0022-538X/79/11-0507/10$02.00/0
Polyprotein
Precursors to Mouse Mammary Tumor Virus
Proteins
S. J.ANDERSON,' R. B. NASO,'*J. DAVIS,2AND J.M. BOWEN2
DepartmentofBiology'andDepartment of Molecular Carcinogenesis andVirology,2 The University of Texas
System
CancerCenter,
M. D.AndersonHospital
and TumorInstitute, Houston,Texas77030Received forpublication16February1979
Mouse mammary tumor virus (MMTV) derived from the culture medium of
GRcellscontainedseven proteins, identifiedasgp55, gp33, p25, pp2O, p16, p12,
andplO. The major viral phosphoproteinwas the 20,000-molecular-weight pro-tein, pp2O. Immunoprecipitation of cytoplasmic extracts frompulse-labeledGR cells identified three MMTVgag-specific proteins, termed Pr78WaF, Prl1110F, and Prl80V'9. These intracellular polyproteins were precipitable from cytoplasmic
extractsbyantiseratovirionsp25and p12 butnotbyantiseratogp55.Themajor
intracellulargag-specific precursorpolyprotein, Pr78gag, contained antigenic de-terminants andtryptic peptides characteristic ofp25, p12,plO, and presumably pp2O. Thisprecursor ispresumably derived fromnascentchain cleavageorrapid posttranslational cleavage of the larger intracellular precursor-like protein, des-ignated Pr11099. Pr11099 containedall but oneoftheleucine-containingtryptic peptides ofPr789"9, plus several additional peptides. Inadditionto Pr78gw and Pr1109`9, monospecific antisera to virion p12 and p25 were also capable of precipitating from pulse-labeled cells a small amount of a 180,000-molecular-weightprecursor-like protein, designated Prl80gaF. Thislarge polyprotein con-tainednearly all of the leucine-containing tryptic peptides of Pr789, and
Prll0g10
plusseveral additionalpeptides. By analogy to type C viral systems,Pr180Y9 is
presumedtorepresent agag-polcommonprecursorwhichisthemajorpathway forsynthesis ofMMTVpolymerase. Immunoprecipitationofcytoplasmicextracts frompulse-labeled cells with antiseratogp55identifiedtwoenv-specificproteins, designated gPr76en" and
gp79env.
The major env precursor, gPr76en", could be labeled with radioactive glucosamine andwasshowntocontainantigenic deter-minants andtryptic peptides characteristic ofgp55 andgp33. Aminor glycopro-tein,gP79env,
contained both fucose andglucosamine andwasprecipitable from cytoplasmicextractswithmonospecificserum togp55. Itissuggested thatgP79env
represents fucosylated gPr76en" which is transiently
synthesized
and cleavedrapidly intogp55andgp33.
Theprecursor-product relationships of
virus-specific
proteins during the replication ofmurine and avian type C retroviruses have been well established (2, 6, 13). The presence of mouse mammary tumor virus (MMTV) polyproteinprecursors in mouse mammary tumorcells has
also beenreported (8-11,28, 30,34,35). Inthis
paper,we describethe furthercharacterization
of MMTVproteins andtheintracellular
precur-sorsfrom whichthey are derived. Specific
im-munoprecipitation of intracellular precursors andanalysisoftrypsin-digestedMMTVproteins
bycolumnchromatographyconfirmed that viral
proteins p25,p12, and plO are derivedfroman intracellular precursor polyprotein, Pr789ag,
which migrates as a doublet ofapproximately
78,000 molecular weight. Inaddition, however,
we report here on the presence oftwo larger
polyproteins, termed
Prllg0
andPr1809'9+,
whichalso contain the
antigenic
properties
and peptide sequencescharacteristic of the MMTV core proteins. Both of these precursors are shown to contain extrapeptide
sequences not accounted for by their content of known core proteins. We have also identified what we be-lieve to be a fucosylated form of the MMTV glycoprotein precursor,gPr76en".
