Tryptic peptide analysis of gag and gag-pol gene products of Rauscher murine leukemia virus.

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JOURNAL OF VIROLOGY, May1979,p.610-623 Vol.30,No. 2 0022-538X/79/05-0610/ 14$02.00/0

Tryptic Peptide

Analysis of gag and

gag-pol

Gene

Products

of

Rauscher Murine

Leukemia

Virus

J. J. KOPCHICK, W. L.KARSHIN, AND R. B. ARLINGHAUS*

Departmentof Biology, The University of Texas System Cancer Center, M. D. AndersonHospitaland TumorInstitute, Houston, Texas 77030

Received forpublication19January1979

[3H]tyrosine-labeled viral precursor polyproteins and knownmatureviral pro-teinsderivedfrom the Rauscher murine leukemia virus gag and pol geneswere

examinedby two-dimensional tryptic peptide mapping.

PrMy0gaP"

wasfoundto

containpeptidesequences of the viralcoreproteins p30, p15, p12, andp1O,aswell

aspeptidesequences found in the cell-associated reversetranscriptase.

Interme-diate reverse transcriptase precursor Pr125P'° lacked peptide sequences of the four-core proteins but contained reverse transcriptase-specific tryptic

peptides

plus twoadditionaltyrosine-containing tryptic peptidesnotrelatedtogag orpol

geneproducts. Methionine-containingtryptic peptide analysis also suggested the

presenceofadditionalprotein material in Pr125,"' (Kopchick etal., Proc. Natl. Acad.Sci. U.S.A.75:2016-2020, 1978).

Pr20&gagP"',

althoughcontaining bothviral core and reverse transcriptase-associated methionine and tyrosine tryptic pep-tides, also contained additional tryptic peptides. These are of two classes: (i)

tryptic peptides associated with the Pr125P'l but not

Pr8"0"'

and (ii) tryptic

peptidesnotfound inPr125"'P orin any known viralprotein. Oneinterpretation

of these results is that

Pr200g'g-1°'

contains additional gene products aside from the gagand pol genes.Pr80QagandPr659'9peptide maps were also examinedand

found tohave sequences ofall four core proteins. Pr65gagwasfound to contain

two p30 tyrosine tryptic peptides that were absent in

Pr808'g,

suggesting that

Pr8Og`gmaynot be the precursortoPr65. Pr8Og",asexpected from itslarger

size, alsocontainedtryptic peptidesnotfound inPr659"(.Twoof theseadditional

Pr8g5ag

tryptic peptideswerefoundinPr80P°o aswell butnot in any of theviral

coreproteins, suggestingthatPr8fgagand Pr80I may have overlappingpeptide

sequences. ConSistentwith thisfindingis the conclusion that

Pr80g'9

terminates within the pol gene. A model that describes the relationship of these recent

findingstoviralgene productsispresented.

The genome of type C nondefective retrovi-ruses is knownto containthreegenetic regions required for replication, known as gag (group

antigens, or core proteins),pol [viral

RNA-di-rected DNA polymerase, or reverse

transcrip-tase(RT)],andenv(envelope proteins) (4). The

proteins encoded in these genetic regions are

known to besynthesized by way of high-molec-ular-weight precursor polyproteins that are cleaved and processed in infected cells to form

matureviral proteins (1-3, 13, 20, 25, 31, 32). In

cells infected with Rauscher murine leukemia

virus(R-MuLV), gag gene-derived core polypro-tein precursors of 80,000 daltons (Pr80ag) and 65,000 daltons (Pr659a') are found (1-3, 20), along witha 200,000-dalton RT precursor poly-protein

(Pr2009a9P")

thatcontains core protein, RTantigenicdeterminants, andp30tryptic pep-tides (1, 2, 10).

Theseprecursorpolyproteinsaresynthesized

in vivo (10), as well as in a cell-free protein

synthesisprogrammedwith 35S R-MuLV RNA,

inaratio of about1 mol of

Pr200ga'.Pol

to20mol

of

Pr8Ogas

plus Pr659'9 (10, 19). A translational

control modelhasbeenproposedtoexplainthe

differential level ofsynthesisofcoreprotein as

comparedwith theviralpolymerase (10, 13, 19).

Peptide mapping studies of precursors and

matureviralproteinshavebeenperformed (13).

Tosimplifythecomplexityof thepeptide maps

of higher-molecular-weight proteins,

methio-nine-labeledviralproteinswereexamined;

how-ever, it was found that the only core proteins that contain methionine are

p30

and

p12

(11).

Thelattercontainsanacidic methionine tryptic

peptide that does not bind to the

cation-ex-change column (1) usedto generate thepeptide

maps previously published (1). Under reducing

conditions,two

p30

methionine-containing

tryp-tic peptides, aswell asseveral

methionine-con-610

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PEPTIDE MAPS OF gag-pol GENE PRODUCTS 611

taining tryptic peptides derived from the viral

polymerase (13),areresolved bythisprocedure.

Inthis regard, M00ga9P°wasfoundtocontain

both p30 methionine-containing tryptic

pep-tides, as well as the viral polymerase-specific

tryptic peptides (13). However, the viral RT

(p8OPoI) contained one methionine-containing

tryptic peptide that comigrated with one of the

p30methionine-containing tryptic peptides. To

investigate the protein content of Pr2009a9-Po

further, we labeled virus-specific proteins and

theirprecursors with radioactive tyrosine,since

tyrosine was found to be present in all of the

core proteins and RT (1). We present results

whichshow that the core proteins p30, p15, p12,

and plO, as well asthe DNApolymerase of

R-MuLV, are containedinthe

200,000-molecular-weight polyprotein RT precursor, Pr200f'.

Most importantly, we found that

Pr2009g-P`

contains additional tryptic peptide sequences

notfoundinthemature corestructuralproteins

or virion polymerase. Furthermore, these pep-tides mappingstudies suggestthat Pr809agand

cell-associatedRThavepartialoverlapping

pep-tidesequences. In addition, a comparison map

ofPr809agandPr659aR suggeststhatPr809agmay

notbeprocessedtothe coreproteinPr65Rag.

MATERLALS AND METHODS

Cells and virus. NIH Swiss mouseembryo cells

(JLS-V16) infected with R-MuLV were used in this study (20). The culture mediumwasamodifiedEagle

formulacontaining 10% fetal calf serum,asdescribed by Syrewiczetal. (28). Cellswere grownin 2-ounce (ca.60-ml) prescriptionbottlesor2-quart(ca.1.9-liter)

roller bottles. Cellsweresubconfluent beforeuse.

Vi-rus waspurifiedasdescribedpreviously(28).

