Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides.

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Vol.22,No.1 Printed in U.S.A.

Analysis of Intracellular Feline Leukemia Virus Proteins

II. Generation

of Feline

Leukemia Virus

Structural Proteins from

Precursor Polypeptides'

GREGORY F. OKASINSKI AND LELAND F. VELICER*

Department ofMicrobiology and Public Health, Michigan State University, East Lansing, Michigan 48824 Received for publication 17 August 1976

The synthesis and processing of feline leukemia virus (FeLV) polypeptides

werestudied inachronically infected feline thymustumorcellline, F-422, which produces the Rickardstrain ofFeLV. Immune precipitation with antiserum to FeLVp30 andsubsequent sodium dodecylsulfate-polyacrylamidegel

electropho-resis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible

precursor polypeptides. SDS-PAGE of immune precipitates from cells

pulse-labeled for2.5min with[355]methioninerevealedthe presenceof a 60,000-dalton

precursorpolypeptide(Pp6O)aswell as a30,000-daltonpolypeptide. Whencells

were grown inthepresenceof theproline analogueL-azetidine-2-carboxylicacid,

a70,000-daltonprecursorpolypeptide (Pp7O)wasfoundinadditiontoPp6Oafter

a 2.5-minpulse. The cleavage of Pp6O could be partially inhibited by the general

protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial

inhibi-tionwasfoundto occuronly if PMSFwas presentduring pulse-labeling.

Intra-cellular Pp7O and Pp6O and FeLV virion p70, p30, p15, p1l, and p10 were

subjectedtotrypticpeptideanalysis. The resultsof this trypticpeptideanalysis demonstrated that intracellular Pp7O and virionp70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p1l, and plO. Pp6O contained thetrypticpeptides of FeLVp30, p15, andplO,butlacked the tryptic

peptidesofp1l.The results of pactamycingeneordering experimentsindicated thatthe small structural proteins of FeLVareorderedp11-pl5-plO-p30. The data indicate that the small structuralproteins of FeLV are synthesizedas partofa

70,000-daltonprecursor. Acleavage schemefor thegenerationofFeLVp70, p30,

p15, p1l, andplO fromprecursorpolypeptides isproposed.

The generation of stable virion proteins from large precursor polypeptides is a well-docu-mented phenomenon observedinmanyanimal virus-infected cells. Post-translational cleavage

of precursor polypeptides was initially

de-scribed forpoliovirus-infected cells (8, 23). This phenomenon has been showntobewidespread, occurring in cells infected by coxsackievirus

(11), Sindbisvirus (21, 22),Semlikiforestvirus

(4, 12, 15),mengovirus (20),and

encephalomyo-carditis virus (3, 14). The synthesis of large

precursor polypeptides has beenproposedas a

mechanism by which polycistronic mRNA is

translatedineukaryotic cells (2).

During the past 3 years several reports of

precursorpolypeptidesinoncornavirus-infected

cells have appeared. Avian myeloblastosis

vi-rus-infected primary chicken fibroblasts

con-tain a76,000-daltonprecursorpolypeptide(27).

Trypticpeptide analysis of thisprecursor poly-'Articleno. 7743from theMichiganAgricultural Exper-imentStation.

peptidereveals thepresence of four virion

pro-teinswithin the76,000-daltonmolecule(27, 28).

Immuneprecipitation of Rauscher leukemia vi-rus (RLV) proteins from infected cellswasused

to demonstrate 82,000- and 65,000-dalton

pre-sumptive precursor polypeptides in JLS-V9 cells (26) and 200,000-, 90,000-, 80,000-, and

65,000-daltonpolypeptides inJLS-V16cells (1, 16).Jamjoometal. (9)demonstratedthat RLV

contains anon-glycosylated 70,000-dalton

poly-peptide thatisidentical to anintracellular

pol-ypeptide,immuneprecipitable byantiserumto

RLV. The virion and intracellular

70,000-dal-ton polypeptide contain RLV p30 tryptic

pep-tides, and the authors suggest a possible

pre-cursor-productrelationship.

Recently, we identified a 60,000-dalton

pre-cursor polypeptide (Pp6O) of feline leukemia

virus (FeLV) p30 (18). We employed both

im-muneprecipitation ofFeLVpolypeptidesfrom

pulse-labeled cells and trypticpeptide analysis

ofPp6OandFeLVp30todemonstratea

precur-74

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sor-productrelationship. In thisreportwe

pre-sent evidence that: (i) there exists a

70,000-dalton precursor polypeptide (Pp7O) of FeLV

p30, p15, p1l, andplO; (ii)thecleavageofPp7O

results in the generation of Pp60 and p1l; (iii)

the cleavageofPp6O can bepartiallyinhibited

by the general protease inhibitor phenyl

methyl sulfonyl fluoride (PMSF); (iv)

intracel-lular Pp7O and FeLV virion p70contain

identi-cal tryptic peptide profiles; and (v) the gene

order of the low-molecular-weight FeLV

struc-turalproteinsis pll-p15-plO-p30.

Our evidenceindicates that the small

struc-tural proteins of FeLV differ from those of

avianvirusesinbotharrangement withinand

cleavagefromprecursorpolypeptides. FeLV

ap-pears tobesimilartoRLV inthat bothcontain

uncleaved precursors of structural protein (9;

present publication). The inhibitionof precur-sor cleavage by a general protease inhibitor reported here seemstobe uniqueforFeLV.

(Most ofthis work was submitted by G. F.

Okasinski inpartial fulfillment of the

require-ments for the degree ofDoctor of Philosophy.

This work was presented in part at the ICN-UCLA Winter Conferences on Molecular and

Cellular Biology, 14-19 March 1976, Squaw Valley, Calif., and at the Cold SpringHarbor

meeting on RNA Tumor Viruses, 26-30 May

1976, ColdSpring Harbor, N.Y.)

MATERIALS AND METHODS

Source of cells and virus. Thechronicallyinfected feline thymus tumor cell suspension (F-422) was

used throughout these experiments. This cell line

produces the Rickard strain of FeLV andwas

propa-gatedaspreviouslydescribed (7).

