Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

42, No. 0022-538X/82/040194-06$02.00/0

Carboxy-Terminal Analysis

of

Poliovirus Proteins:

Termination of Poliovirus

RNA

Translation and Location of

Unique Poliovirus

Polyprotein

Cleavage

Sites

EMILIO A. EMINI,l*MARSHALLELZINGA,2AND ECKARDWIMMER1

DepartmentofMicrobiology, School of Medicine, State UniversityofNew York at Stony Brook, Stony

Brook, New York11794,1and Department of Biology, Brookhaven National Laboratory, Upton, New York

l19732

Received 3November1981/Accepted3December 1981

The carboxy-terminal amino acids of a number of poliovirus proteins were

determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and theirprecursor,P3-tb,werefoundtobe coterminal withasequenceof -Ser-Phe-COOH. As these proteinsarecodedforattheextreme3'endof the viral

RNA, itis possible toestablish the termination site of translation atnucleotide 7,361, 73 nucleotides before thestartof the polyadenylic acidtractof the RNA.

Twoadditional nonstructural proteins, P2-Xandits precursor, P2-3b, werealso foundtobe coterminal witha sequenceof -Phe-Gln-COOH. This result confirms the existence ofat least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirusgenome. Theonlyexceptions arethecleavage sitesat

thecarboxy termini of the structural proteins VP4 andVP1.Carboxypeptidase A

analysisofVP1 establishes aterminal sequenceof -Thr-Tyr-COOH, and similar analysis ofVP4shows Asn to be theterminal amino acid residue, observations thatprovetheexistenceof theexceptional C-terminal amino acids.In noneof the

analyzedcases has C-terminaltrimmingaftercleavagebeen observed.

Poliovirus, a member of the Picornaviridae family of animal viruses, containsasitsgenome asingle-strandedRNA(2.4 x 106daltons) which

acts as mRNAuponhostcellinfection (10). The

RNA is covalently attached at its 5' end to a

small protein (17-19), and its 3' end is polyade-nylated (3, 26).

Recently, the complete nucleotide sequence

of the genomic RNA of a poliovirus type 1

(Mahoney) strain was determined (11, 12, 20). The RNA contains a singleopen readingframe which spans89% of its length. Upon translation in theinfected hostcell,theviral mRNAyieldsa

large polyprotein (NCVPOO) which represents the RNA's totalcoding capacity. This

polypro-tein is post-translationally cleaved toproduce,

via anumber of intermediateprecursor polypep-tides, the virus' structural and nonstructural proteins (Fig. 1) (8, 9, 24). The exact coding

location of each ofthe proteins on the mRNA wasestablished by comparingthe

amino-termi-nalamino acidsequenceofeachproteinwith the

nucleotide codonsequences in the openreading frame (11, 15, 22, 24). Analysis ofthe RNA's

nucleotidesequence andoftheproteins'

predict-ed amino acid sequences yields the following

tentative conclusions.

(i) The termination site of translation ofthe

viral mRNA occurs at nucleotide7,361, 73

nu-cleotides before the start of the polyadenylic acid tract. Several nonstructural proteins are

predicted toterminate atthis site: P3-2,P3-4b, and theirprecursor, P3-lb.

(ii) Viral protein cleavages occur at specific sites characterized by the presence ofGln-Gly pairs. Available evidencesuggests thata virus-specific protease(s) is responsible for breaking thepeptide bond between thesetwo amino ac-ids, yielding proteins with Gly at the amino terminus and Gln atthe carboxy terminus (for references, see reference 11). The only excep-tions are thecleavages between the viral struc-turalproteins, VP4andVP2, and between VP1 andP2-3b. Theformer apparently occurs at an Asn-Sersite, andthelatteroccurs at aTyr-Gly site. The cleavage between VP4 and VP2 is functionally different fromthe other viralprotein cleavages. It occurs atviralRNAencapsidation

and virion maturation (reference 21 and

litera-turecitedtherein). However, the substitution of Tyr for Gln at the carboxy terminus of VP1 cannotbeaccounted forinfunctionalterms.

