A cellular cofactor facilitates efficient 3CD cleavage of the poliovirus P1 precursor.

JOURNAL OFVIROLOGY, Apr.1993, p.2336-2343 0022-538X/93/042336-08$02.00/0

Copyright

X) 1993, American

Society

for Microbiology

A

Cellular Cofactor

Facilitates Efficient 3CD

Cleavage

of the Poliovirus

P1 Precursor

WADE S.BLAIR, XIAOYULI, ANDBERT L. SEMLER* Departmentof Microbiology and Molecular Genetics, College of Medicine,

University

of California,

Irvine,

California

92717-4025

Received6 November1992/Accepted 11January 1993

The production of poliovirus capsid proteins from a capsid protein precursor (P1) is mediated by virus-encoded proteinase 3CD and involves a complicated set ofproteinase-substrate interactions. In addition to

substrate and enzymatic determinants required for this interaction, we describe a cellular cofactor, which facilitates 3CDrecognitionof thePlprecursor.Cellular cofactoractivityis 3CDdependentand saltdependent. Ouranalysisshows thatproteolytic cleavageof the P1precursorattheVPO/VP3cleavagesite exhibitsagreater

dependencyonthecellular cofactor thancleavageattheVP3/VP1site. Suchagreaterdependencyoncellular cofactoractivity canbe relieved (in part) bythe substitution ofanAla residue for the Pro residue at the -4 positionof theVPO/VP3 cleavagesite.However,mutantvirusescontaining Pro-to-Alasubstitutions at the -4 position oftheVPO/VP3 site exhibit defects in viralgrowth.

Poliovirus, like all members of thefamily Picornaviridae, depends entirelyonprotein processingforthe productionof

mature gene products. In the infected cell, the poliovirus

genomic RNA (7.5 kb in length) is translated into a single

polyprotein (240kDa insize)whichis cleaved into functional viral polypeptides atY. G and Q. G amino acid pairs by virus-encoded proteinases 2A and 3C, respectively (8, 10, 18, 25, 26). Viral proteinase 3C exhibits highly specific activitywhichrecognizes few cellularproteins (15, 27) and cleaves the viral polyprotein onlyat a subset of the Q G aminoacidpairstoyieldauthentic viralgeneproducts.Such 3Cproteolytic fidelity requiresacomplexsetofinteractions, whichdependon (i) recognition ofQ. Gcleavage sites, (ii)

otherprimaryamino acid sequence determinants surround-ing Q. G sites,and(iii)structural determinants surrounding Q. Gcleavagesites.

Thecomplexityofpoliovirus proteinase-substrate interac-tions ismostapparentforcleavageofthe P1(capsid protein)

precursor (Fig. 1), which requires polymerase sequences

(3D) in addition to 3C proteinase sequences in the form of proteinase 3CD (13, 29). 3CD recognition of P1 dependson a complete and intact substrate. Carboxy-terminal trunca-tion (31, 32), incomplete processing ofP2sequences from the P1 carboxy terminus (22), or disruption of P1 ,B-sheet

secondary structures by amino acid insertion mutagenesis (30) results in the disruption of P1 processing in vitro. Interestingly, disruption of ,3-sheet secondary structuresin

any onecapsid protein presentin the P1 precursordisrupts

cleavage of the entire P1 molecule. These observations suggest that P1 tertiary structure may be important for

correctpresentation of the Q. G cleavage sitestoproteinase 3CD or, alternatively, that P1 tertiary structure may be

specifically recognized by proteinase 3CD. In addition to

structural determinants, primary sequence determinants

other than the Q. G cleavage sites have been defined for proteinase 3CD recognition of the P1 precursor. Efficient

cleavage of the VP3/VP1 cleavage site depends on the

presence of an Ala residue in the -4 position (4 residues

proximal to thecleaved Q - G scissilebond) (3). The latter

*Correspondingauthor.

primarysequencedeterminant alsoseemstoberequiredfor efficient cleavage of P2 and P3 precursor polypeptides. Synthetic peptides containing authentic poliovirus (24) or

rhinovirus (6) P2 or P3 cleavage sites are cleaved more

efficiently in the presence of homologous purified 3C

pro-teinases whenanAlaresidue is presentatthe -4positionof theQ Gcleavagesites.

Despite a preliminary understanding of the nature of 3CD-P1 interactions, fundamental questions, such as the role ofpolymerasesequences(3D)in3CDrecognitionofP1 and the composition of the 3CD-P1 proteolytic complex, remainlargelyunanswered. Inan attemptto further under-stand 3CD-P1 recognition,weexaminedanadditional

deter-minant required for 3CD-mediated P1 processing. In this study,wedemonstrate that efficient 3CD-mediatedcleavage of the P1 precursor requires a cellular cofactor. We also showthatproteolytic cleavageattheVP0/VP3 cleavagesite exhibits a greater dependencyon cellular cofactor activity

than cleavage at the VP3/VP1 site. Such an increased

dependencyoncellular cofactoractivitymayresult from the lack of an Ala residue at the -4 position of the VPO/VP3

cleavage site. We also demonstrate that the single amino acid substitution ofanAla residue for theProresidueatthe -4 position of the VPO/VP3 cleavage site results in an

increasedefficiencyofproteolytic cleavageatthat site under conditions limiting for the cellularcofactor in vitro.