Radioactive labeling of cells. F-422 cells were pulse-labeled with [35S]methionine (1 uCi/106cells)

or3H-aminoacid mixture(2jiCi/106 cells).Thepulse wasterminated and cells werechased in the pres-enceof10timesthe normal concentration of

methio-nineaspreviously described (18).

Purification of FeLV. 14C0 or3H-amino acid-la-beled FeLV and unlaacid-la-beled FeLVwerepurifiedfrom F-422 culture fluids by discontinuous sucrose gra-dientcentrifugationaspreviouslydescribed (18).

Preparation of subcellular fractions. Particulate

fractions (PF) and cytoplasmic extracts (CE) were

preparedfromNonidet P-40(NP-40)-disruptedcells

aspreviouslydescribed(18). Briefly, cellswere

dis-rupted withlysisbuffer (0.5% NP-40-0.15 M NaCl-0.01 MTris [pH7.4]) andcentrifugedat2,400rpm for5 min in anInternational PR-6centrifuge. The supernatantwasremoved andcentrifugedat100,000

x g for 1 h. The100,000 xgsupernatant (cytoplas-micextract)wasremoved,andthepelletwas solubi-lized withlysisbuffercontaining0.2%deoxycholate (PF). Both the CE andPF weresonicallytreated for 2 to 3 minin a 150-W Bransonultrasonic cleaner (Branson Instruments Co., Stamford, Conn.) and

centrifugedagainat100,000 xg for1h.

Immuneprecipitation.Rabbit antiserumtoFeLV

p30 was prepared and clarified as previously de-scribed (7, 18). Rabbit antiserumto bovine serum albumin (BSA) was obtained from E. Sanders

(Michigan State University). Clarified antisera wereaddedtosubcellularfractionsordisrupted vi-rusandincubated for30minat37°C andthen

over-nightat4°C.Immuneprecipitateswerecollectedby

sedimentation through 1mlof 10% sucrose (wt/wt) inlysisbufferat2,000rpmfor 20 min inan Interna-tional PR-6centrifuge. Thiswasrepeatedone addi-tionaltime,and thefinalprecipitatewassolubilized for sodiumdodecyl sulfate-polyacrylamidegel

elec-trophoresis (SDS-PAGE) as previously described (18).

Doubledetergent disruptionofFeLV. Labeledor unlabeledpurified FeLVwasdisrupted by suspen-sion of viral pelletsinlysisbuffercontaining0.5%

deoxycholatefollowedby5min ofsonictreatmentin a 150-WBranson ultrasonic cleaner. Thedisrupted

viruswascentrifugedat100,000xgfor1hin anSW 50.1rotor(Beckman),andthe supernatantwasused as a source oflabeled or unlabeled soluble FeLV

polypeptides.

SDS-PAGE. Electrophoresis in the presence of SDS was.doneaspreviouslydescribed (18), usinga 9%polyacrylamidegeldescribedbyFairbanksetal. (5). Thegelswerefractionated and assayed for

ra-dioactivity, using 3a70b scintillation cocktail (RPI

Corp., Elk Grove Village, Ill.) as previously de-scribed (7).

Tryptic peptide analysis. Tryptic peptidesof

im-mune precipitated polypeptides and labeled FeLV

polypeptides, eluted fromSDS-polyacrylamidegels,

werepreparedaspreviouslydescribed (18). A1-mg

amount ofBSA wasadded to FeLVproteins sepa-ratedby gelfiltration in the presence of 6 M guani-dinehydrochloride (GuHCl),and theproteinswere

precipitatedbydilution of GuHClto0.6Mfollowed

by the addition oftrichloroacetic acid to a final concentration of 25%. Tryptic peptides were then

prepared as previously described (18).

Cation-ex-change chromatographyoftryptic peptides wasdone

byusingahigh-pressurecolumn of type P

chromo-beads (Technicon) as previously described (18).

Tryptic peptides were lyophilized and stored at

-76°C untilchromatography.

Pulse-labeling in the presence of pactamycin.

Pactamycinwasobtained from George B. Whitfield

of theUpjohnCo. Thedrugwasstored as a 5 x 10-5 Msolution in1 mMacetic acid at-20°C.

A total of 250 x 106 cells wereincubated for 30s

withpactamycinat afinal concentration of 5 x 10-7

M andpulse-labeled with 3H-amino acid mixture(2

,4Ci/100

cells)or"4C-aminoacidmixture (0.21iCi/106

cells)inafinal volume of 5 ml.

RESULTS

EffectofPMSFonthecleavage of Pp6O.To determine the effect of PMSF oncell viability

andprotein synthesis duringtheshortperiod of exposuretobe employed in these

experiments,

cellswereincubated withPMSFfor 2 min and

thenpulse-labeledfor 2.5 min in the presenceof

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76 OKASINSKI AND VELICER

PMSF. Both hot trichloroacetic acid-precipita-bleradioactivity and trypan blue dye exclusion weremonitored. The results of this experiment (Fig. 1)indicated that PMSF reduced trichloro-aceticacid-precipitableradioactivity by 40% (at

50 jug/ml)during the short period of exposure used in these experiments and reduced cell via-bility by 10to 15%.

Pulse-chase-labeling experiments weredone inthe presenceof PMSFtodeterminewhether PMSF, a general protease inhibitor, could in-hibit the cleavage of Pp6O. In experiment 1

(Fig. 2)cells werepulse-labeledfor2.5 minand

then chased for 30 min in the presence or

ab-senceof PMSF. SDS-PAGE of immune

precipi-tatesfrom subcellular fractions ofpulse-labeled

cells demonstrated the presence ofPp6O in the

PFandp30inthe CE (Fig. 2A andE)as previ-ouslyreported(18). When cellswerechased for 0.5 hinthe presenceorabsence ofPMSF, Pp6O

wasabsentfrom both subcellularfractionsand p30wasfoundmainlyinthe CE(Fig. 2B, F, C,

andG), indicatingno inhibition ofPp6O cleav-age.

Theeffect ofPMSFonPp6O cleavagewasalso

examined with the protease inhibitor present duringpulse-labeling. Inthis experiment(Fig.

