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,
Utah84132Received 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. Thiscorrelates 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"
concentrationdependence 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 andEhren-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 METHODSCells 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 [lIreaction 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
particlesweregrownandpurified
from mixed stocks of standard virus andDI-1, -2,
or -3. Virus particles banding at adensity
of 1.32g/cm3(compared
with1.34g/cm3
for standard virus) were rebanded in CsCl and
usedtoinfect HeLacellsintheabsence of
stan-dard
helper
virus. ['4C]uridine-labeled viral RNAwaspreparedfrom standard and DIvirus-infected cells andanalyzedon a
methylmercury
agarose
gel. Figure
1 shows that the RNA fromallthreedefectivevirus 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 withpreviously reportedvalues (5).
Theproteinssynthesizedin vivoduring
infec-tion
by
defective viruses were labeled by theaddition of[35S]methioninetothemedium,and
the proteins were analyzed by
polyacrylamide
gel electrophoresis(Fig. 2a).Withoneexception,
theproteins
synthesized by thedefective viruson November 10, 2019 by guest
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IN VITRO TRANSLATION OF POLIO DI RNA
v STD STO
42+S
POLIODI(
1(2)
DI(3)POLIO
28S 18S31 SVIRUS
VIRUS
rRNA
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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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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-weightpolypeptidepredominates,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
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IN VITRO TRANSLATION OF POLIO DI RNA 485
DI(1)
j3Sj
fmet4.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,
butin-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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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
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[image:6.497.258.447.66.363.2]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
isnotsimplyadirectcleavageproductofNCVPla,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,
whichdoesnotmakeVP4, synthesizesthe smallfmnet polypeptide.
An alternative candidate for thesmall fmet-labeled polypeptide is VPg, the
protein
that isfound 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 onlysugges-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.
4. Celma, M. L., and E.Ehrenfeld. 1975.Translation of poliovirus RNA in vitro: detection oftwo initiation sites. J.Mol. Biol. 98:761-780.
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