Labelingof cells and virus. Cellswererinsedin warmHanks balanced salt solution andpulse-labeled at37°C.RadioactiveR-MuLVwasobtainedby label-ingaroller bottle ofJLS-V5 R-MuLV-infected cells (28) with 50 mCi of

[3H]tyrosine

for48h ingrowth

mediumcontaininglxEagle amino acids.

Immunoprecipitation of viral proteins from cells. Thepreparationofcytoplasmicextractswas as

described previously (20). Briefly, cell lysiswas per-formed in lysisbuffer containing 0.5% Nonidet P-40

(Particle Data Laboratories, Ltd.) and 0.5% sodium

deoxycholate (Schwarz/Mann) (1).Monospecificgoat antiserapreparedagainstR-MuLVp30,p15, p12,plO,

gp69/71, and

p80IW0'

wereobtainedthroughthe Office ofProgram Resources andLogistics, ViralOncology,

National Institutes of Health. Pulse-labeled cellswere

lysedby homogenizationin2to 10ml oflysisbuffer. All antisera were absorbed with excess uninfected

JLS-V16 cellcytoplasmicextracts(2).Also,antiserum

directed against

p80P"

wasabsorbed with disrupted virus as describedpreviously (10).For indirect

immu-noprecipitation,themonospecificantiseraweremixed

withcytoplasmic extracts. Incubation was for15min at roomtemperature and overnightat 4°C. For the second immunereaction, 20volumes of rabbit

anti-goat serum (relative to the volumes of antiserum used in thefirst immune reaction) was added for 15minat room temperatureand 4°C overnight. In a few exper-iments, indirectimmunoprecipitation was carriedout by using the staphylococcal protein A method as de-scribedby Kessler (12). The indirect immunoprecipi-tates and the staphylococcal protein A precipitates werecollected by centrifugation at 10,000 rpm for 20 min through a1.5-ml cushion of immune buffer (0.5% Nonidet P-40, 0.5% sodium deoxycholate, 0.02 M Tris-hydrochloride [pH 7.5], and 0.05 M NaCl) containing 1 M sucrose, 1% Triton X-100, and 1% tyrosine ina Sorvall GSAswinging bucket rotor.

Gelelectrophoresis. Sodium dodecyl sulfate-poly-acrylamide gelelectrophoresis (SDS-PAGE) was per-formed on gel slabs, using the buffer system described by Laemmli (14). Viral proteinsp30, p15, p12, and plO were separated on an 11.25% gel, and precursor pro-teins

MW0"",

Pr80Y"'9, pMY5, and Pr80/"' were resolved on a 6 to 12% gradient gel. The gels were subjectedtofluorography as described previously (5). Toobtain a linear response to radioactivity, the X-ray films werepreflashed (15).

Purification of viral proteins. Viral proteins p30, p15, p12, and plO were first purified by guanidine-hydrochloride agarose chromatography (9). The pro-teins were purified further by SDS-PAGE as part of thetrypticdigestion procedure.

Peptide mapping. Tryptic digestion was carried

outbyincubating the dry slab gel band at 37°C in 2 to

3mlof0.05 MNH4HCO3 (pH 8.5) containing 50jigof trypsin per ml. After 15 h, an additional 50 jig of trypsin per ml was added, and the incubation was continued for 8 h.Tolylsulfonyl phenylalanyl chloro-methyl ketone (TPCK)-trypsin (Worthington Bio-chemicalsCorp.) wasstoredat -20°Cat 5mg/ml in

0.01M HCIcontaining1mMCaCl2. Fragments of gel wereremoved by filtration through0.45-jimcellulose nitrate filters, and the supernatant fluids containing the soluble tryptic peptides werelyophilized. Radio-activity recovery from thegel varied from60 to80%. Thepeptidesweredissolved in0.2mlof bufferA(33), and the sample was clarified by centrifugation at

10,000 x g for 10 min. The tryptic peptides were resolved by cation-exchange chromatographyas de-scribed previously (1) orbytwo-dimensional finger-printing. The latter procedurewasperformed by spot-ting the[3H]tyrosine-containingtrypticpeptidesona

cellulosethin-layer plate (20 by20cm). Thepeptides were separated in thefirst dimension by electropho-resis at 150 V in a 28% formic acid solution. After drying, the peptides wereresolved in the second di-mensionbyascendingchromatography ina butanol-pyridine-acetic acid-water (6.2:1:3.3:2.8) buffer system. 2,5-Diphenyloxazole (PPO) wasapplied to the cellu-losethin-layer platesbyascending chromatography in

anacetonesolutioncontaining10% PPO(wt/vol).To obtain a linear response to radioactivity, the X-ray filmswerepreflashed (15).

RESULTS

Immunoprecipitation of pulse-labeled

cell extracts with

monospecific

antisera

preparedagainst

gag,pol,

andenvproteins.

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612

KOPCHICK, KARSHIN, AND ARLINGHAUS

Wereportedpreviouslythatviralstructural

pro-teinsaremadeininfected cellsby synthesisand

proteolytic cleavage oflarger-molecular-weight

precursorpolyproteins (1-3, 10, 13, 20). In Fig.

1, infected cells were pulse-labeled for 15 min

with[3H]tyrosine and immunoprecipitated with

antiserum to the core, RT, and envelope

pro-teins.Anti-ovalbumin was used as a control(Fig.

1, lane G). Theimmunoprecipitates were

ana-lyzedon a 6 to 12%gradient gel. These

experi-ments have identified several rapidly labeled

polypeptides termed

Pr200g'g-P"

(Fig.1, a,b, and

c), Pr145P°Z-Pr125POI,

Pr809as,

Pr65ag,

and

gPr9oen. It is evident that anti-p30, anti-p15,

anti-p12, and anti-plO recognized Pr20Og'g-P°

(Fig. 1,lanesAthrough D). This indicatesthat

Pr200ea'PoI shares antigenic determinants with

all of the viral

group-specific

antigens, as

re-ported

previously

(10). Antiserum made

against

the viral DNA polymerase also

precipitated

Pr2Offas'Po (Fig. 1, lane E), as previously

re-ported,aswell asPr145Po,

Pr135P'1,

andPr125PI

(10, 13). Antiserum directed against the major

viralglycoprotein did not recognize

Pr200eaggPo

but didimmunoprecipitate a 90,000-dalton

en-velope precursordesignated gPr9oen', whichhas

beenshowntocontainp15(E) and gp69/71

tryp-tic peptides (11). Two additional polyprotein

precursors were precipitated by antiserum to

core proteins. They are termed Pr80Sa9 and

Pr65sas

(Fig. 1, lanes A through D) and have been shownbytryptic mappingtobe

structur-ally relatedtothe fourviral coreproteins (1, 3).

