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Characterization of a 170,000-dalton polyprotein encoded by the McDonough strain of feline sarcoma virus.

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Characterization

of

a

170,000-Dalton

Polyprotein

Encoded

by

the

McDonough Strain of Feline Sarcoma Virus

WIM J. M. VAN DEVEN,' FRED H. REYNOLDS, JR.,'ROBERT P.NALEWAIK,2 ANDJOHN R. STEPHENSON2A

Carcinogenesis Intramural Research Program, Frederick Cancer ResearchCenter,'andLaboratoryof

Cellular and MolecularBiology,National Cancer

Institute,2

Frederick,Maryland21701

Inthisstudy,we demonstrated theexpression ofa 170,000-M,polyproteinin

eachof several McDonough feline sarcomavirus (FeSV)-transformedmink cell

clones and oneMcDonough FeSV-transformedratclone.Thispolyprotein,

des-ignated McDonough FeSV P170, contained feline leukemia virus (FeLV) p15, p12,and p30immunological determinantsand shared two of its five [3S]methi-onine-labeledtryptic peptides with FeLVPrl .Both of thesepeptideswere shownto be specific to the p30 componentof Prl8O'1"1'. The remaining

Mc-DonoughFeSV P170 methionine-containing peptideswere notrepresentedwithin

either FeLVPr180&aY9°1or Pr82env. Of interest, of the three peptides specificto

thenonstructuralcomponentofMcDonoughFeSV P170,one wasalsorepresented in the 115,000-Mr polyproteins encoded by the Gardner and Snyder-Theilen strains of FeSV. These findings raise the possibility that the nonstructural

componentsof polyproteins encoded by each of the threeindependentlyderived

felinetransforming viruses contained bothcommonandunique regions. Moreover, if the sequencesencodingthese components are involved intransformation, as appears tobethecase, ourfindings establish thatthepositionof their insertion

within the gag-pol region of the FeLVgenome can vary among individual isolates.

Severalindependent isolatesof feline sarcoma

virus (FeSV)have been described (4, 10, 17). In

the presence of an appropriate type C helper virus, each of these isolates has been shown to induce fibrosarcomas in vivo and

morphologi-cally transform embryofibroblasts in vitro. The

Gardner and Snyder-Theilen strains of FeSV

encode 115,000-Mr polyproteins and less well defined, highly relatedproteins of around 72,000

and80,000daltons, respectively (6, 7, 11, 13, 17;

J. R. Stephenson, W. J. M. Van de Ven, A. S.

Khan, and F. H. Reynolds, Jr., Cold Spring

Harbor Symp. Quant.Biol., inpress). Such

pol-yproteins contain immunological determinants

incommonwithtranslational products of the 5'

terminus of the feline leukemia virus (FeLV) gag gene (p15, p12, and a portion ofp30). In

addition, nonstructural components lacking

either immunological cross-reactivity or

struc-tural relatedness to known FeLV translational

products andcontaining probable transforming

function have been identified. The latter com-ponents are acidic (5), highly phosphorylated (20), and, atleast in the case of Gardner FeSV, known to possess an associated protein kinase

activitywhichrecognizes P115 as its major sub-strate(20). The demonstrationthat three of five

[3S]methionine-labeledtrypticpeptidesspecific to Gardner FeSV P115 are alsorepresented in

Snyder-TheilenFeSV P115 (19)argues that the

number of feline cellularsequences, whose

ac-quisition by FeLV results in the generation of

replication-defective transforming virus (3), is

relatively limited.

An additional, although less well

character-ized, isolate of FeSVwasoriginallydescribedby McDonough et al. (10). Cells nonproductively transformed by this virus have been shownto

express FeLV p15, p12, and p30 cross-reactive

antigenic determinants (11, 19). Inthe present

study, we report the isolation of several new

clones of mink cells nonproductively

trans-formed byMcDonough FeSV and theanalysis of eachwithrespect toexpression of virus-spe-cific translational products. A polyprotein of

around 170,000 daltons containing FeLV p15, p12, andp30immunologicaldeterminants anda

55,0004Mr possible cleavage product are de-scribed. The170,000-dalton polyproteinwas

an-alyzed withrespect tophosphorylation, protein kinase activity, and relatedness to Gardner FeSV P115, Snyder-Theilen FeSV P115, and FeLVPr1809a9P°'.

MATERIALS AND METHODS

Cellsand viruses. Cellsweremaintained in the

Dulbecco modification ofEagle mediumsupplemented

with 10% calf serum (Colorado Serum Co., Denver, Colo.).Afetalminklung cellline,CCL64, andanormal

rat kidney cellline NRK, have beendescribed (17).

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166 VAN DE VEN ET AL.

McDonough FeSV-transformed subclones of CCL64

and NRK, designated G2-MINK and G2-NRK,

re-spectively, were generously providedby E. M.

Scol-nick, National Cancer Institute. Aline ofminkcells

productively infected with an FeLV pseudotype of

McDonough FeSVwaskindly provided byP.Reddy, National Cancer Institute.AGardner FeSV

nonpro-ductively transformed CCL64 mink subclone, desig-nated 64F3, andaclonal isolate of FeLV subgroupA

have beendescribed previously (19).

Competition immunoassays. Competition

im-munoassays wereperformedbyanalysis ofunlabeled antigensatserial twofolddilutions for abilityto

com-pete with 125I-labeledFeLV subgroup A p30, p15,or p12 for binding limiting amounts ofgoatantiserum directedagainst detergent-disrupted FeLV subgroup

C. Proteins were purified and labeled with "2I by

previously described procedures(6).Competition

im-munoassayreaction mixtures contained0.01 M

Tris-hydrochloride (pH 7.8), 1.0mM EDTA, 0.4%Triton

X-100,1%bovineserumalbumin, and0.2M NaCl in a total volume of 0.2 ml. Antiserum andunlabeled competing antigen were incubated at 370Cfor 1 h before the additionof10,000cpmof "nI-labeled anti-gen.Afterfurther incubationfor3 h at370C and18h

at4°C,antigen-antibody complexeswere

immunopre-cipitatedaspreviously described (6).