How-ever, such a single amino acid substitution haspleiotropic

effects. We showthatmutantvirus, which contains a

Pro-to-Ala substitution at the -4 position of the VP0/VP3 cleavage site, exhibitsdefects in virus growth.

MATERIALS ANDMETHODS

Construction ofthesingleaminoacid substitutionmutation pT7P1-1B(P4A). Synthetic oligonucleotides 20 residues in lengthwereused inthe heteroduplex methodof site-directed

mutagenesis describedby Inouye and Inouye (12) to intro-duce a single amino acid substitution in poliovirus

subge-nomic cDNA pT7-P1 (30) at poliovirus nucleotide 1754 (C-to-Gtransversionmutation), resultinginplasmid pT7P1-1B(P4A). Plasmid pT7P1-lB(P4A) was digested with

endo-nucleasePflMI,anda3,129-bp fragment containing

poliovi-2336

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POLIOVIRUS P1 CLEAVAGE COFACTOR 2337

P1 _{I P2}

I

VPO I VP3

I

VPI I

N@ -,*

97

kD-63 kD

-60 kD

-37 kD

-26 kD-34kD

-I1CD

|1

VPO

I

I

IEv

VP3_]

lJ

FIG. 1. Diagram of potential cleavage products resulting from 3CD-mediated cleavage of P1 precursors in vitro. P1 precursors

synthesized in vitro from subgenomic poliovirus cDNApT7-P1and incubated in thepresenceof proteinase 3CDarecleaved into capsid

protein VPO, VP3, and VP1 orpolypeptide intermediate 1ABC (a

VPO-VP3 fusion) or 1CD (a VP3-VP1 fusion). Molecular sizes of

capsid proteins and polypeptide precursors areshownonthe left.

(+), 3CD-mediatedcleavageevents. NCR, noncodingregion.

rus sequences 496 to 3625 was introduced into a T7

transcription vector containing a full-length poliovirus

cDNA(pT7-PV1) (9), resulting in plasmid pT71-1B(P4A). In vitro transcription of subgenomic and full-length polio-virus cDNAs.SubgenomiccDNAswerelinearized by

diges-tionwithHindIII, while full-length cDNAs were linearized

by digestion with EcoRI at sites located 3' of poliovirus

sequences. In vitro transcriptions of subgenomic and full-lengthcDNAswerecarriedoutwithbacteriophage T7 RNA polymerase asdescribed previously (19, 30).

In vitro translation of RNAs derived from subgenomic cDNAs and cleavage of in vitro-synthesized P1 precursors.

Translation of RNAs derived from subgenomic cDNAs pT7-P1 and pT7P1-1B(P4A) was performed as described

previously (3, 30). Either 1 or 2.5

RI

of the resulting P1 in

vitro translation reaction mixture was incubated in the presence of either 1

p,l

of PV1-infected HeLa cell extract

diluted 10-fold in HD buffer (20 mM N-2-hydroxyethylpi-perazine-N'-2-ethanesulfonic acid [pH7.4]-1 mM dithiothre-itol) or 1.5,ul ofpoliovirustype 1 (PV1)-infected HeLacell

extract, respectively.The above cleavage reactionmixtures

weredilutedtoafinalreactionvolumeof5,10, 25,50,or100

,ulin thepresenceof HDbufferandincubatedfor 3 hat30°C, orwhen indicated, cleavage reaction mixtureswere diluted

100-fold inHDbuffercontaining 150 mMpotassium acetate

(KOAc), HeLaS10cellextract(totalprotein, 400,ug), HeLa S200 cell extract (total protein, 400

p,g),

or fractionated

HeLa S200 cell extracts (total protein, 50

p,g)

prior to

incubation. Cleavagereactionswereterminated by addition

of equal volumesof 2x Laemmlisamplebuffer, andcleavage

productswere analyzed by sodium dodecyl sulfate

(SDS)-polyacrylamide gelelectrophoresis (17). Autoradiogramsof SDS-polyacrylamide gels were analyzed with an LKB-2

Ultrascanlaserdensitometer.

Fractionation ofHeLa S200 cell extracts. HeLa S200 cell

extracts were prepared bycentrifugation of HeLaS10 cell

extracts, preparedasdescribedpreviously(4, 7), in a

Beck-man 50 Ti rotor at 200,000 x g for 1.5 h. HeLa S200 cell

extracts were dialyzed in buffer C (Tris-hydrochloride

[pH

7.9]-1 mMdithiothreitol) and loadedon aphosphocellulose

column.Afterelutionwith bufferC, cellular cofactor

activity

was detected by using the above-described cleavage assay with theflowthroughfraction, whichwassubsequently sub-jected toDEAE-cellulose(DE52)chromatography. Cellular cofactor activity was then eluted from the DEAE-cellulose columnwith 0.4 MNaCl in buffer C. All fractionated HeLa S200extracts weredialyzed inHDbufferpriortoanalysis.

Transfection of RNAs derived from full-length poliovirus

cDNA pT71-1B(P4A) and mutant virus stock preparation.

RNAs derived from pT71-1B(P4A) were used to transfect subconfluent HeLa cellmonolayersasdescribed

previously

(3, 28). Transfected HeLa cell monolayers were overlaid

witheitherDulbeccomodified Eagle mediumcontaining 10%

fetal calf serum or a semisolid medium consisting of

Dul-becco modified Eagle medium, 6% fetal calf serum, and 0.45% agarose. The transfectedmonolayerswereincubated at 33°C until cytopathic effects or plaques were visible. Mutant virus stocks were prepared by either picking well-isolated plaques 3days aftertransfectionor

harvesting

liquid

overlays 2days after transfection. Liquid

overlay

harvests were usedtoinfectfresh HeLa cellmonolayers,whichwere thenoverlaidwith semisolid medium.Well-isolated

plaques

were then picked as described above and were

clonally

purified by asecond round ofinfection andplaque isolation.