Anti-plOserum (Fig. 1, lane D) recognized two

protein bands with approximate molecular

weights of 170,000 and 145,000,respectively,the

smaller of which

migrated slightly

slower than

Pr145PoI

(comparelane D with lane E, Fig. 1). Wehaveshownpreviously by pulse-chase

ex-perimentsthat

pM20fyasPOI, Pr8099a,

and

gPr9Oenl

areunstableproteins (1-3, 10, 11, 13, 20,21). In

pulse-chase incubations with various

monospe-cific antisera, we have shown that the mature

structural proteins, as

exemplified

by p30 and

p15,

accumulate

under chase conditions, with

theconcomitant deceasein

Pr80Sa9

and

Pr65SaS

(2, 10, 20). Theintermediate stable polymerase

precursors

Pr135PoI

and

Pr125Po,

aswellasthe

intracellular 80,000-molecular-weight

RT-re-latedprotein

Pr8OPoI,

whichhasbeen shown to

benearly identicalto virionpolymerase

(p8OPoI)

(13), also accumulate during the chase at the

expense of

Pr200gagPo

(10, 13) (see Fig. 2 of reference 13). The virion envelope proteins

gp69/71

and

p15(E)

also appear in the chase

incubation and are derived from

gPr90en',

as

previously reported (21).

Comparison of the tryptic

peptide

se-quencesof thecoreprecursor

polyproteins

with thematurestructural

proteins.

Cation-exchangechromatography of tryptic digestshas

revealed thatmethionine-containing

p30

tryptic

peptides are present in

Pr89ya9,

Pr65SaS,

and

Pr200easgIg

(2). Also, by the same procedure,

Pr809as

and

Pr65sas

were shown to share

tyro-sine-containing tryptic peptides with the viral

structural proteins

p30,

p15, p12, and plO (1).

Pr8O9a9

wasshowntocontainone extra

tyrosine-containing tryptic peptide (1). To examine

fur-ther the relationship of thegag precursors to

A B C D E F G

p

20flgag

rpol

Pri2200I

Pr

t2;5P°'

Pr8

gfag

P,65gag

a

Pt145po PO

'35P'

-gPr9O

en-.

-am _0Memm~

FIG. 1. Kineticsofformation of R-MuLV precursor polypeptides. R-MuL V-infected JLS-V16cells (=5 x

10' cellsin l-quart [ca. 0.9-liter]prescription bottle) were pulsed-labeled for 15min with 20 ml of Hanks balanced salt solution containing[3H]tyrosine (150juCi/ml). Thecytoplasmic extracts were treated with 50

Al

ofthefollowingantisera: (A) anti-p30, (B)anti-p15,(C)anti-p12, (D) anti-plO, (E) anti-RT, (F)anti-gp69/71,

(G)anti-ovalbumin. Theimmunoprecipitates were analyzed by SDS-PAGE on a 6 to 12% slab gel.

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B

p15

A

p30

279 *

96

4

0

PlO

10

D

p12

FIG. 2. Two-dimensional trypticpeptide maps of[:H]tyrosine-labeled viralcoreproteins.

[;'Hityrosine-labeled viral structuralproteinsp30(A), p15, (B), plO (C), and p12 (D)werepurified by guanidine-hydrochlo-ride-agarose chromatography, followed by SDS-PAGE. The purified proteins were digested with TPCK-trypsin andseparatedon cellulosethin-layer plates in the first dimension by electrophoresis (left toright), followed by ascending chromatography. PPO was applied to the plates by ascending chromatography. Approximately 30,000 cpmofeachtryptic digestwereappliedtothethin-layer plate. The plateswereexposed

topreflashed X-rayfilms for2 to 3weeks.

the virionstructural

proteins

p30,p15, p12, and

plO, wedigestedthese

tyrosine-labeled

proteins

with trypsin andfractionated themby

electro-phoresisinonedimension andchromatography

in a second dimension.

Figure

2A

through

D

shows theresults ofanalyses oftryptic digests

ofp30, p15, plO,andp12,respectively.Viralp30

wasfoundtocontain sixmajor

tyrosine-contain-ing tryptic peptides (no. 2, 4, 5, 9, 27, and 28)

and several minor spots. Viralp15andp12each consisted oftwomajorandtwominor

tyrosine-containingpeptides.Themajor p15peptidesare

numbered6and8; those fromp12arenumbered

7 and 25. Viral plO contained one major and

severalminorpeptides.The minor

tyrosine-con-taining trypticpeptidespotswere notidentified

because ofvariability in detection from

experi-ment to experiment. Mixing experiments were

performed with p30-p15, p3O-p12, and p15-plO

tryptic digests (data not shown). This allowed

the location of the trypticpeptides of the indi-vidualproteins with respectto each other. For

example,themajorplOtryptic peptide (spot 3)

liesdirectly beneath spot 6, which isoneof the

two p15 tyrosine-containing tryptic peptides. The plO peptide (spot 3) was also found to lie

on adiagonalbetweenp30spots2and 4, andso

on(Fig.2).

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614 SOPCHICK, KARSHIN, AND ARLINGHAUS

Figures 3 and 4 show the two-dimensional

peptidemaps ofPr8099aand

P65gas,

respectively.

Analysis of the results indicated that Pr809'9

(Fig. 3) and Pr65,as (Fig. 4) contained

p30-spe-cific peptides (spots 2, 4, 5, and 9), one p15

peptide (spot 6), p12 peptides (spots7 and

25),

andthe plOpeptide (spot 3).These

two-dimen-sional maps confirm our findings that

Pr80Sa9

and Pr65sasshare much of theirproteinsequence

information (1, 2, 10) and that they contain

sequences of all four viral core proteins.

Al-though spot 8 ofp15 appears tobe present in

both Pr8O9a9 andpM5 ag, thisregionof themaps

is not well resolved. This lack of resolution,

combinedwith ourpreviousstudieswith column

chromatography (1), which indicated thatonly

one ofthe two p15 tyrosine-labeled peptides is

present in Pr809ag and

pM55ag,

raises some doubt as to whether both spots 6 and 8 of p15 are present in these precursors. We marked spot 8

with a question mark to indicate this

uncer-tainty. We note that both Pr809ag and

Pr65sag

contained

tyrosine-containing

tryptic peptides

(spots 26 and 34) that are not found in any of

the core proteins. Another interesting point is

that

Pr65sag

contained tryptic peptides(spots 27,

28, and 33) not found in Pr809ag.

