Partial nucleotide sequence of Rous sarcoma virus-29 provides evidence that the original Rous sarcoma virus was replication defective.

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Partial

Nucleotide Sequence of Rous Sarcoma

Virus-29

Provides

Evidence that the Original Rous Sarcoma Virus Was

Replication Defective

ANINDYADUTTA, LU-HAI WANG, TERUKO HANAFUSA, AND HIDESABURO HANAFUSA*

The Rockefeller University, New York, New York 10021

Received28February 1985/Accepted 13 May 1985

Rous sarcoma virus-29 (RSV-29) is the strain ofRSV that has the least number of passages beyond its isolationfrom chickentumorno. 1amongallcurrentstrains ofRSV.Biologicalcharacterization indicated that it was replication defective. RNA analysis of nonproducer clones of RSV-29-infected chicken embryonic fibroblastsshowed thepresenceofasubgenomicmessageof 2.6 kilobases containingsrcandagenomicRNA of 7.7 kilobases that containsgag,pol, and src, butnotenv. Thesrc-containing EcoRIfragmentofRSV-29

proviralDNAwasmolecularly cloned. Sequenceanalysisoftheregionsflankingsrcrevealed that theenvgene

wascompletely deletedinRSV-29andthatthesequence acrossthe deletionwasexactly thesame astheBryan high-titer strain of RSV.Thesequenceimmediately 3'tosrcinRSV-29wascloselyrelatedtothatofthePrague strainof RSV.Thefactthat the strain ofRSV whichhas theminimalnumber ofpassagesbeyond itsisolation isreplication defectivesupports thehypothesis ofLernerandHanafusa (J. Virol. 49:549-556, 1984) that the original RSV is a defective transforming virus. This defective transforming virus is postulated to be the precursortoother defectiveRSVs like theBryan high-titer strainand tonondefective RSVs like the Prague strain. Theparticularclone ofRSV-29 thatwestudiedalso hadashort stretchofsequenceduplicationatthe 3' end ofthepolgene,whichwaspresumably created byan errorofreversetranscription.

In most acutely transforming retroviruses, acquisition of anoncogenefrom host genomicsequences byahelper virus hasresulted in the deletion ofone ormore genesessential for replication. Asaresult,these virusesareusually replication defective (11, 17, 24). Thecommonstrainsof Roussarcoma virus (RSV), Schmidt-Ruppin (SR-RSV) (22) and Prague (PR-RSV) (12), are anomalous in that they contain an

oncogene andyetpreserveall thegenesforreplication (34). However, the Bryan high-titer strain of RSV (BH-RSV)has been found to lack the envgene, rendering the virus repli-cation defective (4, 8, 21). BH-RSV could have been a deletion variant derived from nondefectiveRSV. However, the possibility that BH-RSV arose by an independent transduction ofc-src by a nondefective helper virus could

not be ruled out. Sequence analysis ofmolecularly cloned BH-RSV proviral DNA hasdefined theexact nature ofthis deletion. The DNA sequence has also revealed extensive

homology between thesequencefromthe3' end ofsrctothe long terminal repeat in BH-RSV and the corresponding

sequence at the 3' end ofa helper virus, Rous-associated virus 2 (RAV-2). This observation suggested the steps by whichrecombination betweenthehelpervirus and the3' end ofc-src message produced the3' end of BH-RSV (1, 13).

RSV-29 is avariantofRSVobtained fromastock of Rous chickentumorno. 1thatwasdried andstored since 1929. In 1963, one ampoule of this dried tumor was suspended in bufferandinjectedinto chickens. Extracts of thetumorsthat developed contained viable tumorigenic virus, which was called RSV-29 (26).

We found that RSV-29 is also replication defective. We therefore decided to examine RSV-29 for the nature of its defectivenessandfor thestructureofits3' endtodefine its

* Correspondingauthor.

relationship to other strains of RSV and possibly to refine

our views regarding the putative recombinations that took

place between the helper virus and the genomic c-src se-quences togive rise tothe RSVs.

MATERIALSANDMETHODS

Cellsand viruses.Chickenembryofibroblasts(CEF)were from gs-chf-embryos (SPAFAS, Inc., Norwich, Conn.) and were grown before and after transformation in Scherer medium as described previously (7), but the 1:1 mixture of HamF-10 andDulbecco modified Eaglemediumorminimal essential medium were also used in some experiments.

RSV-29, provided by R. M. Dougherty, is only two tissue culturepassagesbeyondachickentumorinduced withadried preparation storedby W. J. Purdyin 1929(26).

