Common Precursor for Rauscher Leukemia Virus gp69/71, p15(E), and p12(E)

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Vol. 23, No. 3 Printed inU.S.A.

Common Precursor for Rauscher Leukemia Virus gp69/71,

p15(E), and p12(E)

W. L. KARSHIN, L. J. ARCEMENT, R. B. NASO, AND R. B. ARLINGHAUS*

Department ofBiology, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025

Received for publication 24 May 1977

Rauscher murine leukemia virus glycoprotein gp69/71 and non-glycosylated

p15(E) aresynthesized by way of a 90,000-dalton precursor glycoprotein, termed Pr2a+b. Peptide mapping experiments showed that Pr2a+b contains all the tyrosine-containing tryptic peptides of gp69/71. Two additional tyrosine-contain-ing tryptic peptides in Pr2a +b that are not detected in gp69/71 are found in p15(E). Thus, gp69/71 andp15(E) peptide sequences account for all the tyrosine trypticpeptides ofPr2a+b. Thegeneorderof the two proteins wasdetermined by pulse-labeling infected cells in the presence and absence of pactamycin at concentrations ofthe inhibitor that prevent initiation oftranslation, but not elongation. The gene order was found to be: 2HN-gp69/71-p15(E)-COOH. A

newly identifiedmajorviralprotein,termed p12(E),migrates insodiumdodecyl sulfate-polyacrylamide gelsinthe"p12"region. It isrelatedtop15(E)as

deter-mined by tryptic mappingexperiments. p15(E)andp12(E) are not phosphoryl-ated, and both canbe separatedfrom phosphoprotein p12 by guanidine

hydro-chloride-agarose chromatography. p12(E) and p15(E) elute in the void volume

fraction, whereas phosphoprotein p12 elutesbetweenp15andplO. The twop12

proteins can also beseparated fromeachotherby two-dimensional gel

electro-phoresis involving isoelectric focusinginthefirst dimensionand sodiumdodecyl sulfate-gel electrophoresis inthe second dimension.

RNA tumor virus (type C)genomes code for several viral proteins, including the reverse transcriptase, the envelope proteins, and four

lower-molecular-weight structural proteins

termed p30, p15, p12, and plO. The regions of theviralgenomicRNAcoding for these classes ofproteins have been termed

"pol,"

"env," and "gag,"respectively(3). Previous work fromthis

laboratory hasprovided evidence (la,2, 21) that the envelope protein gp69/71 and a non-glyco-sylated envelope protein termed pl5(E)are

con-tainedinafucose-deficient glycoprotein precur-sortermed Pr2a+b (90,000

daltons).

A similar

precursor has been observed by Shapiro et al.

(25)and Famulari et al. (7). In this report, we

provide definitive evidence that shows that

gp69/71andpl5(E)areformedbysynthesis and cleavage ofPr2a+b and that pl5(E) is

appar-entlyCterminaltogp69/71inPr2a+b. A

newly

identified viral protein,p12(E), isderived from

p15(E),

probablyby proteolytic cleavage.

MATERIALS AND METHODS

Cells andvirus. Rauscher murineleukemiavirus (RLV)-infectedNIHSwiss mouseembryo cells (JLS-V16) and BALB/cspleen-thymus RLV-infectedcells (JLS-V5)wereusedinthesestudies(20).

Intracellu-lar precursors were isolated from infected JLS-V16 cells, whereas mature viral proteins were obtained from virus produced in JLS-V5 cells. The culture medium was a modified Eagle formula containing 10% fetal calf serum (29). Cells were grown in 2-quart (ca. 1.89 liters) roller bottles and were 80% confluent before use. Virus was purified as described previously (29).

Labeling of cells and virus. Cells were rinsedin warm Hanks solution and pulse-labeled at 37°C as indicated. For chase incubations, the radioactive medium was removed, the cell sheet was rinsed with Hanks solution, and incubation was continued in complete growth medium. For tryptic mapping ex-periments, precursors were isolated from a roller-bottle culture, whichwaspulse-labeledfor20 min in 25mlofHanks solution. Virus waslabeled for48h inaroller-bottle culture in growth medium contain-ing 1x Eagle amino acids. Under these conditions, lowerconcentrationsof aminoacids severelydepress virus production. To obtain sufficient quantitiesof labeled polypeptides for tryptic mapping, infected cells andvirus were labeled as follows: 2.5 mCi of 3H-labeled amino acids (reconstituted protein hy-drolysate) supplemented with 0.625 mCi each of

[3H]lysine (10 Ci/mmol) and [3H]arginine (10 Ci/ mmol)or 1.0mCiof'4C-labeled amino acids (recon-stituted protein hydrolysate) supplemented with 0.25 mCi each of ['4C]lysine (300 Ci/mol) and ['4C]arginine (312 Ci/mol).To obtainarginine-and 787

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788 KARSHIN ET AL.

lysine-labeled precursors and matureproteins, 0.5 to 1.0 mCi each of['4C]arginineand ['4C]lysine or 2.5 mCi each of [3H]arginine or [3Hllysine were used. For tyrosinelabeling,3.0mCi of ['4C]tyrosine and18mCi of[3H]tyrosine wereused. For methio-ninelabeling,3.0mCi of[35S]methionine and 20 mCi of[3H]methionine were used. 32p labelingof virus was performed atone-halfEaglephosphate concen-tration in a2-quart (ca. 1.89 liters)roller bottle for 48 hwith25 mCi Of[32P]PO4.

