Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation.

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Vol. 44, NO.2 JOURNALOFVIROLOGY, Nov. 1982, P. 683-691

0022-538X/82/110683-09$02.00/0

Site-Directed

Mutagenesis

of the src Gene of Rous Sarcoma

Virus:

Construction and Characterization

of a Deletion Mutant

Temperature

Sensitive for Transformation

DEBRA BRYANT AND J. THOMAS PARSONS*

Departmentof Microbiology, Universityof Virginia Medical School, Charlottesville, Virginia 22908

Received 10 May1982/Accepted7July 1982

Transformationof cells by Rous sarcomavirus results from the expression of

the viral src gene product, pp6Osrc. Site-directed mutagenesis techniques have

been usedtoconstructdefined deletion mutations within thesrcgene of Prague A

strain of Rous sarcoma virus. The deletion of DNA sequences at the BglII

restriction site in the src gene yielded both transformation-defective mutants

(tdCH4, 64, and 146) and a mutant temperature sensitive for morphological

transformation (tsCH119). The genomeof tsCH119 containsanin-phasedeletion

ofapproximately 160base pairs, which mapped to theimmediate 3' side of the

BglII restriction site. Upon infection of chicken cells, tsCH119 encoded a

structurally altered src protein, pp53src, containing a deletion of amino acid

residues202 to 255. Immunecomplexes containingpp53srcisolatedfrom

tsCH119-infected cells grown at 41°C exhibited only 50% less tyrosine-specific kinase

activity than immune complexes isolated from cells grown at 35°C. pp53src

immunoprecipitated from tsCH119-infected cells grown at either 35 or 41°C

containedphosphoserine and phosphotyrosine. We suggestthat tsCH119

repre-sents a class of mutants containing mutations mapping within a functionally

important domain ofthe src protein, distinct from the domain specifying the

protein kinase activity.

Transformation of cells by Rous sarcoma

vi-rus(RSV) results from the expression ofasingle

viral gene, the src gene (14). Genetic and

bio-chemical experiments have shown that the src

gene encodes a 60,000-dalton phosphoprotein,

pp60 src, whose primary site of localization

ap-pears tobethecytoplasmicmembrane(2,18-20,

22, 23, 29). In vitro, pp6src exhibits a unique

phosphotransferase activity, catalyzing the

tyro-sine-specific phosphorylation

of either

immuno-globulin heavy chain, contained in immune

com-plexes, or avariety of other substrates(4,5, 16,

23, 31). In vivo, the expression of pp6Osrc

initi-ates acascade ofeventsleading toalteration of

cellularmorphology, changes in growth

proper-ties of the cells, and modulation of cellular

metabolism (14). Among the earliest events

fol-lowingpp6Osrc expressionis thephosphorylation

oftyrosine in several specific cellular proteins

(1, 6, 10, 30, 33, 35). These early

phosphoryla-tion events andthe unusual phosphotransferase

activity associated with pp60src have focused

attentionon phosphorylation of defined cellular

targetproteinsas acritical step incellular

trans-formation.

The src protein,

pp60src,

contains two major

sitesofphosphorylation, aserine residue

locat-ed in theamino-terminalportion of the molecule

andatyrosine residueataminoacid

position

416

(3, 5, 16, 36). (The numbering of both the src

nucleotide sequenceand pp6osrc amino acid

se-quence is based on the DNA sequence of

Schwartzetal.[D. Schwartz, R.

Tizard,

andW.

Gilbert, inJ. Tooze, ed.,RNA Tumor Viruses:

MolecularBiologyofTumorViruses, 2nded.,in

press]. Tyr416 is the same as Tyr419 referredto

by Smart et al. [36].) The available evidence

suggests thatintransformedcells,

phosphoryla-tion of the serine(s) residue of

pp6Osrc

is carried

outby a

cyclic

AMP-dependent kinase system, whereas phosphorylation oftyrosineappears to

be cyclic AMPindependent (11, 23).