3) cells were incubated with PMSF for 2 min

and thenpulse-labeledfor 2.5min inthe

pres-ence of PMSF. SDS-PAGE of immune

precipi-tates from subcellular fractions of cells

pulse-100

III

4) 0

al

100 E

.0

50

75 o

.6

0..

50 <

25

250 0

0 10 20 30 40 50

1sg PMSF

FIG. 1. Effect ofPMSFoncellviability and

pro-teinsynthesis. Six aliquots ofcells (50 x106cellsper

aliquot)wereincubatedwith0, 5,10,15,20,or50pg

ofPMSFpermlfor2minandthenpulse-labeled for

2.5min with 10 uCiof[35S]methionineperaliquot in

thepresenceofPMSF. The pulse-labelingwas

termi-nated, and the cells were chased for 3.0 h in the

presenceofPMSFasdescribed inthetext.Cell via-bility was determined by trypan blue dye exclusion immediately afterthepulse-label (4.5-minexposure

toPMSF) andat1.5and3.0h. Theeffect of PMSF

onprotein synthesiswasdeterminedbyassayinghot

trichloroacetic acid-precipitable radioactivity from NP-40-disruptedcells.

labeledinthe presence of PMSFdemonstrated the presenceofPp6Oinboththe PF and CE and

little, if any, p30 (Fig. 3A andD). When cells

pulse-labeled in the presence of PMSF were

chased in the presenceor absence of PMSF for

0.5 h, Pp6O was present in both subcellular fractionsaswas asmallamountofp30, indicat-ingpartial inhibitionofPp6Ocleavage.

Nonspecific immune precipitationwas

moni-tored by incubating labeled subcellular frac-tions with5,ug of BSA and 200 ,ul of anti-BSA. Figures 2D and H depict typical results

ob-tained from subcellular fractions of pulse-la-beled cells. We routinely detect only two poly-peptides (labeledaandb) in all control experi-ments. Identical results were obtained from subcellular fractions of cells incubated with PMSF (data not shown).

Effect of L-azetidine-2-carboxylic acid on

the synthesis ofFeLV precursor polypeptides. Wehave previously demonstrateda precursor-productrelationship between Pp6O and p30 (18). Several amino acidanalogues have been tested for their effectonthe appearance and process-ing of FeLV precursor proteins. Of the

ana-logues tested (fluorophenylalanine,

canavan-ine, andL-azetidine-2-carboxylic acid), only

L-azetidine-2-carboxylic acid has yielded any de-tectable effect. SDS-PAGE ofimmune

precipi-tates from subcellular fractions of cells pulse-labeledinthepresence of L-azetidine-2-carbox-ylic acid demonstrated the presence of large amounts of70,000-daltonpolypeptide (Pp7O)as

wellasreducedlevels ofPp6O and p30 (Fig. 4).

A comparison of these results (Fig. 4) with those seeninFig. 2AandEindicated that the 70,000-daltonpolypeptideseen inrelatively low amounts in the absence ofanalogues was now

the principal immuneprecipitable polypeptide whenpulse-labeling was donein the presence of L-azetidine-2-carboxylic acid. This suggests that theprolineanalogue may be inserted into the initial translation product and subse-quently inhibit the cleavage step, resultingin

theformation of Pp6O. Control experiments em-ploying immune precipitation of BSA with

anti-BSA yielded results similartothose shown

inFig. 2Dand H (data not shown).

Immune precipitation of p30 and p70 from disruptedFeLV.The presence ofan intracellu-lar 70,000-dalton polypeptide that is both im-muneprecipitable by anti-p30 and electropho-retically identical to virion p70 led us to exam-ine virion p7O for p30 antigenic determinants.

In aprevious report(18)wedemonstrated that the anti-p30 serumcouldonlyimmune

precipi-tateviralp30 from 0.5% NP-40-disrupted virus.

Subsequent experiments have shown that 0.5%

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20 40 60 80

I I

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E

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20 40 60 80

FRACTION NUMBER

FIG. 2. SDS-PAGE of immune precipitates fromPFand CEof cells incubated with andwithoutPMSF afterpulse-labeling.Atotalof 200 x106cells werepulse-labeledwith 200 pACiof[35S]methioninein4 mlof growth medium. Thepulse-labeledcellsweredivided intofour aliquots,twoof whichwerechasedfor 0.5 h

either withorwithout PMSF(50 pg/ml)and theremaining aliquots whichwereusedasasourceof

pulse-labeled polypeptides. PF and CE were prepared from both pulse-labeled andpulse-chase-labeled cells.

Polypeptides were immuneprecipitated from each fraction with anti-p30 and co-electrophoresed with

3H-aminoacid-labeled FeLVasdescribed in the text. Solidarrows indicate thepositions ofnon-glycosylated

FeLVpolypeptides. (A) and (E) PF and CE, respectively, from pulse-labeled cells; (B) and (F)aPFandCE,

respectively, from cells chased in the absence of PMSF; (C) and (G) aPFandCE, respectively, from cells

chasedinthepresenceof50pgPMSFperml; (D) and (H) SDS-PAGE of immuneprecipitates fromaPF and

CE,respectively, of pulse-labeled cells incubated with5 pgof BSA and200 piof anti-BSA.

NP-40alone doesnotsolubilize viralp70 toany

considerabledegree (datanot shown).

"4C-amino acid-labeled FeLV was disrupted

bytreatmentwith 0.5% NP-40 and sodium

de-oxycholate and then immuneprecipitated with

anti-p30. The detergent disruption procedure

employed in these experiments solubilizes the

major FeLV polypeptides (Fig. 5A), although

only 60 to 70% of the FeLV p70 is solubilized

(data notshown). SDS-PAGE of the resulting

immune precipitate demonstrated two labeled

FeLV polypeptides. Both FeLV p30 and p70

wereimmuneprecipitated (Fig. 5B). FeLVp70

wasonlypartially immune precipitated (30% of

thetotalsolubilizedp70 countsper minute) in

thisexperiment. The dataindicated that FeLV

p70 contained some p30 antigenic

determi-nants. Control experiments using5 ,4gof BSA

and 200 1.l of anti-BSA showed virtually no

precipitation of labeled viral proteins,

indicat-I

CY

x

c

0

h-Li

N'.

