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0022-538X/79/05-0481/08$02.00/0

Capsid Protein Precursor Is One of

Two Initiated Products of

Translation of Poliovirus RNA In Vitro

STEVE HUMPHRIES,`* FRED KNAUERT,' ANDELLIE EHRENFELD'2

Departmentsof Cellular, Viral and MolecularBiology'andBiochemistry,' University of UtahMedical Center,Salt Lake

City,

Utah84132

Received forpublication 28 November 1978

Previous studies in ourlaboratory have demonstrated that cell-free systems

translating the Mahoney strain of poliovirus type I RNA utilize two unique

initiation sites. In this study, defective-interferingparticles ofpoliovirus, which

contain deletions in the regionencodingthe capsid proteins,areshownto initiate translationofproteins in vitroatthesesame twosites. Both the standard virus

and the defective-interfering virus RNA direct the synthesis oftwopolypeptides labeled withn-formyl-methionine (fmet)attheiramino termini. The size of the

smaller fmet polypeptide synthesized in vitro by the defective virus appears

identical in size to that of the standard virus. However, the

larger-molecular-weightfmet

polypeptide

is reduced in size from 115,000 to69,000 daltons. This

correlates exactly with the reduced size of the precursor to thecapsid proteins

synthesized by the defective virus in vivo andwith the size of the deletion in the

defective virus RNA (1,200 bases). This provides genetic evidence that the 115,000-dalton fmet polypeptide synthesized in vitro by the standard virus is NCVPla,the precursor tothecoatproteins. Although the identity ofthesmall

(5,000 to 10,000daltons) fmetpolypeptide is not clear, severallines of evidence

enableus toexclude thepossibility that it is VP4, the smallest viral capsid protein.

Thegenerally acceptedmodel forpicornavirus

RNA translation is that initiation occurs at a single site at or near the 5' end of the RNA. However, using n-formyl-[3S]methionine

([3S]fmet) donated from [35S]fmet-tRNAfmet to specifically label the amino termini of initiated polypeptides, Celma and Ehrenfeld (4) demon-strated that in a cell-free system made from poliovirus-infected cells, two different amino-terminal tryptic peptides are synthesized. The relativeproportionsofthetwopeptides synthe-sized variedas afunction of the

Mg2"

concentra-tion in the reacconcentra-tion.

Recent work in our laboratory (13) has

re-solvedtwomajor[3S]fmet-labeled polypeptides

by sodium dodecyl sulfate-polyacrylamide gel electrophoresiswhichresult from in vitro trans-lation of viral RNA. The relativeproportions of thesepolypeptidesshowan

Mg2"

concentration

dependence which correlates with that shown bythetwoamino-terminaltryptic peptides

iden-tified by Celma and Ehrenfeld. Synthesis ofa 115,000-dalton polypeptide predominates at

Mg2"

concentrations between 1.0 and 2.5 mM,

whereas synthesis of a 5,000 to 10,000-dalton

polypeptide predominates at higher

Mg2"

con-centrations(2.5to4.0mM).Knauert and

Ehren-feld further showedthatfmettryptic peptideII

(4) is derived from the high-molecular-weight

polypeptide,andfmettrypticpeptideIisderived from thelow-molecular-weight polypeptide.

Thepresentstudyuses ageneticapproachto

obtain informationon theidentity of thesetwo

fmet polypeptides and uses a mutantof polio-virus, thedefective-interfering (DI) particle first isolated by Cole et al. (6). DI particles infect cells, shut off host cell protein synthesis, and

replicate their RNA as efficiently as the

stan-dard virus(5), butnoprogenyparticlesare

pro-duced because the defectivegenomeRNAlacks the information coding for some of the capsid

proteins (5). In DI particle-infected cells,

NCVPla, the precursor to the capsid proteins,

isreduced in sizebybetween 30,000 and 40,000 daltons. Since thesequencescodingforNCVPla have beenmappedatthe 5'end of thepoliovirus

genome RNA (21, 22), NCVPla is likely to be

the amino-terminal portion of the translation

product and, therefore, tobelabeled with fmet

in vitro. If the high-molecular-weight,

fmet-la-beled polypeptide synthesized in vitro is

NCVPla,we would expect the size of the fmet

polypeptide in DI-infected cell extracts to be

reducedby30,000to40,000 daltons.