Immunoprecipitation and SDS-PAGE.

Expo-nentiallygrowingcellswerelabeledwith

'Pi

(carrier-free;NewEnglandNuclear Corp., Boston,Mass.) or

[35S]methionine (1,075 Ci/mmol; Amersham Corp., Arlington Heights, Ill.) aspreviously described (19).

Afterlabeling,cellmonolayerswerewashed twice with

serum-freeDulbecco-modified Eagle medium and dis-ruptedat40Cin PBSTDSlysis buffer (10 mM sodium phosphate[pH7.2], 0.9%NaCl,1%Triton X-100, 0.5%

sodiumdeoxycholate,and0.1%sodium dodecyl sulfate [SDS]).Extracts(1 ml)wereincubatedat40C for18 haftertheaddition ofasolution containingRNase A

(200 ug/ml), RNaseT2 (0.1,ug/ml), DNase I (4.0 Ag/

ml),normal goatserum (5

p1),

andprotein

A-Sepha-rose C1-4B (1.0 ml of a 10% [vol/vol] suspension)

(Pharmacia Fine Chemicals, Inc., Piscataway, N.J.)

and clarifiedby centrifugationfor 1 hat 100,000xg.

Virus-specific proteins were immunoprecipitated by

incubation for 18 hat40Cinthepresenceof 5

pl

ofa

specific antiserum,addition of 50p1ofa10%(vol/vol) protein A-SepharoseC14Bsuspension,and incuba-tion foranadditional 2 hat40C. Immunocomplexes

were collected bycentrifugation at2,000 xgfor 10

min,washed threetimesinPBSTDSlysisbuffer and analyzed bySDS-polyacrylamide gel electrophoresis (PAGE)on5to20%polyacrylamide gradient slab gels by the method of Laemmli (8). Radioactivity was

visualized by scintillation autoradiography as

de-scribedbyBonnerandLaskey(2).

Tlwo-dimensional tryptic peptide analysis. Vi-rus-specific proteinswerepurified by

immunoprecipi-tation and SDS-PAGE. After electrophoresis, gels

werewashedextensivelywith10% trichloroacetic acid,

followedbyasolutioncontaining10% aceticacid and

10%methanol,and dried undervacuum.Labeled

pro-teinswerelocalizedby autoradiography andcutfrom

the dried gel, and selected gel slices were further

washed in 10% methanol and lyophilized. Proteins weredigested byincubationofgelslicesintolysulfonyl

phenylalanyl chloromethylketone-trypsin (Worthing-tonBiochemicals Corp., Freehold, N.J.) (50gLg/mlin 0.05 M ammonium bicarbonate, pH 8.0) for 6 hat

370C.Supernatant fluidswereremoved andreplaced by a fresh tolylsulfonyl phenylalanyl chloromethyl ketone-trypsin solution for additional digestion at

roomtemperaturefor16h.Supernatant fractions were pooled, filtered, lyophilized, and washed twice.

[35S]methionine-labeled digestswereincubated for 2

h at00Cin 0.1mlof chilled performic acid (30% H202-90%formic acid [1:9] preincubated for 2h at room temperature).Samplesweresubsequentlydiluted with

2mlof water,lyophilized,suspended in electrophoresis buffer (acetic acid-formic acid-water [15:5:80]), and spotted on cellulose-coated thin-layer chromatogra-phyglassplates (10 by 10 cm; EM Laboratories,Inc.,

Elmsford, N.Y.). Electrophoresiswasperformed for 30 minat500V, andchromatographywasconducted in the second dimension in buffer containing butanol,

pyridine,acetic acid, andwater(32:25:5:20). Radiola-beled tryptic peptides were visualized by

autoradi-ography with Kodak X-Omat R film. Detection of

[35S]methionine-labeled peptides was enhanced by

spraying the thin-layerchromatography plateswith ethercontaining7%2,5-diphenyloxazole(2).

Assayforproteinkinaseactivity.Tissue culture cells (107) were disrupted by repeated aspiration

througha25-gaugeneedle in5ml ofasolution of10

mMTris-hydrochloride (pH 7.2),100mMNaCl,1mM EDTA, and0.1%Triton X-100and clarified by

cen-trifugation at 2,000 x g for 10 min. Virus-specific

proteinswereimmunoprecipitatedasdescribedabove, andproteinkinaseactivitywasassayed by suspension

ofimmunoprecipitatesin 20 mM Tris-hydrochloride

(pH 7.2)-5 mMMgCl2buffercontaining1 utCi of [y-32P]ATP(2,000Ci/mmol;NewEnglandNuclearCorp.,

Boston,Mass.) and incubation of reaction mixtures for

10min at300C.Reactionswereterminated by addition of3 ml of ice-cold PBSTDS lysis buffer, and

non-protein-bound32P-labelwasremovedbyrepeated

cen-trifugation inexcessPBSTDSlysisbuffer. After the final wash,immunoprecipitatesweresuspendedin 20

pl

ofasolution of0.65MTris-hydrochloride(pH 6.7),

1%SDS, 10% glycerol, 2.5% 2-mercaptoethanol, and

0.1% bromophenolblue and heated for2minat90°C. Samples were analyzed by SDS-PAGE with a 5%

acrylamide-0.133% bisacrylamidestackinggelanda

9-cm, 5 to 20% linear polyacrylamide separation gel (acrylamide/bisacrylamide ratio; 30:0.8)ina

Tris-gly-cine-SDS buffer systemat20to 30mAasdescribed by Laemmli(8). Radioactivitywasvisualizedby au-toradiography with Kodak X-OmatRfilm.