The goal of this study was to determine the

carboxy-terminal amino acid sequences of four polioviral proteins: VP4, VP1, P2-X, and P3-2. Thecarboxy terminus ofP3-2provided evidence

194

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948 3381 5106 (TERM.)7361

VPg-PU- ' Po(4y'' (A,

NCVPOO (247)

fo(97) 3b(65)

VP0(37) VP3(26) VPI(34) Sb(46)

lb (84)

9(12) 2(72)

X(38) 7c(20) 4b(52)

VPg(2)

FIG. 1. Poliovirusprotein processing pathways.The viral RNA is denotedbyaheavy line,and theproteins

aredenotedbyfiner lines. Thefigureisnotdrawntoscalewithrespecttorelativeproteinsizes.P1, P2,andP3

representthe threemaincleavage regionsof the viralpolyprotein.Theproteinsdenotedbytheenlarged letters

and numberswereusedforcarboxy-terminalamino acidanalysisin the studiesreportedhere. The numbersin

parentheses are the molecular weights (x1,000) of each of the individualproteins as calculated from their

predicted amino acidsequences(11).The numbersonthe RNAgenomerepresent theexactsitesatwhichsome

proteolytic cleavages of the protein products occur as established from datapresented in this paperandin

reference 22. Theexactsite ofthe termination of translation is also indicated(TERM.)

for the exact termination site oftranslation on

the viral mRNA. The terminus of P2-X

con-firmed the existenceofGln-Gly cleavage sites,

whereas the termini ofthe two structural

pro-teinsconfirmed the nucleotidesequenceswhich

predict unique cleavage sites between VP4 and

VP2 and between VP1 and P2-3b. In addition,

evidence wasprovidedfor the cotermination of

P3-2, P3-4b, and theirprecursor, P3-tb, aswell asfor cotermination of P2-X and its precursor,

P2-3b.

MATERIALSANDMETHODS

Labeling andpurification of viral proteins. (i)

Non-structural proteins(P3-2, P34b, P3-b, P2-X, P2-3b).

Approximately3.0 x 10'HeLa S3cellswereinfected

with poliovirus type1 (Mahoney) atamultiplicity of

infection of 50 PFU/cell. At 2.5 hpostinfection, 1.0to

2.0 mCiof the 3H-labeled amino acid in questionwas

addedinasmallamountof medium. At5.0h

postin-fection,the infected cellswereharvestedby pelleting,

washing with phosphate-buffered saline, and pelleting

again. The final pellet was resuspended in Laemmli

sample buffer (62.5 mM Tris [pH 6.8], 10%glycerol,

2.0%osodium dodecyl sulfate [SDS], 5.0%o

2-mercap-toethanol, 0.005% bromphenol blue).

Agreateryieldoftheprecursorproteins (P3-lband

P2-3b)was obtained by treatingthe infected cells at

2.75 h postinfection with 0.8 mM ZnC12, a specific

inhibitor of the viralproteolytic reactions (6).Inthese

cases, labeling was carried out from 3.0 to 4.0 h

postinfection, followed by harvesting ofthe cells as

described above.

Proteinpurification was carried out by subjecting

thepreparationtoSDS-polyacrylamide gel

electropho-resis ina12.5% Laemmli gel (14). With

[35S]methio-nine-labeled viral proteins as markers, the protein

bands of interestwereexcised from the driedgel.The

gel pieceswererehydrated, and the proteinwas

elec-troelutedfrom the gel by anISCO 1750 sample

con-centratorwithSDS-free buffers (0.05MNH4HCO3in

the outer chamber and 0.01 M NH4HCO3 in the

sample compartment) containing100 ,ug of myoglobin

in the sample compartment as carrier. The eluted

proteinwaslyophilized, dissolved in 0.2 M

N-ethyl-morpholine acetate (Pierce Chemical Co.) (pH

8.5)-0.1%SDS and storedat-20°C until used.