Cloning of RSV-29-infected chicken cells. A secondary culture of CEFwasinfected withRSV-29 and itshelpervirus athighvirusdilution(multiplicityof infection of10-4to10-5

focus-forming units per cell). After 5 h, the infected cells weretrypsinized andplatedoutat adensityof106 cellsper

100-mm dish and overlaid with minimal essential medium containing0.36%agar.Individualcolonieswerepickedafter 14 days of incubation and were cultured separately on a feeder layer of uninfected CEF. Clones of cells that were morphologically transformed but did not produce focus-forming virus were superinfectedwith helper virusRAV-2. Clones that consistently produced infectiousfocus-forming virus on superinfection with RAV-2 but that did not do so without helper virus were designated nonproducer (NP) clones and were used for studies on viral RNA. Proviral DNAwas preparedfrom NP clone 1.2.

Preparation of DNAprobes. Viral DNA probes (see Fig. 1B) were prepared from the following viral DNA plasmid clones: pSRA-2, which contains the entire SR-RSV-A (SF)

genome (3); pFC3, which contains the 2.5-kilobase-pair

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(kbp)EcoRI DNAfragment of SR-RSV-A (NY)containing

the 5' region ofgag (obtained from F. Cross); pLTR-L6,

which contains the PvuII-to-EcoRI DNA fragment of the SR-RSV-B U3 region (obtained from T. Takeya); and

pTT107, which contains the 3.1-kbp EcoRI fragment of SR-RSV-A (NY) containing the env-src region (31). DNA

fragments representing defined regions ofthe viral genome were prepared bydigestion oftheaboveplasmidDNAs with appropriate restriction enzymes followed by agarose

gel

electrophoresis and electroelution. The purified viral DNA

fragmentswere labeled with [32P]dCTP by nick translation (14).

Viral RNA analysis. Totalpoly(A)-containingRNAswere

prepared from two tofour 10-cm dishes of RSV-29-infected CEF accordingto theprocedures described

previously (33).

Portions (5 to 10 ,ug) of each poly(A)+ RNA

preparation

were used for analysis by agarose gel electrophoresis, Northernblotting,andhybridization with variousviral DNA

probes (Fig. 1). RNase T1 resistant

oligonucleotide

finger-printing hasbeen describedpreviously (32).

Molecularcloning of RSV-29proviralDNAfragment. Total cellular DNA was extracted from RSV-29 clone 1.2 as

described previously (25) and was

digested

to

completion

with was e tonit

fragn

A

7-7E

2-6ki

PROS E

FIC nonpr RSV-virus, izatio Probe SR-R thesr are ir SR-R tovir Ba, I

Hincl exten

genor

bottoi

ol

> 1

A u, a: B

5-0kb- -

-_.kb-._-_...

3 2

kb-FIG. 2. (A) Southern

hybridization

oftotal cellular DNA from SR-RSV- and RSV-29-infected CEF. The

probe

used is shownin

panelB. (B)Size of

src-containing

EcoRI

fragment expected

froma

nondefective RSV

(SR-RSV

or

PR-RSV)

andfromadefective RSV withadeletion ofenv(BH-RSVorRSV-29).Thesizes of theEcoRl

fragmentsareindicatedinkb belowthe

figures. Enzymes:

R, EcoRI;

H,

HindlIl;

K, KpnI.

an excess ofEcoRI. A fraction ofthe

digested

DNA the src gene of SR-RSV-A

(NY)

and

hybridized

with a

.lectrophoresed througha0.8%agarose

gel,

transferred

5.0-kbp

EcoRI

fragment

of RSV-29

proviral

DNA

(Fig.

2).

trocellulose, and

probed

with the0.8-kilobase

(kb)

PvuII The rest of the

digested

DNAwasfractionated under iden-nentofpTT107. This

fragment

containedthe 3' third of tical conditionswith

appropriate

molecular

weight markers,

and the

5.0-kbp

DNA

fragment

wasrecovered

by

electroelu-tion. This DNA was cloned into lambdagt

WES,

and the

recombinant

plaques

were screened

by

hybridization

with

thesame0.8-kbPvuII

fragment

of

pTT107

describedabove. p 'Ppp n pn p n P p n p p n P One of the positive recombinants was purified by three rounds of plaque purification, and the 5-kbp src-containing EcoRI

fragment

was subcloned into the EcoRI site of

lb-e

3#

3 3| ' -

'w

pBR322

to construct

plasmid pR29.

All

procedures

used I

Ww were essentially as described previously (14).

:b- * j " _ - Restriction

mapping.

pR29

was

digested

with

single

and

multiple

restriction enzymes, and the DNA

fragments

were fractionated by electrophoresis through agarose or

acryla-E 2 3 4 5 b. 7 B ,ol midegels and stained with ethidium bromide. Themap was

confirmed

by partial digestion

of32P-end-labeled DNA with

appropriate

restriction enzymes.