This minor polyprotein, termed gP79env, contains both the antigenic properties and peptide sequences ofMMTVgp55 andgp33 andcanbelabeledwith
either
[3H]glucosamine
or[3H]fucose.
MATERIALS
AND METHODSCellculturesandvirus.MMTV waspurifiedfrom
the culture medium ofaGRmousemammary adeno-carcinomacellline(23, 32).Thecellswerepropagated 507
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Island, N.Y.) with a glucose concentration of 4,500 mg/liter, supplemented with 10% fetal calf serum (GIBCO), insulinat 10,g/ml, dexamethasoneat 1 Lg/ ml, and gentamicinat 50jLg/ml (DHG+DI medium). Production of MMTV was monitored routinely by sodiumdodecylsulfate-polyacrylamidegel electropho-resis (SDS-PAGE) of sucrose gradient-purified virus (density, 1.16 to1.18 g/ml) labeled with [3H]leucine. Virionpolypeptide labeling. The GR cells grown to confluency in490-cm2 plastic roller flasks were used 14 days aftersubculturing. Cells were labeled by in-cubation in 50ml of methionine-free DHG+DI me-dium towhich had been added 2.5 mCi of [35S]methi-onine (910Ci/mmol). After a24-hincubation, 25 ml of nonradioactive complete DHG+DI medium was added, and the incubation was continued for an addi-tional24h. The medium was then removed from the cells and clarified of debris by centrifugation at 16,300 xg for 10min. Crude virus pellets were obtained by centrifugation oftheculture fluidsat80,000 xg for 2 h.The virus pellets were suspended in buffer contain-ing 0.01 MTris-hydrochloride (pH 7.5), 0.1 MNaCl, and1mMEDTA(TNE) and banded by centrifugation at 38,000 x g for 16 hthrough a 15 to 60%sucrose gradient in TNE. Virus banding at 1.16 to 1.18 g of sucroseper ml was diluted in TNE and pelleted by centrifugationat80,000xg for 2 h.
For comparison, virion proteins were sometimes labeled with 5 mCi of [3H]leucine (30 Ci/mmol) in mediumat 50%theusual concentration of amino acids
orwith[32P]phosphateinphosphate-free medium.
Antisera. Antisera to MMTV proteins were pre-pared in rabbits (1, 13) by injection of preparations of viral proteins. Gradient-purified MMTV disrupted with Nonidet P-40 and sodium deoxycholate (0.5%) was usedto prepare anti-MMTV sera. Viral proteins from detergent-disrupted virus were also separated into glycoprotein and nonglycoprotein fractions by chromatography on concanavalinA (J. Davis, manu-script in preparation). Antisera to viral gp55, p25, and p12 weremade using proteinsisolated by isoelectric focusing of concanavalin A-chromatographed MMTV glycoproteins and nonglycoproteins (Davis, in prepa-ration). Anti-p25 serum was also prepared, using p25 purified by SDS-PAGE. When indicated, antisera were preabsorbed with cytoplasmic extracts of Rauschermurineleukemia virus (RLV)-infected NIH Swiss mousecells (JLS-V16) or with extracts ofGR cells grownin the absenceof dexamethazone (1, 25). Antisera were testedfor specificity byprecipitation of detergent-disrupted [3H]leucinevirus andanalysis of theprecipitates by SDS-PAGE and autoradiofluorog-raphy.
Labeling andextraction of cytoplasmic viral
proteins. Cytoplasmic proteins were labeled by the additionof 2.0 mCiof[35S]methionine(910Ci/mmol), 5mCi of[3H]methionine (40 Ci/mmol), or 5 mCi of [3H]leucine(45Ci/mol)in20 ml ofEarlebalanced salt solution to cellsgrowntoconfluencyin150-cm2plastic culture flasks. Cells were thenlysed immediately or subjectedto achaseperiod by removal of the isotope andsubsequentincubation inDHG+DImedium. Iso-lationofcell-associated viral proteins by
immunopre-previously (1, 25). Immunoprecipitates were pelleted bylow-speedcentrifugation and washed twice in buffer containing NonidetP-40and sodiumdeoxycholate (1, 25). Whenindicated, immunoprecipitation wasaided by absorption of the immune complexes with the Cowan I strain ofStaphylococcus aureus (17, 29). Long-termpulsing of4.5 hwith [3H]glucosamine (38 Ci/mmol)or[3H]fucose (24Ci/mmol)wasdone in 10 ml ofDHG+DI per flask.