Immune precipitation. The preparation of cyto-plasmic extracts and rabbit anti-RLVserum were as described previously (la). Cell lysiswasperformed inlysisbuffer containing0.5%Nonidet P-40(NP-40; Particle Data Laboratories, Ltd.) and0.5%sodium deoxycholate (la). Forpreparative purposes, pulse-labeledcellsin2-quart (ca. 1.89liters)roller bottles were lysed in 10 mloflysis buffer. For analytical experiments, 2-ounce (ca.60ml)prescription bottles wereused,and lysis was donein 2ml oflysisbuffer. The intracellular precursors, including Pr2a+b, were isolated by direct immuneprecipitation with anti-RLVserum. A2-mlportion ofcytoplasmic ex-tractwasmixed with0.4ml of antiserum. The im-mune precipitates were collected by centrifugation at10,000xgfor10 minand washed three times with immunebuffercontaining0.5%NP-40,0.5%sodium deoxycholate, 0.02 M Tris-hydrochloride (pH 7.5), and0.05MNaCl (20).

Gelelectrophoresis. Sodiumdodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) was doneon 1.5-mm-thickgel slabsbyuseof thebuffer system described by Laemmli (14). The gels were subjectedtofluorographyasdescribed (4). To obtain alinear response to radioactivity, the X-ray films werepreflashed (15).

Purification of viral proteins and precursors. Viral proteins gp69/71 andp15(E)werepurified by SDS-PAGE fractionation ofviruslabeled for48h.In

addition, p15(E),p12(E), and p12werepurified from virus by guanidine hydrochloride-agarose column chromatography(9);pl5(E) and pl2(E) elutedinthe void volume. The column fractions were dialyzed against0.05 MNH4HCO3(pH 8.5),freeze-dried, and further fractionated by SDS-PAGE. Pr2a +b from infectedcells was purified bySDS-PAGE (8%) from immune precipitates obtained by direct immune precipitation with anti-RLV serum from 20-min pulse-labeled infected cells.

Tryptic mapping. Tryptic digestion was carried outbyincubating a dried slab gel band, which was cutupintosmall piecesin 2mlof0.05 MNH4HCO3 (pH8.5)containing50,ugoftrypsin per mlfor16h at37°C. Anadditional50

jig

oftrypsin per ml was added, and the incubation was continued for an additional 4 h. Tolylsulfonyl phenylalanyl chloro-methyl ketone-trypsin (Worthington Biochemicals Corp.) was stored at5mg/ml in 0.01 M HCl contain-ing 1 mM CaCl2 at -20°C. The gel pieces were removedby filtration through0.45-,umcellulose ni-trate filters, and the supernatant containing the soluble tryptic peptides was lyophilized. The pep-tidesweredissolvedin0.2to 0.4mlof buffer A(31), andthe sample was clarified by centrifugation. A yieldof 60to70% wasobtained from the dry slab gel

throughthisstage of theprocedure.Thesamplewas

appliedtoajacketedcolumn(1by23cm) of Chromo Beadtype P resin(Technicon),and the columnwas

eluted withanexponential pyridine-aceticacid gra-dient with increasingpH. Thefractions of 3.33 ml werecollectedat50°Cat aflowrateof20ml/h. The fractionsweretakentodrynessat100°C, dissolved in0.5ml of0.01NHCl,andthen mixed with5ml of Triton X-100containing countingfluid(29).

Gener-allyan80to90%yieldofradioactivitywasrecovered from thecolumn.

Two-dimensional gels. Two-dimensional PAGE wascarriedout asdescribedbyO'Farrell (22). The first-dimension isoelectric focusing was performed in 2.5-mm cylindrical diameter gels. The second dimension was performed on an 11.25%, 1.5-mm-thickpolyacrylamide slabgelby the Laemmli sys-tem(14).

RESULTS

Purification ofprecursor and mature viral

proteins.

The glycoprotein precursor Pr2a+b

was purified from infected cells

pulse-labeled

with [14C]tyrosine for 20 min. A cytoplasmic

extract was prepared, and immune

precipita-tion with antiserum to disrupted virus was

used to isolate virus-specific precursors. The

immune precipitate was fractionated by

elec-trophoresisonapreparative 8%polyacrylamide

slabgel (Fig. 1). The anti-RLV serum

precipi-tates viral structural proteins and their poly-protein precursors (1, la). It also precipitates several proteins present in uninfected cells identifiedinFig. 1 as hl, h2, and h3 (la).

Anti-bodies to these proteins can be removed from

the anti-RLV serum by prior treatment with

excess uninfected cell proteins (la). Normal

serum precipitates 1/20 to 1/1o the amount of radioactive protein from such a pulse-labeled extract. A few oftheradioactivebands seen in normal serum precipitates comigrated with viral precursors, but their amounts were 1/20to 'hothatobservedinanti-RLV immune

precipi-tates.

Viralglycoproteingp69/71wasobtainedfrom

purified virus preparations labeled for48h with

[3H]tyrosine. The viral proteins were

fraction-ated by preparative electrophoresis in 11.25%

polyacrylamide slab gels (Fig. 2). The gp69/71 fraction and the Pr2a +b fraction werefoundto be greater than 95% pure as determined by reanalysis on anSDSgel.