MATERIALS

AND METHODS

Cells and virus. Thegrowthand maintenanceof HeLa S3 cellswas asdescribedby Celmaand Ehren-481

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feld (4). Virus stocks enriched forDI-1, -2,and -3 (a kind gift from E.Wimmer) wereused toinfect HeLa cells, and the progeny virus was purified from the mixed infection as previously described (5, 6). The defective virus particleswereseparated from standard virusparticles bytwocyclesofcentrifugationonCsCl density gradientsinNTE buffer (10 mMNaCl,10mM Tris[pH7.4],2mMEDTA) containing1%Brij58(6). Centrifugation was carried out in cellulose nitrate tubes (0.625 by3 inches [ca. 1.6 by7.6cm], spunin the angle 65rotor at30,000 rpm for15hat4°C. The visible virus bands were collected and rebanded in CsCldensity gradients. Purified viruswasdiluted with NTE buffer and pelletedbycentrifugationinthe angle 65 rotor at 60,000 rpm for 1 h. Virus particles were suspended in NTEbuffer and stored at -70°C. An aliquotwasdiluted into water, and the absorbanceat 260 nm was measured using a Beckman model 25 spectrophotometer. Theconcentrationofvirus parti-cleswascalculated assuminganabsorbance of1at260 nmequals10'3standard virus particlesand 0.85x1013 DI-1defective virus particles (5).

Invivolabelingofviral RNA. To prepare viral

RNA, HeLacellsinfected with 1,000 particles of either DI or standard virus per cell were labeled in the presence of 5 ,ug of actinomycin D per ml, by the addition of5,uCi of[14C]uridine (50mCi/mmol; New EnglandNuclear) perml from2.5 to 4.5h postinfec-tion. CellswerewashedonceinEarle saline and lysed in RSB (10 mMTris [pH 7.4], 10mMNaCl, 1.5mM MgCl2) made 0.5% in Nonidet P-40. After removal of cellulardebris bycentrifugation,the supernatantwas made 0.5% sodiumdodecyl sulfate and the RNAwas deproteinated with phenol and precipitated with ethanol.

Invivolabelingofviral proteins. Cellsinfected

witheither standardorDI virusat1,000particlesper cell were labeled by the addition of20

ACi

of[35S]_ methionine (>500 Ci/mmol; New England Nuclear) perml2.5 to4.0 h postinfection. Cells werelysedas described above, andpostmitochondrial supernatant was made 1% sodium dodecyl sulfate and stored at -20°Cuntilrequired.

In vitro protein synthesis. Cell-free extracts (S10)ofeither standardorDIvirus-infected cells were preparedaspreviously (4) 3.75 to 4 hafterinfection.

FormyI-["S]methionine-tRNAf"'

prepared from total rabbitliver tRNA (Grand Island BiologicalCo.) was charged and purifiedas previously described. Condi-tions for the incorporation of[35S]fmet by cell-free extracts were as described by Celma and Ehrenfeld (4). The incubation also contained 15 Ml per 100 [lI

reaction mixture ofribosomal salt wash containing initiation factors, prepared from uninfected HeLacells by the method ofKaufman et al. (12).

[35S]fmet-la-beledtrypticpeptides wereprepared and analyzedas previously described(4).

PreparationofVPg. Preparation ofVPg was es-sentially as described by Lee et al. (16). At 2.5 h postinfection witheitherstandard ordefective virus, cells were washed in saline and resuspended in me-dium minuslysine.A100-,uCi/ml amountof[3H]lysine