The useof RSVmutants(both conditional and

nonconditional) has provided important

infor-mation indefining the roleof

pp6Osrc

in cellular

transformation(14). However, the lackof

infor-mation regarding the nature ofthe primary

nu-cleotide sequence changes insuch mutants has

made it difficult to deduce exactly how the

mutations have altered

pp6O`rc

structure and

modulated enzymatic function. The molecular

cloning oftheRSVgenome and its

characteriza-tionby restriction endonucleasemapping (9, 15,

17) and DNA sequencing (7; Schwartzetal., in

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press) have provided a new dimension to the

genetic analysis of RSV-mediated cellular

trans-formation. Here we report on the isolation of several defined deletion mutations within thesrc geneof the Prague A (PrA) strain of RSV. These deletion mutationswere constructed at theBglII recognition site (nucleotides 606 to 611) within

the cloned DNA genome of PrA RSV. Among

the mutants isolated was a viable deletion mu-tant (tsCH119) that induces

temperature-sensi-tive transformation of chicken embryo cells. Some of the biological and biochemical proper-tiesof this mutant are described.

MATERIALSANDMETHODS

Cells, viruses, and plasmids. Cultures of primary

chicken embryo cellswereprepared from gs-negative, chf-negative embryos (Spafas) and maintained in cul-ture asdescribed (26). ViralDNAusedfor mutagene-sis experiments was derivedfromamolecular clone of

PrARSV, originally cloned inalambdavector(15) and later subcloned in pBR322 at a Sall restriction site (pSL102). Transfection of chicken cells with cloned viral DNA was carried out by firstremoving the viral

DNAinsert from the pSL102plasmid and digesting it with SalI, followed by purification of the 9-kilobase pair (kbp) viral insert by agarosegel electrophoresis. Purified viral DNA was ligated withT4 DNA ligase (NewEngland BioLabs), and 50to100 ngofDNA was

applied to cultures of chicken cellsasdescribed

previ-ously (15).

Restriction enzyme digestionandagarose gel

electro-phoresis. Restriction enzymes EcoRI, HaeIII, Hinfl, SmaI, and PvuIIwere purchasedfrom Bethesda Re-search Labs. HincII, SalI, BglII, and PstIwere pur-chased from New England BioLabs. All restriction enzyme digestions were performedas recommended by the supplier. DNA restriction fragmentswere re-solved by agarose gel electrophoresis as described previously (15). Small DNAfragments wereresolved by electrophoresis through 5% polyacrylamide gels

(24).

Constructionof deletion mutants.pSL102DNAwas incubated withBgIIIrestriction enzyme under condi-tions for

partial cleavage

(10 mM

Tris-hydrochloride

[pH7.4]-60mMNaCl-4mMMgCI2containing10,g of DNA and 0.6 U ofBglII).After 30min,EDTAwas

addedto 15mM,and the DNA wasprecipitatedwith 2

volumes of ethanol. The 13.5-kbp DNA fragment resulting from cleavageatoneof thethreeBglII sites inpSL102wasresolvedby agarosegelelectrophoresis

andrecovered from thegel slice. ForBAL31

(Bethes-da Research Labs) exonuclease treatment, the DNA wassuspended in12 mMCaCl2-12mMMgCl2-0.6M NaCl-1 mM EDTA-20 mM Tris-hydrochloride (pH 8.1) at a final concentration of10 ,ug/ml and a final volumeof 10 ,ul. Two units of BAL31 was added, and thereactionwasincubatedat15°C for 20s.EDTA was

addedto afinal concentration of 50mM,and theDNA

was precipitated with ethanol. To repair uneven or

ragged ends generated bydigestion with BAL31, we

suspended theDNAin18,ul of 50mM Tris-hydrochlo-ride(pH7.8)-5mMMgCl2-10mM2-mercaptoethanol

containing 50 ,ug of bovine serumalbuminper mland

1.8 ,uM concentrations of each deoxynucleoside tri-phosphate. Nine units of DNA polymerase I (New England BioLabs) was added, and the reaction mix-ture wasincubatedat15°C for 60min. The DNAwas

precipitated withethanol, and residual triphosphates wereremoved bychromatographyon aSephadex G-100 column. Fractions containing DNA were pooled andprecipitated with ethanol. The DNA was ligated with T4 DNA ligase and used to transform Escherichia coli HB101 asdescribedpreviously (8). The resulting ampicillin-resistant colonies were screened for dele-tion mutadele-tions by isolatingDNAfrom1-ml cultures, digesting with BgllI, and analyzing the resultant DNA fragments by agarose gel electrophoresis.