LTR g° Fe e^: ,~ re ~2 DNA sequencing. The KpnI-to-NcoI fragment measuring 300 base

pairs (bp)

onthe 5'sideofsrcwassubcloned from

R 53 8 pR i -A 83 pR29 into replicative forms of

M13mpl8

and

M13mpl9

+

^____

j

4 w.

between the

KpnI

and

HinclI

sites. The

450-bp

PstI-to-EcoRI

fragment

onthe3' side ofsrc was subclonedbetween the PstI and EcoRI sites of the

M13mp8

and

M13mp9

Pj;SAT!VE

P.JA

_GENDM

RSV-29 RNA4

S _NA,

_IC

IN NP

_

CLIRN

ONS

E _ F .O

replicative

_forms.

_The

_recombinant

plasmids

_were

_used

_to

SUB

SGNB-EiM.,-" transform Escherichia

coli

JM101,

whichwas

plated

on YT

26Kb-s->j

<-~--

--$~XE- - - --

-agar

(16)

containing

isopropylthiogalactoside

and

dibromo-i.

1

dichloro-indolylgalactoside.

White

plaques

were screened oducing

(n)

and producing

(p)

clones of CEF

infected

with for the presence of insertsby gelelectrophoresis analysisof

*29 alone and a mixture of RSV-29 and RSV-29-associated the size of phage DNA from culture supernatants (16). respectively,wereanalyzed byNorthernblottingandhybrid- One recombinant virus of each type was selected and on with nick-translated probes indicated below the lanes. (B)

sequenced by

the

dideoxy

method

(20)

with both

[32P]dATP

-s used for theNorthernanalysis. SR-A, Representationof the and

[35S]dATP (New England

Nuclear

Corp., Boston,

.SV-A genome.The arrow indicates thesplice acceptorsite for Mass.). The products of the sequencing reactions were

,cmessage. IC, Intercistronic regionof RSV. Theprobesused fractionated on 8 and 5% polyacrylamide sequencing gels in ndicated by lines below the corresponding segments of the buffer consisting of 50 mM Tris base, 50 mM boric acid, and

aSV-A

genome.A+ belowaprobe indicatesthat it

hybridizes

2 mM disodium EDTA. Each fragment was sequenced at -alRNA;a- indicatesnohybridization. Enzymes: R,EcoRI, leasttwice on either strand.

5amHI;

K,

KpnI;

X,

XhoI;

S, Sall; N, NcoI; A, AccI; H

the

Dn

enes

werand.

[I;Bg,BglI;P,PvuII. The hatched box indicatestheminimum The DNA sequences were analyzed with the computer

Itof thedeletion thatwouldexplain the results. Theputative programsin theSequence

Analysis Package designed

bythe mic and subgenomic RNAs from RSV-29 are shown at the Biomathematics

Computation Laboratory

of the

University

m. of

California,

SanFrancisco.

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RESULTS

Defectiveness of RSV-29. Intheearly 1960s, Simons found thatRSV-29isreplication defective (P. J. Simons, personal communication). Toconfirm the defectiveness of thevirus, CEFinfected withhighdilutions(multiplicityof infection of 10-5)of the initial RSV-29 stock wereplatedin softagarfor the isolation ofcolonies. About 70% of the isolatedcolonies, which originally did not produce transforming virus, re-leasedhigh titers of transforming virus upon superinfection

withhelper viruses (RAV-1 orRAV-2). By the interference assaywith avianleukosis virusof knownsubgroups (6, 34),

we found that the original RSV-29 stock is of subgroup A. Since the virus appears tobedefective intheenvgene(see

below),this subgroup specificity representsthatofahelper

virusassociated with theoriginal stock of RSV-29.

Analysis of RSV-29 RNAs. The total poly(A)-containing RNAs obtained from RSV-29-infected NP clones and the clone doubly infected with both RSV-29 and RSV-29-associated virus were analyzed. Representative results of oneNPclone andoneproducercloneareshown inFig. 1A. The NP clones contained two species of RSV-29-specific RNAs, measuring 7.7 and 2.6 kb, detectable by several probes. The doubly infected producer clones contained an additional 2.8-kb species ofRNA. The 7.7-kb RNA ismost likely thegenomic RNA, and the2.6-kb RNA is thespliced src mRNA ofRSV-29. The producer clone also contained the helper viral genomic RNA that could not be separated from the 7.7-kbRSV-29genomicRNA but thatwas revealed by hybridization with probes derived from env and the intercistronicregionbetween envand srcof the SR-RSV-A

genome. The 2.8-kb RNA appeared to be the RSV-29-associated virus env mRNA. Neither the 7.7-kb nor the

2.6-kb RNA ofRSV-29hybridizedwithprobesderivedfrom env and intercistronic regions. The result of the hybridiza-tion ofRSV-29 RNAs withvarious SR-RSV-A-derived DNA probes is summarized in Fig. 1B. Sinceprobes 5, 6, and 7 (Fig. 1B)failedtohybridizetoRSV-29genomicRNA while allotherprobes did,it is concluded that theRSV-29genome

containsadeletion in theenvregion. The 5'boundaryof the deletion islocated between the BamHI and XhoIsites,since thegenomicRNAhybridizedtoprobe4.The3'boundaryis locatedbetween the AccI site and thespliceacceptorsite for src mRNA(position -76 from the initiation codon ofsrc), because probe 8 hybridized with the 7.7-kb genomic RNA and with the 2.6-kb mRNA. There was no evidence of deletions in theotherreplicative genesorinthesrcgene.

