A poliovirus replicon containing the chloramphenicol acetyltransferase gene can be used to study the replication and encapsidation of poliovirus RNA.

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0022-538X/92/085040-07$02.00/0

A

Poliovirus

Replicon

Containing

the

Chloramphenicol

Acetyltransferase Gene Can

Be

Used

To

Study

the

Replication and

Encapsidation

of

Poliovirus RNA

NEILPERCY, WENDY S. BARCLAY,MICHAELSULLIVAN, ANDJEFFREY W. ALMOND* DepartmentofMicrobiology, UniversityofReading, Whiteknights, P.O. Box228,

Reading RG6 2AJ, UnitedKingdom

Received28February 1992/Accepted10May1992

Apoliovirus replicon, FLC/REP,which incorporates the reporter genechloramphenicolacetyltransferase

(CAT) in place of the region encoding the capsid proteins VP4, VP2, and part of VP3 in the genome of poliovirus type3, hasbeen constructed. Transfection of cells indicates that theFLC/REP repliconreplicates

efficiently

and thatactive CAT enzyme is producedas aCAT-VP3 fusion protein.ThelevelofCATactivityin transfected cells broadly reflects the level ofFLC/REP RNA. A series of mutationsinthe5' noncodingregion

ofpoliovirus type3 were introduced intoFLC/REP,and theireffectsweremonitoredbyasimple CAT assay. These experiments helped to define further the stem-loop structures in the 5' noncoding region which are essential forRNAreplication. TheCAT-containing poliovirus repliconcould also bepackaged into poliovirus capsids provided by helpervirusand wasstableas asubpopulation of virusparticlesover atleastfour passages. Thelocationof the CAT gene in FLC/REP excluded the presence of an encapsidationsignalintheregion of the

poliovirusgenome comprising nucleotides756to1805.

The genome of poliovirus is a single-stranded

positive-sense RNAmolecule ofapproximately7,500nucleotides. It

hasavirus-codedprotein, VPg, covalently attachedtoits 5' terminus andapoly(A)tract atits3' terminus. The body of the RNA comprises a 5' noncoding region (NCR) of 743 nucleotides, a large open reading frame encoding a virus

polyprotein with an Mr of 220,000 and a 3' NCR of 72

nucleotides (10, 21, 24). The openreadingframe is divided into 3 regions on the basis oftheprimary posttranslational

cleavages of the virus-coded polyprotein. Thus, the P1

regionencodes thestructural proteins of the virus capsid and

the P2 and P3 regions encode the nonstructural proteins, which include two proteases and an RNA polymerase(10). The 5' NCR of the poliovirus genome is highly conserved in

primaryandsecondarystructures,suggestingthat itplays a central role in virus replication (26). Indeed, it has been

demonstratedthatthecentralapproximately300nucleotides

promote the internal entry ofribosomes required for

cap-independent

translation

(18).

Inaddition,the 5'-terminal 100

nucleotideshave been shown to form a cruciform structure essential in the plus strand for RNA synthesis (1).

Although the replication of poliovirus RNA has been

successfullystudied in classicalgenetics, these studies have beenlimited by the need to generate infectious virus parti-cles. Nowadays, highly infectious RNAs can be generated from cloned cDNAs in vitro (27), so that it is possible to study a single cycle of replication of mutated genomes createdby reverse genetics. Using these methods, Kaplan andRacaniello(9)constructed aseries of subgenomic polio-virus replicons from which large regions ofP1weredeleted. These experiments demonstrated that the P1 region is not essential for genomereplication. This conclusion is in agree-mentwith studies on naturally occurring (8) and artificially

engineered

(7) defectiveinterfering (DI) particles which also suggest that both the P2 and the P3 regions must be

con-* Correspondingauthor.

served and translated for deleted RNAstoretain replication competence. Inprinciple,similar transfectionmethodscould be used to characterize further the role of the 5' NCR in virusreplication following site-specific mutagenesis.In prac-tice,however, mutations whichseverely affect translation or replication are difficult to assay over a single cycle,

espe-ciallywhencomplementing helpervirus is present. Further-more,it isoftenimpossibleto recoverRNAswhicharepoor replicons asDI particles because of theirinability to com-petewithhelper virus.