RESULTS

FeLVgaggeneproteinexpressionincells nonproductively transformed by the Mc-Donough strain of FeSV. We previously

re-portedtheexpressionof FeLVp15, p12,andp30

in aminkcellclone,G2-MINK,

nonproductively

transformed bytheMcDonoughstrain of FeSV (19). To further determine the specificity and significance of such reactivities,we obtained a

cell line chronically infected with the original McDonoughFeSV seed stock froman

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McDONOUGH FeSV

ent source and, by endpoint cloning on CCL64 minkcells,successfullyderivedtwo new

nonpro-ductively transformed cell lines.Bycompetition

immunoassay,both of thesetwonewly derived cell lines andaMcDonough FeSV-transforned

ratcellclone,G2-NRK,provided byE.M. Scol-nick, were found to express levels ofp15, p12, and p30immunological reactivity comparableto

thosepreviously reported for the G2-MINKcell

line (datanotshown).

Forfurthercharacterization of these reactivi-ties, extracts of the McDonough FeSV C115-1 and G2-MINK cell clones were subjected to

SDS-PAGEanalysis afterlabelingin vivo with [3S]methionine and immunoprecipitation by

serawith specificity for individual FeLV

struc-tural proteins. As shown inFig. 1, proteins of around 170,000, 160,000, and 55,000 Mr were

immunoprecipitated from McDonough FeSV C115-1 cellsbygoatantis rundirectedagainst detergent-disrupted FeLV (laneA) andbysera

withspecificity forpurifiedFeLV structural

pro-teins, including p15, p12, and p30 (lanes B through D), butnotbygoat anti-FeLV plO or

gp7O (lanesEandF).That theseproteinswere

McDonough FeSV specific was indicated by their absence in immunoprecipitates ofnormal

minkcells. Similar analysis of the G2-MINK cell lineindicated thepresenceofa170,000-Mr pol-yprotein immunoprecipitable by antisera di-rected againstFeLV and byseraagainst FeLV

p15, p12, and p30. A second major protein of around 160,000 Mr was precipitated from

G2-MINKcellsbyanti-FeLV andto alesserextent by antisera directed against individual FeLV

structural proteins(lanesHthrough M). ComparisonofMcDonoughFeSV P170to

Gardner FeSV P115 by immunoprecipita-tionandSDS-PAGEanalysis. Inview ofour

previous demonstration ofstructural relatedness betweenpolyproteinsof around 115,000Mr

en-coded by the Gardner and Snyder-Theilen strains ofFeSV, it was of interestto test Mc-Donough FeSV P170 and Gardner FeSV P115 forpossibleserologicalcross-reactivity.Forthis purpose, we utilized an antiserum prepared in

goats and previously shown to recognize both the structural (p15 and p12) and nonstructural

componentsofGardner FeSV P115. Thisserum,

designated anti-GardnerFeSV P115, is of partic-ular interestinthat, inadditiontoFeSV P115,

it

recognizes

a150,000Mrcellular

phosphopro-teinwithassociatedprotein kinase activity and bindingaffinity forFeSVP115(F. H.Reynolds, Jr., W. J. M. Van de Ven, and J. R.Stephenson, submittedforpublication).

As shown inFig. 2, precipitation of Gardner FeSV P115 and McDonough FeSV P170 from the 64F3 and McDonough FeSV C115-1 cell lines,respectively, wasobtained withgoat anti-FeLV butnotwithnormalgoat serum(lanesA,

D, E, andH).Although both polyproteinswere

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

P170- "a_ _

P160r5- 9

Pr55-*0 Ebmqm*

4-98K

* 4-69K

* -46K

_ -4-30K

;_m, -4-12K

FIG. 1. Immunoprecipitation and SDS-PAGE analysis ofpolyproteinsencoded by theMcDonough strain ofFeL V.Celllinesincluding McDonoughC115-1(Athrough G), G2-MINK (H throughM),and controlCCL64

mink(N)werepulse-labeled in[3S]methionine(100

tCi/ml)-containing

mediumfor 30min,

immunoprecipi-tated with goat antiserum directed against detergent-disrupted FeLV (A, H, andN), with antisera with specificity forFeLVp3O(B andI), FeLVpJ5(C and J), FeL Vpl2 (D andK),Rauscher murine leukemia virus plO(E and L), and FeLVgp7O (F andM), and withcontrol goat serum (G), and analyzed bySDS-PAGE. Molecular weight standards included "4C-labeledphosphorylase B (98,000), bovine serum albumin(69,000),

ovalbumin(46,000), carbonicanhydrase(30,000),andcytochrome c (12,000). VOL. 35,1980

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168 VAN DE VEN ET AL.

A B C D E F G H J K L

gagpol

P170-P160 P150O

P115-P,65gag

a*

4

.w.

Ii. _U -69K

[image:4.508.125.406.79.283.2]

- -46b

FIG. 2. Comparison ofMcDonough FeSVP170 and Gardner FeSVP115 byimmunoprecipitationand SDS-PAGE analysis. Cells, including the Gardner FeSV-transformed mink clone 64F3 (A through D), McDonough

Cl15-1(E through H), and CCL64 mink cellsproductivelyinfected with FeLV subgroup A (I through L), were

pulse-labeled in [36Slmethionine (100

lCi/ml)-containing

medium for 30 min, immunoprecipitated, and analyzedbySDS-PAGE.Sera included anti-FeL V (A, E, and I), anti-Gardner FeSVP115(B, F,and J), anti-Gardner FeSV P115extensively absorbed with FeLV (C, G, and K), andnormalgoat serum (D, H, and L). Molecularweight markersare asdescribed in thelegendtoFig. 1.

also efficiently precipitated by anti-Gardner FeSV P115 (lanes B and F), this could reflect recognition of FeLVgaggene-codedstructural

components, asindicated by

immunoprecipita-tion of FeLV Prl80""9' and Pr659'9, but not

Pr82env, from FeLV subgroup A-infected mink cells (lane J). After extensive absorption with detergent-disrupted FeLV, anti-Gardner FeSV P115 still immunoprecipitated Gardner FeSV P115 but no longer recognized either FeLV

Pr180Ya9'P°orPr659ag(lanes C and K).

