• No results found

Polyprotein Precursors to Mouse Mammary Tumor Virus Proteins

N/A
N/A
Protected

Academic year: 2019

Share "Polyprotein Precursors to Mouse Mammary Tumor Virus Proteins"

Copied!
10
0
0

Loading.... (view fulltext now)

Full text

(1)

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

Cancer

Center,

M. D.Anderson

Hospital

and TumorInstitute, Houston,Texas77030

Received 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 that

gP79env

represents fucosylated gPr76en" which is transiently

synthesized

and cleaved

rapidly 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) polyprotein

precursors 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

and

Pr1809'9+,

whichalso contain the

antigenic

properties

and peptide sequencescharacteristic of the MMTV core proteins. Both of these precursors are shown to contain extra

peptide

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 of

MMTVgp55 andgp33 andcanbelabeledwith

either

[3H]glucosamine

or

[3H]fucose.

MATERIALS

AND METHODS

Cellculturesandvirus.MMTV waspurifiedfrom

the culture medium ofaGRmousemammary adeno-carcinomacellline(23, 32).Thecellswerepropagated 507

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

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 viral

proteins contained methionineaswellasleucine.

The SDS-PAGE profiles of MMTV derived

on November 10, 2019 by guest

http://jvi.asm.org/

(3)

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-grated

parallel

to mostofthemajor proteins of

a

[3H]leucine-labeled

MMTVpreparation, with

theexception 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 by

asharp lower band under a hazy upper band.

The Pr1109'9 and

Prl80'ga

bands were minor compared with the Pr789'9 bands. Analysis by

SDS-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

1-1

pis

E

opw

p

15

p

12 E &

PP12

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.507.100.401.72.338.2]
(4)

.

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

was

incorporated

into

gPr76env,

gp55,

and

gp33

(lanes

A and

C),

butthese

proteins

couldnot be

precipitated

by

anti-p25

serum

(lane

B).

These three

proteins

also could be labeled with

[3H]fucose (lanes

D

and

F).

In addition to

gPr76env,

however,

[3H]-fucose

appeared

to be

incorporated

into a pro-tein of

approximately

79,000

molecular

weight,

termed

gP79en". Again,

anti-p25

serum did not

recognize

any

[3H]fucose-labeled

proteins

(lane

E).

Therelativeabsenceofthis

fucosylated

pro-tein in

experiments

labeling

with

[3H]leucine

(see

Fig. 3)

or

[3H]glucosamine

suggested

that

gp79env

is present in very low amounts and is

apparently

fucose rich. It is also obvious that

gp55

and

gp33

labeled better with

[3H]fucose,

than with

[3H]glucosamine

under the

labeling

conditions used.

Tryptic digestion

and

chromatographic

analysis

ofvirion core

proteins

and their

intracellularprecursors.Virionand

cytoplas-mic

proteins

werelabeledwith

[35S]methionine,

[3H]methionine,

or

[3H]leucine

as described

above. Radioactive

proteins

were separated

by

SDS-PAGE and located

by

radiofluorography.

Regions

corresponding

to bands in the

autora-diograms

were cutfrom the

gels,

and the

pro-teins inthese slices were

digested

with

trypsin

(1, 24, 33). Polypeptide

fragments

were

sepa-rated

by

ion-exchange

column

chromatography

(1).

Eluted radioactive counts per fraction are

shown

(Fig.

5and

6).

Figure

5A-Cshows the

peptide

maps of

p25,

p12,

and

plO cochromatographed

with Pr789'9

peptides.

Theprecursor-product relationshipis obvious from themapsofoverlapping

peptides.

The

region

of eluent with comigration

peaks

corresponding

to all three viral

proteins

may

represent

methionine-arginine

or

methionine-ly-sine

dipeptides

present in each protein. One

major peak

and several minorpeaksin

Pr78sas

areunaccountedfor

by

thethreeviral

proteins

analyzed.

These

regions,

inadditiontothe void volumeof the

column,

representpeptideswhich suggest the presence of a fourth viral

protein,

presumably pp2O,in

Pr785a.