E

0.

I0

A2.5 min Particulate Fraction E 2.5min CytoplasmicExtract

puSe0 P30 pulse p30

B 0.5 hr chose MS F 0.5 hrchose;PS

p70

p3

p15

plO p70 f p15 plO

2 ; and * janid

l~~~~~~~~1

11

3 0.5 hr chase Fontrol}0.5 hrchose +3

p70 p30 p15 plO p70 * p15plO 2- p70 p30 ~p15andl 7 3 1 andp~l

JQ Pulse

control)

H Pulse control

,mp30 p30

p70O p15plO p70 p15pIO

2 l and land 2

S I3

4m;

046

a,

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FIG. 3. SDS-PAGE of immune precipitates from PF and CE of cellspulse-labeledwith[35S]methioninein the presenceand absence of PMSF. A total of 300 X 106 cells weresuspended in 6 ml of growth medium deficientinmethionine but containing 50 mgofPMSF per ml for2min. The cellswerethenpulse-labeledfor 2.5 minand divided into three aliquots, and two aliquots werechasedinthe presenceorabsenceofPMSF. PF and CEwerepreparedand then immuneprecipitatedwith anti-p30 andelectrophoresedasdescribed in the text.(A) and(D)aPFandCE,respectively, from cellspulse-labeledinthe presenceofPMSF; (B) and (G)a PFand CE, respectively, from cells pulse-labeled and chased in the presenceofPMSF (50pglml);(C) and(F)

aPF andCE, respectively,fromcellspulsedinthe presenceofPMSF and chased in the absenceofPMSF.

20 40 60 80 20 40 60

FRACTION NUMBER

FIG. 4. SDS-PAGE ofimmuneprecipitates the CE and PF of cells pulse-labeled [35S]methionine inthepresenceofL-azetidine-2 boxylicacid.Priortopulse-labeling,100 X 106 were incubatedfor45 min ingrowth medium

cient in methionine and proline but containing azetidine-2-carboxylic acid (021 mglml). The

were pulse-labeled for 2.5 min with 100 [35S]methionine in thepresenceofL-azetidine-2

boxylicacid(021 mg/ml),and thenaCEand

_.

ing little or no nonspecific trapping (data not

3 shown).

°2

Tryptic peptide analysis of intracellular

2 C

Pp7O,

Pp6O,

and FeLV virion

p70,

p30,

p15,

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p1l, and p10. Intracelular 3H-amino

acid-la-| beledPp7OandPp6Otrypticpeptideswere sub-jected to high-pressure cation-exchange

chro-e

matography

on acolumn of

type

Pchromobeads

(Fig. 6). The trypticpeptidefractionsare

iden-from tified

by

the elution

pH

of the

peak

fraction

with

(Fig.

6).

Pp7O

contained 29 detectable

peptide

S-car- fractions, whereas Pp6O was resolved into 25

cells

defi-ng

L-cells Ci of

?-car-aPF

were prepared. The fractions were incubated with

300piofanti-p30and5 pgofdisruptedFeLV. The

immune precipitates were collected and co-electro-phoresed with 3H-amino acid-labeled FeLV as de-scribed in the text. Solid arrows indicate the

posi-tionsof labeled FeLVpolypeptides.