Absorp-tion ofanti-GardnerFeSV P115also resulted in loss ofreactivity directed against McDonough FeSV P170 (laneG). Figure 2 shows alack of detectable immunological cross-reactivity

be-tweenMcDonough FeSVP170and the 150,000-Mr mink cellular phosphoproteinP150.

Immu-noprecipitation of P150 from Gardner FeSV, McDonough FeSV, and control minkcellswas

observed with anti-Gardner FeSV P115 both before and after absorption with FeLV. This

finding, in combination withalack ofdetectable

precipitation of McDonough FeSV P170 by FeLV-absorbed anti-Gardner FeSVP115,argues

that, ifthesetwoproteins shareimmunological determinants, they are not recognized by this

antiserum.

Comparison of

[3"S]methionine-labeled

tryptic peptide compositions of FeLV

Pr1809-°IIo`

andMcDonoughFeSV P170. The

above resultsdemonstrate significant

immuno-logical cross-reactivity between McDonough FeSV P170 and FeLVPrl80"'-P andthus raise the possibility that synthesis of McDonough FeSV P170might result from a small in-phase deletion in the gag-pol region of the FeSV ge-nome. Totestthispossibility, [3S]methionine-labeled McDonough FeSV P170, the160,000Mr protein P160 expressed in the G2-MINK cell

line, and FeLV Pr180OI)9Plwerepurified by

im-munoprecipitation and SDS-PAGE and sub-jectedtotwo-dimensionaltryptic peptide anal-ysis. As shown inFig. 3,McDonoughP170and

P160each contained fivewell-resolved [3S]me-thionine-labeled tryptic peptides, designated a

through e. Although a number of minor, less

intensely labeled peptideswerealsoseen, their presence wasvariablebetween individual

exper-iments, indicatingtheirprobablecellular,rather thanviral,origin (datanotshown). Incontrast, FeLVPr180g""P'wasfoundtocontainatotal of 12labeledpeptides,only2of which (peptides2

and11)appearedtocorrespondinmapposition

to peptides represented in McDonough FeSV

P170.

Inviewof the above resultsindicating immu-nological cross-reactivity between FeLV gag

gene-coded proteins and McDonough FeSV

P170, weundertooktofurthercharacterize the

two peptides common to McDonough FeSV

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A

a tar

d

:;.

a

b**

.. ~~~~~C

B

d

* 0

*..

.

--.S X~~~~

.. a

C

thionine-containing peptides specific to p30,

both of whichwererepresentedinPr6M"' (Fig.

4).

Tofurthertestwhether the [3S]methionine-labeled tryptic peptides common to FeLV

Pr1809'"9P`

and FeSV P170 were FeLVp30

spe-cific, we performed additional mixing

experi-ments.In aninitial experiment,

[3S]methionine-labeled FeLV Pr180'9°' and Pr65"" were

mixedat a1:1ratiobeforetryptic peptide anal-ysis. The fact that the 2Pr65W'9-specific peptides (peptides2and11) comigrated with 2 of the 12

Prl80"g"g'peptides confinns that the

180,000-Mr proteinexamined was, in fact, FeLV Prl80

(Fig. SA). As shown in Fig. 5B, in a mixing

experiment involving FeLV Prl80mI)-pI and

FeSV P170, the only comigration was between

peptides 2 and 11 and b and c, respectively. Finally, in a mixing experiment between

Mc-Donough FeSV P170 and FeLV p30, the two

[3S]methionine-labeled FeLV p30 tryptic pep-tides were shown to correspond in position to

McDonough FeSV P170 peptides b and c (Fig. 5C).

A

7

*8

Sag

Xi.

6- 9

W8

42

0 *11 12

_"

2

0 'I

FIG. 3. Comparison of

IS]methionine-labeled

tryptic peptide compositions of McDonough FeSV P170 (A), McDonough FeSV P160 (B), andFeLV

PrI80gap' (C). [35S]methionine-labeled P170 and P160wereisolatedfrom G2-MINK cells by immuno-precipitation and SDS-PAGE as described in the legend toFig.1.FeLVPr1809'9 wasisolated from

[35S]methionine-labeled FeLVsubgroup A-infected minkcells. Proteinsweredigested with tolylsulfonyl

phenylalanyl choloromethyl ketone-trypsin as de-scribed in thetext. Tryptic digests (6/000 to 10K000

cpm) were suspended in electrophoresis buffer and spottedoncellulosethin-layer glass plates (o). Sepa-ration inthefirst dimension was byelectrophoresis,

and that inthe second dimensionby ascending chro-matography. McDonough FeSV

[3SJmethionine-la-beledpeptidesaredesignatedathrough e,and the12 major[35S]methionine-labeledpeptidesrepresented inFeL VPr1809`'"Plare labeled1through 12.

P170 and FeLV

Prl80?agPo.

For this purpose,

FeLV

Pr65Mag

and p30 were analyzed with

re-spect to

[35S]methionine-labeled

tryptic peptide composition.The resultsdemonstratedtwo

me-B

D

2

[image:5.508.53.241.76.452.2]

0

4II

S

FIG. 4. Two-dimensional[35S]methionine-labeled

trypticpeptide mapsof FeLVPr65S""(A) and FeLV p30 (B). Proteins were immunoprecipitated from FeLVsubgroupA-infected mink cells by an antiserum againstFeLVp30, furtherpurified bySDS-PAGE,

and subjected to two-dimensional tryptic peptide analysisasdescribedinthelegend to Fig. 3. Peptides

arelabeledaccordingtotheirmappositions relative

toPrl80g'-P"lpeptides shown in Fig. 3.

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170 VAN DE VEN ET AL.