Viral proteinsP15(E) and pl2(E) were puri-fiedbyatwo-stepprocedure, the firststepbeing

guanidine hydrochloride-agarose column

chro-matography. In such a procedure, pl5(E) and pl2(E) elute in the void volumeofthe column eluate. The void-volume peak was dialyzed against 0.05 M NH4HCO andfreeze-dried.The

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COMMON PRECURSOR FOR RLV ENV PROTEINS 789

-hi

L~~~~~~A

~~~-h2

iF_ ,>Prla+b

.. ,

>Pr2a+b -Pr3

-Pr4

X-h3

FIG. 1. PreparativeSDS-PAGEof the immune precipitated intracellular glycoproteinprecursors.

Virus-infected JLS-V16cellswerepulse-labeled for20minwith 3.0mCi of['4C]tyrosinein 5ml ofHanksbalanced salt solution. The cytoplasmic extract was precipitated with antiserum to disrupted RLV. The immune precipitatewasisolated and fractionatedon an8% preparativepolyacrylamideslabgel containingthreewells. The immuneprecipitatefromonerollerbottlewasdistributedamongthe threesamplewellsafterboilingin electrophoresissample buffer. Theproteinbands labeledhl, h2,andh3arenormalcellularproteins (la).

materialwasdissolved in1%SDS and1%

mer-captoethanol and thenfractionatedby electro-phoresis in 11.25% polyacrylamide slab gels. Proteins p15(E) and p12(E) werethe only

low-molecular-weightcomponents of the guanidine hydrochloride void-volume fraction, although smallquantities ofunidentifiable high-molecu-lar-weight proteins were also present. This procedure enabled us to effectively separate

p12(E) from p12. The latter quantitatively

elutes between p15 and plO on the guanidine

hydrochloride-agarose column. Our studies in-dicated that p12(E) purified in this way was

freeof thephosphorylatedp12proteinas

deter-mined by 32p labeling (see Fig. 13 and 14). When viruswas applied directly to SDS-poly-acrylamide gels, the "p12" region of the gel did containp12(E), butnotphosphorylatedp12 (not shown). Trace amountsofthep12

phosphopro-tein, seen as two bands, migrated slightly

slower than p15(E). It is not known where in thegel fractions the remainder of the p12 phos-phoproteinmigrated. Thus, phosphoprotein p12 isapparentlynotsolubilized by boilingvirus in 1% SDSand 1%2-mercaptoethanol.

It should be mentioned that p15(E) purified bySDS-PAGE only (as in Fig. 2)wasaspure as

thatpurifiedbythe above two-step procedure,

asdeterminedby trypticmapping experiments.

Presence of gp69/71 and p15(E) peptide

se-quencesinPr2a +b. We havepreviously shown thatPr2a+b and gp69/71 containmanytryptic

peptides that comigrate on ion-exchange col-umns(21).However,theelutionprofiles of each

tryptic digest were complex. To simplify the

complexity of the tryptic digests and the subse-quentcomparisons of thepeptidesequences,we

chose to label proteins with radioactive tyro-sine. Figure 3 shows ion-exchange profiles in which[14C]tyrosine-labeledPr2a+btryptic

pep-tides were mixed with tryptic peptides of

[3H]tyrosine-labeled gp69/71. The results (Fig. 3) clearly showed that Pr2a +b shares many

tyrosine-containing trypticpeptideswithgp69/ 71. Figure 3 also shows that thereare atleast twoadditionaltyrosine-containing tryptic

pep-tides in Pr2a+b that are notfound ingp69/71

(see arrows in Fig. 3). An analysis of a

['4C]tyrosine-labeled p15(E) tryptic digest mixed with a [3H]tyrosine-labeled Pr2a+b

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790 KARSHIN ET AL.

FIG. 2. Preparative SDS-PAGE ofmatureviralproteins.ViruswasgrowninJLS-V5 cellsinthepresence

of18mCiof[3H]tyrosine for48 h.Viruswaspurified byisopycnic gradientcentrifugation,and viralproteins

wereseparatedon an11.25%preparativepolyacrylamideslabgel. The labeled viruswasdistributedamong the threesample wells afterboilinginelectrophoresis sample buffer.

800

E

0.

l4C-IYrosinelobe/ed Pr2o+b 3H-lyroslnelobe/edgp 6917/

600

C

3400

200

80 120 FractionNumber

FIG. 3. Ion-exchange chromatography of tryptic digests of tyrosine-labeledPr2a +b andgp69171.The

arrowsshow thetwoadditionaltyrosine-containing tryptic peptides in Pr2a+b that are not found in gp69171.

gest(Fig. 4)clearly showedthatthesetwo addi-tionaltryptic peptide fractionsarederived from

p15(E). We conclude from these results that p15(E) and gp69/71 are cleavage products of Pr2a +b.

We have previously reported thatgp69/71 is deficientinmethionine relativetotheenv pre-cursorPr2a+b (21). Further studies have clari-fied this. The results show that Pr2a+b

con-tains an acidic methionine-containing tryptic

peptide fraction characteristic ofgp69/71 (data not shown) and a neutral methionine-tryptic

peptide fraction characteristic of p15(E) and

H--.5-tfyrosinelobe/ed Pr 2a+b

?4C--tyrosinelabeled pl5E

20 40 60 80 100 120

Fraction Number

,,

'IT.

,6 ",.

I.

:6

ll

I,,w

1

ll

i 4l

140 160 180

FIG. 4. Ion-exchange chromatography of tryptic digests oftyrosine-labeledPr2a +bandpl5(E). The p15(E) used in this experiment waspurified from virus by SDS-PAGE as described in the legend to Fig.2.

pl2(E) (la, 21). We note,however,thatgp69/71

did contain small amounts of the methionine-trypticpeptide fraction that is characteristic of

p15(E). Also, the labelingofgp69/71 with me-thionine doesvary from experimentto

experi-ment. Inaddition,preparations ofanti-gp69/71

also vary in their ability to recognize

p15(E).

Someanti-gp69/71 sera,