The plaque-purified stocks were expanded

by

two serial passagesthrough HeLa cell monolayers at33°C. The titers ofthestocks were determinedon60-mmplatesof HeLa cell monolayers under semisolid mediumat33°C.

Sequencing of mutant viral RNA. Viral RNA was har-vested by Nonidet P-40 lysis as described by Campos and Villarreal (5). RNA was sequenced by extension of a 20-nucleotideprimercorrespondingtonucleotides1810

through

1830ofpoliovirus RNA by using

[a-32P]dATP,

dideoxynu-cleotides, and reverse transcriptase.

[35SJmethionine

pulse-labeling of infected HeLa cells.

[35S]methionine pulse-labeling of HeLa cells infected with either PV1 or Sel-lB-06 wascarriedout as described

previ-ously(1). Briefly, HeLacellswere infectedat a

multiplicity

ofinfection of 30 and incubatedat33°Cfor 6 h. After the 6-h incubation, infected cells were labeled with 60

pCi

of

[35S]methionine,

incubated for 1 h at either 33 or

39°C,

and then harvested. Pulse-labeled viral proteinswere diluted in Laemmli sample buffer and analyzed on SDS-containing

12.5% polyacrylamide gels.

One-stepgrowth curve

analysis.

HeLa cell

monolayers

on

60-mmplateswereinfected witheither PV1 orSel-lB-06 at a multiplicity ofinfection of 30. After a 30-min

adsorption

period, infected monolayers were rinsed with

phosphate-buffered saline and overlaid with

liquid

medium. The in-fectedmonolayerswere incubated at33or

39°C.

After

2,

3,

4, 5, and 6 hat39°Cor2, 4,

6, 8,

and 10 h at

33°C,

cells and supematants were harvested. Plaque assays were used to determine PFU percellat eachtime

point.

RESULTS

Proteolytic processing ofin vitro-synthesized P1 precursor polypeptides under diluted cleavage reaction conditions. To examine efficiencies of

cleavage

atthe

VP0/VP3

siterelative totheVP3/VP1 site, P1 cleavage assayswerecarriedout in vitro under various dilution conditions. The rationale for these experiments was that under dilute

cleavage

reaction conditions, the kinetics of

virus-specific

cleavage

of P1

(a

trans-cleavageevent)would be

effectively

reduced,

allowing

a careful analysis of relative

cleavage

efficiencies at the

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2338 BLAIR ET AL.

M 1 1//1/5V10 125 /50 1/100

1CD-

vPo-

VPl-

VP3P-1 2 3 4 6 7

FIG. 2. 3CDcleavageof in vitro-translated P1precursorsunder

diluted reaction conditions. As describedinMaterialsandMethods, RNAs transcribed from awild-type poliovirus

subgenomic

cDNA (pT7-P1)weretranslated in vitro in thepresenceof[ 5S]methionine. In vitro P1 translation reaction mixtureswerediluted5-, 10-, 25-, 50-,or100-fold(asindicatedatthetop)in HD buffer and incubated in the presence of PV1-infected cell extract for 3 h at 30°C. Following incubation, cleavagereaction mixtureswerediluted in 2x

Laemmlisample buffer,and theresulting productswereanalyzed by

SDS-polyacrylamide gel electrophoresis. M,marker lane ofextracts

from[35S]methionine-labeled, PV1-infected cells.

VPO/VP3 andVP3/VP1sites. As describedin Materials and Methods,P1precursorsweresynthesizedinvitrofrom RNA transcriptsderived from awild-type poliovirus subgenomic

cDNA (pT7-P1) in the presence of [35S]methionine and incubated in thepresenceof PV1-infected HeLa cellextract

(a source ofproteinases 3C and 3CD) at various cleavage

reaction mixture dilutions in HD buffer. As shown inFig. 2, virus-specific cleavageatthe VPO/VP3cleavagesiteappears tooccurlessefficientlyrelativetocleavageat theVP3/VP1 site under dilutecleavagereaction conditions. With increas-ing cleavagereaction mixture dilutions (5-, 10-,and25-fold) (Fig. 2,lanes 3to5),acorrespondingdecrease inproteolytic

cleavage efficiency at the VPO/VP3 site relative to the VP3/VP1sitewasobserved. This is shownbyan accumula-tionofintermediate 1ABC(aVPO-VP3 fusionproduct

[Fig.

1]) and a decreasedproductionofcapsid proteinsVPO and

VP3relativetoVP1. Atcleavagereactionmixture dilutions of 10- or 25-fold virus-specific cleavage occurred almost

exclusively at the VP3/VP1 cleavage site, and at cleavage reaction mixture dilutions of 50- or 100-foldvery little P1

cleavagewasobserved(Fig. 2). The decreased efficiency of cleavage at the VPO/VP3 site relative to the efficiency of cleavage at the VP3/VP1 site is more pronounced than

appears in Fig. 2because ofunequal capsid protein methi-onine content. The methionine contents ofcapsid proteins VPOandVP3are1.8- and2.3-foldgreater,respectively, than that ofcapsid proteinVP1.Therefore, capsid proteins VPO

and VP3 appear to be present in greater molar quantities

thanVP1 when labeled with [35S]methionineand analyzed

on

SDS-polyacrylamide gels.