A

4

6..

8 9 3 5 7 10

2' 1

11

12

B

a

"'e

64, 8 q

3 7 10

2(i)

0 * 12

C

possibility that the threeP170-specific [3S]me-thionine-labeled peptides might have been en-codedby sequences contained within the FeLV

envgene. To test thispossibility, we compared

themajor FeLVenvgene translationalproduct

Pr82env and McDonough FeSV P170 with re-spectto[3S]methionine-labeled tryptic peptide composition. As shown in Fig. 6A, Pr82env con-tained only two labeled peptides. In a mixing experiment, neithercorrespondedto any ofthe five major [3S]methionine-labeledpeptides

spe-cifictoMcDonough FeSVP170 (Fig.6B).

Comparison of

[5S]methionine-labeled

tryptic peptides specific to McDonough

FeSV P170 and Gardner FeSV P115. We

havepreviously shownthe

115,000OMr

polypro-teinencoded by the GardnerstrainofFeSVto contain, within itsnonstructuralcomponent,five well resolved tryptic peptides, three of which

(Fig. 7,peptides2, 4,and5) correspond to

pep-tidespresentinSnyder-TheilenFeSV P115 (19).

A

a

.

0

2*(

(b)

0

B----...

..

.

..

....~~~~~~~~~~~~~~~~...

B

[image:6.508.61.259.59.466.2]

11 (c)

FIG. 5. Identification of[36SJmethionine-labeled

tryptic peptides sharedby McDonough FeSV P170 and FeLVPr)809w"9"'. [35S]methionine-labeled

pro-teins, including McDonough FeSV P170, FeLV

Pr180O9'W-', and FeLVp30, werepurified by

immu-noprecipitation and SDS-PAGE as described in

the legends toFig. 3 and 4.Labeledproteins were

mixed at 1:1 ratios in thefollowing combinations:

Pr1809`IPol and Pr655'9 (A); Pr1809'9-Pol and Mc-Donough FeSV P170 (B); and McDonough FeSV

P170 and FeLVp3O (C). Theseproteinsweresubjected

to tryptic peptideanalysis, and individualpeptides

werelabeledasdescribed in thelegendtoFig.3. Lack of [35S]methionine-labeled tryptic peptides common to FeLV Pr82v and

McDonough FeSV P170. The above results established that only a portion of the coding

capacity for McDonough FeSV P170was

rep-resented within thegag-pol region of the FeLV

genome. Thesefindings, however,leftopenthe

d

a e

*~~~

b

0*

FIG. 6. Comparison of [35SJmethionine-labeled tryptic peptide compositions of FeLV Pr82enV and McDonough FeSV P170. [35S]methionine-labeled proteins werepurified by immunoprecipitation and

SDS-PAGEandsubjectedtotryptic peptide analysis as describedin the legend toFig. 3. FeLV Pr82e

was analyzed either alone (A) or mixed 1:1 with McDonoughFeSV P170(B). The twomajorPr82en" [35S]methionine-labeled peptides are designated *

andMcDonoughFeSVPl70peptidesaredesignated

athroughe. 0

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It was thus of interest to compare the tryptic peptidecompositionsofMcDonoughFeSV P170 and Gardner FeSV P115. Figure 7 shows that,

although the tryptic peptide compositions of

[3S]methionine-labeled isolates ofMcDonough FeSV P170fromtwo independent transformed clones, McDonough C115-1 (Fig. 7A) and G2-MINK (Fig. 7B), were indistinguishable, both

weredistinct from Gardner FeSV P115(Fig.70).

Ina mixingexperiment, however, McDonough FeSV P170peptide a wasshowntocorrespond

in position to Gardner FeSV P115 peptide 2. The remaining four [3S]methionine-labeled peptides, specifictoeach, exhibitedno detecta-blecorrespondence in trypticmapposition.

Determination of relatedness of Mc-Donough FeSV P170tomink cellular P150.

As discussed above, we have previously de-scribeda150,000-dalton mink cellular phospho-protein with associated protein kinase activity and binding affinity for Gardner FeSV P115

(Reynoldsetal., submitted forpublication). Al-though P150 has been showntolack structural

A

a

d

lb.

b

0.

C

C

3

240

4

0o 'p

0

relatednesstoGardnerorSnyder-Theilen FeSV P115 (19), the possibility that it might be

par-tially relatedtoMcDonough FeSV P170wasnot

excluded. Tryptic peptide analysis of P150

re-vealedatotal of11methionine-containing

pep-tides (Fig. 8A), noneof whichappearedto

cor-respondtothe5major

[3S]methionine-labeled

peptides specific to McDonough FeSV P170 (Fig.8B). To confirm the apparentlack of

cor-respondence between the trypticpeptidemaps ofthesetwoproteins, amixing experimentwas

perforned (Fig. 80). Again, none of the 11

[3S]methionine-labeled peptides specific to

P150,labeled1through11,correspondedin map

positiontothose contained withinMcDonough FeSV P170 (labeledathrough e).

Analysis ofMcDonough FeSV P170 for labeling in vivoby

"Pi

andfor associated protein kinase activity. High-molecular-weightpolyproteins encoded by the Gardner and Snyder-Theilen strains of FeSV have been showntocontainmultiple sites of phosphoryla-tionintheir nonstructuralcomponents(19, 20).

w

..U

B

d

4, a

_ e

b

c

0

D

3

El

28

1I b

d

^O

e

4

[image:7.508.48.447.319.581.2]

5 c 0

FIG. 7. Comparisonof[3S]methionine-labeledtrypticpeptidecompositions ofMcDonoughFeSVP17Oand

GardnerFeSVP115.