supplied by

the Virus Cancer Program, do notrecognize

pl5(E) (21),

whereas others doprecipitatesmallamountsof pl5(E) (R.B.Naso, unpublisheddata). We con-clude from these studies thatgp69/71does

con-tain methionine, but that pl5(E) [or

pl2(E)]

sequences are still associated with

gp69/71

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COMMON PRECURSOR FOR RLV ENV PROTEINS

small butvariable amounts.

Itwas also determined that

glucosamine-la-beledtryptic peptides of gp69/71 eluted from the Chromo Bead cation-exchange column before fraction 30, the majority of the radioactivity

elutinginthe flow-through material (data not

shown). Thus,noneof the glucosamine-contain-ingtryptic peptides contributed to any similari-tiesbetween Pr2a+b and gp69/71. This is also important to consider since pl5(E) is not

la-beled with either glucosamineor fucose. The orderof theproteins in Pr2a +b was

ex-amined by labeling cells in the presence and

absence ofpactamycin, adrug thatselectively

inhibits theinitiationoftranslationat 5 x 10-7 M, but notelongation(5, 28, 30). Byincubating cells with radioactive amino acids in the pres-ence of5 x 10-7 M pactamycin, one

preferen-tially labels the C-terminal ends of nascent

polypeptide chains. Thus,it ispossibleto

deter-mine whether pl5(E) is N or C terminal in Pr2a+b. Figure 5 shows thatpactamycin treat-mentfor30 to 60 s beforeand duringthe pulse-labeling reduced gp69/71 formation in the

chase, whereas p15(E) labeling was increased relativetothat in the absence of drug (Fig. 5E

toG).Thedrug also inhibitedprotein synthesis

about 50%, which was compensatedfor in the

gel analysesby applying equalamounts of

ra-dioactivityinthepulse (17,000 cpm) and chase (10,000cpm).

Pulse-labeling experiments showedthat pac-tamycin treatment significantly affected the patternofsynthesis of precursorproteins (Fig. 5B to D), as indicated by the increase in Pr3

relativetoPr4and Prla+b relative toPr3 and

Pr4. Since Pr3 (-80,000 daltons) and Pr4 (==65,000 daltons) have been shown to share tryptic peptide sequences and antigenic

deter-minants with all ofthe gag proteins (1) and thatgag and pol antigenic determinants and p30 tryptic peptides are present in Prla+b (=200,000daltons) (la, 12), theseresults could beinterpretedasfollows: (i)thattheadditional peptide sequences in Pr3 are C terminal to

those ofPr4 and (ii) that Pr3 is Nterminalto

pol in Prla+b. However, no firm conclusions

can be drawn from these results since

pacta-mycin mayhave someeffect onthe

processing

of precursors during short

pulse-labeling

ex-periments.

The results of another pulse-chase experi-mentperformedinthepresenceand absenceof

pactamycin are shown in Fig. 6. The results were quantitated by preparing densitometer

tracings of the autoradiographs. The results

clearly showedthatpl5(E) is increased relative to gp69/71 inthe presence ofpactamycin

com-A B C D E F G

A^ -.. .\ ---_"' A^E

.

Prla+b

-Pr2o+b

--Pr3 -- _ A

Pr4---- -

-p30--_

p15---

---Pr2a+b -gp 69/71

-

p30

_p15E

- - '.p15

FIG. 5. Synthesis of RLV proteins inthe presence ofpactamycin. Virus-infected JLS-V16- cells in 2-ounce (ca. 60 ml) glass bottles were pulse-labeled with 200,Ci of"4C-aminoacidhydrolysatein 4mlof Hanks balanced salts for15 minat37°C. Pactamy-cin, when present,was addedat30or60 spriorto the label at a concentration of5 x 10-7 M. Chases werefor 3 h in 8 ml of complete growthmedium. (A) Marker virus; (B) 15-min pulse; (C) 15-min pulse with 30-spreincubation with pactamycin;(D)15-min pulse with 60-spreincubation with pactamycin; (E) 15-minpulsefollowed by 3-h chase; (F) 30-s preincu-bation with pactamycinfollowed bya15-minpulse anda3-h chase; (G)60-spreincubation with pacta-mycinfollowed by a 15-min pulse and a 3-h chase. Cytoplasmic extracts were prepared, and immuno-precipitation was performed with anti-RLV serum. The immune precipitates were analyzed by SDS-PAGE on a6to12%gradient slab gel.The driedslab gel was exposed topreflashed X-ray film (DuPont Cronex2DC).Equalamountsof radioactivityas im-muneprecipitates wereappliedtoeachsample well: (B, C,and D) about 20,000 cpm; (E, F, and G) about 10,000 cpm.

pared with that obtainedintheabsenceof pac-tamycin. The ratioofpl5(E)togp69/71was 0.39 inthecontrol and 0.76 in the presence of pacta-mycin.

Relationship ofp15(E) and p12(E). As dis-cussed above, p15(E) and pl2(E) areboth pres-ent inthe void volume ofthe

guanidine

hydro-chloride-agarosecolumn. Previousstudies have shownthatthey shareamethionine-containing tryptic peptide fraction that isalso present in Pr2a+b (la, 21). To examine further the

rela-tionship between pl5(E) and

p12(E),

the pro-teins were labeled with arginine and

lysine,

and their tryptic digest chromatograms were

compared (Fig. 7). The results indicate that pl5(E)andpl2(E) share manytryptic

peptides.

pl5(E) is slightly more complex than

p12(E)

andcontains atleasttwoadditional

tryptic

pep-tides (Fig. 7, fractions =105 and =125). These observations are consistent with the derivation ofpl2(E) from pl5(E) by proteolytic cleavage.

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792 KARSHIN ET AL.

dalton polyproteinprecursortothegagproteins

(26). We examined the possibility that there