A +PMSF Particulate Fraction D+PMSF Cytoplasmic Extract

2.5min pulse plO 2.5 min ~ 1

an u Pp60 |a

p70 - 30

pi5

ir pulse Pp60 and

2- :p70 p30 p11pil

Pp60

Lasa

a ~~~~~~~~~~~~b

B +PMSF E+PMSF

Pp60

0.5 hr chase

casd

hre

i

and

2 p70 p30 p15 pil p70 p30 p15p1l

Pp60

C -PMSF,. F -PMVSF Pp6O

0.5hr chase plO 0.5 hr

and chase andl

2- p70 p30 pI5 piI p70 p30 pI5 p~l

Pp60

b

~~~k~~ b

20 40 60 80 20 40 60 80

1.5

.2 1.0

IL E 05

on

W6

A Particulate Fraction 8Cytoplasmic Extract

p70 P30 p15 PlO p70 p30 pi5pIO

i iand j and

Pp70 1 P070 i

P060 PL60

abb

ir

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0

u_ 8

U BImmunePrecipitatedp30 p70

E p7O p30p15 plO

i' 6- p

1,J

4-2

0 20 40 60 80

FRACTION NUMBER

FIG. 5. SDS-PAGE of immune precipitatedp30 andp70 fromdetergent-disrupted FeLV. "4C-labeled FeLV wasprepared from 25 x 106cells incubated

with25 uCiof "4C-labeled amino acid mixture for24 h in12.5 mlofgrowth medium. The viruswas

puri-fied as described in the text. The viral pellet was

suspendedin 0.6 mlof lysis buffer containing0.5% NP-40 and 0.5% deoxycholate by 3 min of sonic oscillation in a150-W Branson Ultrasonic Cleaner

(Branson Instruments Co., Stamford, Conn.). The disrupted virus was then centrifuged for 1 h at 100,000 x g. (A) FeLV polypeptides (20,000 cpm) wereprecipitated with 9 volumes ofacetoneand 50

pgofBSA ascarrier and thenelectrophoresed; (B) FeLVpolypeptides (20,000cpm) wereimmune

pre-cipitatedwith 100

41

of anti-p30 and electrophoresed

asdescribed in thetext.

peptide fractions. Twenty-three of the 25

pep-tidefractionspresentinPp6Owerealso foundin

Pp7O. Two Pp6Otrypticpeptide fractions, with elution pH values of 3.85 and 3.94, were not

detectableinthetryptic peptide profileofPp7O. Pp7Ocontained,inadditiontothe23Pp6O

tryp-tic peptide fractions, 6 peptide fractions, with

elutionpHvalues of3.28, 3.62, 3.73, 3.81,4.12,

and 4.14. Because Pp7O and FeLV vision p70

were both immune precipitable with anti-p30

(Fig. 4 and 5)and because Pp70 contained the

tryptic peptidefractions ofPp6O (Fig. 5), tryptic peptide analysis of the structural proteins of FeLVwasdonetodetermine: (i)whether

intra-cellular Pp7Oand FeLV virion p70 were

simi-lar, and(ii)whether the additional tryptic pep-tide fractions seen in Pp7O, but not in Pp6O,

could be assigned to any of the FeLV structural proteins.

Tryptic peptide analysis of FeLV virion p70 yielded29 peptide fractions (Fig. 7) thatwere

indistinguishable from the 29 peptides of intra-cellular Pp70. The results demonstrated that intracellular Pp7O andvirion p70 (Fig. 6 and 7) were identical by tryptic peptide analysis. A total of 13 of 15 p30 tryptic peptide fractions were present in virion p70 and intracellular Pp60and Pp7O (Fig. 6 and 7). Virion p15

con-tained 9 tryptic peptide fractions, of which 7

were present in virion p70 and intracellular Pp60 and Pp70 (Fig. 6 and 7).The assignmentof the p15 tryptic peptide fractions with an elution pH of 3.79 remains uncertain due to the low level ofradioactivity found in the p15 tryptic

pepide fractions and thehigh level of radioac-tivity present in the p70 tryptic peptide frac-tions, withelution pH values of3.77and 3.80.

The pH values are accurate to ±0.01 pH unit

and thus could allow assignment of this p15

peptidetoeither the p70peptide fractions 3.77 or3.80.Wechose to consider thispeptideas not

.s

A Pp70

EY0-0020'wt4q 60 80 0 20 14060- ISO

FRACT ION NUMBER

FIG. 6. Tryptic peptideanalysisofPp70and Pp60 eluted from SDS-polyacrylamide gels. Pp70 was prepared by SDS-PAGE of immune precipitates from the PF and CE of 500 x 106cells pulse-labeled for 2.5 min with 500 ,uCiof3H-amino acid mixture in the

presence of L-azetidine-2-carboxylic acid. Pp6O was

prepared in a similar manner from the PF of 500 x 106 cells pulse-labeled(without L-azetidine-2-carbox-ylic acid) with 500 ,uSCi of 3H-amino acid mixture. Both polypeptides were eluted from gels, and tryptic peptides were prepared asdescribed in the text. Tryp-tic peptides were eluted from a column of type P chromobeads, with a linear gradient of pyridine ace-tate (pH3.1to 4.9) at a flow rate of 30 mlh. Various

peptide peaks are identified by the pH of elution determined from reading pH values with a PHM 26 expanded-scale pH meter(Radiometer, Copenhagen, Denmark). Percent recovery ranged from 70 to 80% of the radioactivityelutedfrompolyacrylamide gels.o,

ElutionpH valuesofPp6O trypticpeptidesabsentin

Pp70.

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Cl

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FRACTION

NUMBER

FIG. 7. Tryptic peptide analysis ofFeLVvirionp70,p30,pl5, pll, andp1O.FeLVp7O,p30,and pl5were prepared by SDS-PAGE of3H-aminoacid-labeled FeLV. FeLVp1l andplOwerepurified by gel filtration of labeled FeLVin GuHCl. Tryptic peptides of thefive virion proteins were preparedasdescribedinthetext. Tryptic peptides were elated and pH values weredetermined as describedin the legend to Fig.6.Percent recovery ranged from 60 to 80% of theradioactivity elated from polyacrylamide gelsorgelfiltration columns. Symbols: A, elation pH values of tryptic peptides absentinvirionp70andPp7O ofFig. 6; ( ),p70tryptic peptides presentin bothp70andvirionproteinsp30, p15,p1l,andplO.

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being present within p70 because of the rela-tively low level of radioactivity. The tryptic

peptidefractions of p1l had elution pH values of

3.29, 3.61, 3.74, and4.11 (Fig. 7). Three of the

p1l tryptic peptide fractions were present in

both virion p70 and intracellular Pp7O, but

wereabsentinthePp6Otryptic peptide profile

(Fig. 6 and 7). Although the p1l peptide

frac-tion eluting at pH 4.11 could not be assigned

unequivocallydue to lowerradioactivity and a

variation of ±0.01 pH units, the presence of a

pH4.12peptidefraction in Pp7O andp70that is

absent in Pp6O is consistent with the other three p1l fractions. These data show that both

virionp70 and intracellular Pp7O contain p1l,

whereasPp6O lacks p1l. Tryptic peptide

analy-sis of virion plO revealed fourtryptic peptide fractions, withelution pH values of 3.28, 3.48,

3.67, and 3.86. Two of the plOtryptic peptide

fractions (pHvalues 3.48and3.67) were present inp7O, Pp7O, andPp60,indicating the presence

of plO in all three molecules. The plO tryptic

peptide fraction with an elution pH value of