Suchproblemscould be partlyovercomebythe insertion of a reporter gene into the mutated poliovirus replicon,

which would allow it to be distinguished unambiguously from helper virus and would enable its replication to be monitoredbya simple enzyme assay. Ourprincipal objec-tive inthe presentstudywas todeterminewhetheraforeign gene, chloramphenicol acetyltransferase (CAT), could be inserted intoanonessentialregion of the poliovirus genome and replicated and expressed following transfection. We thenexploitedtheCAT-containinggenometoinvestigatethe role of certain 5' NCR stem-loop structures (20, 22, 23) in virusreplication. Finally,wedetermined whetherthe

CAT-containing genome could be packaged efficiently into the

capsids ofsuperinfecting helpervirus. We propose that the

poliovirus replicondescribedherewillbe ofvalue in

study-ing the replication andpackaging of poliovirus RNA.

MATERIALSANDMETHODS

Construction of plasmids. Theplasmid pT7FLCcontainsa full-lengthinfectiouscDNAof thepoliovirus type 3 genome in a modified pBR322 vector. The virus sequences are derived fromaclone ofpoliovirus type 3 P3/Leon/37 (pOLIO Leon [25]), except that the sequences between N278 and N2766weretaken fromafull-length clone of Sabin poliovi-rus type 3 (28), in which the SstI site at N1900 had been removedby mutagenesis(unpublisheddata).Inaddition, the AatIIsite of pBR322(N4286) had been replaced byaNotI

5040

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a

b

T7

ampp4K1

pT7FLC/REP

ori

Sal

CAT

GG5'NCR

IF77

i

inframe fusion

c

CAT

M G AO T G Y NTEMI ATG GGA GCT CAA ATCACT GGATAT N-TERMIN

743 756

CAT VP3

0 G G A T S D N H C-TERMIN CMGGA GGT GCG ACG TCA GAC MCCAC

1805

POLIOVIRUS-CAT REP

[image:2.612.79.298.74.355.2]

FIG. 1. (a) The structure of plasmid promoter(black triangle) precedesafull-ler

rus type 3 genome (filled) into which has encodingCAT(hatched region)betweenpo

TheRNAproduced in vitro followingtransc

linearizedwithSalI. (c) Thesequencesatthi proteinandtheCAT/VP3 boundary after in into the P1 region of the poliovirus genon

REP. Atthe Nterminus of the CATgene, created.Thisallowed the CATgenetobeii fragment so that the first fouramino acids

protein replaced those of CAT. At the C terminationcodonwasreplacedby thesequ

site. These two modifications result!

uniqueNotI and SstIsites. CATsequei

pT7FLCwere derived from thevecto

tated such thatanSstI sitewasintrodui

of the CAT gene. These manipulatic

amino acid changes at the N terminu, (Fig. lc). Mutations were also introc

termination codon, to incorporate a

toward the carboxyl end of the CAT duce anAatII siteat the 3' end of the The CAT sequences werethen introdi

replacing the SstI-AatIIfragment byth togenerate plasmid pT7FLC/REP (Fii Plasmid pT7pol-FLC/REP, which c

tion in 3Dpol, was constructed by

dil

DNAwiththeunique enzymesXbaIai

the vector fragment, and religating pT7FLC and pT7FLC/REP

harboring

NCR were generated by site-directe

NotI-SstI subclone of the 5' NCR in M13. Mutations were CAT

ligated

into the

parent

plasmids

via the

unique

NotI and SstI

sites.

Cells and viruses. Ohio HeLa cells were grown in Eagle's

I

minimalessentialmedium (EMEM) containing 5% fetal calf serum.For plaqueassays,6-wellplateswereinoculatedwith 200

,l

ofdilutedvirus and overlaid 30

min

later withEMEM plus 2.5% fetal calf serum and 2% agarose. After incubation at

34°C

for 3 to 4 days, the cell monolayers were stained with crystal violet.