Figure

6A-Cshows the peptide map of

[3H]-leucine-labeled

Pr18099a+, Pr1109ya,

and

Pr789'9,

precipitated from cytoplasmicextractswith

anti-p25

serum. The maps show that Prl1019

con-tained all but one of the peptides (panel

C,

asterisk)

characteristic of

Pr789as

plus seven

otherpeptides (panel B,asterisks) notfound in

Pr78'as. Pr18099a+

likewisecontainedallbutone ofthe

leucine-containing

peptidescharacteristic ofPrllOgag (missing peptideinfraction11,

panel

B)

andwascharacterized

by

approximatelyfive

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.507.82.232.56.310.2]
(5)

MMTV PRECURSOR PROTEINS 511

A

B

*"MI_

C

~~~~~~~~~~~~-:

D

E

F

G

H

I

4'-.

Prl8Og9g+

Pr

789ag

v.

*

gPr

76env

.

4w11:---,.

gp

[image:5.507.58.456.74.352.2]

55-p25

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 MMTV

proteins

la-beled with

[3S]methionine

indicates that all the viral proteins contain methionine. Of the sixto

seven

virus-specific proteins

identified,

the

pro-tein of 20,000 molecular

weight,

termed

pp2O

(Anderson andNaso, Abstr. Annu. Meet. Am. Soc. Microbiol. 1978,

S270,

p.

257)

wasshownto be most

heavily

phosphorylated,

in agreement with the results of other

investigators (27, 33).

Most,

ifnot

all,

of the other viral

proteins

are also

phosphorylated,

butto alesserextentthan

pp2O

(28,33; Anderson and

Naso,

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

precursor

gPr76env

can be labeled

wellwith[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 fucosylated

gPr76env

which is transiently made and cleaved rapidlyinto gp55

andgp33.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 with

radioactivefucose, whereas the major

glycopro-teinprecursor

gPr83env

lacks fucose. The

leuke-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

0 . . .qmm

on November 10, 2019 by guest

http://jvi.asm.org/

(6)

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'),

is

phos-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 1

K0

P

1;

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.507.116.405.71.304.2]
(7)

A

-3

4

B

3-0

' 2

m

1-B

913

C

x6

E

0

'_

._c

4

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 elutionof

the columnwith2Mpyridineacetate(13).

513

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.507.145.397.27.664.2]
(8)

'r-x

(U 0

-o cc

8

6~~~

*A

16 - C

>. 14

, 12

o

1o0

2

P 7gag

CU

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+, in

in-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 type

C 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

on November 10, 2019 by guest

http://jvi.asm.org/

[image:8.507.71.240.55.522.2]
(9)

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'"',

which

con-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.

LITERATURE CITED

1. Arcement, L. J., W. L. Karshin, R. B. Naso, and R. B. Arlinghaus. 1977. 'gag' polyprotein precursors of Rauscher murine leukemia virus. Virology 81:284-297. 2. Arlinghaus,R.B.,R. B.Naso,G. A. Jamjoom, and L. J.Arcement. 1976. Biosynthesis of Rauscher leukemia viralproteins, p. 293-309. In Anima,l virology, vol. 4. ICN-UCLASymposium on Molecular andCellular Bi-ology.AcademicPress,New York.

3. Barbacid, M.,J. R.Stephenson, and S.A.Aaronson. 1976. 'gag' gene ofmammalian type C RNA tumor viruses.Nature(London) 262:554-559.

4. Bolognesi, D.P.,R.Luftig, and J. H. Shafer. 1973. Localization of RNA tumor virus polypeptides.I. Iso-lationof furthervirus substructures. Virology 56:549-564.

5. Bonner,W.M., and R. A. Laskey. 1974. A film detection method fortritium-labeledproteins and nucleic acids in polyacrylamidegels.Eur. J.Biochem.45:83-88. 6. Butterworth,B. E. 1976.Proteolytic processing of animal

virus proteins. Curr.Top. Microbiol. Immunol. 77:1-41.