(ii) Structural proteins (VP4, VP1). HeLa S3 cells

wereinfectedasabove,exceptthatlabeling with the

3H-labeled amino acidwasfrom 2.5to7.0h

postinfec-tion. Thecellswereharvestedasabove, suspended in

lysis buffer (0.01 M NaCI, 0.01 M Tris [pH 7.35], 1.5

mM MgCI2) and lysed by several cycles of

freeze-thawing. The cell nucleiwerepelleted. Viruswasthen

pelletedfrom thesupematantby spinningat80,000x

gfor 5 hin1.0%SDS. The viruswassuspendedin 0.1

buffer (0.1 M NaCI, 0.01 M Tris [pH 7.5], 1.0 mM EDTA) and purified by velocity sedimentation through

agradient of 15to30%o(wt/wt) sucrose-0.1 buffer and

0.5%SDS. The viral bandwascollected, and the virus

waspelletedasdescribed above and then suspended in

Laemmlisample buffer. The virioncomponentswere

dissociatedby heatingto100°C for 2 min.

Protein purificationwascarriedoutexactlyas

out-lined above.

Carboxypeptidase A analysis of carboxy-terminal

amino acids. Carboxypeptidase Awas obtained from

Worthington Diagnostics. The procedure used was

basicallythatof Bhownetal. (4). Briefly, the protein

in 0.2 M N-ethylmorpholine acetate (pH 8.5)-0.1%

SDSwasplacedat 80°C for10min. Aftercoolingto

roomtemperature, carboxypeptidase Ain 0.2 M

N-ethylmorpholine acetate (pH 8.5) was added at an

enzyme/protein ratio of 1:4. (The amount ofprotein

involvedwas essentially that of the carrier

myoglo-bin.) The reaction was allowed to proceed at room

temperaturefor the desired time and then terminated

bytheaddition of2drops ofglacial acetic acid. The

samplewasimmediately lyophilized.

Theliberated amino acidswereconclusively

identi-VP2(30)

T

VP4(7)

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5 10 30 60

REACTION TIME

(min)

fied and quantitated by amino acid analysis. The lyophilized samples were dissolved in 0.5 ml of buffer

(0.2 Msodium citrate [pH 2.1] containing 15%

polyeth-ylene glycol) and run on athree-buffer, single-column

amino acidanalyzer with the ninhydrin pumpturned

off. Thefirst two buffers were those described earlier

(24), andthe third was 1.5 Msodium-0.1 M citrate (pH

4.6). The column was 22by 0.6 cm,the resin was

Bio-RadAminex A-7, the buffer flow rate was 20 ml/h, and

the temperature was 54°C. Fractions (0.5 ml) were collected every 1.5 min and assayed for radioactive

counts.The total counts in the peak fractions

charac-teristic for a given amino acid (determined by

compar-ing to elution times for known standards) were used

forquantitating that amino acid (see below).

RESULTS

The strategy employed in theseexperiments

was dictated by the difficulty encountered in

obtaining enough highly purified virus-specific proteins todetect,by UVlight absorbance, the release of amino acids after treatment of the protein with carboxypeptidase.Hence, the

pro-tein to be analyzed was radiolabeled with the specific amino acidspredicted, from the nucleo-tide sequence of the viral RNA, to be at the carboxy terminus. Since poliovirus effectively turnsoff host cell protein synthesis (reviewed in reference 7), radiolabeled amino acidsare

incor-poratedonly in viralproteins. Individual

prepa-rations were made for each amino acid. The

labeled protein was then purified from other labeled viral proteins and from the bulk of nonlabeled host cell proteins. The purified pro-teinwassubjectedtocarboxypeptidaseA treat-ment for the specified times, and the released amino acids were identified by an amino acid analyzer. Thepercentageofreleased,freeamino acid was quantitated by comparing the total radioactive counts in the amino acid peak from theanalyzer with the calculatedcountsexpected from complete release ofa single residue. The latter valuewascalculated from the totalcounts

incorporated in the purified protein and the

total number of residues of the amino acid in

question predicted to be in the protein by the nucleotide sequence. Thisprocedure,of course, assumes equivalent incorporation of an amino acid within the entireprotein.