For superinfection studies, confluent 6-cm dishes of Ohio HeLa cells were infected with 200

,ul

of diluted virus to achieve a multiplicity of infection of approximatelyS PFU

P2 P3 3'NCR per cell. After 30

min,

the inoculum was removed and A(30) replacedwith

EMEM.

Analysis

of RNA by dot-blot hybridization. Transfected cells were harvested and lysed by being resuspended in a solution containing 0.1 M NaCl, 0.1M Tris (pH 7.5), and 1% Nonidet P-40 on ice for 15

min.

Cell debris and nuclei were removed by centrifugation, and the

supernatants

were ex-tracted with an equal volume of phenol-chloroform. After precipitation with ethanol, the nucleic acid was collected by centrifugation, resuspended in water, and combined with an

IUS OF POLIO-CAT FUSION equal volume of

10x

SSC

(1

x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) plus 17% formaldehyde. The RNAs were

then heated at

65°C

for 15

min,

dotted onto presoaked nylon filters (Hybond

N;

Amersham), and cross-linked by UV

IUS

OFCAT-POLIO FUSION irradiation. The filters were then prehybridized for 2 h in 5 x SSPE(lx SSPE is 0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA

[pH

7.7]), 50% deionized formamide, 5 x Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS) containing 500

'LICON

,ug

of denatured salmon sperm DNA.

pT7FLC/REP.

The

17

Forgenerationof aCAT-specificprobe, 100 ng of a510-bp

igthcopy of thepoliovi-

SstI-NcoI

fragment

from

pT7FLC/REP

was labelled with been inserted the gene [a-

P]CT?

(50

,uCi;

Amersham).

The

probe

was denatured

isitions 743 and 1805. (b)

by

boiling

and

hybridized

tofilters for 8to18 hat

50°C.

The -riptionof

pT7FLCIREP

filters were washed at

65°C

with diluted SSPE containing

e Nterminusof the CAT 0.5% SDS until no further radioactivity was detected in the isertion of the CAT gene wash buffer and then air dried and subjected to autoradiog-ne in plasmid pT7FLC/ raphy.

, a unique SstI site was Analysis of

[3SJmethionine-labelied

polypeptides.

Trans-nserted

on an

SstI-AatII

fected or infected Ohio HeLa cells on 60-mm dishes were

ofthe

poliovirus

Ppoly-

labelledat 4 to 6 h postinfection by

adding

1 ml of methio-terminus of CAT, the

ience encodingGin-Gly. nine-free

EMEM

containing

50,uCi of

[35S]methionine.

At 6 h, the cells were harvested by scraping and lysed in 1 ml of radioimmunoprecipitation assay buffer and cell debris was removed by centrifugation. To 400

Rl

of this extract, 4,u of ed in a vector with a 1:10 dilution of anti-CAT rabbit polyclonal antibody nces for insertion into (SPrime-3Prime Inc.) was added, and the mixture was incu-irpRSVCAT (6), mu- bated at

37°C

for 1 h. A

100-,u

volume of a 10% solution ced at the5'terminus containing prewashed Staphylococcus aureus cells

(Pan-)ns

resulted in three sorbin) was added, followed by incubation for 1 h further at s of the CAT protein

37°C

and washing three times with radioimmunoprecipita-luced to remove the tion assay buffer. After being pelletted, the

mixtures

were

3ln-Gly

cleavage site boiled in 20,ulofLaemmlibuffer for 3minand analyzed by

protein, and to intro- autoradiography following electrophoresis on an SDS-poly-CAT gene (Fig. lc). acrylamide gel (12% acrylamide).

aced

into pT7FLC by Analysis of CAT enzyme

activity.