[3SlJmethionine-labeled

McDonoughFeSVP170and Gardner FeSVP115 were isolated

byimmunoprecipitation andSDS-PAGE as described in the legend to Fig. 2. Trypticpeptide maps were

generatedasdescribedinthelegendtoFig.3;polyproteins tested included McDonough FeSVPJ70isolated

from McDonoughC115-1 cells (A),McDonough FeSVP170isolatedfromG2-MINKcells (B), GardnerFeSV

P115 isolated from the 64F3 cell line (C), and a 1:1 mixture of McDonough FeSV P170 isolatedfrom

McDonoughC115-1cells andGardnerFeSVP115isolatedfromthe 64F3cellline(D). The major McDonough

FeSVP170andGardnerFeSVP115trypticpeptides are designated a through e and 1 through 5, respectively.

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172 VAN DE VEN ET AL.

A

:

4

3 {-:,"

..-

A.

1 2 3

6

10

BIII.

...

IIIIIIII.lIIIIIIIIIII"IJ.""I"IIlIUI

~~~~~~~~~~~~~~~..

...1"l111|11-"1"':11111'''1''1'11'''---''---.''''

D

d

e

a.

c.

0

C

a

4 : 5

d

e

6

b

1

0

[image:8.508.76.265.80.512.2]

2 3

FIG. 8. Comparison of McDonough FeSV P170 and mink cellular P150 withrespect to

[35Slmethio-nine-labeledtryptic peptide composition.

[35SJmethi-onine-labeledMcDonough FeSV P170wasisolated

from McDonough C115-1 cells by

immunoprecipita-tion andSDS-PAGEas describedin the legendto Fig.3. Minkcellular P150wassimilarlyisolatedfrom

normalCCL64 mink cells(F.H.Reynolds,Jr., W. J. M. Van deVen,and J. R.Stephenson,submittedfor publication). Tryptic peptide analysiswasperformed

asdescribed in thelegendtoFig. 3;proteinstested included mink cellular P150(A); McDonoughFeSV P170(B); anda 1:1 mixtureofmink cellular P150 and McDonough FeSV P170 (C). The major Mc-DonoughFeSVPl 70 andminkcellularP150tryptic peptidesaredesignateda througheand1 through

11,respectively.

To explore the possibility that McDonough FeSV P170 mightalso be phosphorylated, the

McDonough C115-1celllinewasculturedin3p; containing medium, and labeled proteins were

analyzed by immunoprecipitation and

SDS-PAGE. Asshown in Fig. 9, (lanes A, B, D, and

E), P170 was labeled under these conditions, althoughonlyto alimitedextent.Under similar conditions, 4070-A amphotropic mouse virus

Pr659'9

(lanesDandE),FeLVPr659'9(lanesG

and H), and mink cellularP150 (lanesB, E, H,

J, and K) were each muchmore efficiently

la-beled. Thus, in vivo phosphorylation of

Mc-Donough FeSV P170isconsiderably less than

previously reported for polyproteins encodedby

the Gardner andSnyder-Theilen strains of FeSV (19,20).

Several virus-coded transformingproteins, in-cluding thenonstructural component of Gardner FeSVP115,havebeen showntoexhibit protein kinase activity (20). For a further characteriza-tion ofMcDonough FeSV P170, in vitro phos-photransferaseactivitywasassayed under

con-ditions previously shown to result in efficient phosphorylation of Gardner FeSV P115. As

shown inFig.10 (lanesDandE),nodetectable phosphorylationofMcDonough FeSV P170was

obtained under these conditions. In contrast,

both Gardner FeSV P115 (lanesA andB) and mink cellular P150 (lanes A, B, E, and H) were

labeled atreadily detectable levels. These find-ings thus argue that McDonough FeSV P170

differs from Gardner FeSV P115inthat, when

analyzed under similar conditions, it lacks de-tectable protein kinaseactivity.

DISCUSSION

Evidence thattype C retroviruses constitute natural vectors for cloning cellular genes with transformingfunction isaccumulating(fora

re-view, see reference 18). The acquisition of cel-lular sequences by the viralgenomeappearsto

involvearecombinationaleventresulting in the insertion of suchsequenceswithinapreexisting viral gene andconcomitant loss ofaportion of

theviralgenome.Inseveral cases,translational productsof suchacquired sequenceshave been shown to be

initially

synthesizedaspolyproteins containing amino-terminal gag gene-encoded components (1, 6, 12, 14, 15, 17, 21). The fre-quency of cellular sequences within the feline cellular DNA,whose insertion into the type C viralgenome confersthetransformingpotential, appearsto be limited. This isindicated by the

demonstration that the acquired sequence-en-coded translational products of the two FeSV isolates, Gardner and Snyder-Theilen, are

im-munologically

andstructurally related(18),and

bytheresultsofmolecularhybridizationstudies

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POLYPROTEIN ENCODED BY McDONOUGH FeSV

A B C D E F G H J K L

¶. .W*

0_

nw

--98K

4-69K

40 -4-46K

-30K -4-12K

FIG. 9. Deternination of theextentofphosphorylation ofMcDonough FeSV P170 byimunoprecipitation

and SDS-PAGE analysis. Cell lines, including McDonough C115-1 (A through C), McDonough C115-1

superinfected with4070-A amphotropic wildmousevirus (DthroughF),McDonough C115-1 superinfected

withFeLVsubgroupA(G through I),andcontrolCCL64 mink cells(J through L),werepulse-labeled for2 h in mediumcontaining32Pi (100 MCi/mi) andanalyzed byimmunoprecipitationand SDS-PAGE.Sera were

prepared ingoatsandincluded anti-FeLV (A, D, andG), anti-FeSVP115(B, E,H, J, andK),andnormal

goat(C, F, I,andL).Molecular weightstandardsare asdescribedin thelegendtoFig.3.

A B C D E F G H I

P150-*-P115*

* -4-98K

:1

.0 -4--69K

* 46K

--30K

[image:9.508.105.390.77.287.2]

SMh -4-12K