The 32P-labeled RNAs from cells infected with RSV-29 and its helper virus were also analyzed by RNase

T1-resistantoligonucleotide fingerprinting. Thepatternsof both viralRNAs, except for thesrc-and env-specific oligonucle-otides, superimposed (data not shown). The pattern ofthe non-src oligonucleotides is different from those of several

strains of helper viruses originally isolated from BH-RSV (RAV-1, RAV-2, RAV-3, andRAV-7).

Molecular cloning ofa 5-kbp pol-src fragmentof RSV-29.

Whendigestedwith EcoRI,the RSV-29proviral DNAgave a 5-kbp fragmentthat hybridizedwith aprobefromthesrc gene(Fig. 2). These datasuggestthat thegenomicstructure ofRSV-29 is similarto that ofBH-RSV (13) inwhich the

EcoRIsites ingag and U3arepreservedbut the site in env is lost. This 5-kbp fragmentwaspreparedfrom CEF trans-formed clonally with RSV-29, molecularly cloned into

lambdaphagevector, and then subcloned intopBR322. Restriction site mapping ofpR29. The restriction map of

the EcoRI fragment of RSV-29 is presented and compared

with those of PR-RSV-C and BH-RSV (Fig. 3). In thepol

gene, the map of RSV-29 is the same as that of PR-RSV with twoexceptions: (i) an extra PvuII site is present near the 3' end of pol which is absent in PR-RSV, SR-RSV, and BH-RSV, and (ii) a BamHI site at the 3' end ofpolthatis present in PR-RSV but is absent in RSV-29. BH-RSV also lacks thisBamHI site. In the src gene, the restriction maps of PR-RSV andRSV-29 are similarexcept for the absence of one BglII and two BalI sites in RSV-29. None of the restriction siteslocatedinthe env geneofPR-RSV (23) were found in RSV-29. Furthermore, the restriction fragments

bridgingacrosstheputativedeletion in pR29, e.g., the NcoI

1.2-kbp fragmentand theBalI1.3-kbp fragment, comigrated on electrophoresis with the corresponding fragments from

pBH-beta (13), suggestingthat the sizes of the deletions in the viruses RSV-29and BH-RSV are very similar.

pol-src junctionin RSV-29. Tostudy the deletion in detail, the300-bp KpnI-NcoI fragment of pR29 was cloned into M13 phage vectors in both orientations and sequenced. The sequence from the KpnI site inpolto the Ncol site at the

initiation codonof src ispresentedinFig. 4A.

The sequence across the deletion of env is exactly the same as in BH-RSV (Fig. 4B). Following the termination codon ofpol (asdefined inPR-RSV-C) are 6nucleotidesthat arealsopresent inPR-RSV-C,andimmediately3' tothis are the 91 nucleotides preceding the initiation codon of src in

BH,PR-C,andSR-AstrainsofRSV,aswell asin recovered avian sarcoma virus 1441and c-src(13, 23, 28, 29).

As shown inFig.4A anddiscussed below(seeFig. 7), the RSV-29 clone we studied has amutation in the 3' endofpol.

It has 13 basesreplacing2basesatthe samepositionin the sequenceof PR-RSV(23)whichwouldresultinaframeshift in the

carboxy-terminal

partof the polprotein, replacingthe last 28 amino acids of the PR-RSV pol protein with 43 different amino acids. Thenewreading frame is terminated

by

anopal termination codonat-61relativetotheinitiation

codon ofsrc.

3'end ofRSV-29.The

450-bp fragment

fromthe PstI site

RpoI PR-C I.rI Ps HH

Hp R BH-RSV

I-4.A

A K gp85

I

Xb Bo tBg Ba X B

BlN P

N

+4+ + +

+ (s +

II IIIII,,,I, ,I ++4+ + +4* 4. +

+ 4 +

gp37 src p R

R|P |N

Ps,).sl

l

t

B i B H Ps BINBI BgBp Ps

s

R

4.ll 11 ---- IIII.IA---4.-4r--.