Immunoprecipitationandpolyacrylamide gel analysis

of labeled cell proteins. Labeling of cells with

[3S]methionine was carried out as follows. Cells

grownin 100-mm culture dishes were washed twice with labelingmedium (Dulbecco modified Eagle medi-umwithoutmethionine) containing 1% calfserumand incubated for 30 min in thesamemedium. Cellswere

thenincubated for4h in freshlabeling medium

con-taining 300 ,uCi of [35S]methionine (Amersham Corp.) per ml. Labeling of cells with 32Pwas carriedoutby washing culturestwice withmedium 199 minus phos-phate (Flow Laboratories, Inc.), supplemented with 5% dialyzed calf serum, and incubating them in the same medium for 2 h. Labeling was performed by incubation of the cells for 4 h in the same medium containing 0.5 mCi of 32p, (New England Nuclear Corp.) per ml. Immunoprecipitation of src protein from labeled cell extracts was carried out as described previously (27) except with the following modifica-tions. Cells were washed twice with STE (0.15 M NaCi-50 mM Tris-hydrochloride [pH 7.2]-1 mM EDTA) and suspended in 10 mM Tris-hydrochloride (pH7.2)-0.1 M NaCl-1 mM EDTA-0.5% deoxycho-late-1% Nonidet P-40 (23). Lysates were clarified at 100,000 x g for 30 min and incubated with tumor-bearing rabbit (TBR) sera at 0°C for 30 to 60 min. Immune complexes were adsorbed to protein A-Se-pharose (Sigma Chemical Co.) by incubation at0°C for

60min, with mixing at 15-min intervals. The immune complexes were collected by centrifugation and washed twice in lysis buffer and once in 10 mM Tris-hydrochloride (pH7.2)-iMNaCl-0.1% Nonidet P-40. After afinal wash with the original lysis buffer, the immune complexes were suspended in sample buffer (0.25MTris-hydrochloride [pH6.81-12.5%glycerol-5

mM EDTA-5% sodium dodecyl sulfate), boiled, and subjected to electrophoresis on a 10.5% polyacryl-amide gel (21). Fixed and stained gels were equilibrat-ed in En3Hance (New England Nuclear Corp.) to provide fluorographic enhancement (for gels contain-ing35S-labeledproteins), dried, and exposed to X-ray film.

Measurement ofproteinkinaseactivity. Tomeasure pp60src_associatedprotein kinase activity, infected cell

extracts wereimmunoprecipitated with eitherTBRsera

or normal rabbit sera as described above. Immune

complexeswerecollected, washed in

phosphate-buff-eredsaline, suspended in 50 p.1 of kinase buffer (20mM

potassium phosphate [pH 7.2]-0.1 M NaCl-5 mM

MgCl2-1mMEDTA-1 mM2-mercaptoethanol) with1

p.Ci of [y-32P]ATP (3,000 Ci/mmol; New England NuclearCorp.),andincubatedat37°Cfor30min.The

reactionswerestopped by the addition of25p.lof2x

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MUTAGENESIS OF THE RSV src GENE 685

Partial Bgl II Digestion

Isolate 13.5 kb Linear

B B B B

I

Exonuclease Digest Ligate

clone in E. coli

FIG. 1. ConstructionofBglIImutationsin theRSV

genome. Plasmid DNA from the clone pSL102

(con-taining a complete copy of the RSV genome) was linearized by partial digestion with the restriction

enzymeBglII (B). The linear moleculeswereisolated by agarose gel electrophoresis and treated with the exonuclease BAL31 to generate terminal deletions.

