The resultsfromthe two analyses are sentedi cDNAsar and CDNAgpA are distict, atsuperimposed in thefigure. Symbols: 0, cDNAOVA; least inlarge part, butourdata cannotexclude
0,CDNASR-A someoverlap (see below).
Preparation of
cDNA,Vc.
We have alsopre-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 theenvgenesof
subgroups
AandC ASV(seeTable4).The specificity of
cDNA.,c
for the deletion in0.1
1'
env wasdocumented
as described above forcDNAOPA (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. Constantgroups.
The kinetics of hybridization ofamounts of 3H-labeled
cDNAgOVA
or 3H-labeled cDNAgpA andcDNAisC
with representativecDNA,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 finalex-werechosensothattheratiobetweenthecDNAand
itscomplementarynucleotide sequences in the RNA TABLE 3. Geneticcomplexities of
cDNAUPl
and would range from 10-2 to 10; computation ofthe cDNAaarcaratios 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 weredoneinvolumesof5Pifor40h 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 0.19withcDNAr,.,. 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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[image:5.501.54.224.50.168.2] [image:5.501.71.222.285.404.2] [image:5.501.257.453.468.585.2]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)oruseddirectly(CandD). Variousamountsofviral RNAwerehybridizedwith 800cpm(0.04ng)ofeither 3H-labeled
cDNAs,pAor3H-labeled
cDNA,,,c
and800 cpm(0.01 ng) of32P-labeledcDNABIIforappropriateperiodsat68°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-labeledcDNAB77
wasincluded in reactiongroupAor
subgroup
CandcDNAB77
reactedat mixtures as aninternal standard andhybrid-identical rates with the various viral RNAs. ized
extensively
with RNA from allsubgroups
Apparently,
noneof the virus stocks containedexcept
G. Wealsoanalyzed
reactionsbetweenappreciable
numbers ofmutantsbearing
dele-cDNArep
and viral RNA from several of thetions in env;
otherwise,
the reaction withsubgroups;
these reactionsprovided
amoresat-cDNAgp
would have been slower than that withisfactory
testofhomology
becausecDNArep
hadcDNAB77
(seebelow). beenselectedtobeanearly
uniformcopyoftheHomologies
amongtheenv genesof differ- ASV genome, whereascDNAB77
contained entsubgroups
of avian leukosis-sarcoma vi-highly
reiterated DNA ofverylowgenetic
com-ruses. Weanalyzed homologies
amongtheenvplexity
andasmallamountof DNAtranscribed genesfrom differentviralsubgroups by hybrid-
from the bulk of the ASV genome (8). Theizing
cDNAIpA
to70S RNAfrom all the known results withcDNArep
conformed to those withsubgroups
of avian leukosis-sarcoma virusescDNAB77;
inparticular,
therewasonly
limited (Table4).Allreactionswerecarriedtovalues ofhybridization
with RNA from GPV.By
con-C,t
suffilcienttoensure aplateau
ofhybridiza-
trast,cDNAGpv
hybridized
extensively
(80%)tion.
Hybridization
wascomplete,
or almost with RNAfirom GPV butnotwith RNA fromcomplete,
forsubgroups A, C,
and D, whereas ASV(Table 4).Weconclude that there isexten-reactionswere
incomplete
with RNA from sub- sivehomology
among the genoxnes of all thegroups B (80%), D (88%), and E (70%) and virusestestedexceptGPV,whichappears tobe
barely
detectable with RNA firomsubgroups
Flargely
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 resultsmir-tions in the
adjoining
genesrc (B77-C tdASV rored those withcDNA.PA. In
summary,all theand Pr-C tdASV)
hybridized
cDNAIIPA
com- virus strainsindigenous
tochickens(subgroups
pletely.
A,
B,C,
DandE)share extensivehomology
aton November 10, 2019 by guest
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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 andcould 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 thetwoleast 20% of the ASVgenome,correspondingto genesmayoverlap.The combined
complexity
ofa genetic complexity ofca. 2,000 nucleotides.
cDNAsar
andcDNASPA
isatleast3,000nucleo-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).
Inaddition, 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), andcDNNam
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 ofcDNA.PA
are all included within theauthors)
that have deletions inenv(5).
Wethe 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 ofsomeoverlap.
or all of the envelope glycoprotein; hence,
Frequency
of deletions in env. We havecDNAgPA
mayrepresentmostofenv. shown previously that mutant viruses withThe 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 deletionsspecifically
retards therateofhybrid-suggest twopossible explanations for this dis- ization with
cDNA,ar,,
an effect which canbe crepancy. (i) Some of the nucleotide sequencesreproducibly
detected whendeletions constitutedeleted 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 forpublication).
Sinceon November 10, 2019 by guest
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[image:7.501.48.442.76.257.2]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
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*ri h i o bewe .N and the '
,nontransforming
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