(60Ci/mmol;NewEngland Nuclear) was added, and

theincubationwascontinuedfor afurther2.5 h.The cellswere lysed as described above, andviral RNA

wasextractedusinga25:24:1 mixtureofphenol,

chlo-roform, andisoamylalcohol. Afterethanol precipita-tion, the RNA was dissolved in 50 mM ammonium

acetate,pH 5.0,anddigestedwith 100 U of T2RNase

per ml, 200 U of TI RNase per ml, and 150 U of

pancreatic RNaseA perml (allenzymes from Calbi-ochem)at37'Cfor 90min. The RNA nucleotideswere

thenadsorbedtoDEAE-celluloseasdescribedbyLee

etal. (16),andthe materialthatwasnotretained on

thecolumnwasanalyzedfurther.ProteinaseK

diges-tion was performed in 10 mM Tris (pH 7.4)-i mM EDTA-1%sodiumdodecyl sulfate with 1 mgof

pro-teinaseK(E.Merck,Darmstadt, Germany)permlat

37°C for2 h.

Acrylamide gel electrophoresis. Viral proteins

were analyzed on 6 to 24% sodium dodecyl

sulfate-polyacrylamideslabgelsasdescribedbyLaemmli(14).

Electrophoresis was run at a constant current of 35 mA andamaximumvoltageof 150V untilthe

brom-ophenolbluedyereached thebottomofthegel (ap-proximately16h).Ifnecessary,gelswerepreparedfor

fluorography according to Bonner and Laskey (2). Autoradiographywas on Kodak SB5 film for2 to5

days.

Methylmercury agarose gel electrophoresis.

Methylmercury agarose gelelectrophoresis was

per-formed as described by Batt-Humphries et al. (S.

Humphries,C.Simonsen,and E. Ehrenfeld,Virology,

in press), using a modified method of Bailey and Davidson(1).Agarose gels,0.8%,were runat80 V (40

mA) for 13 h.Fluorographyofgels was asdescribed byBatt-Humphriesetal.(Virology,inpress).

RESULTS

Defective

poliovirus

particlesweregrownand

purified

from mixed stocks of standard virus and

DI-1, -2,

or -3. Virus particles banding at a

density

of 1.32g/cm3

(compared

with1.34

g/cm3

for standard virus) were rebanded in CsCl and

usedtoinfect HeLacellsintheabsence of

stan-dard

helper

virus. ['4C]uridine-labeled viral RNAwaspreparedfrom standard and DI

virus-infected cells andanalyzedon a

methylmercury

agarose

gel. Figure

1 shows that the RNA from

allthreedefectivevirus stocks issimilar in size

and that none are significantly contaminated

with standard virus RNA. The band width of

theRNA from DI-3 is greater than that for

DI-1 orDI-2 and mayreflectheterogeneity in the

virus stock. The sizes of the defective virus

RNAsrange from 84.2% (DI-1) to81.9%

(DI-3)

ofthestandardgenome.Thisrepresentsa

dele-tion of 1,200 to 1,400

bases,

in agreement with

previously reportedvalues (5).

Theproteinssynthesizedin vivoduring

infec-tion

by

defective viruses were labeled by the

addition of[35S]methioninetothemedium,and

the proteins were analyzed by

polyacrylamide

gel electrophoresis(Fig. 2a).Withone

exception,

the

proteins

synthesized by thedefective virus

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IN VITRO TRANSLATION OF POLIO DI RNA

v STD STO

42+S

POLIO

DI(

1(2)

DI(3)POLIO

28S 18S

31 SVIRUS

VIRUS

rRNA

rRNA

r

asp

U

. :.i;

4.0-0

ao

x

0

CU

O

3.0-1-J

3

a:

J

cJI

2I

o L)

I.

1.5-f 12

7 8 9 10 I 12

b

DISTANCE MisG RATED(cm}

13 14

FIG. 1. (a) Size analysis of RNAs from purified poliovirusDIparticlesonmethylmercurygels. Samples

were run on a0.8%agarosegelin 5 mMmethylmercury hydroxideasdescribed. The molecularweights of the DIRNAswereestimated fromasemilog plot of the molecular weightagainstdistancemigrated,asshownin

(b).Standards usedwere42Svesicularstomatitis virus(VSV) virion RNA (molecular weight [MW] =3.6x