SDS-PAGE. SDS-PAGE of immunoprecipitated cytoplasmic and virion proteins was performed in 11.25% or continuous-gradient 6 to 12% polyacryl-amide slab gels containing SDS and using the Tris-glycine buffer system aspreviously described (1, 25). Gels containing tritiated samples were treated with dimethyl sulfoxide-PPO(2,5-diphenyloxazole) (5)and exposedtopreflashed X-ray film (20).
Tryptic digestion and column chromatogra-phy. Bandsshowingradioactivityonautoradiograms were cut fromgels, and the proteinswere extracted and digested with trypsin (1). The resulting poly-peptide fragments were separated by ion-exchange chromatographyonChromobead typePresin (Tech-niconCorp., Inc., Tarrytown,N.Y.) (1) witha logarith-mic gradient buffer of acetic acid andpyridine. Frac-tionswere collected sequentially,and the bufferwas
evaporated at95°C. TritonX-100-containing scintil-lation fluid (1) was added, and the radioactivity in each fractionwasmeasured inaPackard scintillation counter.
Nomenclature. In this study,we are usinga
re-cently adopted nomenclature for oncornaviral poly-peptides agreed uponat aNational Cancer Institute-sponsored Tumor ViralImmunology Workshopheld inArlington, VA.,on 8-9March1977.Theterminology had been previously introduced (15) andapplied to MMTV proteins (27). The estimated molecular weights of thevirus-specificproteins observed in these studies are in close agreement with the results of Nusse etal.(27) andapproximatethe results of others (8-11, 30). In these studies, gp55 and gp33are often referredto asgp52orgp49 and gp36orgp34, respec-tively.Virionp25 is likewise referredtoasp27orp24, and similar minor variations in thesmaller viral pro-teinscanbe noted. Whenpublished values differ from estimates of theprotein molecular weights presented inthis paper, published values will befollowed by our estimatesinparentheses.
RESULTS
SDS-PAGE of viralproteins.
Electropho-resis of MMTV obtained from the culture
me-dium ofradioactivelylabeled GR cells separates
proteins ofapproximately 55,000 (gp55), 33,000
(gp33), 25,000 (p25), 20,000(pp2O), 16,000 (pl6),
12,000(p12), and 10,000(plO) molecular weights,
asdetermined by comparison with theproteins
ofRLV (Fig. 1). Labelingwith [35S]methionine
or
[3H]leucine
indicated that each of these viralproteins contained methionineaswellasleucine.
The SDS-PAGE profiles of MMTV derived
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A
B
C
D
E
9 i-mago*
p80
(,ol)
gp7O
Pr
65
ga
gp55
gp 33
p25
pp20
gp55
gp33
am.
p30
6U
p16
p12
p25,
pp2O
p16
p12
10
....
<
plO
FIG. 1. SDS-PAGEoftheproteins ofMMTV and RLV. Viruslabeled with[3H]leucine, [35S]methionine,
or[32P]phosphatewaspurified by isopycnic gradientcentrifugation,and viralproteinswereseparatedonan
11.25%polyacrylamide slab gel. (A) MMTV labeled with[3HJleucine(10,000cpm); (B)MMTV labeled with
[35S]methionine (10,00X cpm); (C) RLV labeled with[3HJleucine (15,000 cpm); (D) MMTV labeled with
[3H]leucine(10,000 cpm); (E) MMTV labeled with[32P]phosphate (3,000 cpm). Virus bandsarevisualizedby fluorographyasdescribed in Materials and Methods.
from GR mouse mammary tumor cells lacked anyprotein bands migrating
parallel
tothep30 of RLV.Therefore,
weconcludedthat releaseof type C retrovirus by GR cells was negligible. Other minor bands were present in virus, the most evident being those of approximately 50,000 and 29,000molecularweight.Labeling of virion proteins with
[32P]phos-phate yielded bands in SDS-PAGE that mi-gratedparallel
to mostofthemajor proteins ofa
[3H]leucine-labeled
MMTVpreparation, withtheexception ofp12 (Fig. 1,lane E).Judging by
theappearanceof theradiogram corresponding
tothisgel, the protein of20,000molecularweight is themajor phosphorylated protein.We there-fore have designated this protein
pp20
(S. J.Anderson and R. B. Naso, Abstr. Annu. Meet.