7. Cardiff,R.D., N. J.Puentes, L. J. T. Young,G. H. Smith, Y. A.Teramota,B. W.Altrock,and T. S. Pratt. 1978.Serological and biochemical characteriza-tionof the mammary tumor virus with localization of plO.Virology 85:157-167.

8. Dahl, H.-H. M., andC. Dickson.1979.Cell-freesynthesis of mousemammarytumorvirus Pr77 from virion and intracellular mRNA. J. Virol. 29:1131-1141.

9. Dickson,C., and M. Atterwill. 1978.Polyproteins re-lated to the major coreprotein of themousemammary tumorvirus. J. Virol. 26:660-672.

10.Dickson,C.,J. P.Puma, and S. Nandi.1975. Intracel-lular synthesis of mouse mammary tumor virus poly-peptides: indicationofaprecursorglycoprotein.J. Virol. 16:250-258.

11. Dickson, C.,J. P.Puma,and S. Nandi.1976. Identifi-cation of a precursorproteintothemajorglycoproteins ofmousemammarytumorvirus. J. Virol. 17:275-282. 12.Dittmar, K. J., and K. Moelling. 1978. Biochemical propertiesofp15-associated protease inaavian RNA tumorvirus. J.Virol. 28:106-118.

13.Eisenman, R., and V. Vogt. 1978.Thebiosynthesisof oncornavirus proteins. Biochim. Biophys. Acta 473: 187-239.

14.Fleissner, E., and E. Tress.1973.Isolationofa ribonu-cleoproteinstructurefromoncornaviruses. J. Virol. 12: 1612-1615.

15.Jamjoom,G.A.,V.L.Ng,and R.B.Arlinghaus.1978. Inhibition of maturation of Rauscher leukemia virusby amino acidanalogs.J. Virol. 25:408-412.

16.Karshin, W. L., L. J.Arcement,R.B.Naso,and R. B. Arlinghaus. 1977. Common precursor for Rauscher leukemia virus gp69/71,pl5(E) and p12(E). J. Virol. 23:787-798.

17.Kessler,S. W.1975.Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction ofantibody-antigen com-plexes with proteinA.J. Immunol.115:1617-1624. 18.Kopchick, J. J., G. A. Jamjoom, K. F. Watson, and

R. B.Arlinghaus. 1978.Biosynthesisof reverse tran-scriptase from Rauscher murine leukemia virus by syn-thesisand cleavage of a 'gag-pol' read-through viral precursorpolyprotein. Proc. Natl. Acad. U.S.A. 75: 2016-2020.

19.Kopchick,J.J., W. J.Karshin, and R. B. Arlinghaus. 1979.Trypticpeptide analysis of 'gag' and 'gag-pol' gene products of Rauscher murine leukemia virus. J. Virol. 30:610-623.

20. Laskey,R.A.,and A. D.Mills.1975.Quantitativefilm detection of'Hand'4Cinpolyacrylamidegels by fluo-rography. Eur. J. Biochem. 56:335-341.

21. Long,C. W., R.Berzinski, and R. V. Gilden. 1977. Immunologic studies of the low-molecular-weight DNA binding protein of murineoncornaviruses.Int.J. Can-cer.19:843-850.

22. Marcus, S. L.,N.H.Sarkar,andM.J.Modak.1976. Purification andpropertiesofmurine mammarytumor virus DNA polymerase.Virology 71:242-254. 23. Muhlbock,0.1965.Noteon anewinbredmouse-strain,

GR/A.Eur. J. Cancer.1:123-124.

24. Murphy,E.C., Jr.,and R. B.Arlinghaus.1978.Tryptic peptide analysesofpolypeptidesgenerated by prema-ture termination of cell-freeprotein synthesis allowa

determinationof the Rauscher leukemia virusgag gene order. J. Virol. 28:929-935.

25. Naso, R. B., L. J.Arcement, W.L. Karshin, G.A. Jamjoom, and R. B. Arlinghaus. 1976. A fucose-deficientglycoprotein precursortoRauscherleukemia virus gp69/71. Proc. Natl. Acad. Sci. U.S.A. 73:2326-2330.

26. Naso, R. B., and R.L. Brown. 1977. Synthesis and cleavageof Rauscher leukemia virusprecursorproteins

insynchronizedcells.Virology82:247-251.

on November 10, 2019 by guest

http://jvi.asm.org/

(10)

A. M.Michalides,and H.Bloemendal.1978. Trans-lation ofmousemammarytumorvirus RNA:precursor

polypeptidesarephosphorylated duringprocessing.

Vi-rology91:106-115.

28. Pal,B.K.,R.M.McAllister, M. B.Gardner, and P. Roy-Burman.1975.Comparative studiesonthe

struc-turalphosphoproteinsof mammaliantypeCviruses. J. Virol. 16:123-131.

29. Premkumar-Reddy, E., S. G. Devare, R. Vasuder, and P.S. Sarma.1977.Simplified radioimmunoassay for viral antigens:useofStaphylococcusaureusasan

adsorbent for antigen-antibody complexes.J. Natl.