3.86 was not present in p70 or Pp7O, but was

presentinPp60, whereas theplOpeptide

desig-nated 3.28isalso seen in p70 and Pp7O, but is

absent in Pp6O. We judge the presence of the plO peptides3.86 and3.28 and thePp6O peptide 3.85 (Fig. 6) tobe expectedanomalies of tryptic

digestionof precursor andproduct proteins.

The tryptic peptide analysis of intracellular

and virion polypeptides was done in parallel

rather than by a double-label procedure,

be-cause of the relatively small amounts of plO

and p1l available from FeLV and the expense

of generating largequantities ofprecursor poly-peptidesof FeLV. Tohelpinsure uniformityin thisparallel analysis, allof thetryptic

peptides

of the proteins examined (Fig. 6 and 7) were

prepared in parallel and stored as

lyophilized

tryptic peptides until chromatography (no

longer than 3 weeks). Tryptic peptides were

eluted with buffers prepared in large

quanti-ties,and the column oftype Pchromobeadswas

washed with sodium hydroxide,

pyridine,

and

starting bufferbefore each run. FeLVplOand

p1l werepurifiedbygelfiltrationinGuHCl(7).

Todetermine the effect ofGuHCl-treated

pro-teinsonthetrypticpeptideprofiles,tryptic

pep-tides were preparedand analyzed from FeLV

p30andp15 preparedfrombothSDS-PAGE and

gelfiltrationsinGuHCl.Polypeptidesprepared

by bothproceduresyieldedqualitatively

identi-cal results when their tryptic peptides were

analyzed by high-pressure cation-exchange

chromatography (data notshown). The

repro-ducibility of the pH valuesassignedtothe tryp-ticpeptides wasexamined from severaltryptic

peptide profiles of FeLV p30 andwasfound to

vary by ±0.01 pH unit (datanotshown).

Pactamycin gene ordering. The order of FeLVp30, p15, p1l, andplO within Pp7O was

determinedbypreferential labeling of the

car-boxy terminal end of newly synthesized poly-peptides. Ourexperimental approachwas simi-lartothatemployed by other workerstoorder the proteins of poliovirus(24, 25), encephalomy-ocarditis virus (3), and recently avian myelo-blastosisvirus (AMV)(28). Inourexperiments pactamycinwasusedtoselectivelyinhibit initi-ation of polypeptide synthesis, while permit-tingchainelongation. Cellswereincubated for

30 swith5 x 1O-7 Mpactamycin, a

concentra-tionrequiring 10 to 15 min tocompletely inhibit

incorporation oflabeled aminoacids, and then

pulsed-labeled in the presence of pactamycin with3H- or

'4C-amino

acid mixtures for5min.

Aparallel culturewaspulse-labeledinthe

ab-sence of pactamycin with the opposite (i.e.,

either 3H- or 14C-amino acid mixture) labeled

aminoacidmixture. Afterpulse-labeling, both cultureswerechased for20hand released virus

waspurified from thegrowth medium.Labeled

FeLVproteins wereseparated by gel filtration

inthe presence of6 MGuHClto allow

resolu-tion of FeLV p1l and plO, which are not

re-solvedby SDS-PAGE (7).The relativelabeling ratiosweredetermined from thepercentage of label in each protein synthesized in the pres-enceof pactamycinoverthepercentageoflabel

in each protein synthesized in the absence of

pactamycin.

This experimental approach, based on an

analysisofpolypeptidesinextracellularvirions aftera20-h chase, wasnecessitated by the lack ofhigh-titer monospecificantiseratoFeLVplO

andp1l, which is neededtoeffectively

quanti-tateintracellularpolypeptides. Control

experi-ments with immune precipitation by antisera

to whole virusdemonstratedthat the intracel-lular p30, p15, and p11-plO peaks detected on

SDS-PAGE shortly after a pulse were not de-tectable 20h later(G. Okasinski, unpublished

data). These results were consistent with our

publisheddata (18)concerningincorporationof

p30 into extracellular virus in the absence of pactamycin. Immune precipitation, with

anti-serumtop30andp15,ofsubcellular fractions of

cells pulse-labeled inthe presenceof

pactamy-cinandchased for1hresultedinnodetectable

Pp60 (data not shown). The results of these

controlexperiments indicated thatpactamycin didnotdetectably effect eitherthe cleavage of

Pp6O or the release ofviral polypeptides from

these cells.

Table 1 depicts the results ofa pactamycin

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TABLE 1. Effect ofpactamycin on therelative labeling of FeLV virionproteinsa Viral protein Relative labeling ratiob

p11 0.44

p15 0.65

plO 1.06

p30 1.21

a Cells werepulse-labeled inthe presence or

ab-sence ofpactamycin and then chased for 20 h. Puri-fied virus wasdisrupted, and proteins were chro-matographed by gel filtration in the presence of 6 M GuHCl.

bRatios are thepercentage oflabel in each pro-tein synthesized in the presence of pactamycin over the percentage of label in each protein synthesized

intheabsence ofpactamycin.

experiment. An examination of this data

indi-catesthat pactamycintreatmentledto a

prefer-entialloss of labelinp1l and p15 relativetoplO and p30. The relative labeling ratios obtained

inthis experimentindicatethefollowing order for FeLV proteins: N-pll-pl5-plO-p30-C.

DISCUSSION

In this communication we have presented evidence that the majornon-glycosylated

struc-tural proteins of FeLVaresynthesized as part

of a large precursor polypeptide. The largest precursordetected in these experimentswas a

70,000-dalton polypeptide, present both

intra-cellularly (Fig. 4) andas acomponentof FeLV (Fig.5,6,and 7).Trypticpeptide analysis dem-onstrated thatPp7O and virion p70were identi-caland that bothcontainedthe trypticpeptides

of FeLV p30, p15, p1l, and p10 (Fig. 6 and 7).

Thepresenceof p1l tryptic peptides in

intracel-lularPp7O and the absence of these tryptic

pep-tides in Pp6O indicate that the conversion of

Pp7O

-*

Pp6O

involves the release ofp1l from Pp7O. Wehavealsopresented evidence

suggest-ing

that the conversion of

Pp7O

-0

Pp6O

canbe inhibited by L-azetidine-2-carboxylic acid (Fig.

4), whereas the conversion of

Pp6O

-0 FeLV structuralproteinswaspartiallyinhibitedbya

generalproteaseinhibitor(Fig. 2and 3).

Pulse-labeling experiments in the presence of

pacta-mycin indicated that the order of small FeLV

structural proteinsispll-pl5-plO-p30(Table1).

The presence of a virion-associated protein

(p70) with atryptic peptide profile identicalto

intracellular Pp7O indicates the incorporation

of anuncleavedprecursor into mature virions.

A similar finding has recently been

demon-strated for Rauscher leukemiavirus(RLV) and RLV-infected cells (9) as well asevidence fora