maybe, in fact, two proteins with the molecu-lar weight of p12. We have found thistobe the

case and have identified them as p12 and

p12(E). The first indication that this was the

case came from studies with antisera raised

against the guanidine hydrochloride-purified

CS

asw

LJ I\

Cl

02,rech/onofMigrol/,or

FIG. 6. Densitometer tracings of autoradiographs ofviralproteins isolatedfromcells treated withand without pactamcyin. Virus-infected JLS-V16 cells

werepulse-labeled for15min witha 14C-amino acid

proteinhydrolysate (0.5 mCi) in 5mlofHanks bal-anced salt solution. In (B) the cells were

preincu-bated for 30 s in 5 x 10-7 Mpactamycin prior to

pulsing with 14C-labeled amino acids in the same

concentration of pactamycin. In (A) thepulse was

doneintheabsenceofpactamycin.Bothpulseswere

thenchasedfori h incomplete growthmedium in the

absence of pactamycin. Cytoplasmic extracts were

prepared, and immuneprecipitation wasperformed

with anti-RLVserum.The immuneprecipitateswere

fractionatedona6 to12%gradient polyacrylamide gelslab. The driedgelwasprocessed by

fluorogra-phy,and theappropriate X-ray film stripswere

ana-lyzed at 590 nm on a spectrophotometer equipped

withagelscanner.

This conclusion is supported by pulse-chase studies of virus-infected cells. A protein that comigrates with virion pl5(E) is present in rela-tively largeamountsinpulse-labeled cells

com-pared with intracellular p12(E). The relative amountsof pl2(E) increaseduring chasesatthe apparentexpenseofp15(E) (21; Fig. 8of

refer-ence11).Furthermore, pl2(E) is found in larger

amounts than pl5(E) in virions released from infected JLS-V16orJLS-V5 cells.

p12(E), aproteindistinct from

phosphopro-tein p12. Some confusion arose when we

re-ported thataproteininthe p12 region ofSDS

gels shared peptide sequences with the

glyco-protein precursor Pr2a+b (la). The confusion was amplified when Aaronson and colleagues

reported that the p12 protein eluting from the guanidine hydrochloride-agarose column be-tween p15 and plO was present in a

65,000-Fraction Number

FIG. 7. Ion-exchange chromatography oftryptic digests of arginine- and lysine-labeled pl5(E) and p12(E).

1000

800 600 400-200

0o

00

0 20 40 60 80 100 140 160

Fraction Number

FIG. 8. Ion-exchange chromatography oftryptic digests of methionine-labeledpl2andp12(E). Both p12 (A) andp12(E) (B) were isolatedbyguanidine

hydrochloride-agarose column chromatography. They werefurtherpurified by SDS-PAGE and

auto-radiographed, and the bands were cut out and minced into small pieces. The proteinswereextracted

in0.1%SDSin 0.05MNH4HCO3 (pH 8.5)at370C for18to22 h. Theproteins were thenprecipitated,

oxidized,and treated withtrypsin bytheprocedure of Vogtetal. (31).The sampleswerethenappliedtoa

Chromo Bead Pion-exchangecolumn(1 by23cm)as

described in thetext.Theproteinswerelabeled with

[35S]methionine.

A Control

p-30

p15 go69171

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p12protein (termedpl2),kindly suppliedbyC. J. Scherr of the National Cancer Institute. Such antisera precipitatedgag precursors Pr3

(80,000daltons) andPr4 (z=65,000daltons), but not the glycoprotein precursor Pr2a+b

(z-90,000daltons). This sharply contrasted with

our tryptic mapping data (la), which clearly

showed thatap12 protein [pl2(E)], isolatedby

SDS-PAGE, shared a methionine-containing

trypticpeptide fraction withPr2a +b, but which

was not present in gag precursors Pr3 and

Pr4. As indicated above, thegag p12 or

phos-phoprotein p12 of RLV is not solubilized by denaturation in SDS-PAGE sample buffer at

100°C. The netresultisthat p12 doesnot comi-gratewith pl2(E) unlessitisfirstisolatedon a

guanidinecolumn (see Fig. 13 and14).

Wethen set out tocompare the p12 proteins

that are easily separatedby guanidine

hydro-chloride-agarose column chromatography. We first established by radiolabeling experiments that p12 and pl2(E)containedmethionine. The two "pl2" preparations, each labeled with

me-thionine, werepurifiedasfollows.

[35S]methio-nine-labeledviruswasdenaturedin 8M

guani-dineat100°C for5minandappliedto a

guani-dine hydrochloride-agarose column (9). The profile was similar to that shown in the

experimentdescribedinthelegendtoFig. 12A (solid line). The void-volume reaction and the p12 peak were pooled separately, dialyzed

ex-tensivelyversus0.05MNH4HCO3, and

lyophi-lized. The residuewasdenaturedinSDS-PAGE

sample buffer, and the sampleswere

fraction-atedon 11.25% polyacrylamide gels. The

void-volume fraction contained p15(E) and p12(E)

(neitheronebeingphosphorylated;seeFig.14).

The p12 regionof thecolumncontaineda

phos-phoprotein (see Fig. 13). The methionine-con-taining tryptic peptides of these two proteins

were compared (Fig. 8). The results showed

thatp12 contained only anacidic

methionine-containingtryptic peptide fraction that eluted in the ion-exchange flow-through eluate (Fig. 8A), whereas p12(E) contained a moreneutral

methionine-containing tryptic peptide (Fig. 8B). Variable amounts of acidic flow-through materialwerepresentinpl2(E). Ananalysisof thetotal aminoacid-labeledtrypticmapsofthe