Transfected cell pellets te modified CAT gene were resuspended in 100

pl

of

Tris-HCl

(pH 7.8) and lysed

g.

la). by rapid freeze-thawing. After

centrifugation

at 13,000 rpm :arries a 200-bp dele- for 15 min, 2 ,ul of the supernatants was used to measure gesting

pT7FLC/REP

protein concentration with a Coomassie protein assay kit

nd XhoI,blunt ending (Pierce). Samples containing 50

,ug

of protein were then All derivatives of incubated with 0.1

,uCi

of [14C]chloramphenicol (50

mCi/

mutations in the 5' mM) and 0.5 mM acetyl coenzyme A for 2 h. Labelled

ad mutagenesis of a materials were extracted with 1 ml of ethyl acetate,

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0e*

DNA

C

*: REP

24 10 8 6 4 2

FIG. 2. ReplicationofFLC/REP RNA.TotalcytoplasmicRNAs

wereisolated atvarious times (2 to 24 h) aftertransfection with in vitrotranscripts of pT7FLC/REP (REP)orfromuntransfectedcells

(C). An aliquot ofeach RNA was spotted onto nylon filters and detected by hybridization to a CAT-specific probe. pT7FLC/REP

DNA(DNA) was also spottedonto the filteras apositive

hybrid-izationcontrol in 10-fold dilutions rangingfrom 10to0.001ng(leftto

right).

trated byvacuum-drying, and subjected to thin-layer chro-matography with a solvent ofchloroform-methanol at 95:5 (vol/vol). Thin-layer chromatography plates were air dried

andautoradiographed.

RESULTS

Construction ofthe replication plasmid pT7FLC/REP. On thebasis of the structures of naturally occurring defective interfering particles (DIs) of poliovirus (7, 8), the plasmid pT7FLC/REPwasdesignedtoretainsequences essential for

virusRNAreplication andtoincorporatethe foreign marker

gene, CAT. The construction was based on the plasmid

pT7FLC, which contains a full-length cDNA of poliovirus

P3/Leon/37 (25) under thecontrol ofa T7 promoter. When pT7FLCwas linearized by digestionwith restriction

endo-nuclease Sall, transcription by T7 polymerase in vitro

pro-duced a genomelike RNA molecule with 2 additional

gua-nosine residues at its 5' terminus and a poly(A) tail of 30

residuesatits3' terminus (Fig.lb). ThisRNAhasaspecific

infectivity of 105 PFU/,ug following transfection of Ohio HeLa cells (data not shown). Plasmid pT7FLC/REP was

constructed from pT7FLC, with the 657 bases of the CAT codingsequenceinserted inframein place of the1,049 bases of the P1 region between 756 and 1805 encoding proteins VP4, VP2, and part ofVP3 (Fig. lb). The initiation codon andcodons 2to4of the polioviruspolyproteinwereretained

andreplaced by amino acids1to4of CAT, but otherwise the CAT protein forms the amino terminus of the polyprotein. The RNA transcribed from pT7FLC/REP was 408 bases

(5%) shorter than full-length poliovirus RNA. This is well withinthe sizerangeofnaturally occurringDIs, which have deletions of between 4.2 and 13.2% ofthetotalgenome (8).

FLC/REP RNA is amplified. To determine whether RNA

transcribed from pT7FLC/REP could act as areplicon, T7

transcripts were transfected onto Ohio HeLa cells, and at

various times posttransfection total cytoplasmic RNA was

extracted and hybridized to a radiolabelled CAT-specific DNA probe (see Materials and Methods). Figure 2 shows

thattheamountof CAT-containingRNAincreaseswithtime

over a 24-h period following transfection. A similar result was obtained ifa poliovirus-specific probe was used (data

notshown).Noincreaseabove base levelsof CAT-contain-ingRNA couldbe detected in cellstransfectedwith

replica-tion-defective T7 transcripts (see below). These results

illustrate that the CATgeneis replicatedwhen insertedinto

the P1 region of a replication-competent genome, presum-ably by poliovirus RNApolymerase.