Specifically,

tryptic peptide spots 27 and 28 of

p30

origin

weremissingin

Pr809ag,

andspot 33appearedto

beuniqueto

Pr65sag.

Pr80yag

also contained

ad-ditionalspots not found in

Pr65sag

(spots 1, 10,

and 29 and one spot at the top center of the

Pr80ag map). The facts that some p30tryptic

peptidesweremissinginPr80gaG but were

pres-* 29

9 10 9

34

26

*

j t.

0

FIG. 3. Two-dimensionaltryptic peptide map of[3H]tyrosine-labeled Pr8O"'9.R-MuLV-infectedJLS-V16

cellswerepulsed for15minwith 15 mlof[3H]tyrosine(1501LCi/ml)inHanks balanced salt solution at 37°C. Thecytoplasmicextractwastreatedwithanti-p30, and theimmunoprecipitatewasanalyzedbySDS-PAGE.

Pr80w'waseluted anddigestedwithtrypsin,and the[3H]tyrosine-labeledtryptic peptides were resolved as

described inthelegendtoFig.2.Approximately 70,000 cpm were applied, with a 5-week exposure time.

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PEPTIDE MAPS OF gag-pol GENE PRODUCTS

28$

5

94

93

25 2

Q

0

FIG. 4. Two-dimensional tryptic peptide map of[3H]tyrosine-labeledPr65g. PrO5ag was isolated, and thetryptic peptides were resolved as described in the legend to Fig. 3. Approximately 50,000 cpm were applied, witha4-week exposure time.

ent in Pr659'9 and that Pr65Vg contained

an-other tyrosine-containing tryptic peptide (spot

33) not found in Pr80g9g suggest that Pr809'9

may notbe theprecursor to

Pr659'9,

aconclusion

that is indisagreementwith our previous

inter-pretations (1, 2, 10). However, further work is

neededonthispoint.

Peptide

mapsof

Pr2OO9"0-"1

and the

inter-mediate

RTprecursors.

Pr20(gYg-P"

shares

an-tigenic determinants withthe group-specific

an-tigens p30, p15, p12, and plO and the virion

RNA-directed DNApolymerase (Fig. 1 and

ref-erence 10).

Pr200gag9P"

has also been shown to

share methionine-containing tryptic peptides

withthe major viral structural protein p30 (2). In the present study we further characterized the

tryptic

peptide sequences present in

Pr2009ag-P°',

placing particular emphasis on

de-termining whether all four viral core proteins

are present in

Pr200ga"'.

The results (Fig. 5) showedthatp30spots 2, 4, 5, and 9 are present

in Pgr200gagP. Viralp15spot 6, p12 spot 7, and

plO spot 3 werealso present inPr200gag-Po.There

aremanyotherspots in

Pr200gag°"'

that are not

related to thosepresent in Pr809ag (Fig. 3) and inPr659ag (Fig. 4). Some of these spots, i.e., spots

10, 13 to 20, 29, and 36, were found in

cell-associated RT

(Pr80PO')

(compare

Fig.

5and

6).

Thus, these results show that

M009ag-P°

con-tainssequencesfoundin all four viral core

po-teins,aswellas

peptide

sequences found in the

RT; the latter is inagreement withour

methionine-containing tryptic

pep-tides(13). We noted that Pr80MYo didnotcontain

any of the

peptide

spots characteristic of p15,

p12,or plO (compare

Fig.

4 and

6).

Pr8O""'

did

contain one peptide which is characteristic of

p30, i.e., spot 4. However, the other five p30

tyrosine-containing peptides

are not found in

Pr8OP°I.

It remains to be determined whether spot4in

Pr8MOI°

isabona fidep30peptide.

Anotherinteresting fact about

Pr200Og"P°

is

spots 10and29(Fig. 5).These

tyrosine-contain-ing peptides are presentin all RT-related

pro-teins (Fig. 5 through 7)

(Pr2009ag-1°1,

Pr125Po',

and

Pr80IP°),

aswellasinthe gag gene

product

Pr80Vg (Fig. 3). Theyare,however,absent from

Pr65'9andallof the viral coreproteins.These

resultssuggestan

overlap

betweenPr809`9and

Pr80M°o

and also indicate that the gag andpol genes are adjacent onthe viral genome. How-ever,further work is neededto

firmly

establish

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616 KOPCHICK, KARSHIN, AND ARLINGHAUS

26

3..14

S.

I

"P

*24

.35

16 28

36

2

32

23 22

3-,

i8

FIG. 5. Two-dimensionaltryptic peptidemapof [3H]tyrosine-labeledPr20099P°.Amixtureof the b andc

componentsofPr2OO'90P" wasisolated,andthetryptic peptideswereresolvedasdescribed inthelegendto

Fig.3.Approximately 100,000cpm wereapplied,witha12-weekexposuretime.

the presence of shared peptide sequences

be-tweenPr809'9andRT-relatedproteins.

The intermediate RT precursors (Prl45Po-Pr125POI) were also examined by

two-dimen-sional peptide mapping (Fig. 7). Pr125PI

con-tained the tyrosine-labeled RT-specific tryptic peptides 10, 11, 13to21, 24, 29,and 36 (compare Fig. 6 and 7). Pr125Pol also contained spots 12

and21.Spot21waspresentin

Pr2009'9-1°;

how-ever, spots 11 and 12 were not (Fig. 5). The

tyrosine-labeled peptide maps of Pr145PoI and

Pr135PY were verysimilartothemapof Pr125Po/

(data not shown). One cannot readily observe

spots4and36inFig.7;however,ontheoriginal

fluorograph, one candetect these peptides. We

emphasize that Pr145Pol-Pr125Po didnotcontain

anypeptide spots, except spot 4, characteristic ofthe viral core proteins. They did, however,

containspots 12and21notfound inanyof the

viralcoreproteins, RT,orthegaggeneproducts

ofPr80Vg and Pr659as. The possible nature of

thesesequences will be discussed below. These

results indicate that Pr145Po0-Pr125PoI did not ariseby partial removal of thegaggeneproducts orbyaprematureterminationevent. If

prema-ture termination were the cause of

Pr145PoI-Pr125p°' production, thenallof thecoreproteins

would beexpected in

Pr145P°'-Pr125PI'

and

pos-sibly only part of the RT sequences. This is

clearlynotthecase.

Anotherimportant point is that the complex-ity of the peptidemapsof

Pr200g-P°,

Pr125PI,

andPr801P° decreased with the decrease in

ap-parentmolecularweight.