two p12 preparations showed vast differences. The p12 eluting between p15 and plOfrom the guanidine hydrochloride-agarose column (see Fig. 12A, dashed line) contained mostly acidic trypticpeptidesthatelutedintheflow-through fraction, and onebasic tryptic peptide fraction was detected (Fig. 9). This basic peptide was

also present inthegag precursorPr4 (Fig. 9). Peptidemapsofpl2(E) (Fig. 7) and p12 (Fig. 9)

showed large differences, indicating that the

300'

40 80 20 60 200

FRACTION NUMBER

FIG. 9. Ion-exchange chromatography of tryptic digests of aminoacid-labeledp12andPr4. The pro-teins were labeled with a mixture of amino acids supplemented with arginine and lysine, purified, anddigested with trypsin as described in the text.

two proteins are vastly different in chemical structure. We have previously shown, (1) that

p12

shares tryptic peptides with gag precursors Pr3 through Pr5

(z80,000

to 55,000 daltons). Thus, the two pl2 proteins have different

pep-tide sequences, andthey originate from

differ-ent

precursors,

one [pl2(E)] from the env

precursor and the other (pl2) from the gag

precursor.

Analysis of viral proteins by two-dimen-sional gelelectrophoresis. In an efforttobetter separate e t two

p12 proteins,

we fractionated

the viral

proteins by

the two-dimensional gel

procedure ofO'Farrell (22). This

procedure

con-sists ofelectrophoresis through an isoelectric focusing medium in the first dimension fol-lowed by SDS-PAGE inthe

second

dimension. The results yielded a number of interesting findings. Surprisingly,

p30

exhibited a large

amountofcharge heterogeneity, muchmoreso

than

p15, p12,

and

plO.

Fourteen to fifteen

spots, ranging from pH 6 to pH 7+, were ob-servedinthe

p30

molecularweightrange (Fig.

10). Similar charge heterogeneity has been

observed with VP-1 protein of simian virus 40 (23). The structural protein VP-1 satellite bandswere inpartattributedto invivo

altera-tions (23). The

p15(E)

region had about four species, three of which have the same molecu-larsize.

p15

hadonemajorspeciesmigratingin the pH 7 region;

plO

had one species in the pH 7+ region. The

p12

region contained one major species at pH 7, a few minor ones be-tween pH 6.5 and 7, and a faint

p12

species

detectableinthepH5region. This latterminor species was found to be

phosphoprotein

p12,

whereasthose ataboutpH7 wereattributable

to

p12(E).

This was determined by analyzing

the guanidine

hydrochloride

column

void-vol-ume fraction [containing

p15(E)

and

p12(E)]

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794

KARSHIN ET AL.

FIG. 10. Two-dimensional electrophoresis of RLV labeled with '4C-amino acids, supplemented with [14C]arginine and [14C]lysine. The procedure is describedinthetextandisthemethodof O'Farrell (22). The mixtureofampholines usedwas 4partsofthepH5to7and1partofpH3to10,yieldingapH range from slightly below pH5 togreaterthanpH7. Theorigin is attheright ofthefigure.

andthep12 fraction(Fig. 11AandB).It isclear

that the p12 protein species migrating on the

guanidine column between p15 and plO mi-grates in two-dimensional gels in the p12 re-gion, with an isoelectric point of pH 5 (Fig.

liB),

and that thep12protein[p12(E)]

remain-ing in thevoid volumeof theguanidine column hadanisoelectric pointabove pH 7(Fig. 11A). Figure 11Ashowssomematerial from thevoid volumeinp15(E) and p12(E) molecular weight

range that remained at the origin ofthe

first-dimension isoelectricfocusing gel. Thiswasnot the case when intact virus was analyzed di-rectly (Fig. 10). Thus, the void-volume aggre-gate apparently was not entirely solubilized during the first-dimension analysis. These

re-sults indicate that p12 is an acidic protein, whereasp12(E) is moreneutral.

Phosphoprotein p12 and non-phosphoryl-atedp12(E). Since murine type C p12 proteins

have been reported to bephosphorylated (24), we examined the possibility that p12 is

phos-phorylated,asissuggestedby its low isoelectric

point.Inthese studieswetook

advantage

of the fact that RLV p12 contains methionine, whereas p15 andplOaremethionine-free (Fig. 12A). Virus was separately labeled with a

[3H]arginine-lysine mixture and with

[35S]methionine. Appropriate amounts ofeach

purified virus preparation were mixed

to-gether,denatured, andanalyzed by chromatog-raphy on a 6 M guanidine

hydrochloride-aga-rose column (Fig. 12A). The results clearly

showed that p15 and plO lacked methionine,

whereas p12 contained methionine. The pres-enceofmethionineinp12and its absence in p15

andplO allowedustodeterminewhetherp12is

phosphorylated. In this experiment RLV was labeled separately with [3H]methionine and

[32P]PO4

and then mixed together in suitable

proportions. The virus preparation wastreated differentlythanusualprior tochromatography

ontheguanidinecolumn. In thiscaseviruswas incubated for 2 h in 0.5% Triton X-100 contain-ing 25 ,ug of pancreatic RNase per ml. This

procedure hydrolyzes the 32P-labeled viral

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RNA, butalso results in some proteolysis (not

dependent upon the presence of RNase) since virion Pr4 (the gag precursor of -65,000 dal-tons) isspecificallydestroyed by this treatment (11). Analysis of theRNase-treatedpreparation by guanidine hydrochloride-agarose column

chromatographygavethe profile shown in Fig. 12B. 32pradioactivity was present in the