106;24);31SVSV L mRNA(MW=2.1x 106; 19) (bothakind gift from S.Batt-Humphries);standard polio

virion RNA(MW=2.6x 106;Y. F.Lee,A.Nomoto, and E. Wimmer, submitted for publication);28SHeLa

cellrRNA(MW=1.65x10P).Theestirnated molecularweights for the DI RNAswere:DI-1,2.19x106 (84.2%

ofstandard);DI-2 and-3,2.12x106(81.9% of standard).

between 2.5 and4 h afterinfectionare

synthe-sizedby the standardvirus.However,inthe

DI-1-infectedcells, NCVPla, the precursor tothe

capsid proteins, is absent, and a new protein

migrating between NCVP3 and NCVP4 is

ap-parent.Furthermore,asexpected,the viral

cap-sid proteins VP2, VP3, and VP4 could not be

detected, althoughpolypeptideswithmobilities

similar to those of VP1 and VPO, the direct

precursor to VP2 andVP4, canbe seen. From

the known molecular weights of thepoliovirus

proteins,andusingaT4-infected E.coliprotein

marker (a gift from G. Stetler), the molecular

weight ofthe new protein synthesized by the

defectiveviruswasestimated to be69,000.This

reduction of approximately 45,000 daltons is

roughly equivalentto375 amino acids and

cor-relates well with the observed 1,200 bases of

RNA deletedfrom the defectivegenomeof

DI-1.

We nextcomparedtheproteinsmade in vitro

by cell extracts (S10) prepared from DI-1 and

standard virus-infectedcells,using

[3S]methio-ninetolabel theproteins synthesized.Ascanbe

seenin Fig. 2b, theproteinsmade in vitro

cor-respond well with those made in vivo; the

115,000-dalton capsidprecursor is not madeby

the defective S10, and a new polypeptide of

about 69,000 daltons is present. VP3, which is

synthesized bythe standardS10,is notobserved

in the DI-S10; as in the in vivo samples, there

arebands of reduced intensitythat migrate in

theregion of VP1 and VPO. VPO isonly finally

cleavedtoVP2and VP4 afterincorporationinto

virus particles (10), and since only the early

stages of virusassemblyhave been observed in

up

a

VOL. 30,1979 483

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VIRUS

-<T>

3I

em

VP .MCV

w

I

rPRO7f...

.<...

...- .__i _=

.1..

4-MO

Asaw E_

[image:4.497.55.447.66.451.2]

g....,-....

FIG. 2. Sodiumdodecylsulfate-polyacrylamidegelelectrophoresisanalysis ofproteinssynthesized in vivo and in vitroby standardand DIvirus.[35S]methionine-labeledproteins made (a)in vivobetween2.5and4 hpostinfectionand(b)ininfected cell S10extractswere run on6to24%polyacrylamidegelsasdescribed.

vitro (for review, see 20), the absence of VP2 and VP4 in these extractsisnotunexpected.

We next examined the polypeptides

synthe-sized in the S10, which could be labeled with

[35S]fmetdonated from

[35S]fmet-tRNAf`et

Fig-ure 3 shows that two major fmet-labeled

poly-peptidescanbedetected in the standard

virus-infected S10. At low

Mg2"

levels (2.5 mM), syn-thesis of thehigh-molecular-weightpolypeptide

predominates,andathigh

Mg2e

levels(4.0 mM)

thelow-molecular-weightpolypeptideis

prefer-entially made. In the DI-S10, the size of the

small fmet protein appears to be unchanged,

although sinceresolution ispoor inthisregion

ofthegel,smalldifferences inmolecularweight

wouldnotbedetected. However,the size ofthe

high-molecular-weight fmet polypeptide is

re-duced from115,000toabout69,000daltons,

cor-respondingin size totheproteinsynthesized by

thedefective virus invivo. The

high-molecular-weightfmetpolypeptide issimilarlyreduced in

size in DI-3virus-infectedS10lysates,appearing

as a diffuse band of about 69,000 daltons (not

shown). This is presumably a consequence of

theheterogeneityinthis virus stock noted

ear-lier.These data demonstrategeneticallythat the

115,000-daltonpolypeptidelabeledwith

formyl-[35S]methionine in standard virus-infected S10

lysatesinNCVPla, theprecursortothecapsid

proteins.