Pr20099fgPoI

had about

30 major tyrosine-labeled tryptic peptidesthat can beresolved;

Pr125PO'

had 16 peptide spots; Pr80Pol had 14 tyrosine-containing tryptic pep-tide spots. Thus,

PrQ00939aPOI

and the intermedi-ateRTprecursorsarenotaggregatesofPr80PI. Noncore and non-RTtryptic peptides

as-sociated with

Pr2009-1"°.

Pr2009a9P°1

is the

primarygeneproductthat leadstothe formation

of the RT (10, 13). Our previous published

ex-periments have indicated that

Pr2009gaPOI,

as

wellasthe intermedite RTprecursors, contains

methionine-labeledtryptic peptidesthatarenot found ingagorpolgene products (13). Inthis

paper,analyses of two-dimensional [3H]tyrosine-labeled tryptic peptide maps of

Pr2009ag-P",

as

wellasthe intermediate RTprecursors

(Pr125P"I,

Pr135Pol, and Pr145POI) and the viral core pro-teins, gave results that are consistent with a

20

*f 17

3

0

1I

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PEPTIDE MAPS OF gag-pol GENE PRODUCTS

20 29

10

ii

,13

14

24 15 24

is 17

4

36 18

19

[image:8.497.109.396.68.336.2]

0

FIG. 6. Two-dimensionaltrypticpeptide mapof[3H]tyrosine-labeled Pr80". Pr8OP"'wasisolated from R-MuLV-infectedJLS-V16 cells. The cellswerepulsedfor15minwith

[3H]tyrosine

(150,uCi/ml) in25mlof Hanks balanced salt solution at37°C. After pulsing,the cellsheetwasrinsed with Hanks balanced salt solution, and completegrowthmediumwasaddedfor1h.Thecytoplasmicextract wastreatedwith absorbed anti-RT serum, and theimmunoprecipitatewasprocessedasdescribed in thelegendtoFig.1.Digestionwith trypsin and two-dimensionalmappingwere asdescribed in thelegendtoFig.2.Approximately20,000 cpm

wereapplied,witha4-week exposuretime.

similar interpretation. Thus, tyrosine-labeled

peptide 21 was found in

Pr200eaggP"

and in the

intermediate RT precursors, but it was absent

in

Pr80PO'

and in all of the four core proteins

(Table 1). This is the only tyrosine-containing

peptide shared bypr2000'9agPo andPr125POl that

isnotfoundineither

Pr80oI

or thecore proteins.

Also, spot 12is ofinterest since itwas not seen

in

Pr200g'g-P"

but appeared in

Pr145POI, Pr135Pol,

andPr125POI butnotinPr80OP°. Spot 11was not

present in Pr20ffas9Po butwasfound in

cell-as-sociated RT, aswell as intheintermediate RT

precursors.

Inadditiontotrypticpeptides thatareshared

by

Pr200g'g-P"

and theintermediate RT

precur-sors, Pr200g'g-P° has tyrosine-labeled tryptic

peptidesthat arefound onlyin

Pr2)g(a5g-PO

(Ta-ble 1). None of the known viral gene products contains these tryptic peptides (see composite shown inFig. 8). Spots 22, 23, 30 to 32, 35, and 37 are

examples

of suchtryptic peptides, spots

35and37beingminor spots (Table1).

Methionine-containing

tryptic

peptides

of

Pr200"-P°.

Figure9showsa

[35S]methionine

profileobtainedbyfractionatingatrypticdigest

of

Pr2009'9-P'1

(the b component) on a

cation-exchange column, asdescribed previously (13).

A similar profile was obtained from band a,

except that one major methionine-containing

peptide was found atthe position indicated by

the arrow in Fig. 9. Peaks 8 and 10 in Fig. 9

comigrate with p30 tryptic peptides (13). The

other

methionine-containing

core

protein,

p12, has an acidic tryptic peptide that elutes inthe

flow-through

of the column

(1).

Although

the

majority of the methionine-containing tryptic

peptidesin

Pr200g'g-P"

areRT related (13),seven

peptides (peaks 2, 4 5, 12 to 14, and 16) are

neithercoreproteinnorRTrelated(Table1and

reference13). Three of thepeptides(peaks 5, 14, and 16) are found in

Pr2009oa9PO

but not in any ofthe core or lower-molecular-weight RT pre-cursors. Peaks 2, 4, 12, and 13 are found in Pr200gagYPo and thelower-molecular-weight RT precursors Pr145P°',

Pr135P01,

and

Pr125Pol

but

not in Pr80°OP or in any of the core proteins

(Table 1 and reference 13). These latter

se-quences may be derived from the same

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20

29

.. 10

; 11 13

12 14

15 24

16

17 4

36

18

19

0

FIG. 7. Two-dimensionaltrypticpeptidemapof[3H]tyrosine-labeled Pr125PoI. Pr125PoIwasisolated and mappedasdescribed in thelegendtoFig. 6.Approximately20,000 cpmwereapplied; the exposuretimewas

Imonth.

TABLE 1. Radioactivetyrosine-andmethionine-containing trypticpeptidesassociated withgag andpol geneproducts and proposed A, B, and C viralproteins

Viralpro- gag A pol B C

tein tyro' methb tyro meth tyro meth tyro meth tyro meth

Mr20ag-P"' 1-7,8?, 8,10 10,29 4,10, 13-20, 1, 3, 6,7, 9, 21 2, 4, 22, 23, 5, 14,

9, 27, 24, 29,36 10,11, 15 12, 30, 31, 16

28 13 32

Pr125P"' 10,29 4,10, 11,13- 1, 3, 6,7, 9, 12,21 2,4, 20,24, 29, 10, 11, 15 12,

36 13

Pr80P"' 10,29 4,10, 11,13- 1,3, 6,7, 9, 20,24, 29, 10, 11, 15 36

Pr8o""5 1-7,8?, 8,10 10,29 9,25,

26,34 Pr65"-' 2-7,8?, 8,10

9, 25-28, 33, 34

p30 2-5,9, 8,10 27,28

p15 6, 8 None

p12 7,25

plO 3 None

aTyrosine(tyro)-labeled trypticpeptidesarenumberedasshown inFig.5and 8.

Methionine(meth)-labeled trypticpeptidesarenumbered as shown inFig.9.Thepeptidemapsrelating to the methionine-containingtryptic peptides havebeenpublishedelsewhere(13).

quences thatyieldtyrosine-containing peptides

12 and 21. It is possible that they represent a

protein which is cleaved from Pr125po'toyield

Pr801J'.Theputative proteinwould beexpected

tohaveamolecularweight of about40,000.