Themutated DNAwasligatedwith T4 DNAligaseto

regeneratecircularmolecules withadeletion atoneof the BglII restriction sites (A). Mutated DNA was cloned in E. coliHB101. In the sequenceof PrRSV

(Schwartzetal., in press), BglII restriction sitesare

located 1,630 bp (ingagp27), 4,233 bp (inpot), and

7,733 bp (in src)from the 5' end of the viralgenome.

samplebuffer. Labeledsampleswere heatedat100°C

andsubjected toelectrophoresis on10.5%

polyacryl-amide gel,and thephosphorylatedproteinswere

visu-alized by autoradiography. Quantitation of

heavy-chainphosphorylationwascarriedoutby cuttingout

theheavy-chain bandandcounting the gelfragment. Phosphoamino acid analysis. A one-dimensional phosphoamino acid analysis of 32P-labeled proteins was carried out as described by Collett et al. (5). Phosphoamino acidswereresolvedbyelectrophoresis

inpyridineacetatebuffer(pH3.5)oncellulose-coated plates, and labeled amino acids were visualized by

autoradiography. The position of phosphoaminoacid

standardswasdetermined by ninhydrin staining. RESULTS

Theisolationand characterizationofmutants

containing defined sequence changes within the

RSVsrcgenewouldgreatlyfacilitatethe

analy-sis ofthe structure andfunctionof the RSVsrc

geneproduct, pp60sc. The BglII restriction

en-zyme recognition site was chosen as a site for

the construction of deletion mutants for two

principal reasons. First, there are only three

BglIIsites in the viral DNAgenome(15) andno

sites intheplasmid vector(Fig. 1). Second, the single BglII recognition site in the src gene

resides 607 base pairs (bp) from the amino-terminal methionine codon, thereby facilitating theconstruction ofmutations within the5'

one-halfofthe src gene. The scheme for the

con-structionofBglII mutantsisoutlinedin

Fig.

1.A

plasmid containing

a complete copyof the PrA

RSV genome

(cloned

attheSallsiteof

pBR322)

was

digested

with

BglIl

under conditions which

yielded partial digestion

products, and a

13.5-kbp

linear DNA

fragment, resulting

from a

sin-gle

BglII

cleavage,

was isolated

by

agarose

gel

electrophoresis.

After treatmentwith

exonucle-aseBAL31to removeterminalsequences, DNA

was

ligated

with T4 DNA ligase and used to

transform E. coli HB101. Individual

transform-ants were grown, and

plasmid

DNA was

pre-pared.

Mutant

plasmids containing

adeletion of a

BglII

site were identified

by digestion

of

indi-vidual

plasmid

DNAs with

BglII

andanalysis

by

agarose

gel

electrophoresis. Figure

2 shows the

pattern of

BglII

restriction fragments obtained

by

deletion ofa

BglII

restriction site within the

gag,

pol,

and srcgenes. Theunique restriction

patterngeneratedas aresult of the deletionofa

BglII

restrictionsite

(i.e.,

thefusionoftwo

BglII

restriction

fragments)

permittedtheready

iden-tification ofdeletion mutants. To date wehave

7.6 k b

-3.1 kb

-2.6 kb

-wt

gag pol

src

FIG. 2. Agarose gel electrophoresis of DNA from

mutants generated by deletion of a BglII site in pSL102. DNA from individual mutant clones was digested with BglII, and the DNA fragments were

resolved by electrophoresis through 0.85% agarose gels. Representative plasmid DNA containing dele-tionsat each of theBglII sites inpSL102are shown. wt, pSL102; gag, deletion at BglII sitein gag gene; pol, deletionatBglII site inpol gene; and src, deletion

atBglII site insrcgene. The size in kilobases(kb) of the respectiveBglII fragments obtained frompSL102

isshownattheleft. VOL.44,1982

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686 BRYANT AND PARSONS

TABLE 1. BglII deletion mutations in thesrcgeneof

PrARSV

Approximate Biological properties

Mutant size ofdele- Replica- Transforma- src tion(bp)a tionb tion' protein'

63 2,100 - - NDe

64 1,200 + -

-119 165 + + +

146 800 + -

-4 <100 + -

-aDetermined byrestrictionenzyme mapping.

bDetermined by resistanceof cultureto

superinfec-tion withPrARSV andpresenceofreverse

transcrip-taseactivity in culture medium(28).

cDeterminedbymorphologicalalteration.

dDetermined by immunoprecipitation of

[35S]me-thionine-labeled cell extracts(seetext). eND, Not determined.