The twoinitiation sitesutilizedduring

polio-virustranslationinvitrowereoriginally detected

N. -'7

;%.-,' :- -L L.'I"A-y'-'-.

.... j

ID0.

kl-)

.1.-. .-j

'.

i11i,P" " e.

I': -.-.o

-i f,

-,-Wool

V...,.:..

:8

MM

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IN VITRO TRANSLATION OF POLIO DI RNA 485

DI(1)

j3Sj

fmet

4.OmM 2.5mM

Mg++

Mg++

STANDARD

35Sj me.

4.0mM 2.5mM

Mg++ Mg++

NCVP l

vp

--VPI

VP2

-VP3

-VP4_h..

FIG. 3. Sodiumdodecyl sulfate-polyacrylamidegel analysisofpolypeptidesmade ininfectedcellextracts labeledby[35S]finet-tRNA 't Sampleswererun on

6to24%polyacrylamidegels, andthe gelwas

fluo-rographedasdescribed.

by tryptic peptide analysis. The polypeptides

labeled in vitroby the incorporationof [ S]fmnet

werethusdigested with trypsin, and the tryptic

peptides were analyzed by high-voltage paper

electrophoresisatpH 1.9(Fig. 4). As expected,

the DI-infectedS10 directsthesynthesisof two

[3S]fmet-labeled tryptic peptides,which

coelec-trophorese with the two tryptic peptidesfrom

thestandrd virus-infectedS10.Thereappearsto

be no significant differencein the relative

pro-portionsofthetwotryptic peptides synthesized

by the defectiveorstandard virus-infectedS10

atthedifferentMg2+concentrations used.

At this time,we have no direct evidence for

the identity of the low-molecular-weight

[3S]-fmet-labeled polypeptide. Two known

low-mo-lecular-weight proteins synthesized in

virus-in-fectedcellsareVP4andVPg,theproteinfound

covalentlyattached to the 5' end of virion RNA

(9, 15, 16). VP4 is not synthesized in cells

in-fected with DI particlesalone (Fig. 2a). It thus

became ofinterest todetermine if the defective virus makes VPg. To test this,cellsinfected with either standard or defective virus DI-1 were

labeledwith [3H]lysinefor 2.5 to 4 h

postinfec-tion,and RNAwasprepared from a cytoplasmic

extract as described by Lee et al. (15). The

resulting RNAwasdigested to completion with

T2, Ti, andpancreatic RNases, and the

nucleo-tideswere adsorbed to a DEAE-cellulose

col-umn. From both the standard- and DI-infected

cytoplasmic RNA,>96%of theacid-precipitable

radioactivity was not retained on the column

and eluted in the void volume. Figure 5 shows

that this eluted material migrated on a

poly-acrylamide gelas asinglelow-molecular-weight protein ofmobility similar tothat ofthe [35S]-fmet-labeledlow-molecular-weightprotein.

Fur-thernore, the eluted material from both the

standard- and the DI-containing RNAmigrates

toward the cathodeonhigh-voltagepaper elec-trophoresisatpH3.5andis completelysensitive to proteinase K digestion (data not shown). From thiswe conclude that the defectivevirus

both codes for and synthesizes the small protein, VPg, that has been found attachedtothe 5' end ofpoliovirus RNA.

DISCUSSION

Previous work fromourlaboratoryhas dem-onstrated that in poliovirus-infected cell

ex-tracts, two different sites are utilized for the

initiation ofprotein synthesis (4, 11, 13). We havedetermined theidentityof thepolypeptide synthesized from one of these sites, using the defective virusparticlesfirst described byCole

etal. (6). In agreementwith theirobservations,

wefind that thedefective virus doesnotdirect the synthesis of NCVPla, the 115,000-dalton

precursor to the viral

capsid proteins,

but

in-steadaproteinof about69,000daltons is made

(5).

The reduced size of the protein is in good correlation with the observed reduced size of the

genome.Theextentandlocation of the deletion

have recently been determined byTi

oligonu-cleotide fingerprinting and by heteroduplex

mapping in the electron microscope (E. Wim-mer,personalcommunication).Thedeletion

ap-pears to leave intact the first 10%(or about 700

bases) ofthe5'endof the genome and extends

forabout 1,000 to 1,300 bases to a point 25 to

30%fromthe 5' end of the RNA.