Themethionine-containingtrypticpeptides5,

14,and16and the fivemajortyrosine-containing

tryptic peptides (spots 22, 23, 30 to 32) found

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29

270 70

704

35 16%

28U

2

0

IC) FIG. 8. Schematic ofthe tide map ofPr200`9P°. TI

shown in Fig. 5have been gene related (0), polgene i

protein ((). Spots 10 and 2,

related.

0 20 40 60 80

Fractij

FIG. 9. Cation-exchange I component of [3H]methion [3H]methionine-labeled tryl tionsareidentifiedby numb thelocation ofapeak founc

Pr200ga9-°' but missing from

only in Pr2()gagaPol could viralgeneproduct. Two of

(butnotall) could be jun cussedbelow.

DISCUS' The results presented previous fmdings that PI

genic determinants and

quences with the core

Pr2009'9-P°l is comprised labeled "a", "b", and "c'

20 antigenically and

structurally

related based on

immunoprecipitation experiments with mono-specific sera (Fig. 1) andmethionine-containing

32 tryptic peptide analyses on ion-exchange

col-10 umns (2, 13). When oxidized

methionine-con-4M ,13

tainig

tryptic

peptides

were

compared

(2),

the

"a" polypeptide was found to be closely related,

V24

%W15

but not identical, to the "b" polypeptide. Under

reducing conditions, the methionine-labeled

6D 22

tryptic peptide profiles

of al three

polypeptides

d4

p~

UP were similar

(G.

A.

Jamjoom

and R. B.

Arling-(5

haus,

unpublished

data).

Treatmentof

ceUs

with

4

41 4I37

the

protease

inhibitor TPCK caused the "a" polypeptide to accumulate at the expense of the 9§18 "b" and "c"

polypeptides

(10).

3Q

were obtained when the amino acid analog

can-avanine was substituted for arginine during

pulse-labeling experiments (10). Thus, the a

o0

119 polypeptide

appears to be the precursor to the

b and c polypeptides. In this regard, we note

that Evans et al. (6) reported that 220,000- and

3Hetyrosine-labeled

pep- 230,000-dalton polypeptides containing core

pro-ie

tryptic peptide

spots teins bind to lectincolumns.Wehave found that

identified

as eithergag one of the three bands of

Pr2009`9-P°

becomes

related (0), or as extra labeled

with radioactive

mannose

(J.

J.

Kop-9 are bothgagandpoi chick, V. Ng, and R. B.Arlinghaus,unpublished data).

Pr80P') has been shown to be formed by

syn-thesis and cleavage of a high-molecular-weight

lo precursor polyprotein,

Pr2009g-P°

(13).

Antise-rumpreparedagainst RT specifically recognized

Pr200919-Po1

and three intermediate polypeptides,

Pr145P°1,

Pr135POl, and Pr125PIo. Another

poly-89 11 121314 1516 peptide,

Pr80P°1,

identical in size to the mature

lI

viral enzyme

p80P01,

is found in infected

cells

(13).

The two

proteins

appearidentical relative

to their methionine-containing tryptic peptide

profiles, with the exception of one peak (13).

Theresults reportedherefurther emphasizethe

100 120 140 160 180 relationship between

Pr80OP°

and

Pr2Wag-P°l.

All

[image:10.497.48.234.62.275.2]

on Number except one (spot 11) of the tyrosine-containing

elution profile of the b tryptic peptidesfound inPr80(P°'arealsoseenin

ine-labeled Pr2009ag-P°l.

Pr2009ag-P°l.

All ofthese findings, together with

Dtic peptide peak frac- pulse-chase experiments done in the presence of

ers. The arrowindicates cycloheximide (13), indicate that

Pr80°°'

is de-i in the acomponent Of rived by cleavage of

Pr2009agcP°'

through inter-the b component. mediate

pol

precursors

(Pr145P°I,

Pr135Pol,

and

be another unknown

Pr125Io').

'thesetrypticpeptides Our previous work showedthat one of the RT

Lctionpeptides, asdis- methionine-containing tryptic peptides comi-grated with one of thetwo p30

methionine-con-SION

taining

tryptic

peptides

(13).

Inthis

report

one

of the six tyrosine-containing p30 tryptic pep-in this study confirm tides migrated with one of the Pr80PoI

tyrosine-r200g9g-POI

shares anti- containing

tryptic

peptides, i.e., spot 4 (Fig. 2 tryptic peptide se- and 6). Pr80P°, immunoprecipitated with anti-and RT proteins. RT sera from Moloney MuLV-infected cells,

ofthree polypeptides shares no tyrosine-containing tryptic peptides

in Fig. 1. They are withp30,p15,p12, orplO,asdeterminedby ion-VOL. 30, 1979

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exchangechromatography (D. Lyons and R. B.

Arlinghaus, unpublished data). This

observa-tion,along with resultsobtainedwithantiserato all four core proteins (Fig. 1 andreference 10), establishes that RT does not share peptide se-quenceswith any of thefourcoreproteins.

Regarding

Pr200ga1P"

and the core proteins,

tyrosine-containing tryptic peptides

character-istic ofp30, p15, phosphoprotein p12, and plO

canbereadilyresolved in

M009',""

by peptide

mapping(Table 1).

Concerning

thetwo

tyrosine-containing p15 tryptic peptides, spot 6 is

defi-nitely present in

M009`'-P"',

but there is some

doubt about spot8. There isuncertaintyabout

spot8 in Pr809'l- and

Pr659"',

as well. We have

shownpreviouslythatonlyoneofthetwomajor

tyrosine-containing tryptic peptides of p15 is

present in Pr809` and Pr65('Lg (1). It is clear,

however, that

Pr2009a9P°'

does contain p15

an-tigenicdeterminantssinceitcanbeprecipitated

byanti-p15sera(Fig. 1,laneB).

Thedata presented here furthersubstantiate

the results of Arcement et al. (1, 2), in that

Pr80Y'- and Pr65gag showantigenicdeterminants

andtrypticpeptidesequenceswithp30, p15,p12,

and plO and that Pr809ag contains additional peptide sequences relative to pMY5"g'. One

sur-prisingfindingwasthat twop30tryptic peptides

(no. 27and 28)foundinPr65-ga were notfound

in Pr809'-, which couldmean that Pr809` may

not be cleaved to yield Pr65g'l. An additional

tyrosine peptide (no. 33) was also found in Pr65-ag and notinPr809'9.Our previous studies with amino acidanalogsand proteaseinhibitors (10) suggested that Pr80g"'l was the direct

pre-cursor to

Pr65-a*.

However, another

interpreta-tion,which is also consistent withourpastdata,

is that Pr659ag gives rise to core proteins,

whereasPr80ga is modified for another purpose

and never gives rise to mature core proteins.