void-volumefraction and in the p12 region (the lat-ter as twopeaks)aswellas in the low-molecu-lar-weight fraction (less than10,000).Since p15 and plO do not contain methionine and p12

does, [3H]methionine and32ppeaks in the p12 region must be p12 proteins. The smaller peak may result from proteolysis of the

higher-mo-lecular-weightgag precursor protein Pr4. We notethatp30appears intact and lacks 32p radio-activity, asdo p15and plO.

Fractions fromthevoid-volumepeak and the major p12 peak were pooled, respectively, and

analyzed on 11.25% SDS-polyacrylamide gels

(Fig. 13 and 14). The results showed that the major p12peakelutingfromtheguanidine col-umn in fractions 96 to 101 was homogeneous and contained radioactive phosphorous (Fig.

13). p15(E) and p12(E) from the void-volume fraction of the column were resolved by

SDS-PAGE, and neither containedradioactive

phos-phorous (Fig. 14). The minor p12 peak from

fractions102to 108of theguanidine

hydrochlo-'s

m.

5E

*

c 2E

L] ;2

6.5

7 7+

pH

FIG. 11. Two-dimensionalelectrophoresis oftheguanidine hydrochloride-agarose column

chromatogra-phy-purified void and p12 regions. The virus was labeled with 14C-amino acids supplemented with [14C]arginineand['4C]lysine. (A)isthevoidfraction,and(B) isthep12 regionthat elites betweenp15and

plO.

The column fractions were dialyzed against0.05 MNH4HCO3 (pH8.5) and lyophilized. The two-dimensionalprocedureisdescribedinthetextandis themethodofO'Farrell(22). Theoriginisattherightof

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796 KARSHIN ET AL.

°,;

2.5 ,I5 1KO

0 2E

. 20 4 ,,

X51 0p69/71+Pr4

1.0 Void- 2 X

05~~~~~~~~P5pl5120p <0.5

i25 B p30 2X0

0 20 40 60 80 00 120 40 160

Fraction Number

FIG. 12. Guanidine hydrochioride-agarose col-umn chromatography of labeled virus. In (A) one virus preparation was labeled withf3H]arginine and [3H]lysine and the other was labeled with [35S]methionine, and the two were mixed in appro-priate amounts. In (B) one virus preparation was labeled with [3H]methionine and the other was la-beled with[32P]P04,and the two were mixed in ap-propriate amounts. The latter preparation was pre-treated prior tochromatography with 0.5% Triton X-100 andpancreatic RNase, as stated in the text.

1200

1000

800-2

600-E

- 400

200

p12

1.

,

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1

240

200

160

120 a

85

80 40

0 10 20 30 40 50 60 70 80 90 100

[image:10.503.60.249.58.289.2]

GelSliceNumber

FIG. 13. SDS-polyacrylamide gel ofp12 region from a guanidine hydrochloride-agarose column.

p12, labeled with[3H]methionine and [32P]PO4 and isolated from theguanidine hydrochloride-agarose column above, was electrophoresed on an 11.25%

SDS-polyacrylamide slab. After slicing into 1-mm cross-sectionstrips, eachgel slicewastreated with5

mlof NCS-basedcountingfluid(20) andanalyzed. ride column (Fig. 12B) migrated on SDS gels

slightly faster than did the major p12 peak and also contained 32p radioactivity (not shown). Thesedata indicate thatp12isphosphorylated, confirming earlier work of others(24), and that p15(E) and p12(E)are notphosphorylated.

DISCUSSION

The results presented in this paper

provide

strongevidence that RLVglycoprotein

gp69/71

and a

non-glycosylated

viral protein termed

pl5(E) areformed by synthesis and cleavage of

a

90,000-molecular-weight glycosylated

precur-sorthatwehaveidentifiedasPr2a+b.

Similar

results by others (7, 25) have confirmed our initial findings (la, 21).

SDS-PAGEpatternsofviralproteins

labeled

with radioactive glucosamine and fucose showed that gp69/71 is

readily

labeled, butno

evidencewasfoundfor the incorporation of la-bel into proteins migrating in the pl5(E) or pl2(E) region of the gel (10, 21). Thus, pl5(E) and pl2(E) appear tohave little orno carbohy-drate.

Thedatapresentedinthispaper andin our

earlierreports (la, 21)havemadeitpossible for

us to propose a model (Fig. 15). Inthis model

70

60

> 50

40-'O

> 3 0

20

In

12E

2800 2400

2200

1600

,-1200

800

400

0 10 20 30 40 50 60 70 80 90 100 Gel SliceNumber

FIG. 14. SDS-polyacrylamide gel of void region isolated from aguanidinehydrochloride-agarose col-umn. The void region, labeled with [3H]methionine and[32P]P04and isolated from the guanidine hydro-chloride-agarose column above, was electrophoresed on an 11.25% SDS-polyacrylamide slab gel. After slicinginto1-mmcross-section strips, each gel slice wastreated with 5 ml ofNCS-based counting fluid (20) andanalyzed.

Virus-specific mRNA

ITranslation and glycosylation

gp69/71 pl5E

Pr2ao-

b 2HN COOH

|

Cleavage

and glycosylotion

gp69/71 + pl5E

I Cleavage

p12E

FIG. 15. Modelshowing the order of the env pro-teins within theprecursor Pr2a+b and the subse-quent cleavage pattern from this precursor to com-pleteenvproteins.

I

0L---I -I n

.L

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gp69/71 andp15(E) exist in tandem in Pr2a+b. We have tentatively ordered the two proteins within Pr2a+b by using the drug pactamycin. Treatment of cells with 5 x 10-7M pactamycin

allows preferential labeling of the C-terminal ends of growing nascent polypeptide chains (5, 28, 30) by inhibitingthe initiation of new

poly-peptide chains while allowing elongation to