FIG. 10. Analysis ofMcDonough FeSVP170for protein kinase activity. GardnerFeSV-transforrned(64F3)

(A through C), McDonoughFeSV-transforrned(McDonoughC115-1)(Dthrough F), and uninfected control(G

through I) mink cellsweredisrupted, immunoprecipitated, and analyzed for[y-32PJATPphosphotransferase

activityasdescribedin thetextAntiserausedincludedanti-FeLV (A,D, andG),anti-FeSVPZ15(B, E, and

H),andnormalgoat(C, F, and I). Molecularweightstandardsare asdescribedin the legendtoFig. 3.

VOL. 35, 1980 173

P170-4-P150- w

gag Pr65

-*-7.

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[image:9.508.129.368.372.610.2]
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174 VAN DE VEN ET AL.

(3). The acquired sequence-encoded

transla-tional product of an independent mammalian

RNA transforming virus, Abelson murine leu-kemia virus (12, 21), is distinct from the analo-gous component of theFeSV-encoded

polypro-teins (Stephensonet al., inpress). Whether such

differencesreflect evolutionarydivergence or

re-latetothedifferences inthe diseases induced by

these virusesis, however, as yetunresolved.

In the present study, we demonstrated the

expression ofa170,000-Mrpolyproteinin three

independently derivedMcDonough FeSV-trans-formed mink cell clones and one McDonough

FeSV-transformed rat clone. This newly

de-scribed polyprotein, designated McDonough

FeSV P170, containedP15,p12,andp30 immu-nological determinantsandshared two

[3S]me-thionine-labeled tryptic peptides with FeLV subgroup A Pr1809a9P°, both of which were

showntobespecifictoFeLVp30. The

remaining

threeof the five[3S]methionine-labeled tryptic peptides contained within McDonough FeSV

were not represented within FeLV Prl80ga*-° (Fig. 11). These could have corresponded to

acquired cellularsequences responsible for the

McDonough FeSV transforming function. Alter-natively, McDonough FeSV P170 could have

arisen as a result of deletions within FeLV Pr180q9-P°O, and the peptides unique to Mc-Donough FeSV could have been encoded by genomicsequenceslocatedatpositionsadjacent

tothe sitesof such deletions. This latter possi-bility, however,seemsless likely since such dele-tionsmustbemultiple, relatively small, and in phase andmustspecificallyencompassthe

sup-pressible termination codon atthe 3' terminus of the FeLV gag gene. If McDonough FeSV P170 does contain acquired sequence-coded

components with tranforming potential, they

must be situated at positions different from thosepreviously reported for other feline

trans-forming viruses. This is indicated by the fact

thatMcDonoughFeSVP170containstwo p30-specific [35S]methionine-labeled tryptic

pep-tides, neither ofwhich is representedin either

Gardner or Snyder-Theilen FeSV P115. The

relationship of McDonough FeSV P170 to a

previously reported 120,000-Mr polyprotein

ex-pressedinMcDonough FeSV-transformedmink

cellsis notresolved (11).

Thepresentfindings alsoshowthatoneofthe three tryptic peptidesspecificto the

nonstruc-tural component ofMcDonough FeSV P170 is alsorepresented in the115,000-Mrpolyproteins encoded by the Gardner and Snyder-Theilen

strains of FeSV (Fig. 11). The possibility that comigrationofthissinglemethionine-containing tryptic peptide in two-dimensional electropho-retic and chromatographic separations of all

A

a

4D

d

e

1G 7 8 9

4

38 6 10 12

*b5 C

2(b) *

11(c)

0

B

10

0

d

e 4

0

b o

c

FIG. 11. Schematicdrawingdepictingrelatedness ofMcDonough FeSV P170[35Slmethionine-labeled trypticpeptidestothosespecificto

FeLVPrl809'',

FeLVp30, and Gardner FeSV P115. (A), the two-dimensionaltrypticmappositions of3

[35S]methio-nine-labeledMcDonoughP170-specific peptides (3)

and 10FeLVPr180"g'°'-specific peptides (0) are shown. Inaddition, thepositions oftwoFeLV

p30-derivedpeptides (0) common to both are shown.

Trypticpeptides specific to McDonough FeSVP170 (0) and Gardner FeSV P115 (0) and the single

peptidecommontoboth

(0)

areshownin(B). three isolates is fortuitous, although

unlikely,

cannotbeexcluded.Alternatively, the possibil-ity that polyproteins encoded by each of the three transforming viruses contain a common

regionmustbeconsidered.If thelatter modelis correct, this common region is apparently

en-codedbysubsets ofthe sequences

acquired

by

the independently derived transforming virus

isolates.

Todate,wehavebeenunabletodemonstrate

autolabelingproteinkinaseactivityin

immuno-precipitates ofMcDonoughFeSVP170, indicat-ing that P170 lacks an associated or intrinsic proteinkinase forwhichit isanactivesubstrate.

Moreover, McDonough FeSV P170 is not an

apparent substrate for mink cellular phospho-protein P150,which also hasassociated

protein

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kinaseactivityandbinding affinityfor Gardner

FeSV P115 (Reynoldsetal., submittedfor

pub-lication). McDonough FeSV P170 thus differs from the P115polyproteinencodedbyGardner FeSV, which exhibits an associated protein ki-nase activity recognizing P115 as amajor

sub-strate inspecific immunoprecipitates (20). The

possibilitythatMcDonoughP170possesses

pro-tein kinaseactivitybut lacks necessaryacceptor sites to act as substrate cannot, however, be

excluded. It is also notpossible to exclude the

possibility thatthenonstructuralcomponent of