Pr809'gmight be similar to theglycosylated

sur-face gag gene polyproteins first observed by Tungetal. (30) in 1976. Inagreementwith this

conclusion, we have observed recently that

Pr80Y'- became radioactive during pulse-chase

experiments, using [3H]mannose, whereas

nei-ther Pr65-a- nor p30 was labeled with mannose (Kopchick and Arlinghaus, unpublished data).

In this regard, Evans et al. (6) have shown recently that in Friend MuLV-infected

cells

a 75,000-dalton core polyprotein is slowly proc-essed to form a glycoprotein with an apparent molecular weight of 93,000.

Regarding theorigin of the core protein

pre-cursor

Pr659'l,

itispossible that Pr659'9 may be generated by nascent chain cleavage and that Pr80"' is the first genuine termination product

ofthe gaggene. Thisinterpretation is consistent

with our

previous findings (10)

and those of

Philipson

etal.(22),whofound thatyeast amber suppressor tRNA increased the frequency of

Moloney-MuLV

Pr180Ya"P°I synthesisatthe ex-pense ofa PR789a. Theamount ofPr65-ga syn-thesized was not affected by the suppressor tRNA. Inadditiona 135,000-daltonproteinwas alsoincreased inamount.Thelatterwas

thought

to be similar to Pr135Y''' and could be the re-mainder ofPr200,g-l"'afternascentchain cleav-age ofPr65,9'9.

Anotherpointthat bearsmentioningpertains

to

spliced

mRNA's. The splicing mechanism is believed to generate the envelope mRNA in avian andmurinetype C viruses (16, 23). Such a mechanism couldreadily explain theabsence ofsome p30 sequences in

Pr809't9,

those same sequences,however, beingpart ofPr659a.Thus, Pr809a9 would arise from one class of 35S viral RNA,whereasPr65-' would be translated from another 35S class. However, it is difficult to

reconcile the results previously obtained with protease inhibitors and amino acidanalogs with

suchasplicingmechanism (10).

Another interesting point concerns Pr80gag and the RT precursors. Wefound that twomajor tyrosine-containing tryptic peptides (spots 10 and 29) are shared by Pr80g'l and all the RT-related proteins, yet spots 10 and 29are notin Pr65gagorin any of the viral coreproteins. One interpretation of these results is thatPr809ag is terminated within thepolgene. Thefunction of

this putative shared polypeptide is

unknown,

butaprotease would be a possiblecandidate for suchapolypeptide fortworeasons.First, the C-terminalportion of avian sarcoma virus

Pr76-"(I

appearstocontain a protease in the form of viral protein p15 (34; K. Von der Helm, W. Wile, and K. Willeck, Hoppe Seylers Z. Physiol. Chem. 358:1216-1217, 1977). Second, MuLV RT prep-arationsundergoproteolysisduring purification (18). Thus, the possible shared polypeptide within Pr80g9g and Pr80OP) could be a protease. It is of interest that Pr2009g',P° contains six majortyrosine-containing tryptic peptides and

seven methionine-containing tryptic peptides

notfound in the core structural proteins and the virionpolymerase (Table 1). One of the tyrosine-containing peptides and four of the

methionine-containing peptides can be found in the

inter-mediate pol precursors Prl45Po/, Pr135Po/, and Pr125Pol, but not in Pr80O°o (Table 1). These peptide sequences may represent a new gene productasyetunidentified.

Pr2009'9-°Ialso containstrypticpeptides not

found in the core structuralproteins,pol,orthe intermediate RT precursor (Table 1). In the methionine map, there arethree such peptides

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(no. 5, 14,and 16in Fig.9). The tyrosine

finger-printshows five such major peptides(no.22, 23,

30,31,and 32),aswellasminorspots (no. 35 to

37),whichare neithergag,pol,orintermediate

RT precursor related. Two of these peptides may representsequencesatthe junctures of the

proteolytic cleavage sites which, when mapped

in themature protein, migrate differently. The

remaining tryptic peptides couldrepresent still

another geneproduct, but this requires further

study(seebelow).

The results presented above have prompted

the formulation of a working model (Fig. 10)

thatdescribes the gene content ofR-MuLV

ge-nomic RNA,withparticular emphasis given to

thegagand polgeneproducts. The main point

of the working modelis notonlythat

Pr2009'9"P"

is theprecursor to RT, butalso thatitleadsto

the synthesis of at least two other viral gene

products thatwehave identifiedasB andC.We

emphasize that additional proteinsequences in

Pr2009ag-P"

are not env gene-related products,

since

M009ag-P"

does not cross-reactwith anti-gp69/71 serum, nordoes it contain the

methio-nine-containingtrypticpeptidescharacteristicof

p15(E) (2, 13) (see Fig. 1). The natureofthese

viral gene products is unknown, but at least

three possible virion enzymes are good

candi-dates. It iswellknown thattypeC viruses

con-tainaprotein kinase (8,27). AviantypeC viruses

also contain an endonuclease (7, 24). A third

possible virus-coded enzyme is a protease (34,

35; Von derHelmet

al.,

Hoppe

Seylers

Z.

Phys-iol. Chem. 358:1216-1217, 1977). Otherenzymes

are also found in the mature particle (17, 29).

Noneof theseenzymeshasasyet beenfoundto

T

R-MuLVGenomicRNA LA. Pr200ga9-Pd

,gag'

Pr80gag

' Pr6598

--__________

pl5,p12,p30,plO

A

beviruscoded, except for recent evidenceonthe

avian32,000-dalton endonuclease(24).

The evidence supporting this hypothetical model is of two kinds.First,themolecularweight

of

Pr2009'9-P"

isestimated between200,000and

220,000, based on its comigration in SDS gels with myosin (2, 19); since the core proteins

(65,000 daltons) and the polymerase(80,000

dal-tons)onlyaccountfor about150,000daltons,one

is left with about 50,000 to 70,000 daltons of unidentified protein sequences. Second, addi-tional protein material is detected in. peptide

maps of Pr2009'9-I° and the intermediate RT

precursors(e.g.,

Pr125P1I)

that are not gagorpol generelated.

Because of the recentfindingof an endonucle-asein the avian RT

,8

subunit (24), it ispossible that the non-pol sequences in

Pr145P'1-Pr125PoI

that wehave identified as B in Table 1 andFig. 10 are in fact the Rauscher analog of a viral

endonuclease. We haveplaced the B protein C

terminal to pol for two reasons. One is that Pr809ag andPr8MYPI share two tyrosine-contain-ing tryptic peptides, which places gag andpol adjacent to each other. Second, in the avian system, the endonuclease sequences in the ,B-polymerase subunit are C terminal to the a-polymerase sequences(24, 26).