A functional CAT protein is expressed from FLC/REP RNA. The CAT gene inFLC/REP is positioned so thatthe CATprotein willform theaminoterminus of thepolyprotein

produced in transfected cells. Since the FLC/REP RNA replicates, itseemed likely thatprocessing of the P2 and P3 proteins would occurnormally, but the extentofP1 protein

processing could not be predicted. Processing ofP1 protein into the component capsid proteins occurs at certain of several Gln-Gly motifs (10) and is effected by the viral

protease, 3CD (30). A Gln-Gly motif wasincorporatedvery close to the Cterminus of the CATprotein of

pT7FLC/REP

(Fig. lc), but because of the replacement of VP4, the myristoylation signal was lost. Analysis of myristoyl-defi-cient mutantshassuggested thatmyristoylation of the

poly-protein is necessary for processing (11), although Moscufo et al. (16) have described a mutant with reduced levels of myristoylation that processed P1 normally in vivo. It was therefore of interest toinvestigate the extent ofP1 process-ing of the FLC/REP polyprotein. The predicted Mr of the unprocessed P1 from FLC/REP is 84,000, whereas P1 pro-duced during infection with P3/Leon/37 has anMrof 97,000. Processing of the FLC/REP P1 protein would give rise to proteins with Mrs of 51,000 and 34,000 if processing occurs only at authentic poliovirus cleavage sites and proteins with Mrs of 25,000, 26,000, and 34,000 if the Gln-Gly motif near the CAT/VP3 boundary is also used (Fig. 3A). Cells trans-fected with FLC/REP RNA were therefore pulse-labelled with

[35S]methionine,

and cytoplasmic extracts were immu-noprecipitated with a rabbit polyclonal anti-CAT serum. Polyacrylamide gel electrophoresis revealed a band with an Mr of approximately 51,000 from cells containing FLC/REP

but not from poliovirus-infected or mock-infected cells (Fig. 3B). This suggests that theFLC/REP P1protein is cleaved to produce a containing fusion protein, probably CAT-VP3 and VP1, presumably by protease 3CD, but that the Gln-Gly motif near the junction of the CAT andVP3proteins is not cleaved (Fig. 3A and B).

The CAT-VP3 fusion protein was then monitored for enzymicactivity by the standard CAT assay. Figure 4 shows the increasing levels of CAT activity present in equal amounts of cell extracts over time, reflecting the increase in levels of RNA presented in Fig. 2. On the basis of a visual comparison with dilutions of a known amount of CAT enzyme, the 10-h sample was estimated to contain approxi-mately 2 U of CAT activity per mg of total extracted cellular protein (Pierce protein assay kit). Thus, the CAT-VP3 fusion protein is enzymatically functional and is expressed at high levels in transfected cells.

Todemonstrate that the level of CAT activity was depen-dent on replication of FLC/REP RNA, a polymerase-defi-cient mutant,pol-FLC/REP, which has an intact 5' end and is therefore translation competent but carries a 200-nucleo-tide deletion in polymerase 3D, was transfected into Ohio HeLa cells. No CAT activity was detectable after transfec-tion of pol-FLC/REP RNA, even after 10 h. In

addition,

CAT activity was not detected after transfection of a T7 polymerase reaction mixture from which the enzyme had beenomitted, indicating that template DNA alone could not give rise to functional enzyme under the conditions de-scribed.

CAT assays can be used to study RNA replication of FLC/REP mutants. Since the increase in CAT activity cor-relates well with the increase in

FLC/REP

RNA in the cell following transfection, the enzyme assay can be used to

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[image:3.612.102.256.75.175.2]

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A

25kDa

T

26kDa T 34kDa t

a CAT VP

VP1

g!n gly

51kDa

v

34kDa

v

CAT VP3 l VP1

85kDa

v

f CAT VP3 VP1

CAT 2 4 6 8 10 24 C

FIG. 4. CAT enzymeactivityproducedfrom FLC/REP. Cyto-plasmic proteins were extracted atvarioustimes (2to 24h) after transfection with in vitro transcripts of pT7FLC/REP or from untransfected cells (C),and50 Fg wasanalyzedforCATenzyme activityasdescribed in Materialsand Methods.A1-Uamount of commercial CAT(CAT)wasincluded.

B

M 1 2 3

66-CAT-CA/VP3

11.-l- sA. !T

45- 36-29-.