Am. Soc. Microbiol. 1978,S270, p. 257).
Characterization of antisera. Antisera to
viralproteins were used to precipitate
virus-spe-cificproteinsfrompulse-labeled GRcells.Before
this,these sera were characterizedbytheir abil-ity to precipitate isotopically labeled proteins from detergent-disrupted virions. Results of
theseserumcharacterizationsareshown inFig. 2.Anti-gp55serum,undertheseconditions, pre-cipitated only gp55 from detergent-disrupted virions, whereas anti-p25 serum precipitated
only p25, with trace amounts of smaller viral
proteins. Anti-p12 serum precipitated p12 and
trace amounts of p25 fromdetergent-disrupted
virus(resultsnotshown).
Immunoprecipitation of virus-specific proteins from infected cells. Precipitationof pulse-labeled viral proteins from cells with either anti-p25or
anti-p12
serumresulted inrecovery of radioactiveproteins ofapproximately 78,000 (Pr789'9), 110,000(Pr1099),
and 180,000(Prl80ga)
molecularweight (Fig.3,lanesAand D, respectively). The Pr789'9 protein appeared as acloselymigrating doublet characterized byasharp lower band under a hazy upper band.
The Pr1109'9 and
Prl80'ga
bands were minor compared with the Pr789'9 bands. Analysis bySDS-PAGE of the
immunoprecipitable
cyto-plasmic proteins labeledbya 15-min pulse
fol-lowedbyachase of 1 or 3 h revealed agradual
decrease inradioactivityof thelargeprecursors
32,1979
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E
opwp
15
p
12 E &
PP12
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gp55
qp 55
.
gp33
FIG. 2. Characterization of antisera against MMTVproteins. [3H]leucine-labeled MMTV was solubilized, andequalportions were immunoprecip-itated by (A) anti-gp55, (B) anti-p25, or (C)
anti-MMTVserum.Immunoprecipitateswereanalyzed by SDS-PAGEin 11.25%gelsandscintillation
autora-diography asdescribed in Materials andMethods.
(lanes B andC,and E andF),concomitantwith
increased radioactivityinvirions released from
the cells (not shown). Onlyan extremelysmall
amount oflabel appeared during the chase as
intracellular p25 (lane C), and no intracellular
p12 was apparent. Immunoprecipitation ofcell
extractsfromsimilarlypulse-labeledand
chase-incubated cells with antiserum togp55revealed
the presence of a protein of 76,000 molecular weight, termed gPr76en, (lane G). Thisprotein
wasalso reduced inspecific activity duringthe
chase period, but its removal was concomitant
with theappearanceoflabelinthe gp55 region
of thegel.
Several background bandsrepresenting
non-specific precipitation are also evident in this
figure. They can be recognized by their ability
tobeprecipitatedby alloftheseraandby the
rather limited changes in these bands during
chases.
Labeling of MMTV precursor
polypro-teins with [3H]glucosamine or [3H]fucose.
Figure4shows theSDS-PAGE profileof
intra-cellular virus-specific proteins labeled with
[3H]glucosamineor[3H]fucoseand
immunopre-gp55
serum.[3H]glucosamine
wasincorporated
into
gPr76env,
gp55,
andgp33
(lanes
A andC),
butthese
proteins
couldnot beprecipitated
by
anti-p25
serum(lane
B).
These threeproteins
also could be labeled with
[3H]fucose (lanes
Dand
F).