The hypothetical C region of the genome is

generated from the datapresented abovewhich show that

Pr200919-P°

contains three

methio-nine-containing tryptic peptides (no.5, 14, and

16) and five major tyrosine-containing tryptic peptides (no. 22,23, 30 to 32) that are not gag, pol, or B related (Table 1). The C-protein se-quences areplaced C terminaltotheB-protein

T

'pol'

Pr135 RT

Alt---RT

_

1__

-4

B

B C

C

'env'

Viral Precursor Polyproteins

Viral Enzymes

Viral Core Proteins

FIG. 10. Modeldescribingthe translationproductsofR-MuLVgenomicRNA.I,Initiation; T,termination.

The genes and geneproductsdesignated A, B,andCareunidentified.

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sequences. TheCprotein would be generatedby

theprocessing of

Mr0#'"".

Some of these apparently extra amino acid

sequences in

pM2009ag9Po

could be explainedby

wayofcleavage

junctions

between the

gag-pol

genes and the B-C genes. Assuming that these

two junctions do occur in

pg200gag-Po

and are

labeled with methionine and

tyrosine,

onewould

predict the appearanceoftwo new

polypeptides

inthetryptic mapsof

Pr145Pol-Pr125Po'

relative

to

M009'9-P°l.

Two new

tyrosine-containing

tryptic

peptides

are seen in Pr125PoI

(spots

11

and12) whicharenotfound in

PrMgagP°l.

Spot

11 is also found in

Pr801°o,

whereas spot 12 is

foundonlyinPr145POI-Pr125Pol.

Although

twoof

the extra

tyrosine-containing tryptic

peptides

could be explained by

junction peptides,

three

extra

tyrosine-containing

peptides

still remainin

Pr2009'"°. In termsof the

methionine-contain-ing tryptic peptides, one does not see unique

peptidesinPr145Pol-Pr125POI thatare notfound

in Pr2009ag-P° (Table 1 and reference 13). A

simple explanation for this occurrence is that

the amino acid methionine is not located in

thejunction region. Although two ofthe

tyro-sine-containing tryptic peptides could be

ex-plained by this locationatcleavage junctions in

Mr0gag-Pl,

at least three major

tyrosine-con-taining tryptic peptides and three

methionine-containing tryptic peptidesareobserved thatare

notgag, pol, or Brelated. Thus,wehave labeled

theseextrasequencesC (Fig. 10).

This model also includes a gene for a viral

protease.Theprotease gene(termed"A" inthe

model) isplacedatthe 5'end of thepolgene or

Nterminalin

Pr8O!)o.

Theevidencefor this is as

follows. Protease activity has been detectedin

highly purified preparations of Friend MuLV

RT (18). In this regard, since avian retrovirus

p15 has been showntobe associated with

pro-tease activity (34; Von der Helmet al., Hoppe

Seylers Z.

Physiol.

Chem. 358:1216-1217, 1977)

and it is the C-terminalprotein in the aviancore

proteinprecursor Pr769ag (33), ourfindingthat

Pr809agand

Pr801°'

may have apartial overlap

could have some bearing on the location of a

protease in the murine retrovirus system.

Spe-cifically, protease in virions of R-MuLV

de-scribed by Yoshinaka and Luftig (35) could be

generated by cleavageofeither

Pr80°°'

(RT) or

PrW0ag.

ACKNOWLEDGMENTS

Wethank J.Syrewicz forexcellenttechnical assistance. This research was supported in part by Public Health Service contractNO1-CP-6-1017from the VirusCancer Pro-gram ofthe National Cancer Institute and by grant G-429 fromTheRobert A. Welch Foundation. J.J.K. is supported byafellowship from the Rosalie B. Hite Foundation.

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3. Arlinghaus, R. B., R. B. Naso, G. A. Jamjoom, L. J. Arcement, and W. L. Karshin. 1976. Biosynthesis andprocessing ofRauscher leukemia viral precursor polyproteins, p. 689-716. In D. Baltimore,A.S. Huang, and C. F. Fox(ed.), AnimalVirology ICN-UCLA Sym-posia on Molecular and CellularBiology, Vol.4. Aca-demic PressInc., New York.

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5. Bonner, W.M.,and R. A.Laskey.1974.Afilm detection method for tritium-labeled proteins and nucleic acids in polyacrylamidegels. Eur. J. Biochem.46:83-88. 6. Evans, L. H., S.Dresler,and D. Kabat.1977.Synthesis

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7. Grandgenett,D.P., A. C.Vora,and R. D.Schiff.1978. A32,000-dalton nucleic acid-binding protein from avian retrovirus cores possesses DNA endonucleaseactivity. Virology 89:119-132.

8. Houts, G. E., M.Miyagi, C. Ellis, D.Beard, K. F. Watson, and J. W. Beard.1978.Protein kinase from avianmyeloblastosisvirus. J. Virol.25:546-552. 9. Ikeda, H., W.Hardy, Jr., E.Tress,and E. Fleissner.

1975.Chromatographic separation and antigenic anal-ysis of proteins of theoncornaviruses.V.Identification ofa newviralprotein,p15(E).J.Virol.16:53-61. 10.Jamjoom,G.A., R. B.Naso,and R. B.Arlinghaus.

1977.Further characterization ofintracellularprecursor polypeptides of Rauscher leukemia virus.Virology78: 11-34.

11. Karshin, W.L.,L. J.Arcement, R. B. Naso, and R.B. Arlinghaus. 1977. Common precursor for Rauscher leukemia virus gp69/71, p15(E),andp12(E). J. Virol. 23:787-798.

12.Kessler, S. W.1975.Rapidisolation ofantigensfromcells with a staphylococcal protein A-antibody absorbent; parametersof the interaction ofantibody-antigen com-plexes with proteinA.J. Immunol. 115:1617-1624. 13.Kopehick,J.J.,G. A.Jamjoom,K. F.Watson, and

R. B.Arlinghaus.1978.Biosynthesis of reverse tran-scriptase from Rauscher murine leukemia virus by syn-thesis and cleavage of agag-pol read-through viral precursorpolyprotein.Proc.Natl. Acad. Sci. U.S.A.75: 2016-2020.

14. Laemmli, U. K. 1970. Cleavage ofstructural proteins duringassemblyof thehead ofbacteriophageT4. Na-ture(London)227:680-685.

15. Laskey,R.A., andA. D. Mills. 1975.Quantitativefilm detection of3Hand14Cinpolyacrylamide gels by fluo-rography. Eur.J.Biochem. 56:335-341.

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