The DI protein is large enough to code for

someof the viralcapsidproteins,althoughit is

not known whethercorrect cleavage of the

al-tered precursorcan occur.It ispossiblethat VP1

(molecular weight, 32,000) or VPO (molecular VOL. 30, 1979

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-5mM 4 fr2 'K; .-' ;+-*

9q+M*

Mgt0 YV

Fi¢. 5. Sodiumdodecylsulfate-polyacrylamidegel electrophoresis analysis ofstandard and DI-VPg. Swaplespreparedasdescribed in thetextwererun

on a10to24%polyacrylamidegel andfluorographed

asdescribed.

FIG. 4. High-voltagepaperelectroph

sisof[35SJfnet-labeledtrypticpeptide,

different Mg2" concentrations. Tryptic

productssynthesizedbystandardvirus extractsandbyDI-1-infectedcellextra

paredandelectrophoresedasdescribe,

fmet was applied as a marker and

electrophoresisby staining with chloro The driedelectropherogram wasexpos

SB-5filmfor 7days. Theexposed film locate the radioactive regions on the gram, whichwere cutout andcountec Mg2+, the relativeamountsoftrypticp, IIsynthesizedin thestandard virus-infe 25and 75%and,in theDI-1S10,23anc

weight,39,000)maybecleavedfromthe

69,000-dalton DIprotein,since bandsreducedin

inten-sityare seen in the appropriate regions of the

gel. Wehave observed in some gels, however,

that theputativeVPObandsynthesizedby the

DI virus in vivo does not comigrate with VPO

fromstandardvirus (datanot shown), andthe

identity of thispolypeptidebandinthedefective

virus-infected cellsisnotclear.Cole and

Balti-oresisanaly- more (5) could findnoevidence forany

capsid

sinitiatedat proteins andconcluded that the DI protein is

cpeptides of highlyunstableand israpidlydegradedtoamino

¢-infectedcell acids.

Ictswere pre- The proteinssynthesizedbyinfected cell

ex-!d. Unlabeled tracts in vitro mirror those made in vivo. As located after expected, the standardvirus-extractdirects the platinate(7). synthesisofNCVPla, which is absentfrom

DI-sedtoKodak infected extracts. Probablybecause it isnot so

twasusedto rapidly degraded, the new DI protein can be

oZettrnnherm-d.At 2.5mM

eptidesIand !ctedSlOwere d77%,

respec-tiuely. At 4.0mMMg2+therelativeamounts synthe-sizedwere86and14%(standard) and84and 16% (DI-1).

-rm

5mM 40 m .

i; F. `. y '..:

7-1.

'Y

".".p2

" 'I. vF

-..'r .: .r

.... 1, :'

W.

'V'

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IN VITRO TRANSLATION OF POLIO DI RNA 487

moreclearlyseenin vitro than in vivosamples.

Twomajorpolypeptidespeciescanbelabeled in vitro with [35S]fmet, from both standard- and DI-infected cell extracts. The size of the small fmetpolypeptide appearsidentical in both cell

extracts, but the size of the large fmet

poly-peptide is reduced in the DI virus-infected

ex-tractandcorresponds insize tothe DI protein

of69,000daltonssynthesizedinvivo, which

rep-resents the shortened NVCPla. This

demon-stratesconclusively that the 115,000-dalton large

fmet polypeptide observed in standard

virus-in-fectedcelisextractsisNCVPla, theprecursor to

theviralcapsid proteins.

The fmet-labeled tryptic peptides from the

DI-infectedS10 coelectrophorese with the

stan-dardtryptic peptides from both high- and

low-molecular-weight polypeptides. This isnot un-expectedsince thepeptidesare small,probably

13 to 15amino acids (11), and thus apparently

do not extend from the initiation site for NCVPla into thedeleted region of thegenome.