Pr A

350/410

Normal

,

350/410

identified in excess of 130 BglII mutants, 33 of which contain mutations in the srcgene.

To determine the transforming potential of individual mutant genomes, we removed the viral DNA insert from the plasmid by digestion with SalI; the insert was ligated with T4 DNA ligase and used to transfect chicken embryo cells. The replication of the mutant virus ge-nomes was determined either by resistance of transfected cultures to superinfection by PrA RSV or by measurement of reversetranscriptase activity in the culture medium (Table 1). The mutantCH63 was replication defective. Restric-tion enzyme mapping of this mutaRestric-tion showed that the 2,100-bp deletion extended into the carboxy terminus of the env gene sequence. Mutants CH64, 146, and 4 did not produce foci of transformed cells but didreplicate, indicating

tsCH

1

19

350

~ '

s}~t . - .O

tsCH

119

410

_f P

FIG. 3. Scanning electron micrograph of uninfected chicken cells and cells infected with PrA RSV or

tsCH119 RSV. Chickenembryo cellswereinfected withPrARSVortsCH119andmaintainedat35°Cuntil the cultures werefully transformed. Cultures of uninfected cellsor PrA RSV- and tsCH119-infected cells were incubated at41°C for24 h.Cellsweresubjectedtocritical-point drying and examined inaJSM-35C scanning

electronmicroscopeat amagnificationofx1,100. Upperleft,PrARSV-infected cellsgrownateither 35or41°C;

lower left, uninfected chicken cellsgrownateither35 or41°C; upperright, cellsinfected withtsCH119and grownat35°C; lowerright,cellsinfectedwithtsCH119and grownat41°C.

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_160 base pairs

Hint Sma

I

Hae III Bgl 11 Hae III Hae III Hinf Hint

Xho *N*Nae P

Start src

I I I I III

,st Bgl 11 Pst Taq Taq Bgl

src gene

1578 base pairs

FIG. 4. Mapping of the deletion present in tsCH119 DNA. The partial restriction map of the src gene

sequenceofpSL102is indicated.The extent of the deletion intsCH119DNAwasdeterminedbyendlabelinga Hinfl fragment (top line) followed by digestion with HaeIII. The dots ( ...) represent the uncertainty in positioning the termini of thedeletedregion.

adeletion of only src-specific sequence.

Immu-noprecipitation with TBR seraofextracts from [35S]methionine-labeled chicken cells, infected with either CH64, 146, or 4, revealed no

src-specific proteins. We therefore conclude that these mutants contain deletions that result in expression of an aberrant src protein and are

therefore transformation defective (Table 1). Transfection of chicken cells with CH119 viral DNA (containing a160-bp deletion) resulted in

cellular transformation and the production of infectious virus (Table 1). The morphology of CH119-infected cellsgrownat35°Cwas

indistin-guishable from wild-type PrA RSV-infected cells. However, upon shift of CH119-infected cells to 41°C, the cell morphology altered,

re-sembling that of normal cells. This change in morphology is illustrated in the scanning

elec-tron micrographs of Fig. 3, which compare the

morphologies of CH119-infected cellsgrown at 35 and 41°C with those of uninfected cells and

PrA RSV-infected cells grown at either 35 or

410C.

The deletion within the srcgene of tsCH119 was mapped by first isolating the 3-kbp EcoRI

fragment containingpartofenvand all of thesrc sequence and digesting this subgenomic frag-ment witheither SmaI, HincII, PstI, PvuII, or

Hinfl. Fine-structure mapping was carried out

by further digestion ofapurified Hinfl fragment

spanning the BglII site (Fig. 4)..,An analysis of the resulting restriction fragments showed that

the 5' end of the deletion is locatedator nearthe

BglII site and extends about160bptothe3' side of theBgIIIsite(Fig. 4). The mutation therefore results in the deletion ofapproximately 53to54 aminoacids, 202to255 residues from theamino terminusof pp60src.