proceed. The results (Fig. 5 and 6) indicate that p15(E) is C terminal to gp69/71.

Viral protein p15(E) has beenreported to be present on the virion surface (8). Thus, the antigenic sites of p15(E)exposed on the virion

envelope are sufficient tomediate immunopre-cipitationofintactvirus (7, 8). High-titer anti-serum againstthis protein, however, does not

strongly neutralize virus (8). p15(E)has

simi-laritiestoa proteintermed HA2 associated with

the envelope of influenza virus (16). HA2 is a

subunitofinfluenzavirushemagglutininandis

poorly glycosylatedincomparison tothesecond subunit HA1 (16). Furthermore, HA2, like p15(E)andp12(E), aggregates in guanidine

hy-drochloride (16), and both HA1 and HA2 are

derivedfrom a common precursor (13, 17). Itis not atallclear why therearetwo

poly-peptides in the precursor fraction Pr2a+b as

well as in the mature viral glycoprotein. Our initial tryptic mapping studies indicate that Pr2a is very similar to Pr2b. The same can be

saidfor gp69and gp7l (L. J. Arcement, unpub-lisheddata). The observedminordifferencesin

thetrypticdigest ion-exchange profiles of Pr2a and Pr2b may notbeattributable solelyto gly-cosylationdifferences because the glucosamine-labeled tryptic peptides of these glycoproteins eluteinthe void volumeof the cation-exchange

column under the conditions employed

(Arce-ment, unpublished data).

Strand and August (27) originally isolated and purified the RLV glycoprotein gp69/71. This material is treated by them as a single

protein. Our resultssuggestthatonlythe gp7l

is labeled with fucose (21; Naso, unpublished data), suggesting that the only difference be-tweengp69 andgp7l isthedegree andamount

ofglycosylation. Our tryptic

peptide

mapping resultsarealso consistent with this

interpreta-tion. Our

peptide

mapping results also show thatrelatively small butsignificantamountsof

p15(E) can be present in thegp69/71regionofa

polyacrylamide

gel.Wehavealso observedthat

theintensityofthegp69/71regionin an

autora-diogramvaries fromexperimenttoexperiment

(21) when labeling infected cellswith

radioac-tivemethionine. These data

together

with the fact that differentpreparations ofanti-gp69/71

serum varyintheir

ability

torecognize

p15(E)

suggest that

p15(E)

canbecome

variably

associ-ated with gp69/71 during isolation of proteins. Viral glycoprotein gp45 (6, 19) was not stud-ied in this report. In our hands this glycopro-tein ispresent in small but variable amounts in virions, and it is labeled to a minorextent with

radioactive glucosamine (10, 20, 21). Long-la-beled virus (16 h with ['4C]glucosamine) had a

significantpeak of gp45 (10), whereas 5- to 6-h glucosamine-labeled virus contained little or no gp45 (21). McLellan and August (18) could not

convincinglydetectgp45 onthe virusenvelope,

whereas gp69/71 was readily observed.

Mole-cules of45,000molecularweight were observed,

but they appear to be minor components and

were precipitated by anti-gp69/71 serum (18).

Other studies haveindicated that gp45 may be a subglycosylated form of gp69/71 (H.

Mar-quardt and S. Oroszlan, Fed. Proc. 35:1610, 1976).

Another interesting finding inthese studies

is the existence of two viral p12 proteins. One

viral protein (pl2) is acidic, having an isoelec-tricpoint ofabout pH 5, and it is phosphoryl-ated; the other [pl2(E)] has a more neutral

isoelectric point and isnot phosphorylated. We havetermedtheacidic phosphoprotein p12 and the neutral protein pl2(E) because ofits

rela-tionship top15(E). We havebeen able to sepa-ratethetwop12 proteins bychromatographyon 6Mguanidinehydrochloride-agarose columns. p12elutes betweenp15and plO, whereas pl2(E) elutes inthe void volume of the column as an aggregate. However, p12 is not readily ob-served in the SDS-PAGE system, except for minoramountsinthe 17,000-molecular-weight

region. This minor component can readily be observed with 32P-labeled virus. If virus is treated with NP-40 and RNase prior to

SDS-PAGE, minor amounts of this phosphoprotein

migrate in the pl2(E) region of the gel (W. L.

Karshin, unpublished data). Also, p12 isolated by chromatography on guanidine

hydrochlo-ride-agarose columns migrates in the same re-gion aspl2(E)onSDSgels(Fig. 13and14). The question remains as to why large amounts of

p12 are obtained by guanidine

hydrochloride-agarose column

chromatography,

whereas di-rectSDS-PAGE ofvirions

yields

littlep12.

Pos-sibly

p12 is

incompletely

solubilized or

dena-turedby treatment withSDS-mercaptoethanol

at 100°C, whereas the conditionsof the guani-dine hydrochloride column are

capable

of de-naturingp12.

Viral protein p12 is present in the gag pre-cursor polyproteins Pr3, Pr4, and Pr5 (1),

whereas pl2(E) shares tryptic

peptide

se-quences withpl5(E) andenvprecursor Pr2a+b (la, 2, 21).Ourresults(Fig.7and reference11)

indicate that pl2(E) is a cleavage

product

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798 KARSHIN ET AL.

p15(E).

The importance of this

cleavage

in virus structure or function isnotknown.

ACKNOWLEDGMENTS

This work wassupported byPublic Health Servic con-tractCP-61017 and grant G-429 from The Robert A. Welch Foundation.

Wethank Elena Leroux and JamesSyrewiczforexpert technical assistance.

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