Termination site of translation.Thekineticsof

release ofPhe and Serfromthecarboxy

termi-nus ofP3-2 are shown in Fig. 2A. The fast, exponential releaseofPheishighly characteris-tic of a C-terminal amino acid, whereas the

FIG. 2. (A)Release of[3H]Phe (0)and[3H]Ser(0)

fromcarboxypeptidaseA-treated P3-2.(B)Releaseof

[3H]Gln (0)and [3H]Phe (0)fromcarboxypeptidase

A-treated P2-X. (C) Release of [3H]Asn (0) from

carboxypeptidase A-treated VP4. (D) Release of

[3H]Tyr (0) and[3H]Thr(0) fromcarboxypeptidase

A-treated VP1. Proteins were labeled with a single

amino acidatatime. Eachsetofreactionswascarried outonidenticalprotein samples. Reactionswere ter-minatedat5,10, 15, and 60minafter the addition of

thecarboxypeptidasetotheprotein samples.

0

100

80

60

40

20

80

w

0

w

cr

w

z

0

IL

0

cQ

w

0-x

w

C.

LI

0

w

w

o-a

wO

0

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slower releaseofSerischaracteristicofa

penul-timate amino acid residue (1). As predicted by thenucleotidesequence,these results confirma

carboxyterminus forP3-2of-Ser-Phe-COOH.It

shouldbenoted that theveryslow release of Ser

seen here is probably due to the somewhat refractorynature of thisamino acidto carboxy-peptidase A. Furthermore, the less than com-plete releaseofboth amino acids by60minmay

be dueto aminor inhibition ofcarboxypeptidase action by the presence of SDS in the reaction buffers. Nevertheless, the relative rates of

re-lease ofthe amino acids supportthe prediction

of the nucleotide sequence.

If the translation is shifted from thepredicted reading frame by one or two nucleotides, the

newamino acid sequences of P3-2 predict the

presenceofanIleresidueator nearthecarboxy terminus. Hence, [3H]Ile-labeled P3-2 was di-gested withcarboxypeptidase A, and the release of Ilewasmonitored. Thiswasdonetoeliminate thepossibility that the predicted termination site might bewrongdueto a nucleotide sequencing

errorinvolving the insertionordeletion ofone or twonucleotides nearthe3' endofthe RNA. No

Ile was released after 90min of

carboxypepti-daseA treatment(datanotshown).

ReleaseofmorethanonePheorSer from the carboxy terminus of P3-2, even at prolonged

times of incubation, would not be expected

because oftwoArg residuesatpositions -6and

-7ofthepolypeptide (Fig.3). These arginines,

similartolysineresidues, block further

degrada-tion of P3-2 by the exopeptidase (2); the next

Phe in P3-2occursonlyatresidue -30(11);the

next serine occurs at residue -11 (Fig. 3).

Similarly, Ileresidues wouldnotbeexpectedto

be released as the first Ile occurs at position

-20.

The cleavage scheme ofthe poliovirus

poly-protein predicts that polypeptides P3-lb, P3-2,

andP3-4b share identical amino acid sequences

(Fig. 1) (11 and 22). We therefore labeledP3-lb

and P3-4b with

[3H]Phe

[3H]Ser.

min each, and the release of the labeled amino acids was quantitated. These results (Table 1)

show that approximately equivalent relative

amountsof Phe and Serarereleasedbyall three proteins. This indicates that the three proteins

are coterminal at the carboxy ends and that

carboxy-terminal trimming does not occur

dur-ing processdur-ing fromprecursor to products.