24-

20-

14-FIG. 3. CAT expression from FLC/REP RNA. (A) Predicted cleavage productsof P1 translated frompT7FLC/REPRNA. Cleav-ageattheP1/P2, VP3/VP1boundaries andattheengineered Gln-Gly siteattheCterminus of CAT would liberate threeproteinswithMrs of25,000, 26,000,and34,000 (a),whereascleavage onlyattheP1/P2 and VP3/VP1boundaries wouldliberate two proteinswithMrsof 51,000 and 34,000 (b). The uncleaved P1 precursorwith anMr of 85,000 (c)is also shown. (B)ACAT-specific proteinwithanMrof 51,000 (CAT-VP3 fusion) indicated by the arrow is present in

extracts of cells transfected with FLC/REP RNA (lane 3) but is absent from mock-transfected (lane 1) orvirus RNA-transfected (lane 2) cell extracts, as detected by immunoprecipitation with anti-CAT rabbitpolyclonalantisera andpolyacrylamide gel electro-phoresis.

measure RNA replication. A series of mutations of FLC/ REP containing modifications or deletions in the first 250 bases of the 5' NCR were therefore constructed. These mutations were based on the secondary-structure model proposed bySkinneretal.(23) (Fig. 5)andweredesignedto

assess the role of individual stem-loops inpoliovirus RNA replication. We first obtained evidence that the mutations did notaffect the translatabilityof the RNAby placingthe modified 5' NCRs downstream of a reporter gene in a

bicistronic mRNA(19)andby assayingthe amountofCAT produced from monocistronic RNAs in vivo (unpublished data). Therefore, any differences in CAT enzyme activity

after transfection of the variousmutantRNAs should indi-cate aneffect of the mutationonRNAreplication. Figure 6

shows the CATactivity produced byeachmutant. Itcanbe

seenthat deletion ofstem-loops designatedL(N10-34)orA (N51-78)inmutantsL- and A-, respectively(Fig. 5), result in a complete loss of CAT activity, suggesting that these structuresareessential for RNAreplication.Inversion of the

whole of stem-loopL(mutant L-inv) resulted ina replicon

producing a much-reduced level ofCAT activity, whereas

inversion of the stem only, leaving the loopintact (mutant L-sinv), had little effect. These results suggest that the primarynucleotidesequenceof theloopbut notthestemis importantfor replication of the RNA. Deletion of theloop N188-222 (mutant 220-) had little effect on CAT activity

comparedwith thewild-typeFLC/REP,suggestingthat this stem-loop is not essential for RNAreplication. To confirm these conclusions on the role of stem-loops, each of the mutationswasthen built into the infectious full-length

po-liovirus cDNA, pT7FLC, and the viability of the mutant

genomeswastestedbydirectplaqueassayofthe transfected RNAs. Repeated transfection ofmutant RNAs L- or A-failed to produce infectious virus, confirming that these deletionsarelethal. Mutants L-sinv and 220-producedvirus with a normal plaque size phenotype. Mutant L-inv

pro-duced a virus with a minute plaque size, as would be expectedfrom the level of CATactivity producedfrom the corresponding replicon (Table 1).

FLC/REPRNAispackagedintopoliovirus capsids. Except for the inclusion of the CATgene,theFLC/REPrepliconis

0

220 _DI

A

DII DIII

FIG. 5. Thesecondarystructureof the 5' NCR ofpoliovirus type 3 asproposed bySkinneretal. (23).Thestem-loopsreferred to in

thetext asL(N10-34), A(N51-78), and 220 (N188-222) are

indi-cated.

b

C

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A

.5

* *.o_ * *

B

a

CAT FlCR LJR L R L R A-R 220-R

FIG. 6. CAT enzymeactivityproduced from 5' NCRmutantsof FLC/REP. A50->xgamountofcytoplasmic proteins extracted 10 h aftertransfection with 5' NCRmutantsofFLC/REPwasanalyzed for CATenzyme activity asdescribed in Materials and Methods. Thepositionsofindividual mutationsare asdescribed in Table1.

similarin structure to thegenomeof apoliovirusDIparticle. In order to be propagated, DI genomes require capsids provided in trans. It was therefore of interest to examine

whether FLC/REP could be packaged into poliovirus cap-sidsprovided by helper virus. Cellsweretherefore superin-fectedwith5 PFU of virus per cell at4 hposttransfection with RNA from pT7FLC/REP and incubated overnight. Supernatantswerethen treated with RNase A(1

mg/ml)

to digestanyCAT-containingRNAwhichwasfreein solution

and used to infect fresh cells. Cytoplasmic extracts from these cells contained abundant CATactivity, indicatingthat

theyhad been infectedbyencapsidatedFLC/REP (Fig. 7).