The location of the initiation site for thesmall

fmetpolypeptideis unknown. Villa-Komaroffet al. (23) have also reported that both standard and DI-1 virus RNA direct thesynthesis of the

samefmet-labeledtryptic peptide inaHeLacell

extract, although they only detected a single

initiation peptidefor both RNAs.

Theidentity of thelow-molecular-weight fmet

polypeptide isnotclear. In this gelsystem res-olution oflow-molecular-weight polypeptides is

poor,andcomigrationisnot agoodcriterion for

identification. Although VP4, the small capsid protein, does migrate in thisregion of the gel, we feel that it is unlikely that the small fmet

polypeptide is VP4. First, although VP4 is de-rived from the amino-terminal region of NCVPla (18), the amino acid-terminal tryptic

peptide of the small-molecular-weight poly-peptideinitiated in vitro is different in its

pe-nultimate amino acid from that of the high-molecular-weightpolypeptide (4, 13). This

indi-catesthatthesmall

polypeptide

isnotsimplya

directcleavageproductofNCVPla,aswould be

expected for VP4. Second, the small

fmet-la-beledpolypeptideisdetected inastandard virus-infected extractunderconditions where VPO is

notcleaved(10,20),andnoVP4canbedetected bygelanalysis.Third,thedefective

virus,

which

doesnotmakeVP4, synthesizesthe smallfmnet polypeptide.

An alternative candidate for thesmall fmet-labeled polypeptide is VPg, the

protein

that is

found covalently linked to the 5' end of viral

RNA. ThefunctionofVPgand thelocation of

itscoding sequencesarecurrentlyunknown. One

characteristicofVPgisthat itcannotbelabeled

in vivo withmethionine (8), and we have thus

examined the polypeptides made in astandard

virus-infected S10 that can be labeled with

[3H]lysine and [3S]methionine. Using

polyacryl-amidegel analysis,wehaveobserved that a

low-molecular-weight polypeptide that comigrates

withthe small[3S]fmet-labeledprotein can be

specifically labeledwith[3H]lysineand not with

[3S]methionine (datanotshown). Furthernore, synthesisof this polypeptideisenhancedat high

Mg2e

concentrations, which correlates with the observed increased level of synthesis of the small fmet polypeptide. This evidence is only

sugges-tive that the smallfmet polypeptide might be

VPg, and the identity of this polypeptide is

currently under investigation inourlaboratory.

We have demonstrated that the DI virus

syn-thesizes a protein in vivo that appears to be identicaltotheVPgfoundon the 5' end of the standard virusRNA. Thepositionofthecoding

sequencefor thispolypeptidemusttherefore be

in the first 10% or the last 70 to 75% of the poliovirus RNAgenome.

ACKNOWLEDGMENTIS

This work was supported by grants from the National Science Foundation and from the National Institutes of Health (AI 12387). F.K. is therecipient ofaNational Institutes of Healthpostdoctoralfellowship.

Wealso thank Oliver C. Richards for advice in the prepa-ration ofVPg.

LMRATURE CITED

1. Bailey, J. M., and N. Davidson. 1976. Methylmercury as areversible denaturing agent for agarose gel electro-phoresis. Anal. Biochem. 70:75-85.

2. Bonner, W. M.,and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in

polyacrylamidegels. Eur. J. Biochem. 46:83-88. 3. Celma,M.L,and E.Ehrenfeld. 1974.Effect of

polio-virusdouble-stranded RNA on viral andhost-cell pro-teinsynthesis. Proc. Natl. Acad. Sci. U.S.A. 71:2440-2444.

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Figure

FIG.1.DIofwerevirioncell(b).106; standard); (a) Size analysis of RNAs from purified poliovirus DI particles on methylmercury gels
FIG. 2.hand postinfection Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis ofproteins synthesized in vivo in vitro by standard and DI virus
FIG. 3.rographed6labeledanalysis to Sodium dodecyl sulfate-polyacrylamidegel ofpolypeptides made in infected cell extracts by [35S]finet-tRNA't Samples were polyacrylamide gels, run on 24% and the gel was fluo- as described.
FIG. High-voltageoresissis 4. paper electroph of [35SJfnet-labeled tryptic peptide,s

References

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