To examine the expression of the mutantsrc

gene, we infected chicken cells with either

tsCH119 or PrA RSV. The transformed cells

were grown at 35 or 41°C and labeled with

[35S]methionine. Labeled cellextractswere

pre-pared, immunoprecipitated with TBR antisera, and then analyzed by polyacrylamide gel elec-trophoresis. Cells infected with tsCH119 and

grownateither 35or41°C synthesizeda

53,000-daltonprotein thatwasimmunoprecipitated with

TBR antisera (Fig. 5, lane c). The amount of pp53src produced in tsCH119-infected cells

grownat35°C appeared similartotheamountof pp6jSrcpresentin PrA-infected cellsgrownat35

or 41°C (lane b). The apparent decrease in pp53src levels observed at 41°C represents the smaller number of cells presentinthe tsCH119 culturesgrownat41°C (duetothe lower satura-tion density of these cells) and doesnotappear

torepresentadecrease in therateofsynthesisor

increase in the rate of degradation of pp53src (unpublished data).

Since a number oftemperature-sensitive src

mutantsexhibitdecreased protein kinase activi-ty at the nonpermissive temperature (4, 31, 32, 34),wecompared theamountsof

pp53src-associ-PstII4 Eco RI

End

src ...vlrlr.F.Jw

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a

b

c a

350

41° 35° 41° 35° 41°

b

350 41 35 41

c

35 4 1

-IgG

H

-p60

*

W

[image:6.491.48.246.72.274.2]

-

p53

FIG. 5. Immunoprecipitation of src protein from uninfected cells and cells infected with PrA RSV or

tsCH119. Chicken cells transformedwith PrARSVor

tsCH119 and maintained at either 35 or 41°C were labeledwith[35S]methionine asdescribedin the text.

Cells were harvested and immunoprecipitated with TBR sera, and labeled proteins were analyzed by polyacrylamide gel electrophoresis.Molecularweights

of the src proteins were determined relative to the

positionof known molecularweightstandards(92,000, phosphorylase B; 66,000, bovine serum albumin; 45,000, ovalbumin; and31,000, carbonicanhydrase). Though labeled p60, PrA RSV pp6Osrc has anMr of approximately 59,000 in this system. a, Uninfected

chickencells; b,PrA RSV-infectedcells;c,

tsCH119-infected cells.

ated immunoglobulin G (IgG) kinase activity

present in tsCH119-infected cells grown at 35

and 41°C. Infected-cell lysates were incubated

with TBR antisera, and the immunecomplexes

were collected on Staphylococcus aureus

pro-teinA-Sepharoseand assayed for their abilityto

phosphorylate the heavy chain ofIgG. Figure6

shows thatimmune complexes from cellextracts

ofCH119-infected cellsgrownateither

permis-sive (35°C)ornonpermissive(410C)temperature

(Fig. 6, lane C) readily phosphorylated the

heavy chain of IgG. Quantitationof the amount

oflabel in theheavy-chain bandshowed that the

level of heavy-chain phosphorylation at 41°C

wasapproximately 50% ofthe value observedat

35°C (Table 2). Immune complexes from cell extracts of PrA RSV-infected cells grown at

either35or41°C contained similar levelsofIgG

kinaseactivity (Fig.6,laneB;Table 2). Immune

complexes fromtsCH119-infected cellsgrownat

35 and41°C also phosphorylated the exogenous

substrate casein (data not shown). Therefore, thedeletionpresentin tsCH119 doesnotappear

FIG. 6. Protein kinase activity of

pp60src

and

pp53src.

Chicken cells transformed with PrARSV or

tsCH119weregrownat35 or41°C. Cellswere harvest-ed,immunoprecipitated with TBR serum, and assayed for kinaseactivityasdescribed in thetext.a, Uninfect-ed cells; b, PrA RSV-infected cells; c, tsCH119-infected cells. ThepositionofIgGheavychain(IgGH), 55,000Mr, isindicated.

to substantially alter the proteinkinase activity

associated with pp53src.

Phosphorylation of pp6Osrc occurs on both

serine and tyrosine (3, 5, 16, 36). To determine whether the pattern of pp53src phosphorylation is altered as a result of growth at 41°C, cells infected with either PrA RSV or tsCH119were

grown at 35 and 41°C and labeled with

32p,.