Laser densitometer analysis

and correction forcapsid proteinmethioninecontentshowed thatcleavageofP1is three-tofivefoldmoreefficientatthe

VP3/VP1site thanat the

VPO/VP3

site in cleavagereaction mixturesdiluted 10-fold(datanot shown).These data

dem-onstrated that

virus-specific proteinase

interaction is less

efficientat the

VPO/VP3

site thanattheVP3/VP1site under

dilute

cleavage

reaction conditions. Such differential cleav-age efficiencies may result from an increased

stability

of

virus-specific

proteinase

interaction atthe

VP3/VP1

cleav-age site relative to the

VPO/VP3 site, possibly

due to the presenceofanAlaresidueatthe -4

position

of the

VP3/VP1

site

(see below).

Identification ofacellularcofactor

required

for 3CD-medi-atedcleavage ofP1. The data

presented

above are reminis-centofaloss in3CD-specific activitywithincreasing cleav-age reaction mixture dilution.

3CD-specific activity

can be

definedbyefficient

cleavage

at the

VPO/VP3

andVP3/VP1 cleavage sites as

opposed

to

3C-specific activity,

which

mediates inefficient

cleavage

at the

VP3/VP1

site

(21).

To

determine whether such a loss in

3CD-specific activity

resulted from thedilution ofsomefactor

endogenous

tothe cleavagereaction other than enzymeor

substrate, cleavage

reactionmixtureswerediluted100-fold andincubated under various conditions. At 100-fold

cleavage

reaction dilution

conditions,

the

background

levels of 3C

activity (detectable

with lower dilutions of

cleavage

reaction

mixtures)

are reduced

sufficiently

such that

only

3CD-specific

activity

(relatively

efficient

cleavage

at both the

VPO/VP3

and the

VP3/VP1

sites)

is detected under

optimal

conditions. In

addition, in P1 cleavagereaction mixtures diluted

100-fold,

the contributionsof

putative

cofactors present inthesources of enzyme andsubstrate areminimized

(thus allowing

fora more sensitivecleavage

assay).

As shownin

Fig.

2

(lane 7)

and

Fig.

3A

(lane 4),

in

vitro-synthesized

P1 precursors diluted100-foldin HD bufferorHDbuffer

containing

KOAc

(at

afinal concentration of 150

mM) (Fig. 3A,

lane

6)

and incubated inthepresenceofPV1-infectedcellextract were

cleaved very

inefficiently

at either the

VPO/VP3

or the

VP3/VP1

site

by proteinase

3CD.

However,

when

cleavage

reaction mixtureswere diluted 100-fold in HD buffer

con-taining

150 mM KOAc and HeLaS10extract

(400

,ugof total protein per 100 ,ul of reaction

mixture)

P1 precursorswere cleaved

efficiently

atboth the

VPO/VP3

and the

VP3/VP1

cleavagesites

(Fig.

3A,lane

9).

These data demonstratethat acellular cofactor presentin HeLaS10

cytoplasmic

extracts

facilitates efficient

3CD-specific activity

onP1 precursors. A

preliminary

characterization of the cellular cofactor revealed thatitsactivityisdependentonthe presence of salt

(150

mM

KOAc)

and the presence ofproteinase 3CD. As shownin

Fig.

3A, in

vitro-synthesized

P1 precursorsdiluted

100-fold in HD buffer containing HeLa S10 extract (total protein,400

,ug)

andincubated in the presence ofanextract ofPV1-infected cellswerecleaved veryinefficiently by 3CD

inthe absence of salt

(150

mMKOAc) (lane 7).

Similarly,

P1

precursorsdiluted 100-fold in HD buffercontaining150 mM KOAc and HeLa S10 cell extract and incubated in the absence of PV1-infected cellextract (thesourceof

protein-ase

3CD)

remaineduncleaved(Fig. 3A,lane8). Thecellular cofactor, therefore, does not appear to exhibit proteolytic

activity.

Thecellular cofactor is also heat labile andphenol soluble. HeLa S10 cellextract that was incubated at 600C

(Fig.

3A,lane

10)

orphenol-chloroformextracted(Fig. 3A,

lane

11) prior

to analysis exhibited no cofactor activity. Cellularcofactor

activity

was retained in the S200 fraction

following centrifugation

of a HeLa cell cytosolic extract. J. VIROL.

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[image:3.612.112.254.78.314.2]

POLIOVIRUS Pl CLEAVAGE COFACTOR 2339

A

zC:

0 + M z

Kl IN

U o o

+ - +

0 Co

CO

C

_ o

co 0 Co) co

P1-

1ABC-

VPO-VP1-- _

V P3- &

B XL DE52

M K CiW U- U- o o o

P1=5

1ABC- di

1CD-X v

vPO-VP1-.

VP3- _

1 2 3 4 5 6 7 8 9

UV~~~..

1 2 34568~~~~:7

9 10 11

Fractionation of HeLaS200extractsbyionexchange chro-matography showed that the P1 cleavage cofactor activity

waseluted in theflowthrough fraction ofaphosphocellulose

column (Fig. 3B, lane 4) and was eluted with 0.4 M NaCl

from a DEAE-cellulose column (Fig. 3B, lane 7). Such a

preliminary characterizationsuggeststhatthe cellular cofac-torisaspecific proteinorprotein complex ofayetunknown molecularweight.