possibleuncleaved precursoras acomponentof

the feline leukemia virus pseudotype of

Molo-ney sarcomavirus (19). Subviral particles

pre-paredfrom FeLV containp70within the core of

FeLV rather than as an envelope component (Behnke and Velicer, manuscript in prepara-tion).

Although immune precipitates (using anti-p30) frompulse-labeled cells routinely contain

low levels of a labeled 70,000-dalton moiety (Fig. 2and3) (18),thispolypeptide becomes the principal immune precipitable polypeptide

when cells aregrowninthe presence of

L-azeti-dine-2-carboxylic acid. Weattribute the routine presence of Pp7O at lowlevels inimmune pre-cipitates during normal pulse-labeling

condi-tions(Fig. 2A andB)and withlong-term

label-ing conditions (18) to reflect an intracellular

level of uncleaved Pp7O that eventually

be-comes incorporated into FeLV as virion p70.

However, a portion ofthis Pp7O may also be destined for furthercleavage. Theproline

ana-logue L-azetidine-2-carboxylic acid could affect cleavage of Pp7O in two ways. The analogue could beincorporated directlyintothe cleavage enzymeand alter theactivity of the enzyme,or the analogue could be incorporated into the

substrate (Pp7O). In our experiment the cells

were preincubated with L-azetidine-2-carbox-ylic acid for45min prior topulse-labeling (Fig.

4). Similarresults areobtained when thecells

arepreincubatedfor 0, 5, and 10 min with the

analogue (datanotshown). Ifthe proline

ana-logue were altering the cleavage enzyme

di-rectly, the enzyme would have to have an

ex-tremely short half-life. Themostlikely

alterna-tive is that L-azetidine-2-carboxylic acid is

in-corporateddirectly into Pp7O and thus prevents

the conversion of

Pp7O

-0

Pp6O.

The effect of the general protease inhibitor

uponthe cleavageofPp6O does notprovide an

unequivocal interpretation. We cannot

attri-butethe ratherdramatic changesinthe levels

and distribution ofPp6O (Fig. 3) to a general

effect of PMSF on cell viability because the

decrease in cell viability is rather small.

Al-though PMSF does reduce the level of protein

synthesis, thisreduction appearstobe

quanti-tative rather than selective for viral protein

synthesis. Furthermore, Fig. 3 shows that a

changeinthe totallengthofexposure from 4.5 min (Fig. 3Aand D plus C and F)to34.5 min

(Fig. 3B and E) did notcause any significant

changesintheelectrophoreticprofiles. Figures

2and3show thatapartialinhibitionofPp6O by

PMSF occurs only if PMSF is present during

and shortly after the synthesis ofPp6O. It is

difficult to attribute the inhibition of Pp6O cleavage to asimpleinactivation ofaprotease

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these experiments. Figure 2 shows that Pp60 becomes committedtocleavagewithinthe first few minutes after synthesis, whereas Fig. 3 indicates that PMSF inhibition requiresonlya

shortexposure toPMSFduring pulse-labeling.

BecausePMSFinhibition requires thepresence

of theprotease inhibitor onlyduring

pulse-la-beling, we interpret its activity to an aberra-tionofthe normal juxtaposition ofasubstrate Pp6O and enzyme, possiblyinduced by the in-teraction of PMSF with either Pp6O or a

pro-tease. In support ofour interpretation is the

change in distribution ofPp6O within the PF and CE. Under normal conditions Pp60is lim-ited to the PF, whereas, in the presence of PMSF, Pp6O is foundin both the PF and CE immediately after pulse-labeling. Lindell has recently reported (13) that the addition of PMSFtoisolated nuclei inhibits the release of RNA polymerase, which also indicates that PMSF may have additional activities aside from theproteaseinactivationactivity. Further experimentationinvolvingisolation of intracel-lular membranes and precursor polypeptides

will be required to more clearly elucidate the activity of PMSF on Pp6O cleavage. Previous work with both AMV-infected cells(27,28)and RLV-infected cells (26) has not provided evi-dence forany inhibition ofprecursor cleavage bygeneral proteaseinhibitors. However, RLV

precursor cleavage may be inhibited by

tolyl-sulfonylphenylalanylchloromethylketoneand

carbobenzyloxy-phenylalanyl chloromethyl

ke-tone(R. Arlinghaus, personal communication). Our results with FeLV-infected cells may

re-flect differences in experimental design (i.e.,

concentrations ofprotease

inhibitor)

or in

dif-fering susceptibilities of avian, murine, and feline oncornavirusprecursor

polypeptides.

Trypticpeptide analysis of intracellular

pre-cursorpolypeptides and virion structural

pro-teins(Fig.6and7) demonstratesthat themajor

structural

non-glycosylated

polypeptides

of FeLVaresynthesizedaspartofa70,000-dalton

precursor. The presence ofp30, p15, p1l, and

plO tryptic peptides within the Pp7O molecule and the absence of the

p1l

tryptic

peptides

in

Pp6O indicate that the conversion of Pp70 --Pp6Oresultsinthe formation ofp1l.These data

wouldsuggest that

Pp7O

containsthep1l

moiety

at either its NH2-terminal or COOH-terminal end andthat removalofp1l results in the

for-mation ofPp6O.

The results ofpactamycin gene ordering

ex-periments(Table 1) confirm thesuggested

posi-tionofp1l withinPp70. These results combined with the trypticpeptide analysisand immune

precipitation experiments lead us to propose

(Pp 70)

p11 p15 plO, p30

,/ ,' " (Pp60)

..1'p1 + p15 p O. p30

(Pp25?) '

p15 plO + p30

(p70) i X

pil p15 plO, p30 ,' p15 + p1O

FIG. 8. Proposed scheme forthegeneration of the non-glycosylated structural proteins of FeLV. The order of FeLV proteins within Pp7O was that ob-tained from pactamycin gene ordering experiments. The existence of Pp25 is uncertain.

the cleavage scheme shown in Fig. 8 for the

generation ofFeLV non-glycosylated structural

proteins. Thefirst step in this scheme involves

thegeneration ofp1l and Pp6Ofrom Pp7O.

Be-cause p1l is the NH2-terminal moiety, this

cleavage may occur on a nascent polypeptide

andthus could explainthedetection of only low

levels of Pp7O undernormallabeling conditions

(Fig. 2A and B). The absence of any anti-p30

immune precipitable intracellular polypeptides

otherthan Pp6O and p30 (Fig. 2) argues that

p30is generatedbydirectcleavage ofPp6O.The

order of FeLVstructural proteins also supports

this interpretationin thatp30is

carboxy-termi-nal onthe Pp6O moiety. The generation ofp30

from Pp6O should result in the formation of a