Carboxy terminus of P2-X. The kinetics of release of Gln and Phe from the carboxy termi-nusofP2-Xis shown inFig. 2B. As predicted by

the nucleotidesequence, only one Gln and one Phe are expected to bereleased because of two

Arg residues at -13 and -14 blocking further

digestion. The results confirm a terminus for

P2-Xof -Phe-Gln-COOH. Since Glyisknownto

bepresent attheaminoterminus of the following protein, P3-lb(22),the existence ofatleast one Gln-Gly proteolytic cleavagesitehas been

prov-en.

In addition, [3H]Phe- and [3H]Gln-labeled P2-3b, the precursor protein to P2-X, was

car-boxypeptidase treatedfor 30 min;49.7% of the

[3H]Gln counts per minute per residue and

37.3% of the [3H]Phe counts per minute per

residue were released. These results show that P2-Xand P2-3b are coterminalattheircarboxy endsand thatP2-3b isnottrimmed from this end while beingprocessed to P2-X.

Unique cleavage sites of VP4 and VP1. Of particular interest to us are the cleavage sites

between capsid proteins VP4 and VP1 and

be-tweencapsidprotein VP1andthenonstructural

polypeptide P2-3b. As predicted by the nucleo-tide sequence,these sitesareAsn-Ser and

Tyr-Gly, respectively, and differ from all other known Gln-Gly cleavage sites (11, 15, 22, 23).

The kinetics of release ofAsn from the

car-boxy terminus of VP4 is presented inFig. 2C. The resultclearlyconfirms the prediction of Asn

asthe carboxy-terminal amino acid of VP4 and provides supporting evidence for the unique proteolyticcleavage site betweenVP4andVP2.

Similarly, VP1 labeled with [3H]Tyr or

[3H]Thr was subjected to carboxypeptidase A

digestion. The kinetics ofreleaseofTyr and Thr

from VP1 is shown in Fig. 2D. The results

conform with theamino acidsequencepredicted from the nucleotide sequence and establish a

carboxy terminus for VP1 of -Thr-Tyr-COOH. Since the unique cleavage site between VP1

and P2-3bappears tohavenofunctional signifi-cance, a further test for the presence of Tyr

instead of Gln at this cleavage site was

per-formed. [3H]Gln-labeled VP1 was treated with

carboxypeptidase A for 30 min. No release of Glnwasnoted (datanotshown).

Ascanbeseenfrom Fig. 3 basic amino acids

neartheCterminuspreventrelease ofa second

molecule of the respective labeled amino acids

in VP4 aswellasinVP1. DISCUSSION

Polynucleotidesequenceanalysisissubjectto

errors no matter how carefully the work is

carried out or what method is used. Theamino acid sequences predicted from the nucleotide sequencemustthereforebeconsideredas

tenta-tive, and they should be verified, at least for

areasofspecialinterest,byamino acidanalysis.

The C-terminal amino acids of P3-tb, P3-2,

andP3-4bpresentedhere prove thattermination of viral translation occurs at . . . Ser-Phe, 73 nucleotides beforethestartofthepoly(A)tail of the RNA. Thus, in poliovirus translation the

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-20

-15

-10

-5

--PROSERLYSPHETHRGLUPRO

I

LELYSASPVALLEUILELYSTHRAL

OMETLEUASN

VP4S

-20

-15

-10

-5

--GLYV

ALASPTYR LYSASPGLYTHR

LEUTHR PROLEUSERTHR LYSASPLEUTHRTHRTYR

-20

-15

-5

--I

LEILEASNGLUARGASNARGARGSERASNILEGLYASNCYSMETGLUAL ALEUPHEGLN

SER PR

OASN--IL,

GLYPHEGLY--I

*o

P2

3b

GLYPR

oLEU--P3-lb

-20

-15

-5

--GLYAR

GAL

A.EULEULEUPROGLUTYRSERTHRLEUTYRAR

GARGTRPLEUASPSERPHE-COOH

P3-ib,P3-2,P3-4b-

FIG. 3. Predictedcarboxy-terminal amino acidsequences(11)andcleavage sites of proteins analyzed in this

study. Theexactproteolytic cleavagesitesaredenotedbyarrows.

ribosome travels throughanopenreadingframe of2,207 consecutive triplets and is then released from the mRNA at two adjacent stop codons. These are the only stop codons located in the

openreading frame atthe 3' end ofviral RNA.

