0022-538X/80/07-0165/11$02.00/0
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,Maryland21701Inthisstudy,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),
andproteinA-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
ofaspecific 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
a150,000Mrcellularphosphopro-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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[image:3.508.110.393.396.592.2]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 FeLVPr1809-°IIo`
andMcDonoughFeSV P170. Theabove 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 FeLVp30spe-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), andFeLVPrI80gap' (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 withre-spect to
[35S]methionine-labeled
tryptic peptide composition.The resultsdemonstratedtwome-B
D
2
[image:5.508.53.241.76.452.2]0
4II
SFIG. 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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[image:5.508.253.447.324.586.2]170 VAN DE VEN ET AL.
A
4
6..
8 9 3 5 7 102' 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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[image:6.508.272.459.298.561.2]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
e4
[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 isolatedbyimmunoprecipitation 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(lanesGand 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 thedemonstration that the acquired sequence-en-coded translational products of the two FeSV isolates, Gardner and Snyder-Theilen, are
im-munologically
andstructurally related(18),andbytheresultsofmolecularhybridizationstudies
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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]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, althoughunlikely,
cannotbeexcluded.Alternatively, the possibil-ity that polyproteins encoded by each of the three transforming viruses contain a commonregionmustbeconsidered.If thelatter modelis correct, this common region is apparently
en-codedbysubsets ofthe sequences
acquired
bythe 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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[image:10.508.272.459.84.368.2]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).
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