Fractionationof the HeLa S200 cellextract also revealed cellular proteases with enriched specific activities, which cleaveP1toyield nonauthentic viral polypeptides of

appar-entmolecularweightssimilartothoseofviral proteinsVPO and VP3 (Fig. 3B, lanes 8 and 9). The activities of such cellularproteases are distinct from the 3CD-dependent P1 cleavage cofactor (discussed above) and seem to be 3CD independent (data notshown). Cellular protease activity is notdetectedwith HeLaS10orS200 cellextractpresumably becauseofverylowspecificactivities in theseextractsprior to fractionation. It is unlikely thatthese cellular proteases haveroles in P1processing.

Proteolyticprocessingof P1precursorscontaininga

Pro-to-Ala substitution atthe -4 position of the VPO/VP3 cleavage site. Todetermine whetherdifferential 3C-or3CD-mediated

cleavage efficiencies at the VPO/VP3 and VP3/VP1 sites under conditions limiting for the above-described cellular cofactorcanbe attributedtothepresenceorabsence ofa-4

Alaresidue,aPro-to-Ala singleamino acidsubstitutionwas

introduced atthe -4position oftheVPO/VP3cleavage site [pT71-1B(P4A)] (Fig. 4).P1precursorsderived from pT7P1-1B(P4A) or pT7-Pl (wild type) were synthesized in the

presenceof[ 5S]methionine andincubated in thepresenceof

FIG. 3. In vitro 3CD-Pl cleavage reaction mixtures incubated undervarious dilutionconditions. Pl precursors were synthesized in vitroin the presenceof[35S]methioninefrom wild-type poliovirus in vitro-synthesized mRNAs. (A) In vitro Pl translation reaction mixtureswerediluted100-fold inHDbuffer(lanes 4 and 5) ordiluted 100-foldin HD buffercontaining150mMKOAc(lane 6), HeLaS10 extract(totalprotein,400pLg)(lane 7),150mM KOAcand HeLaS10 (total protein, 400 p.g) (lanes 8 and 9), 150 mM KOAc and heat-treated(60°C)HeLaS10extract(total protein, 400ptg)(lane10),or 150mMKOAc andphenol-chloroform-extracted HeLaS10extract (lane 11). Diluted Pl precursors were incubated either in the presence(lanes4, 6, 7,and 9 to11)orin the absence ofPV1-infected (INF) cellextract(lanes5 and 8). Lane 3, in vitro-synthesized Pl precursors incubated in the presence of PV1-infected HeLa cell

extractunder undiluted(U) cleavage reaction conditions.No RNA, in vitro translation reaction mixtures incubated in the absence of RNA.(B)Invitro-translated Pl precursors were diluted100-foldin HDbuffer containing150mMKOAc(lane 2), HD buffer containing 150 mM KOAc and HeLa S200 cell extract (total protein, 400 ,ug) (lane3), orHD buffercontaining150mM KOAc and fractionated HeLaS200 cellextracts(total protein,50,ug) (lanes4 to9). Lane 4, flowthrough (FT)fraction afterphosphocellulose chromatography; lane5, theFTfraction after DEAE-cellulose chromatography;lanes 6to 9,fractions eluted with 0.2, 0.4, 0.6, and1 MNaCl, respec-tively,fromaDEAE-cellulosecolumn. DilutedPl precursors were incubated in thepresenceofanextractfromPVl-infected cells for 3 hat30°C. M,marker lane ofan extractof[35S]methionine-labeled, PV1-infectedcells.

PV1-infected cell extractundervarious dilution conditions. As shown in

Fig.

5, proteolytic cleavage of P1 precursors derived frompT7P1-lB(P4A) atthe VPO/VP3 cleavage site was more efficient relative tocleavageat theVP3/VP1 site (lanes 9 to 12) under conditions limiting for the cellular cofactor thancleavageattheVPO/VP3 site of P1 precursors derived frompT7-P1 (lanes4 to7).This ismostapparentfor

cleavage reaction mixtures diluted 10- or 25-fold. Laser densitometer analysis showed that P1 precursors derived from pT7P1-lB(P4A) are cleaved three- to fourfold more

efficiently at the

VPO/VP3

site than P1 precursors derived from the wild type in cleavage reaction mixtures diluted VOL. 67, 1993

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[image:4.612.349.529.76.330.2]

2340 BLAIR ET AL.

P1

| VPO | VP3 | VP

WT:

< I.L

z Z

0 0

M z z

P1-ij[-.

1ABC-

ICD-* t

. *. Leu Pro Arg Leu Gln Gly LeuPro*.* 268269 270 271 272 1 2 3

1 1/5 1/10 1/25

LL

z

0

z

1B( P4A)

1 1/5 910 /25

..

..

,1g....

.';-'i'

ts

.. _ :

st

_ iilii Xaxief iw.> A,,

-, 7';'.

.j4,,,,..Qts:.

*'':: ;'

JF_*

SO;"

pT7P1-1B(P4A,

. . . LeuAla Arg Leu Gln GlyLeu Pro * *

[image:5.612.63.300.73.230.2]

288269270 271 272 1 2 3

FIG. 4. Diagram of theP1 precursorillustratingthesingleamino acid substitution mutationpT7-lB(P4A).Aminoacid residues span-ning theVPO/VP3cleavage site forwild-type (WT) P1 precursors andP1precursorsderived frompT7Pl-lB(P4A)aredepicted.*, -4 residue;V,cleaved

Q0

G amino acidpair.