No CAT activity was detected following infection with

supernatants from cells which didnotreceive helpervirus.

Similarly, CATwas not detected if the supernatants were incubated with apoliovirustype3neutralizing antibodyorif the cells were pretreated with an anti-poliovirus receptor antibody, monoclonal antibody303 (15). Thus, infection of cells by FLC/REPwas mediated bypoliovirus helper cap-sids infectingvia thepolioviruscellular receptor, indicating

that the CAT-containing RNA was efficiently packaged. Moreover, the poliovirus capsids containing

FLC/REP

ge-nomes could be passaged at least four times by infecting

fresh monolayers with RNase A-treated supematants. The CAT activity remained high and approximately constant with each passage (Fig. 8).

Titration of themultiplicity ofvirus used for

superinfec-tion indicated that efficient propagation of FLC/REP re-quired coinfection of every transfected cell with helper

virus. We therefore routinely used 5 PFU per cell. The

efficiencyofpropagationofFLC/REPdeclinedsignificantly

when themultiplicityofsuperinfectionwas0.1PFU per cell orless(datanotshown).

TABLE 1. Replication and viability of FLC/REP mutants

Deletion Description ofCAT

activity)

(plaque

size)

FLC/REP Wild type +++ Large

L- N10-34 deleted 0 Notviable

L-inv N10-34inverted + Minute

L-sinv N10-18 and N26- +++ Large

34inverted

A.- N51-78 deleted 0 Notviable

220- N188-222deleted ++ Large

CAT -0 +PV +PV

[image:5.612.332.546.75.186.2]

+anti-PV CAT +PV +PV+303

FIG. 7. Encapsidation of FLC/REP RNA. Supernatants from cells transfected with FLC/REP RNA and superinfected without (+0)orwith(+PV) poliovirustype3 weretreatedwith RNase A(1 mg/ml) andused toinfectfresh monolayers. CATenzymeproduced from50 ,ug ofcytoplasmicextract8hafter infectionwasanalyzed. (A) CAT activityproduced after infection with supernatants from cells whichhadreceivedFLC/REP andpoliovirus pretreated witha

polyclonal anti-poliovirustype3neutralizing antiserum(+PV

+an-ti-PV); (B) CAT activity produced from cells pretreated with an

anti-poliovirus receptor monoclonal antibody (MAb 303) before infection withsupernatantsfromcellswhich had receivedFLC/REP andpoliovirus(+PV+303).

DISCUSSION

Apoliovirusgenomecontainingaforeigngeneinplace of aregion encodingcapsid proteins has been shown to repli-cateautonomouslyin cell culture.Thepresenceofthe CAT gene as a marker allows the replication of the RNAtobe

monitored eitherby probing for increases in CAT-specific

RNA or more simply by a CAT enzyme assay. The CAT

activity in transfected cells was not due to accumulating translation productsfrom input RNA,because transfection

of mutated versions of FLC/REP which were translation

competent but deficient in replication did not result in detectable CATactivity. Hence,thissystem canbe usedto investigate the effects of mutations in the 5' NCR, for

example, ongenomereplicationbyasimple enzyme assay. The enzyme activity will be affected by mutations which alter translation as well as by those affecting replication.

However, the sequences in the 5' NCR which affect trans-lation have beenmapped to the region between bases 320 and 631(19).Onthe otherhand,the sequences which control

w

CAT +0 +PV 10 2° 30 40

FIG. 8. Passage ofencapsidated FLC/REPRNA. CATactivity from 50 ,ugofcytoplasmicextractsofcells8 hafter infectionwith supernatantsfrommonolayers whichreceivedFLC/REPalone(+0)

or with poliovirus type 3 (+PV). A total of one-tenth of the supernatantswasused to infect freshmonolayers,and CAT activity produced 8h afterinfectionwasanalyzedoverfour passages(1°, 2,

30,

and

40).