In addition togPr76env,
however,[3H]-fucose
appeared
to beincorporated
into a pro-tein ofapproximately
79,000
molecularweight,
termed
gP79en". Again,
anti-p25
serum did notrecognize
any[3H]fucose-labeled
proteins
(lane
E).
Therelativeabsenceofthisfucosylated
pro-tein in
experiments
labeling
with[3H]leucine
(see
Fig. 3)
or[3H]glucosamine
suggested
thatgp79env
is present in very low amounts and isapparently
fucose rich. It is also obvious thatgp55
andgp33
labeled better with[3H]fucose,
than with
[3H]glucosamine
under thelabeling
conditions used.
Tryptic digestion
andchromatographic
analysis
ofvirion coreproteins
and theirintracellularprecursors.Virionand
cytoplas-mic
proteins
werelabeledwith[35S]methionine,
[3H]methionine,
or[3H]leucine
as describedabove. Radioactive
proteins
were separatedby
SDS-PAGE and locatedby
radiofluorography.
Regions
corresponding
to bands in theautora-diograms
were cutfrom thegels,
and thepro-teins inthese slices were
digested
withtrypsin
(1, 24, 33). Polypeptide
fragments
weresepa-rated
by
ion-exchange
columnchromatography
(1).
Eluted radioactive counts per fraction areshown
(Fig.
5and6).
Figure
5A-Cshows thepeptide
maps ofp25,
p12,
andplO cochromatographed
with Pr789'9peptides.
Theprecursor-product relationshipis obvious from themapsofoverlappingpeptides.
The
region
of eluent with comigrationpeaks
corresponding
to all three viralproteins
mayrepresent
methionine-arginine
ormethionine-ly-sine
dipeptides
present in each protein. Onemajor peak
and several minorpeaksinPr78sas
areunaccountedfor
by
thethreeviralproteins
analyzed.
Theseregions,
inadditiontothe void volumeof thecolumn,
representpeptideswhich suggest the presence of a fourth viralprotein,
presumably pp2O,in
Pr785a.
Figure
6A-Cshows the peptide map of[3H]-leucine-labeled
Pr18099a+, Pr1109ya,
andPr789'9,
precipitated from cytoplasmicextractswith
anti-p25
serum. The maps show that Prl1019con-tained all but one of the peptides (panel
C,
asterisk)
characteristic ofPr789as
plus sevenotherpeptides (panel B,asterisks) notfound in
Pr78'as. Pr18099a+
likewisecontainedallbutone oftheleucine-containing
peptidescharacteristic ofPrllOgag (missing peptideinfraction11,panel
B)
andwascharacterizedby
approximatelyfiveon November 10, 2019 by guest
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A
B
*"MI_
C
~~~~~~~~~~~~-:D
E
F
G
H
I
4'-.
Prl8Og9g+
Pr
789ag
v.
*gPr
76env
.4w11:---,.
gp
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FIG. 3. SynthesisandcleavageofMMTV precursorpolyproteins. CulturesofGR cellswerepulse-labeled for15minwith 100,sCiof[3HJleucineperml(A, D, andG)andchase-incubatedforIh(B, E,andH)or 3h
(C, F,andI).Equal portions ofthecytoplasmicextracts wereimmunoprecipitatedwithanti-p25 (A-C),
anti-p12(D-F),andanti-gp55(G-I)sera.Immunoprecipitateswereanalyzedasdescribedfor Fig.2.Equalvolumes
ofthe solubilizedimmunoprecipitates wereappliedtothe gel.
additionalpeptides not found in either Pr780`9
orPr1109'9.
DISCUSSION
SDS-PAGE
analysis
of MMTVproteins
la-beled with[3S]methionine
indicates that all the viral proteins contain methionine. Of the sixtoseven
virus-specific proteins
identified,
thepro-tein of 20,000 molecular
weight,
termedpp2O
(Anderson andNaso, Abstr. Annu. Meet. Am. Soc. Microbiol. 1978,
S270,
p.257)
wasshownto be mostheavily
phosphorylated,
in agreement with the results of otherinvestigators (27, 33).