_ + _ _ _- __6 A++& __ v +.+ _ +

+^16

R

,, ,, oil II I I

__ -_

+ -+ __-++ + _+

[image:3.612.312.562.468.603.2]

0.2Kb inRSV-29

ProvirolDNA

FIG. 3. Restrictionmapof RSV-29proviralDNAcomparedwith PR-RSV-C(PR-C) (23)andBH-RSV(13) betweentheEcoRI site in

gag and theEcoRI site inU3. A +or- signindicates whetherthe site waspresentorabsentin BH-RSV and RSV-29. Adotted line marking an enzyme site indicates a site present in BH-RSV or

RSV-29 which is absentinPR-RSV. Wheretworestrictionenzyme

sites cannot be resolved in the scale of this diagram, they are

indicated inaverticalarrayatthesamesiteonthe PR-RSV-Cmap;

thisarrayis thesamefor the BH-RSV andRSV-29maps.Enzymes: R,EcoRI;Ps,PstI;H,HindIII; Hp,HpaI;Xb, XbaI; Ba, BamHI; Bl, BalI; N, Ncol; A, AccI; Bg,Bglll;X,XhoI;P, PvuII;B,BgI; K, KpnI. A indicates that PstI sites were not determined for BH-RSV(13).

RSV-29

I

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[A)

KpnI

KpI- 10 20 30

GGTACCCTCT CGAAAAGTrA AACCCACAT

70 80 90

TGAGCCGAG CCTClTG CAGCATITC

I

env splice acc

40 50 60

CACCCAAAAG GATCACGTGA CTAAGAAAGA

100 110 120

TGATTGGATA CCCAGXGAGA AACCCCCAGC pol mutation

130 140 150 160 170 180

AACAAGCAAG AAOGACTCCA GCGAGAAACC GCCAGCAACA ACCAAGAAAG ACCCGGAGAA -91

src

190 200 210 220

GACACCCITT CTCCCAACGA GACTTAATrA TAlTGTCTGT

iT---r---pol Aenv stop in RSV-29 PR-C

230 240

CTCC1~CG AGCIAGCA

src pol

splice acc stop RSV-29

250 260 270 280 290 300

ACTCTCCIGG TGGCCTCGCG TACCACI=i GCCAGGCGGT AGCTGGACA GTGCAGCCCGA start

src

1309 CCACCATGG

NcoI

[B]

RSV- 29 BH-RSV PR(pol) PR(src)

PR-C src R29

pol src spl pol

stop -91 acc stop

__ I_ _

GAAGACACCCI CCA T[

******************** ******C**AT*A*IGtIC

*TAG*CTA**************************A*********

10/15

src start RSV-29 'IXnXX TCCTCC ACI;4CCCCACGCCGTASCTOCCACACTGCACCCGACCACCATCG BH-RSV *****A***************************************-******************* PR(src) *********************************************-*******************

[C]

PR-C src

pol v-src splice

stop -91 acc

RSV-29 CGAGATTAAITATATI77CTGTGT GCMCAGGAGCGAGCTIGAC

PR(pol) *C**AT*A*7UG7=***G *7CAGGA

c-src TCTGACACC C*GTC***CTCG*** **************** 12/19

FIG. 4. (A)DNAsequenceof309-bp KpnI-NcoIfragment of RSV-29.Thepolmutationrefersto 13 basesinthepol geneofRSV-29 which replaced2bases in thesameposition in PR-RSV-Candisdiscussed in detail in the legendtoFig.7.(B)RSV-29sequencefromtheend of poltotheNcoIsitecompared withthatof BH-RSV andwiththe sequence of PR-RSV pol[PR(pol)] and thatof PR-RSV from 104bases

upstreamfromthe initiation codon ofsrc[PR(src)].The box shows aputative recombination point inanondefective RSVthatcouldgenerate

the deletion inRSV-29orBH-RSV. (C) Sequence of RSV-29acrossthedeletion ofenvcomparedwith that ofPR(pol) (seeabove) and the regionupstreamofc-src(c-src).The box shows anotherrecombination pointbetween the 3' endof poland the region upstreamofc-src that

would produceadefective virus like RSV-29.Inpanels B andC,a*indicatesthe same nucleotide as RSV-29, and a-indicates a nucleotide thatispresentinRSV-29 butabsentin the othersequences.

atthe 3' endofsrcto the EcoRIsite in U3 wascloned into

M13mp8andmp9vectorsandsequenced(Fig. 5A). There is

extensivehomologybetweenPR-RSV-C (23) and RSV-29 in theregions designatedasF3(30),FlA,

FiB,

FlD(13),PPT,

and U3 (Fig. 5B). FlC is interrupted in PR-RSV-C by F2 sequences. In contrast, the

FiC

of RSV-29 is uninterrupted andhomologoustothatof SR-RSV-A (13, 28).

The 3' end of the RSV-29 src gene also contains the

38-nucleotide sequence that is presumably derived from the chickenc-srclocus 1 kb downstream from the c-src

termi-nationcodon, called c-src' (30). It also contains box P(Fig. SA), which has been hypothesized to be involved in the recombination betweenc-srcand c-src' (Fig. 6), and boxQ,

which may have been involved in the recombination be-tweenhelpervirus(RAV-2)andc-src' sequencestogenerate the 3' endofBH-RSV (13)(Fig. 6).