Labeled cell extracts wereimmunoprecipitated

with TBR antisera and analyzed by polyacryl-amide gel electrophoresis. Figure 7 illustrates thatpp53srcwasreadilyphosphorylated at both 35 and41°C, and the ratio of labeled pp53src at 35 and 41°C appeared similar to that of pp60src at

the two temperatures. The extentof serine and tyrosine phosphorylation in pp53src was

deter-TABLE 2. Kinaseactivityin PrARSV and tsCH119-infected cells

Kinaseactivityb Ratio Cell

extract'a4O/5C

350C 41-C (4Cf5) Uninfected cells 2,300 6,200 2.7 tsCH119-infected cells 30,600 15,400 0.5 PrARSV-infected cells 26,100 26,800 1.03

a Cell extracts were prepared as described in the

text.

bKinaseactivityexpressedas countsper minute of

32pincorporated intoheavychain permilligramof cell protein addedtoimmuneprecipitation.

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a b c

350 41 350 410 35° 41°

_ _ ->Pp53

FIG. 7. Immunoprecipitationof32P-labeledpp6O0'

and pp53sr. Uninfected cells and cells transformed

withPrARSVortsCH119weregrownat35or41°C. Cellswereharvestedat4°C,immunoprecipitatedwith TBRsera,andanalyzed bypolyacrylamide gel electro-phoresisasdescribedin thetext. a, Uninfectedcells; b, PrARSV-infected cells;c, tsCH119-infectedcells. The molecular weight of the labeled proteins was

determinedasdescribedin thelegendtoFig.5.

mined by acid hydrolysis of eluted pp53src and resolution of the phosphoamino acids by thin-layer electrophoresis. Both phosphoserine and

phosphotyrosinewerepresentin pp53src isolated

fromtsCH119-infected cells labeled eitherat35

or 41°C (data not shown). Therefore, the tem-perature-sensitive lesion in tsCH119 does not appeartosubstantially affect thestateof pp53src phosphorylation atthe nonpermissive tempera-ture.

DISCUSSION

The deletion of DNA sequences atthe BglII restriction site of the RSVsrcgene has yielded

two types of RSV mutant. The first type of mutant, which includes tdCH4, 64, and 146, contains deletions within the src gene and is

defectivefortransformation. Presumably,

virus-es containing such mutations encode src

pro-teins withlarge deletions ortruncated

polypep-tidesarising fromadeletion-induced frame shift

to an alternate reading frame. In contrast, the secondtypeofmutant,tsCH119, containsan

in-phase deletion of about 160 bp, andupon

infec-tionof chickencellsencodesashortened form of

thesrcprotein withamolecularweight of about

53,000(pp53src). Cells infected with tsCH119are

temperaturesensitive for transformation, having

atransformed morphologyat35°C andanormal

(flat) morphologyat41°C. The pp53src immuno-precipitated from tsCH119-infected cellsgrown

at41°C exhibitedapproximately 50% less tyro-sine-specific kinase activity than pp53src immun-oprecipitated from cells grown at 35°C. The pp53src protein appeared to be phosphorylated

onserineandtyrosine in tsCH119-infected cells grownateither35or41°C.

Inexperiments tobe presented elsewhere (J. Cooper, K. Nakamura, T. Hunter, and M.

We-ber, submitted for publication), a number of

parameters oftransformation have been

exam-ined in cellsinfectedwith tsCH119. This

analy-sishas yieldedtwo interestingobservations

re-garding the phosphorylationof cellularproteins.

First, acomparisonof the levels of total cellular

phosphotyrosine in 32P-labeled

tsCH119-infect-ed cells grown at 35 and 41°C revealed only a

40% reductioninphosphotyrosine labeling.

Sec-ond, the level oftyrosine-specific

phosphoryla-tion of 34,000 protein (10, 30) was reduced by

only 40% in tsCH119-infected cells grown at

41°C compared to cells grown at 35°C. These

data, takenwith theapproximate50% reduction

in heavy-chain kinase activity observed in

tsCH119-infected cells grown at 41°C (Fig. 6,

Table2) wouldsuggest thatatthenonpermissive

temperature, pp53src retains, to a significant

degree, the

ability

to

phosphorylate

known

sub-strates for pp6fsrc. Similarly, at 41°C pp53src

retainsthe

capacity

toinduce elevated levels of

cellular

phosphotyrosine,

even though the

in-fected cellspossess anormal

morphology.