Most,
ifnotall,
of the other viralproteins
are alsophosphorylated,
butto alesserextentthanpp2O
(28,33; Anderson andNaso,
Abstr. Annu.Meet. Am.Soc. Microbiol. 1978,p.257).Results
from the immunoprecipitation
analyses
pre-sented here confirm that gp55 and gp33 arise
fromacommonintracellular precursor,termed
gPr76env
(10, 11, 34, 35; Anderson and Naso,Abstr. Annu.Meet. Am. Soc. Microbiol.1978, p.
257).TheMMTVglycoproteinsandtheirmajor
intracellular
precursorgPr76env
can be labeledwellwith[3H]leucine or[3H]glucosamine. How-ever, labelingwith[3H]fucose indicates that syn-thesis ofgp33 and gp55 may involve a minor,
high-molecular-weight
and heavily fucosylated polyprotein termed gp79env. The major glyco-protein precursor,gPr76enV, doesnotlabelaswell with[3H]fucoseasit doeswith[3H]glucosamine andmaybe fucose deficient. Itissuggested that gp79env represents fucosylatedgPr76env
which is transiently made and cleaved rapidlyinto gp55andgp33.Similar resultshavebeen observed in
the synthesis of Moloney leukemia virus gp7O (T.Gordon Wood, personal communication). In thecaseof theleukemia virus glycoprotein, the
higher-molecular-weight
glycosylated polypro-tein (gP93e"v) can be labeled efficiently withradioactivefucose, whereas the major
glycopro-teinprecursor
gPr83env
lacks fucose. Theleuke-mia virus glycoprotein (gp7O) and the MMTV
glycoproteinscontain bothglucosamineand fu-cose.
Similaranalysesconfirm that a
78,000-molec-ular-weight protein doublet, Pr789", has anti-genic deterninantsin common with at least two
32,1979
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A
B
C
D
E
F
::
:
g~.0Pr76en
*
.w
_
p33
gp33
FIG. 4. Synthesis ofMMTVglycoproteins andglycopolyproteins. GR cells were labeledfor 4.5 h with [3H]glucosamine (A-C)or[3H]fucose (D-F). Equal portions ofthecytoplasmicextractswere
immunoprecip-itated withanti-gp55 (AandD),anti-p25(BandE),oranti-MMTV(CandF)sera.Immunoprecipitateswere analyzedasdescribed inFig.2.
ofthe core proteins ofMMTV (p25 and p12).
Tryptic peptide mappings confirm the
precur-sor-productrelationship ofPr789'-'andp25 and,
inaddition, indicate thatPr789a- shares peptides
in common with the p12 and plO ofthe virus.
TheseresultsaresimilartothoseofDickson and
Atterwill (9). Again byanalogytoRLV, thegag
precursorofMMTVmaybe expectedtocontain four viralproteins,includingthemajor
phospho-protein pp2O. We have been unable to obtain
enoughradioactivity inpp2O to analyze its
me-thionine-containing peptides. This is
presum-ably due toits lowmethionine contentrelative
to the other viral proteins (see Fig. 1, lane B). Other workers have shown that the major MMTVgagprecursor,Pr73'a`
(Pr78a'),
isphos-phorylatedtoyieldpPr759ag(27).This
phospho-rylatedgag precursor presumably corresponds
to the upper band in the Pr789ag doublet
re-ported here.This isconsistentwithother results
concerning thephosphorylationof RLVgag
pre-cursor(pMr59ag) andsuggeststhatpp2Omaybe
presentin Pr789ag and phosphorylated initially while in thatprecursorform (R. B.Naso, W. L.
Karshin, Y. H. Wu, and R. B.Arlinghaus,
sub-mitted for publication). Trypsin digestion of
each of the [35S]methionine-labeled Pr789as bands yields identical peptide maps when
ana-lyzed byion-exchange chromatography (results
notshown).