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(A) SRC-U3 JUNCTION OF RSV-29 PROVIRAL DNA src

PstI stop

10 20 30 40 ---50[F3 60

CTOCAOCCCACCTOCTCCC TOCTI'TCT TVCGTC CTOACTAAGT ACAOOCGC

Box P BoxQ

70 80 90 100 110 J(FlA 120

ACCTACAATT CCTCAAATAA ¶IXCTTC'LA GATI:lT ACCATrAOGCCtIV

130 140 150][FlB 160 170 180

CTXXCATG TACCACGTC'ItTrA T AACTCA ACTATrTATGTATAOXA

drl--->

190 200 210 220 ][FlC 230 ][FlD AAOOCOtC TrCACO-OCATAIAGTCCCCTCAC GATATACTAC TITTCCTTTT

dr--->1

](PPT ][U3 270 280 290 300

GCATACAGOCAAATMTA CTCTATICA ATACTCTI1TACC CATOCTATC

310 320 330 340 350 360

TAACGATGIA TTACCAATAT CCTC A AAAA ACCACCG4

ATCCU&TTC

370 380 390 400 410 420

rWAACTAAOC:ACGAT =CCTTATTAAA0T ATCAGACOC TCTAACATOG

430 440

ATTGGACGAA CCACTCAATT C EcoRI

[B] COMARISON OF 3' ENDS OF RSV-29 ANDPR-RSV-C src

PstI stop

__ _ _ _ _ - --tF3

RSV29 CTGCA00CCC AOCTOCTCCC TGCT1-TUG TlAi CTGACTAACT ACGAOOCGTC

PR-C ********** ********** ********** **

F3]JFlA RSV29 ACCTACAATT OCTCAAATAAl rA CAATICiTO AOCATTrAGC CTCTiCCGr-G

PR-C ********** ********** ********** ********** **********

FlA] (FlB

RSV29 CTCCCCCATC TACXXXCCAG

GTGT-ATC--C-GT-_-AACTlAG=O

ACTAT00TATGTATACCCGA PR-C ********** ******T*** **A*A***TC*A**TTG********* **C***A*G* *********T

FlB ][F1C ][FlD

RSV29 -AAM0c0C TTOCT-GTAC

GcXXATAOCA

GTCCCCTCACGATATAGTAG TCTlr PR-C C**-******* ******T**** ********** **C- -

-F2 in PR-C

]tPPT ][U3

RSV29

GCATtkXMAG-CCGAAATCTA

CTCTIATCCA

ATACTCTTGT

ACTCTI'CAA

CATCTTATC

PR-C **********C*** ********** **********

RSV29 TACGATCGA TIACCAATATGCCTIAOCAA

OGAGACAAAA

AGCACcCTOC

AT1CTTG

PR-C G****** **********

RSV29 CXAAAAAOCG=IACGATC A TTACAAT

ATCAGACOOC

TCTAACATOC

PR-C ********** ********** ********** ******

RSV29 ATITGACGAA CCACTGAATT C

PR-C ********** **********

EcoRI

FIG. 5. (A)Sequence ofthePstI-EcoRI fragment of RSV-29. F3 (30) and F1A-D(13) arepreviously defined segmentsofthe sequences

flanking src in RSV. dr refersto the limits of the direct repeatflanking srcinSR-RSV and PR-RSVas originallydefined bySchwartzetal. (23). BoxesPandQarehypotheticalpoints of recombination required to generate the 3' endof v-src (13;legendtoFig. 6). (B)Comparison of3' ends ofRSV-29 andPR-RSV-C (PR-C). A* indicates anucleotide that is present inRSV-29, anda - inagivensequenceindicatesa

base that is absent in that sequence but present in the other sequence. A seriesof -indicates where PR-RSV-C hasacquired anF2(13) sequence.

DISCUSSION andHanafusa (13), thispol-src

junction

could beformed as

aresultofrecombination betweenthehelpervirusandc-src

Deletion of env. DNA sequence data confirmed the con- usinghomologous sequences existingdownstreamfrom

pol

clusionfrom RNA analysisthat RSV-29 containsadeletion in PR-RSV-C(sequencesofpoland its

neighboring

regions of the entire env gene. The deletion involves exactly the of helper viruses have not been determined, but they are samesequences as inBH-RSV (13).Asdiscussedby Lerner probably similar to those ofPR-RSV-C) and upstream of

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Termination codonfor

c-src C-SRC'

54 1 14 17 1

11 5

V-SRC 47

14 17 115

Termination

codon for v-src

3' END OFHELPER VIRUS

3' END OF RSV

FIG. 6. Model for the generation ofthe 3' end of v-src (12).