We have alsoexaminedthe tumorigenicityof

tsCH119 in chickens (D. Bryant, C.Moscovici,

and J.T.Parsons,unpublished data) by

inocula-tion of virus intothewing web ofday-oldchicks.

PrARSVreadily induced largetumorswithin10

to 14days. Incontrast, inoculation of tsCH119

yieldedsmall (micro) tumorsafteraperiod of 18

to 24 days. Most ofthe latter tumors showed

evidence of regression upon sacrifice of the

birds. The diminished oncogenicity oftsCH119

isconsistent with thetemperature sensitivityof

morphologicaltransformation in cell culture.

Thegeneration ofa conditional

temperature-sensitive mutant by deletion ofan amino acid

sequenceencompassing residues202 to 255 indi-catesthat thisregion of thesrcprotein includesa

domain essential foratleastcertain parameters

oftransformation (morphologyand

oncogenici-ty). Additional evidence for multiple functional

domains in the src protein has come from the

genetic mapping of conventional

temperature-sensitivesrcmutations. Fincham et al. (12) have

recently mapped 14 temperature-sensitive src

mutationson thebasisofrecombination with td

mutations containing known deletions of src.

Ten mutationswerefoundto be clustered in the

3'40%of the src sequence,whereas four

muta-tions mapped within the 5' 60% of the src gene.

In addition, td SF/LO 104, which contains a

mutationthat maps within the 5' 60% ofthe src

gene,transformscells to a fusiform morphology (12). Thesemapping data are consistent with the

src protein containing at least two functional

domains. Recent data from several laboratories

have suggested that the functional domain for

protein kinase activity resides within the

car-boxy-terminalhalfofthe srcprotein (13, 22, 25).

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Invitro mutagenesis experiments fromour

labo-ratory have further strengthened this

supposi-tion. Asingle G-Cto A-Tchange within the

BglI

restriction site yielded an alanine to threonine

changeataminoacid

position

431.The resultant

virus was defective for transformation and

en-coded a

pp6Osrc

protein

which lacked functional

kinase activity in vitro (D. Bryant and J. T.

Parsons, submitted for publication). We have

concluded from these data that a structural

domain including

Ala431

is critical for

protein

kinase activity.

We would suggestthat tsCH119 represents a

class of mutants containing mutations which

alterasecond functionally important domain of

the srcprotein. Theexact natureof this domain

remains to be elucidated but may involve the

recognition of specific cellularproteins for

phos-phorylation. It was recently shown that in

tsCH119-infected cells grown atthe

nonpermis-sive temperature,

pp53src

remains associated

with the plasma membrane (D. Bryant, L.

Lip-sich,

and J. Brugge,

unpublished data).

There-fore, the deletion in the tsCH119srcgenedoes

not appear to directly influence the interaction of

pp53src

with cellular membranes at either 35

or 41°C. However, Krueger et al.

(18)

have

recently described two isolates of recovered

avian sarcoma virus thatencode an src

protein

with apparent alterations in the amino-terminal

region. These src

proteins

exhibit an altered

membrane

association; however,

the viruses

re-tain the

ability

to transform chicken cells in

monolayer culture. It would appear that the

mutations in the recovered avian sarcomavirus

isolates and in CH119 define at least two

func-tional domains

residing

within the

amino-termi-nalportion of the RSVsrc

protein.

The

analysis

of additional constructed mutants, as well as a

finer mapping and characterization of

existing

mutations, will be necessaryto definethe

func-tional

properties

of

pp6Osrc.

ACKNOWLEDGMENTS

We thank Betty Creasy and Thomas Rea for excellent technical assistance. We particularly thank Sarah Parsons,

TonaGilmer, and RayEriksonforgenerouslyprovidingthe rabbit antisera usedinthisstudy.

D.B.isapostdoctoralfellow oftheNational Cancer Insti-tute.J.T.P.isarecipientofaFacultyResearch Award from theAmericanCancerSociety.This workwas supported by Public Health ServicegrantsCA29243 andCA27578from the National CancerInstitute,and grant MV-29D from the Ameri-canCancerSociety.

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