Can-cerInst.58:1859-1861.

30. Racevskis, J.,and N. H.Sarkar. 1978.Synthesis and processingofprecursorpolypeptidestomurine mam-marytumorvirusstructural proteins.J. Virol. 25:374-383.

31. Reynolds,R.K.,and J. R.Stephenson.1977. Intracis-tronicmapping ofthe murinetypeCviral 'gag'geneby

useof conditionallethal replicationmutants.Virology 81:328-340.

32. Ringold, G.,E. Y.Lasfargues, M.J.Bishop, and H. E.Varmus.1975.Productionofmousemammary

tu-morvirus by cultured cells in the absence andpresence

ofhormones:assayby molecular hybridization. Virology

65:135-147.

33. Sarkar, N. H., E. S. Whittington, J. Racevskis, and

mammarytumorvirus.Virology 91:407-422. 34. Schochetman, G., C. W. Long,S.Oroszlan, L. Arthur,

and D. L.Fine. 1978. Isolation ofseparateprecursor

polypeptides formousemammarytumorvirus glyco-proteins andnonglycoproteins. Virology 85:168-174. 35. Schochetman, G., S. Oroszlan, L. Arthur, and D.

Fine.1977.Gene order of themousemammarytumor

virusglycoproteins. Virology 83:72-83.

36. Shapiro, S. Z., M. Strand,andA. Billiau. 1977. Syn-thesis and cleavage processing of oncornavirus proteins during interferoninhibition ofvirusrelease. Infect.

Im-mun.16:742-747.

37. Smith, G. H. 1978. Evidence for a precursor-product

relationship between intracytoplasmic A particles and

mousemammarytumorviruscores.J. Gen. Virol.41:

193-200.

38. Tanaka, H.1977.Precursor-product relationship between nonglycosylated polypeptides ofA andBparticlesof

mousemammarytumorvirus.Virology 76:835-850. 39. Van deVen, J. M., A. J. M.Vermorken, C. Onnekink,

H. P. J.Bloemers, and H. Bloemendal.1978. Struc-tural studiesonRauschermurineleukemiavirus:

iso-lation and characterizationof viralenvelopes.J. Virol. 27:595-603.

40. Yoshinaka, Y., and R. B. Luftig. 1977. Properties ofa

p70proteolytic factor ofmurine leukemia viruses. Cell 12:709-719.

on November 10, 2019 by guest

http://jvi.asm.org/

Figure

FIG.1.fluorography[35S]methionine[3H]leucineor11.25% [32P]phosphate SDS-PAGE of the proteins ofMMTV and RLV
FIG. 2.MMTVSDS-PAGEdiographyMMTVsolubilized,itated Characterizationofantiseraagainst proteins
FIG. 3.p12forof(C, the Synthesis and cleavage ofMMTVprecursor polyproteins. Cultures of GR cells were pulse-labeled 15 min with 100 ,sCi of[3HJleucine per ml (A, D, and G) and chase-incubated for I h (B, E, and H) or 3 h F, and I)
FIG. 4.[3H]glucosamineanalyzeditated Synthesis of MMTV glycoproteins and glycopolyproteins
+3

References

Related documents

Similarly, repetitive sequences and genes that are frequently tagged by MMTV in mammary tumors were not preferentially targeted in cell culture either in mouse or in human cells;

glucocorticoid regulatory elements present in the mouse mammary tumor virus long terminal repeat: a synthetic distal binding site can replace the proximal binding domain. Mouse

Partial expression of endogenous mouse mammary tumor virus in mammary tumors induced in BALB/c mice by chemical, hormonal and physical agents. Organization of mouse mammary

Lanes g and h, Mammary tumors arising in C3H/Sm mice infected with MMTV (C3H) and exposed to DMBA. Lane i, Mammary tumor arising in a DMBA-treated

Radioactive 60-70S RNA from the mouse mammary tumor virus (MMTV) produced by the C3H mouse mammary tumor cell line (Mm5mt) hybridized to a greater extent, and at a lower Cotj/2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels comparing purified mouse mammary tumor virus (MTV) and murine leukemia virus.. Migration is from top

Results: Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR), in cultured human mammary cells (Hs578T), ultimately leading to the infection

To further validate an association between MMTV CIS genes and mouse mammary tumor progression, three low frequency MMTV CIS genes ( Phf19, Foxl1 and Sdc2 ) were selected