Boxes P and Q are putative recombination points between c-src,

c-src' (30) and 3' end of helpervirus. The lengths of the relevant

regions in number of nucleotides are indicated. Recombination 1

probably took place duringreversetranscription. Recombination 2

could have happened by a rearrangement at the c-src locus, by

aberrant processing ofahelper virus-c-src hybrid RNA, orby an errorinreversetranscription.

c-srcinthegenome ofchickens (Fig. 4C). Such a recombi-nation wouldcause adeletion of the entireenvsequenceand produceareplication-defective virus. Alternatively, similar homologous sequences existing downstream from pol and upstream from v-src within the PR-RSV-C genome could also be utilizedtocausetheenvdeletionin thenondefective virus (Fig. 4B). Thus, as with BH-RSV, analysis of the sequence across the deletion of env fails to distinguish whether the deletion occurred during the generation of

sarcoma virus inchicken tumor no. 1 or occurred second-arily inanoriginally nondefective PR-RSV-C-like virus.

The exact homologies in the deletion in BH-RSV and RSV-29 suggest that thesetwoviruseshavedescended from

a commonprogenitorvirusand that the differences in their3' sequences might have arisen in BH-RSV by recombination withhelper viruses during subsequentpassages(seebelow). The fact that a strain of RSV with the least number of

passages beyond chicken tumor no. 1 has deleted its env

A RSV-29 #1. 2 PR-RSV-C

lBlr/2][ Xr H A2

R

gene and istherefore replication defective suggests that the

original isolate of RSVwasalso replication defective. This viewis supported by the observations of Hagino-Yamagishi et al. (5), whofound that two independently isolated avian sarcomaviruses(Si and S2), obtained by inoculating chick-ens with avian lymphoid leukosis virus, contained src and were replication defective. This implies that src, like other oncogenes, canbe transduced into avian lymphoidleukosis

virus toform a replication-defective transforming retrovirus. Furthermore, while replication-defective mutants generated

from nondefective virus have been selected by isolating

single colonies (10, 15), it is difficultto explain how a pure stockofadefective virus (e.g., RSV-29 and BH-RSV) could be established bypassageof nondefective virus inchickens

in which defective viruses do not appear to have any selective advantage over their nondefective counterparts.

Taking all these arguments into consideration, we propose that, like all other transforming retroviruses, RSV was

originally isolated as a replication-defective virus, and that the commonly studied replication-competent strains have been generated during subsequent recombinations with helper virus.

Mutation in pol. The discovery ofthe 13 nucleotides in RSV-29 substituting for 2 nucleotides in pol (as compared

with PR-RSV-C [23] and BH-RSV [M. Sudol and H.

Hanafusa, unpublisheddata]) is interesting. We donotknow whether the mutation and theresultingmissensetranslation

arepeculiartotheparticularNPclonethatwassequencedor arepresentinallclonesof RSV-29. It would be interestingto study thefunctionalandstructuralproperties ofthepol gene product in this clone. On the basis of the findings of Hippenmeyer and Grandgenett (9), the mutation in this

domain is expected to leave the DNA polymerase and

RNase-H functionsofthe enzyme intact.

Apossible mechanism forthegenerationof this mutation ispresentedinFig.7, basedsolelyonsequencecomparisons

andidentificationof direct repeats. Severalprevious

hypoth-esespostulated involvement of direct repeatsin the genera-tionoftransformation-defective deletion mutantsfrom RSV

(2, 18, 19). Since PR-RSV and BH-RSV (M. Sudoland H.

Hanafusa, unpublished data)arehomologousin sequence in thepol gene, it is reasonableto assumethat the

PR-RSV-C-like structure,containingthedirectrepeatsAl and A2,isthe

parental virus. After having reverse transcribed

A1-X-B1-I A1-X-B1-I B1I I X II Al

== =_=

CCCAGGGAGAAACCGCCAGCAACAAGCAAGAAGGACTCCAGGGAGAMCCGCCAGCAACAAGCAAGAAAGACCCGGAGAA

.*T**AG_*--T----AG

_---A---

_--T---

---I B2] I A2 I[ B1 ] X ] Al I

DD_r p;

IB21 A2 B181I X Al 1fKL lKform

B21 A2 1131

I

X Al

4U-

Reverse transcription error

B21

A2r

lBir

I

Xr

I

A2

|B11

X

I

Al I

Intermediate

form

~Deletion

renaui

p

~2 Xr I A2 |18| X Al I RSV-29#1.2mutont

FIG. 7. MutationinpolinRSV-29no.1.2.(A)Comparison of the 3' ends ofpol in RSV-29no.1.2and inPR-RSV-C(22). A*indicates thesamebaseas RSV-29,andablankspaceindicates the absence of that base in PR-RSV-C.Al,A2 andBi, B2arepairs of directrepeats presentinthePR-RSV-Csequence.(B)Diagram oftheerrorinreversetranscription and the deletion usingthesedirectrepeatsthat would generatethemutationsequencedin RSV-29no. 1.2.