precursor polypeptide of about 25,000 daltons

(Pp25) containingp15andplO. Immune

precipi-tation of subcellular fractions from pulse-la-beled cells usinganti-pi5under thesame condi-tions used for anti-p30 also yields Pp7O and Pp6O, but not a 25,000-dalton precursor (G.

Okasinski,unpublisheddata). This observation leads us to believe that p15 and plO may be rapidly generated directly from a would-be

Pp25.The finalfeatureof this proposed

genera-tion of structural proteinsisthe incorporation ofPp7O into assembled virusasp70.

A comparison of the arrangement and

pro-posed processing of FeLV structural proteins with that recently reported for AMV reveals

some distinct differences. The order of AMV structural proteins withina 76,000-dalton

pre-cursor(Pr76) isN-p17-p28-p14-C,withthe

posi-tion of plO being uncertain (28). The major group-specific antigen (p28) is located inter-nally within AMV Pr76, whereas the

corre-sponding FeLV protein is COOH-terminal on FeLVPp7O.Vogtet al. (28) have proposed that

the first cleavage ofPr76resultsinprecursors

of66,000 and 12,000 daltons (Pr66 and Prl2),

the latter arising fromcleavageatthe COOH-terminal end and ultimately giving rise to AMVp14. ThusAMVprecursorprocessingmay

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first cleavage is atthe COOH-terminalend in

the former andattheNH2-terminalend in the

latter. Avian Pr76, thus, requires complete or

almost complete synthesis prior to the first cleavage, whereas feline Pp7O could be cleaved

as a nascent polypeptide. These differences

could explain the relative ease of avian Pr76

isolation and the absence of abundant feline Pp7Ounder normallabeling conditions. Pr66is

believed to be further processed to a 60,000-dalton precursor (Pr60). At this point thetwo

proposed schemes become very similar. It is believed that AMV p28, plO, and p17are gener-ateddirectly fromAMV Pr6O.This process may be analogous to the generation of FeLV p30,

p15,andplOfrom FeLV Pp6O. Although Vogtet

al. (28) havedetecteda32,000-daltonprecursor (Pr32), it is notbelievedtobeanessential step

inthe generationof AMV proteins. Recent pub-lished work with RLV precursor proteins has also failed to detect precursors of RLV p30

smaller than 65,000 daltons (1,16, 26),although

this point isstill under investigation and there is some unpublished evidence for smaller

pre-cursors(Arlinghaus, personalcommunication).

Thus FeLVappears todiffer from AMVinboth

the arrangement of structural proteins in

pre-cursorpolypeptides and inthe initial cleavage from precursors, but may be similar in the processing of60,000-dalton precursor polypep-tides. The incorporation ofaprecursor

polypep-tide into assembled virions alsoappears tobea

unique phenomenon of FeLV and RLV (9), while absent from AMV.

The identification of precursor polypeptides

inFeLV-infected cells is consistentwithsimilar

reports ofprecursor polypeptides in

AMV-in-fected cells (18, 27) andinRLV-infected cells (9, 16, 26). Precursorpolypeptides similartothose identifiedinAMV-and RLV-infected cells have also been synthesized in cell-free translation systems (6, 10, 17, 29). Inaddition tothe

pres-ence of precursor polypeptides in cells

produc-tively infected with oncornaviruses, these

pre-cursorshaveinevery instancebeenindicatedto

have a membrane association (9, 16, 18, 26-28). Although the synthesis of oncornavirus pro-teins in productively infected cells results in

the formation ofhigh-molecular-weight,

mem-brane-associated precursor polypeptides, the size of detectable precursors and their

subse-quent processing appears to vary. Whenamino

acid analogues have been utilized to inhibit

precursor processing, the results have varied

fromno effect in AMV-infected cells (27)with

fluorophenylalanine andcanavanine to

inhibi-tionofformation ofa65,000-daltonprecursorin

RLV-infected cellswith canavanine andno

ef-fect withfluorophenylalanine and

L-azetidine-2-carboxylicacid (26).Inourhands L-azetidine-2-carboxylic acid resultsinanaccumulationof Pp7O (Fig. 5), whereas canavanine and fluoro-phenylalanine yield no detectable alterations

inelectrophoretic profiles of immune precipita-ble precursor polypeptides (G. Okasinski,

un-published data). Our finding of partial inhibi-tion of Pp6O cleavage by a general protease inhibitor (Fig. 3and 4) is, to ourknowledge, the first suchpublishedreport andserves topoint

out what may be fundamental differences in

various oncornavirus precursor polypeptides

and/orinhost cell influencesonprecursor proc-essing.

The synthesis and processing of RLV

precur-sorpolypeptides as discerned by two different groupsofworkers usingdifferent host cellsmay bestexemplify the effects of host cells on

pre-cursorprocessing (16, 26). One groupof

work-ers, using JLS-V9 cells infected with RLV,

could identifypresumptive precursor polypep-tides with molecular weights of 82,000 and

65,000. These precursor polypeptides are

be-lieved to be precursors of RLV p30, p15, and

pl2b (26). Results ofimmune precipitation of

intracellular protein with anti-RLV indicated that very little, ifany, RLV p30 was present intracellularly and suggested that production

ofp30from precursor polypeptides is

immedi-ately followed by assembly of RLV p30 into extracellular virus. When JLS-V16 cells were examined for the presence of RLV precursor

polypeptides,four large polypeptides were iden-tified, with molecular weights of 200,000, 90,000, 80,000, and 65,000, respectively (1, 16). JLS-V16 cells, however, do contain readily identifiable intracellular RLVp30, suggesting relatively larger pools of intracellular RLVp30.

These differences inthe number of identifiable precursor polypeptides, their size, and their processing rates (as judged by intracellular

RLVp30levels) all argue that due caution must be exercised in developing a general mode of oncornavirusproteinsynthesis and processing.

ACKNOWLEDGMENTS

We thankAlice Swanson for technical assistance. This research was supported by Public Health Service grant CA-12101 from the National Cancer Institute and grant DRG-1230 from the Damon Runyon Memorial Fund forCancerResearch, Inc., and a grant from the Elsa U. Pardee Foundation. L.V. is a recipient of research career development award CA-70808 from the National Cancer Institute.

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