10-fold (data not shown). Additionally, incubation of P1

precursors derived from pT7P1-1B(P4A) diluted 25-fold in

HDbuffer in the presenceofanextractfrom PV1-infected cellsresulted inproteolytic cleavageattheVP0/VP3site and theproduction ofcapsid proteinsVPOandVP3(Fig. 5,lane 12), while very little cleavage at the VPO/VP3 site was

observed when P1 precursors derived from the wild type

were incubated under the same conditions (Fig. 5, lane7). These data demonstrate thatanAla residue introducedatthe -4 position of the VPO/VP3 site increases 3C or 3CD cleavage efficiency atthat siterelative totheVP3/VP1 site under conditionslimitingfor the cellular cofactor.Theyalso suggestthat the cellular cofactorserves acompensatory role tofacilitate efficient 3CDcleavageatthewild-type VPO/VP3 cleavage site in the absence ofa -4Alaresidue.

[35Sjmethionine pulse-labelingofcells infected withmutant virus Sel-lB-06 or PV1. The Pro-to-Ala single amino acid

substitution mutation at the -4 position of the VP0/VP3 cleavage site was introduced into a full-length poliovirus

cDNAto construct plasmid pT71-B(P4A). After transfec-tion ofRNAs derived frompT71-1B(P4A)into cell

monolay-ers,mutantvirusSel-lB-06, which contained the Pro-to-Ala amino acidsubstitution, was recovered. Mutant virus

Sel-1B-06 displayed a small plaque phenotype at both 33 and 39°C. [35S]methionine pulse-labeling analysis was used to examinetheproduction ofvirus-specific proteins in

Sel-1B-06-infected cells at 33 and 39°C. Cells were infected with either Sel-lB-06 or PV1 for 6 h at 33°C, labeled with [35S]methionine, and incubated ateither33or39°C for 1 h.

After the 1-h incubation, cells were harvested, and

virus-specific polypeptideswereanalyzedonSDS-polyacrylamide

gels. [3S]methionine pulse-labeling analysis showed that protein processing in cells infected with mutant virus Sel-1B-06at33°Corin cells infected with Sel-lB-06at33°C with

ashiftto39°Cimmediately following the pulse with

[35Slme-thioninewasphenotypically similartothat for the wildtype

(Fig. 6). However, a slightly decreased accumulation or

production of 1ABC (a VPO/VP3 fusion product) in cells infectedwithSel-lB-06 compared with that forthewildtype

was observed. Such a slightly decreased accumulation of 1ABCwasconfirmed byimmunoprecipitationanalysis using

antiserum directed against capsid protein VP3 (data not

shown) and presumably results from anincreased cleavage

efficiency attheVPO/VP3 site. These data, therefore,

dem-1 2 3 4 5 6 7 8 9 10 11 12

FIG. 5. 3CDcleavageofPlprecursorsderived frompT7-Plor

pl7Pl-1B(P4A). Pl precursors were synthesized in vitro in the

presenceof[35S]methioninefrom mRNAs derived fromsubgenomic pT7-Pl (wildtype[W+])orpT7Pl-lB(P4A)cDNAasdescribedin

Materials and Methods. PlprecursorsderivedfrompT7-Pl (lanes4

to7)orpT7Pl-lB(P4A) (lanes9to12)werediluted5-,10-,or25-fold (asindicatedatthetop)in HDbuffer and incubated in thepresence of PV1-infected cellextract.Lanes 3 and8, Plprecursorsderived

frompT7-PlorpT7P1-1B(P4A)wereincubated in the absence(no

INF) of an extract from PV1-infected cells. No RNA, in vitro

translation reaction mixtures incubatedinthe absence of RNA.M, marker lane of an extract from [35S]methionine-labeled,

PV1-in-fected cells.

onstratethataPro-to-Alaaminoacid substitutionatthe -4 position of the VP0/VP3 site results ina slightly increased

cleavage efficiency atthat site andareconsistent withwhat

wasobserved invitro(Fig. 5).

One-step growth analysis of mutant virus Sel-1B-06. To determinethe effects of the-4Pro-to-Alasubstitutionatthe VPO/VP3 siteonviralgrowth,one-stepgrowthanalysiswas

carried out at 33 and 39°C as described in Materials and

Methods. The results of suchananalysis showed thatmutant virus Sel-lB-06 exhibited delayed kinetics of virus produc-tioncomparedwith thatof thewild typeat33°C (Fig. 7A).In addition, 10-fold and 100-fold reductions in maximum viral yield from cells infected withmutantvirus Sel-lB-06

com-pared with the yield for the wildtypewereobservedat33°C (Fig. 7A) and 39°C (Fig. 7B), respectively. These data, therefore, demonstrate thata Pro-to-Ala single amino acid substitutionatthe -4positionof theVPO/VP3 cleavagesite results in a temperature-sensitive defect in viral growth,

mostlikelyatsomestepof the viral replication cycle other thanproteolytic processing.It ispossiblethat suchanamino

acid substitution interferes with capsid protein function at thelevel ofprotomer-protomer interactions.

vP0-~

tw

VP - 1 _

.~

VP3-_NOW

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[image:5.612.320.556.75.380.2]

POLIOVIRUS P1 CLEAVAGE COFACTOR 2341

(0.