Chase experiments generally indicate that
gPr76enL ismorerapidlyturnedoverincultured
GR cells than is Pr78enl. Chasing permits the
appearance of isotopically labeled gp55 in the
cell, presumably via cleavage of gPr76enl or
gp79en , or both. Chase incubation, however,
doesnot result inthe appearance ofsignificant
amountsof p25orothergagproteinsinthecell,
eventhough Pr789a5 appearsto chase andviral
proteins appear in virus particles in the chase
medium. These results suggestaninefficient pre-cursorprocessing, buttheymayalsoreflectthe
precursor-product relationship between the A
particlesandBparticlesofMMTV(37). Tanaka
(38)has shown thatintracytoplasmicAparticles represent the pronucleocapsids of MMTV. A
particles are composed of polypeptides of
ap-proximately 70,000 molecular weight which
carry the antigenicities ofthe virion core
pro-teins, p25,p15(p12),andp7 (plO).Incubation of
Aparticlesresulted inthegenerationofthecore
proteins of MMTV (38). The P70 observed by
TanakapresumablyisanalogoustothePr789'9 identified in this study. The cleavage products of thisprecursor,therefore,wouldnotbepresent in the cell until the A particles mature to B
particles. The extremely low levels of p25 in
infected cells suggest that thismaturationoccurs
coincident with or shortly afterbudding ofthe virus.ThepreviousworkbyDickson and
Atter-'Pr-Ti'~fV
-gp3
""e
5,
I 1K0
P
1;
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E
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'_
._c
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3
2
D17
-Pr780413Hmet)
---p25(35S-met)
180 Fraction Number
FractionNumber
p25 p12
p10 -Pr789U9(3H-met)
---plO(35fSmet)
20 40 60 80 100 120 140 160 FractionNumber
FIG. 5. Ion-exchange chromatography oftrypsin-digested
[3H]methionine-labeled
Pr78`9 cochromato-graphed with similarly digested[35S]methionine-labeledp25
(A),p12(B),orplO(C). Identities ofsomeofthe peptidesas totheirpresencein viralproteinsareindicatedabove eachpeak.Thearrowsmarkthe elutionofthe columnwith2Mpyridineacetate(13).
513
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, 12
o
1o02
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6 25*p5
2~~~~1
20 40 60 80 100
120140160
2M
Fraction Number
FIG. 6. Ion-exchange chromatography of trypsin-digested [3H]leucine-labeled Pr7&RC, PrJJ1f15, and Prl80"g+. Results wereplotted on the samegraph basedon alignment of["4C]tyrosine-labeled tryptic
peptides of RLVp3O (arrows) whichwereusedas an
internal marker in each chromatography run (12).
Theheavy bars mark elution of thecolumn with 2 M pyridineacetate.
will also suggests this possibility (9). In these
characteristics, thereplicationof MMTV is
dis-tinguishedfrom that of type C virusreplication.
In thelatter,intracellular levels ofvirionp30are evidentevenduring relativelyshort chase
incu-be shown toproceed normally in the cell even
when virus assembly or budding is prevented
withinterferon treatment (36; Y. H. Wuand R.
B. Naso,manuscript in preparation). Similarly,
typeC gag- andenv-specificprecursors
synthe-sized during the S phase ofsynchronized cells
arecleaved in the cell before virus releaseduring
thesubsequentmitosis(26).
Continuing the analogy with murine type C
viruses, Long et al. (21) have observed that
MMTV p14 (p12) binds efficiently to
single-stranded DNA and may,therefore,beanalogous
tomurine type C virus plO (4, 14). The further
comparisons of MMTV p25 with RLV p30 as
themajorcoreprotein,MMTVpp2Owith RLV
ppl2 as the major virion phosphoprotein (16,
28), and MMTV plO with RLV p15astheonly
virion envelope-associated polypeptide coded for
by the gag regions of their respective virion
genomes indicate further analogy (7, 36). The
order of gagproteinsin the RLV gag precursor,
Pr65sas, has been shown to be H2N-p15-ppl2-p30-plO-COOH (3, 24, 31). An analogous order
forMMTV gag precursor, Pr78aS, ispredicted
tobeH2N-plO-pp20-p25-p12-COOH.