C-SRC

14

zI,I

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RAV-O

KI_-SR-RSV

DJ-E

BH-RSV

D_-E

GAG

POL

liz'

IZZHID

ENV

GAG

POL

ENV

SRC

4

I

MH2

GAG POL

SRC1

I

RAV-2

GAG

POL

SRC

RSV-29

m

I

PR-C Minus F-2

FIG. 8. Comparison of the 3' ends of several avian retroviruses to show the regions across which certain pairs of viruses are related. The

arrowed box ineachviruscorrespondsto box Q of Fig. 5 and 6.

A2, the enzyme could have fallen off the template and then reattached, possibly to a second template, at

Al

because of thehomology betweenAl and A2. This would generate the

intermediate form shown in the figure, with repeats of

X-Bl-A2 (Xr-Blr-A2r). A subsequent deletion, using the

direct repeats Blr and B2, could generate the variant we have identified.

3' endof RSV-29. Comparison of the sequence atthe 3'

end of RSV-29 reveals analmost exact homology between RSV-29 and PR-RSV-C in this region, suggesting that the two viruses are closely related (Fig. 5B). In line with the

discussion above concerning the deletion in env, this might mean that PR-RSV-C is derived from RSV-29and that the

insertion of F2 in theFlCsequenceof PR-RSV-C occurred at a later date. However, an alternative possibility is sug-gestedby thereportedextensive homology of3' sequences in

SR-RSV-A

and

MH2

leukemia virus (27). The fact that

pairs of viruses like MH2 and SR-RSV-A, which certainly have had

different

origins and different passage histories,

share common 3' sequences, suggests that in the course of

passage in

chickens,

theseviruseshavebeen associatedwith a common pool of helper viruses with which they often exchange their3' sequences inblock. Superimposed on this

v-src

PR-RSV-C

RSV-29 SR-RSV-A BH-RSV RAV-2 Y73 MH2 UR2

c-src'

G¶EGIGGAGGTCGCTGAG TAAGTACGAGGC

__________________

-G-TGCCGCGA---A---A --GTA-ACTT-T

A-A-A---A A-A-A---A--- A--- -A-A---GA-CG

IOB

--GTA-ACTT-T --GTA-GCTT---ACA -GGTA-GCTT-- ATCTGCAC-TC-FIG. 9. Ahighly homologous sequencepresentatthe 3' ends of several avianretroviruses andinc-src'.The box labeledv-srcrefers tothe 3' endofthesrcgenein the RSVs. A-denotes thesamebase

atthatpositionasin PR-RSV-C.BoxQis thesame asinFig.5 and 6.

arepoint mutations, deletions, andinsertions which do not

obscuretherelatedness of the 3' sequences. Thehomology

between RSV-29 and PR-RSV-Cfoundinthisstudy couldbe

analogousandmight merelyreflect the fact that these viruses haveacquiredtheir 3' sequencesby similar exchangeswith a commonhelper virus. Thishypothesiswould also propose that F2 wasinsertedinto PR-RSV-C at alater date.

The relationships discussed aboveand that between

BH-RSV andRAV-2(1, 13),areshowninFig. 8. Acomparison of these 3' sequences revealed a highly homologous short

stretch in all these viruses(Fig.9). Thishomologousregion

is present at the 3' end of the src sequence in theRSVs, from

box Q to the termination codon (Fig. 9). The homology betweenhelpervirus sequence and c-src' inthisregionwas

postulatedtobe responsible for the recombinationatthe 3'

endrequiredtogenerate RSV inchickentumor no. 1(1, 13) (Fig.6and9). Itisinterestingtoobserve that this sequence

isrelativelywellpreservednotonlyintheRSVs, but alsoin RAV-2 (1), MH2 (27), Y73 (11), and UR2 (17) in their 3'

noncodingregions, suggesting thatthis sequenceispresent in severalhelperviruses that have notbeenassociated with

anyofthe RSVs and so its presencecouldhot beanartifact

created by exchange of3' sequences between helpervirus andsrc-containingRSV(Fig. 9).This is inconcordance with our hypothesis, because it affirms that box Q could have been present in the original helper virus that gave rise to RSV inchickentumor no. 1.

ACKNOWLEDGMENTS

Wethank H.Iba forhelpful discussions,andS.Sugano,R.Jove,

and F. Crossforreadingthe paper. Wearealsograteful toR. M.

Doughertyforprovidingastock of RSV-29and to P. J. Simon for communicating earlierresults.

This work was supported by Public Health Service grants

CA14935andCA18213toH.H. and CA29339toL.-H.W.fromthe National CancerInstitute. L.-H.W. isarecipientof PublicHealth

Service Research Career Development Award CA00574from the

National CancerInstitute. A.D. was supportedin partbya grant from R. J.Reynolds Industries,Inc.

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