0

9

(0

0

m

P1 - _ _ g*

P3---3CD

1ABC

-3D- _...

VPO-VP2

-VP3-

_ _

2A-- _

[image:6.612.127.226.77.435.2]

1 2 3 4

FIG. 6. [35S]methionine pulse-labeling analysisof cells infected

with PV1 or Sel-1B-06. As described in Materials and Methods,

cellswere infected with either PV1 (lanes 1 and 3) or Sel-1B-06

(lanes 2and 4), incubatedat33°C for6h, pulsedwith

[35S]methio-nine, andincubatedateither33°C (lanes 1 and 2)or39°C(lanes 3 and

4). Cellswereharvested andresuspended in Laemmli sample buffer,

and viralpolypeptides were analyzed by SDS-polyacrylamide gel

electrophoresis.

It is unlikely that the observed temperature-sensitive defectinSel-1B-06 virus growth results from lesions in the virusgenomethatmayhave beenintroduced during cDNA

manipulations or virus stock preparation other than the

primary mutation. The Pro-to-Ala mutation was

recon-structed in different full-length poliovirus plasmid back-grounds, and a mutant virus which exhibited a growth

phenotype similar to that of Sel-1B-06 was generated. In

addition, multiple plaqueswere isolated during theoriginal

construction ofmutantvirusSel-1B-06, and all virus stocks derived from such plaque isolates exhibited similar growth phenotypes.

DISCUSSION

Proteinase 3CDrecognition and cleavage of the P1

precur-sor is a complex reaction, involving proteinase-substrate

interactions at multiple levels. Structural determinants as

well as definedprimary amino acid sequence determinants

arerequired forthisinteraction. Inthis article, the

require-ment for a cellular cofactor, an additional determinant for efficient 3CD cleavage ofP1, was described. Such a cofactor appears to be a specific protein or protein complex which was eluted in theflowthroughof aphosphocellulose column and was eluted with 0.4 M NaCl from a DEAE-cellulose column. Cellular cofactor activity is 3CD dependent and dependent on salt (150 mM KOAc). Proteolytic cleavage of the P1 precursor at the

VPO/VP3

site exhibited a greater dependency on cellular cofactor activity than cleavage at the

VP3/VP1 cleavage site. We believe that such a greater dependency results from the absence of a -4 Ala residue (which is present at the VP3/VP1 site) at the

VPO/VP3

site. Consistent with this conclusion, the substitution of an Ala for the Pro residue at the -4 position of the

VPO/VP3

site resulted in an increased cleavage efficiency at that site under conditions limiting for the cellular cofactor. However, mu-tant virus

Sel-1B-06

containing such a Pro-to-Ala substitu-tion at the -4position of theVP0/VP3 site exhibited defects inviral growth, resulting presumably from defects in viral capsid proteinfunction rather than defects inP1 processing. The cellular cofactor described in this study seems to increase3CD-specific activity. 3CD-specific activity is func-tionally differentiated from 3C activity on the basis of the efficiency ofP1cleavage. Proteinase 3CD activity mediates efficient cleavage at both the VPO/VP3 and the VP3/VP1

cleavage sites, while proteinase 3C mediates inefficient cleavage only at the VP3/VP1 cleavage site (16, 21). It is probable that in P1cleavage reaction mixtures diluted less than 25-fold (Fig. 2) a sufficient amount of 3C-specific activity is present to mediate inefficient but detectable amounts of VP3/VP1 cleavage. The observed preferential proteolytic cleavage at the VP3/VP1 site relative to cleavage at theVPO/VP3siteunder suchcleavage reaction conditions mayresult from areduction of 3CD-specific cleavage activ-ity to levels near or below background levels of 3C activactiv-ity. However, inP1cleavage reaction mixtures diluted 100-fold (a more sensitive assay system)inefficient 3C-specific cleav-age activity on P1 precursors is diluted to nondetectable levels and the assay detects exclusively 3CD-specific P1 cleavage activity. Under such assay conditions, which allow thedistinction of 3C- versus 3CD-specific activity, very little 3CD-mediated P1 cleavage was detected in the absence of cellular cofactor activity (Fig. 3). Our data, therefore, dem-onstrate that 3CD-specific activity is greatly facilitated by the presence of cellular cofactor activity. Therequirement of acellular cofactor for efficient 3CD-mediated cleavage ofP1 may explain the results of a recent study describing the purification of bacterially expressed 3CD proteinase. Pro-teinase 3CD, partially purified from genetically engineered

Escherichia

coli, exhibited lower than expected levels of 3CD- versus 3C-specific cleavage activity on P1 substrates (11), possibly resulting from the absence of3CD-P1cleavage cofactor activity in bacterial extracts (3a).

As a function of facilitating 3CD-specific activity, the cellular cofactor appears to mediate recognition of a nonideal(VPO/VP3)cleavage site. Asdiscussed above, a -4 Alaresidue is present at most of the cleaved

Q.

Gpairs on thepoliovirus polyprotein (23) and seems to be animportant substrate determinant required for 3C- or 3CD-mediated

substraterecognition (3, 24). On the basis of these criteria, theVP0/VP3cleavage site, whichcontains a -4 Pro residue, might bedefined as a less than ideal(nonideal) cleavage site. Despite the absence of a -4 Ala residue, 3CD-mediated

cleavageof theVP0/VP3 siteappearstobeefficient in vivo (2) and in vitro under nondiluted cleavage reaction

condi-tions (Fig. 2). However, under dilute cleavage reaction

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2342 BLAIR ET AL.