Recently,Dahland Dickson(8)translatedthe genomic RNAof MMTV ina cell-free
transla-tion system derived from mouse L cells and
rabbitreticulocytes. They identified among the
translationproductsproteinsof 77,000, 110,000,
and 160,000 molecular weights. All three
pro-teinswereprecipitable byantiserumtothe
ma-jorcore proteinof MMTV and the 110,000-dal-ton protein contained methionine-labeled
pep-tidescharacteristic of the 77,000-dalton protein
plusadditionalpeptides.Ourresultsindicate the
existence of three similarprecursor-like
polypro-teins,
Pr78sas,
PrI109a9, and Prl809a9+, inin-fected cells. Theprecursor-product relationships
ofthesepolyproteinstothe viralcoreproteinsis
concludedbasedontheirantigenic specificities, peptide compositions, and kinetics of
appear-ance and removal during pulse-chase studies.
The presence ofthecompletecoreprotein
com-plement in Pr78sas suggests that this
MMTV-specific precursor is analogous to the
65,000-dalton precursorprotein,
Pr65Ras,
of murine typeC viruses. The primary translation product of
the typeC virus gag gene,however, isamolecule
of 80,000daltons,Pr809ag,which containsall the
core sequences plus additional information
which may represent a virus-specific protease
(1, 12, 40). We suggest that theMMTV-specific
Prll0gag polyprotein represents the primary
translationproduct of theMMTVgag geneand
is, therefore, analogous to the type C viral
Pr809ag. In addition to core and envelope
pro-teins, MMTV also contains a RNA-dependent
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[image:8.507.71.240.55.522.2]VOL. 32,
DNApolymerase (22). In murine type C viral
systems, the70,000 to80,000-daltonpolymerase
is synthesized via a 180,000 to 200,000-dalton
precursor polyprotein,
Pr20(9'"',
whichcon-tains both core proteins and polymerase
antigen-icities and peptide sequences (18, 19). We
sug-gest that theMMTV-specific Pr1809`9+ is
anal-ogous tothetype CviralPr2009a9-P° and
repre-sents the primaryprecursor to MMTV
polym-erase in the infected cell. Further studies on
MMTV polymerase and Pr180919+ will be
nec-essary to establish this presumed relationship.
ACKNOWLEDGMENTS
Wethank Maria Elena Lerouxand Norma Mindiola for experttechnical assistance.
S.J.A. is therecipientofaRosalie B. Hitefellowship,and R.B.N. is aLeukemiaSocietyof America Scholar. This work wassupported by a grant from the American CancerSociety (NP 245) and a Public Health Service grant(RR5511-14) from theNational Institutes of Health.
ADDENDUM INPROOF
While this manuscript was in press, Dickson andAtterwill (Cell 17:1003-1012, 1979) reported the composition of two GR-MMTV precursorpolyproteins,Pr77,"w andp110"''.They also observedaviralpolyprotein, termedp30,which appar-ently contains all of the peptides of p14 (p12) and additional peptides uniquetoPrl101"'.Duetothecomplexity of the two-dimensional maps ofplI0"l' andPr77,"',however, several of thepeptidespotsreportedtobeuniquetop110"`' appearedto
comigratewithpeptidesofPr77"'',confoundingattemptsto
assign someof thesepeptides uniquely toplil"g". Peptide analysisofp30and other intermediate-sizedpolyproteins sug-gestedthat the gene orderinP110""w isplO-pp21-p27-p14-X, with Xrepresenting the uniquep110""9peptides. Based on the sizedifferential ofPr77"''andplIO1",onewould expect the peptidesrepresentingXtoapproach33,000molecularweight. Ifp30 contains p14, thenonly 16,000molecular weight of protein remains as pl10"ga-specific peptides. This suggests that about 17,000 molecular weight ofprotein unique to p110" remains unaccounted for. Dicksonand Atterwill's map ofp1109'9does appeartocontainseveralpeptidesuniqueto
p1109`"butabsent inp30.Theplacementof these sequences atN-terminalorC-terminal ends ofpllO0' remains specula-tive.
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