Incontrast to the shortnoncoding sequence at

the 3' end, the noncodingsequenceatthe 5'end of the RNA is 741 nucleotideslong and contains several AUGs andstopcodons before the initia-tion signal of translation (11, 20).

All poliovirus polypeptides, with the

excep-tion of VP4 (N terminus blocked; 11) and VP2 (N-terminus is Ser; 15), have Gly at their

N-termini, as shown by amino acid analyses (15,

22, 24). These Gly termini are generated by

cleavage ofaprecursorpolypeptide, leaving, in

allbutonecase, apredicted Gln residueatthe C terminusof the othercleavage product. For the cleavage between P2-3borP2-X andP3-lb(Fig.

1) it hasnowbeenproventhatGln-Gly isabona

fide cleavage site and that the new amino acid terminiare notfurthermodified.

CleavagesatGln-Glyarethoughttobecarried

out by a viral proteinase (for references, see

reference 11). The Tyr-Gly site of VP1/P2-3b

(Fig. 1) may prove as susceptible to the viral proteinase as the Gln-Gly sites. On the other

hand, Tyr-Gly may serve an important

regula-tory function during viral replication. It may

respond with different kineticstothe same

pro-teinase that cleaves Gln-Gly sites oritmay be

susceptibleto adifferentproteinase altogether.

Maturation of virions occurs by cleavage of

VPO inacomplex of (VPO, VP3, VP1)to(VP4, VP2, VP3, VP1). This cleavage appears to

re-quire the presence of the viral genome RNA

(reviewed in reference 21).Sincethe maturation cleavageoccursatasite(Asn-Ser)notinvolved inanyother processingstep,itmaybe mediated byyetanotherproteinase.

Proteolytic processingof viralpolypeptidesis

TABLE 1. Release of

[3H]Phe

[3H]Ser

P3-lb andP3-4bbycarboxypeptidaseA

% of cpmexpectedforone

resi-Protein duereleasedat30min'

Phe Ser

P3-lb 60.9 48.9

P34b 67.6 42.5

P3-2 81.4 34.3

a Proteins were labeled with oneamino acid at a

time. Reactions werecarried outasdescribed in the

textand terminatedat30min.

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acommonphenomenon. Cleavages of the hem-agglutinin glycoprotein of influenza virus by hostcellularproteinase(s) involves the removal ofapentapeptide inthe caseoffowlplaquevirus andofasingleamino acid in all other influenza virus strains analyzed (16). Similarly, the gag geneproductofRous sarcomavirus isprocessed byaviralproteinasetofourpolypeptides where-by nine aminoacidsareremoved between

prod-ucts p27 and pl2, and two amino acids are

removed between p19 and plO (E. Hunter,

per-sonal communication). In contrast, the

cleav-ages reported here and the cleavage between VPgand P3-2 (22)do notresultin anytrimming of termini. Absence oftrimming has also been observed in the proteolytic processing of the

aphthovirus(foot-and-mouth disease virus)

cap-sid polypeptides (5, 13).

ACKNOWLEDGMENTS

WethankNicholas Alonzo for invaluable technical assist-ance.

Thisworkwassupported by Public Health Services grants AI-15122andCA-28146 from the National Institutes of Health andbytheU.S.Department ofEnergy.E.A.E. isa postdoc-toral fellow of theAmericanCancerSociety.

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