B.

6-

4-CD

2-0.

0--2

I . . I . . I . . I . I '

.--2 4 6 8 10 12

time(hrs)

I , . I , . .I I

1 2 3 4 5 6 7

time(hrs)

FIG. 7. One-stepgrowth analysis of cells infected with either PV1 orSel-1B-06. Cellswereinfected with either PV1 (E) ormutant

Sel-1B-06(U)at33'C(A)or39'C (B), andcells and supernatantswereharvestedatspecifiedtimes after infection.PFUpercellateach time pointwere determinedbyusing plaque assays.

conditions (10- or 25-fold) proteolytic cleavage at theVPO/

VP3 site appears to be less efficient than cleavage at the VP3/VP1 cleavage site, which contains a -4 Ala residue.

Cleavage efficiencyatthe nonidealVPO/VP3 cleavagesite is increasedindiluted cleavage reaction mixtures by either the additionofcellular cofactoractivity (Fig. 3)orthe substitu-tion ofanAla residueforthe Pro residue atthe -4position of the VPO/VP3 cleavagesite(Fig. 5). Sucha-4Pro-to-Ala

substitution also results ina slightly increasedefficiency of

cleavageatthe VPO/VP3 site in vivo(Fig. 6). Wepropose,

therefore, that in addition toincreasingoverall 3CD

recog-nition ofP1,the cellularcofactor alsoservestocompensate for the lack ofa -4Alaresidueby facilitatingefficient 3CD interactionspecificallyat theVPO/VP3 site.

Although thepresence ofa -4 Pro residuemay

compro-mise cleavage efficiency at the VPO/VP3 site under condi-tions limiting for cellular cofactor activity, the -4 Pro residue appearstobeimportant forcapsid protein function. Protein processing of virus-specific polypeptides in cells infected with mutant virus Se1-1B-06, which contains a

Pro-to-Alaamino acid substitution atthe -4position of the VPO/VP3 site, is similartothat for the wildtype. However, mutant virus Se1-1B-06 exhibited a temperature-sensitive

defect inviral growthat39°C. It is likely, therefore, that the -4Pro residue(located atthe carboxy terminus of VP2) is structurally important for viral capsid protein function. Previous studies havereported defects in viralcapsid

assem-bly (1) and viral RNAencapsidation and release (14) result-ing from alterations at the carboxy terminus of capsid proteinVP3 andattheamino terminus of VP1,respectively. These observationssuggestafunctional requirementfor the

Pro residueatthe -4position of the VPO0VP3 cleavagesite (conserved in all three serotypes of poliovirus) despite a

resulting dependency on cellular cofactor activity for

effi-cient3CD-mediated cleavage atthat site.

Asaresult ofongoing effortstopurifythe cellular cofactor to homogeneity, preliminary data show that the cellular cofactoractivity is 3CDdependent and, therefore, doesnot

appeartobeaproteaseitself. Cellular cofactoractivity also

has a saltrequirement (100to 150 mMKOAc) for activity. Although the cellular cofactor requires salt for activity and

appears stable at high salt concentrations, salt

concentra-tions above 250 mM partially inhibit its activity (data not

shown). The molecular weight of the cellular cofactor is

presently unknown, butthe cofactor isexpectedtobelarger than 12 kDa in size. Extracts from HeLa S10 (data not shown)and HeLaS200andfractionatedHeLaS200extracts dialyzed in HD buffer priorto analysis (Fig. 3B) by using dialysis tubingwitha12-to14-kDa-molecular-size exclusion retained cofactor activity. The latter observations exclude thepossibilitythat cofactoractivityresults from the addition of small molecules such as divalent cations, nucleotides, NAD+, or other small coenzymes to the diluted cleavage reaction mixtures described in this study. Thepresence of cofactor activityin HeLa S200 extracts (devoidof cellular membranes) and the observed inefficient cleavage of P1

precursors incubated in diluted cleavage reaction mixtures

containing 150 mM KOAc and canine microsomal

mem-branes(atfinal concentrations of 0.2eq/,ul)demonstrate that cofactor activity is not exclusively attributable to cellular membranes(datanotshown). Additionally,the detectionof cofactor activity in specific fractions after HeLa S200

ex-tractsweresubjectedto ionexchange chromatography sug-geststhatthe cellular cofactor isaspecific proteinorprotein

complex. A recent study reported the association of heat shockprotein70 withpoliovirusP1 ininfected cells(20),but preliminary evidence suggests that heat shock protein 70

maynotexhibit cofactor activity(2a). Mechanistically, the cellular cofactormayact tostabilize 3CD-P1 interactionsby facilitating the formation of a cofactor-3CD-P1 proteolytic complex. Alternatively, the cellular cofactormay act inde-pendentlyonP1orproteinase3CDtoinducearecognizable

substrate conformation orto confer P1 cleavage activityto 3CD.

ACKNOWLEDGMENTS

We are grateful to Holger Roehl for critical comments on the

manuscript andtoHung Nguyen forexperttechnical assistance. This workwassupported by Public Health ServicegrantAI22693

from theNational Institutes of Health. W.S.B. wasa predoctoral

trainee of the Public Health Service (grant A107319). X.L. was

supported bytheChineseVisiting Scholars Program of the Irvine HealthFoundation.

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-1

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