P170 has protein kinase activity but that the

enzyme is relatively inactive in an uncleaved polyprotein form.

ACKNOWLEDGMENTS

We thank B. Pincus and E. Ambush forexcellent technical assistance.

This work was supported under contractNO1-CO-75380

from the National Cancer Institute and by a fellowship awarded to W.J.M.V. by the NetherlandsOrganisation for the Advancement of Pure Research (Zuiver Wetenschappelyk

Onderzoek).

LITERATURE CITED

1. Bister,K., M. J. Hayman, and P. K.Vogt. 1977. De-fectiveness of avianmyeloblastosis virus MC29: isola-tion oflong-termnonproducer culturesand analysis of virus-specific polypeptide synthesis. Virology 82:431-448.

2. Bonner, W.M., and R. A.Laskey.1974.A filmdetection method for tritium-labeledproteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem.46:8388.

3. Frankel, A. E., J. H. Gilbert, K. J. Porzig, E. M.

Scolnick, and S. A. Aaronson. 1979. Nature and distribution of feline sarcoma virus nucleotide se-quences. J. Virol. 30:821-827.

4. Gardner, M. B., R. W. Rongey, P. Arnstein, J. D. Estes, P.Sarma, R. J. Huebner, and C. G. Richard. 1970.Experimentaltransmissonoffeline fibrosarcoma tocatsanddogs. Nature(London) 226:807-809.

5. Khan, A.S.,D. N.Deobagkar,and J. R.Stephenson. 1978.Isolation andcharacterization of a feline sarcoma

virus-codedprecursorpolyprotein. J. Biol. Chem.253:

88944901.

6. Khan, A.S.,and J. R.Stephenson. 1977. Feline leuke-miavirus:biochemical andimmunological characteri-zationof gaggene-coded structural proteins. J. Virol. 23:599-607.

7. Khan,A.S.,and J. R.Stephenson. 1979. Feline sar-comavirus-codedpolyprotein:enzymatic cleavage by a type C virus-coded structuralprotein. J. Virol. 29:649-656.

8. Laemmli, U. K. 1970. Cleavage of structural proteins

during the assembly of the head ofbacteriophage T4. Nature(London) 227:680-685.

9. Lowry,O.IL,N. J.Rosebrough,A. L.Farr,and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 192:265-275.

10. McDonough, S. K., S. Larsen, R. S. Brodey, N. D. Stock, and W. D. Hardy, Jr. 1971. A transmissible feline fibrosarcoma of viralorigin. Cancer Res. 31:953-956.

11. Porzig, K. J., M.Barbacid,and S. A. Aaronson.1979. Biologicalproperties and translationalproductsof three independent isolates of feline sarcoma virus.Virology 92:91-107.

12. Reynolds, F. H., Jr., T. S.Sacks, D. N. Deobagkar, and J. R. Stephenson. 1978.Cellsnonproductively

transformed by Abelson murine leukemia virus express ahigh molecular weightpolyprotein containing struc-tural and nonstrucstruc-turalcomponents.Proc. Natl. Acad. Sci.U.S.A. 75:3974-3978.

13. Sarma, P. S., andT. Log.1977.Defectiveness of a feline sarcoma virus demonstrable by the acquisition of a new envelope antigenic type by phenotype mixing. Virology 78:336-339.

14. Sherr, C. J., L A. Fedele, L. Donner, and L. P.Turek. 1979.Restrictionendonuclease mapping ofunintegrated proviral DNA ofSnyder-Theilen sarcoma virus: locali-zation ofsarcoma-specific sequences. J.Virol. 32:860-875.

15. Sherr, C. J., A.Sen, G.J.Todaro, A.Sliski,and M. Essex. 1978.Pseudo-typesoffeline sarcoma virus con-tain an 85,000-daltonprotein withfeline oncomavirus-associatedcell membrane antigen (FOCMA) activity. Proc.Natl. Acad. Sci.U.S.A. 75:1505-1509.

16. Snyder,S. P., and G. H. Theilen. 1969. Transmissible felinefibrosarcoma. Nature (London) 221:1074-1075. 17. Stephenson,J. R., A. S.Khan, A. ILSliski,and M.

Easex1977.Felineoncomavirus-associated cell mem-braneantigen: evidence for an immunologically cross-reactivefeline sarcoma virus-coded protein. Proc. Natl. Acad. Sci. U.S.A. 74:5608-5612.

18.Stephenson, J. R., A. S.Khan,W. J. M. Van de Ven, and F. ILReynolds,Jr. 1979.TypeC retroviruses as vectorsforcloningcellulargenes withprobable trans-formingfunction. J. Natl.Cancer Inst. 63:1111-1119. 19.Van deVen, W. J. M., A.Khan, F. ILReynolds,Jr.,

K.T.Mason,and J.R.Stephenson.1980. Transla-tionalproductsencodedby newly acquired sequences ofindependentlyderived feline sarcomavirus isolates

arestructurallyrelated. J.Virol. 33:1034-1045. 20. Van deVen, W. J. M., F.ILReynolds, Jr., and J. R.

Stephenson. 1970.The nonstructural components of

polyproteinsencodedbyreplication-defective mamma-liantransformingretroviruses arephosphorylated and have associated protein kinase activity.Virology 101: 185-197.

21. Witte,0.N., N.Rosenburg,M.Paskind,A.Shields, and D.Baltimore.1978.Identification ofanAbelson murine leukemia virus-encoded protein present in

transformedfibroblasts andlymphoidcells.Proc. Natl. Acad.Sci. U.S.A.75:2488-2492.

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Figure

FIG.1.plOMolecularspecificityminkovalbuminoftatedFeL Immunoprecipitation and SDS-PAGE analysis ofpolyproteins encoded by the McDonough strain V
FIG. 2.pulse-labeledMolecularPAGEanalyzedGardnerCl15-1 Comparison ofMcDonough FeSVP170 and Gardner FeSVP115 by immunoprecipitation and SDS- analysis
FIG. byandscribedcpm) by [3SJmethionine-la-beledpeptides[35S]methionine-labeledminkP170PrI80gap'legendV Pr1809`'"Pl are labeled 1 through FeLV FeL FeSV that were were and cells
FIG. 5.tryptic Identification of [36SJmethionine-labeled peptides shared by McDonough FeSV P170
+5

References

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