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genome replication are generally believed to lie within the

first 100 nucleotidesof the 5' NCR (1). Even gross deletions within this 100-base region do not decrease translation (19) butwere observed to affect CAT production in transfected cells.

Although a Gln-Gly motif was incorporated near the

CAT/VP3 boundary in FLC/REP, this signal did not appear to be cleaved

efficiently,

since

only

a CAT-VP3 fusion

protein could be detected in transfected cells. In light of recentresultsindicatingthe importance of alanine at the P4

position(2), this result is not surprising. The result suggests, however, that the CAT enzyme is active as a fusion protein.

Since the CAT assay lends itself readily to quantitation, small changes in the replication efficiency of genomes should be detectable by this system. Cells transfected with the

wild-type replicon produced about 2 U of CAT activity per mg of extractedcellular protein. This level compares

favor-ablywith thosereported for some plant RNA viruses used as

expressionvectors(5)and suggests that even poor replicons can be monitored by CAT assay. Indeed, using the CAT assay we were able to detect replication of a mutant which gave rise to minute plaques (L-inv). This mutant had the sequences of the L domain (N10-34) of the 5' terminal clover-leaf inverted. A different mutant, with only the stem sequences inverted (L-sinv) but with wild-type loop se-quences, producedhigh levels of CAT activity and gave rise to large wild-type plaques when built into a complete ge-nome.These observationssuggest that the primary sequence

exposedat the loop of this structure is important for RNA

replication,whereas the sequenceof the stem region is not. When the entireN10-34 loop was deleted (mutant L-), the

replication was so severely affected that no CAT activity

could be detected andviable virus could not be recovered. This was also the case for a mutant that had the A domain

(N51-78) of the clover-leaf deleted (mutant A-), whereas loss of the loop 220(N188-222)had only amarginal effect on CAT activity and no effect on plaque size. This is in agreement with previous reports, which suggested that sim-ilar mutations in this region also resulted in viruses with

essentiallywild-typephenotypes (4). The roles of individual domains of the 5'-terminal clover-leaf structure in RNA

replication are currently underfurther investigation with a series ofCAT-containingreplicons.

It was also possible to demonstrate specific,

receptor-mediated propagation of recombinant genomes indicating that

FLC/REP

RNApackaged efficiently into homologous capsids. This isin contrast to arecent report which describes the construction ofpoliovirus replicons carrying regions of human immunodeficiency virus genes within P1. Although Choi et al. (3) showed that the human immunodeficiency virus sequences were amplified, they were unable to dem-onstrate packaging either by superinfection or by cotrans-fection ofhelper virus RNA. The reasons for these

contrast-ingresultsarenotclear. It ispossiblethat insertion of these

particularforeignsequences has eitherremovedordisrupted an encapsidation signal in the P1 region. However, since

packaging could not be detected for any of four different

replicons, this explanation seems unlikely. Moreover, our own unpublished studies suggest that such signals are not present in theP1region.

Theapproachwe describe here hasbeen used already to mapimportantcis-actingsequences of several other viruses. For example, Levis and coworkers (13) also inserted the CAT gene into Sindbis virus and achieved expression of

biologicallyactive CAT followingtransfectionofinfectious RNAs produced in vitro. Superinfectionwith helper virus

enabled the recovery of CAT-containing genomes which had been packaged into capsids provided in trans. Furthermore, in a separate study CAT expression from a Sindbis virus vector increased over seven passages such that an amplifi-cation of 1018-fold was achieved (29). More recently, the CAT gene has been amplified, expressed, and packaged when inserted into the genomes of the negative-strand RNA viruses influenza virus (14) and Sendai virus (17). However, unlike the CAT replicons described here, those based on influenza virus genes were unstable, and their CAT activities decreased markedly over three passages (14). This was attributed to the rapid emergence of DI particles when influenza virus was passaged at a high multiplicity of infec-tion. Although not confirmed in the present study, we believe that the FLC/REP genome is amplified at a rate similar to that of wild-type poliovirus, since the size differ-ence between the two RNAs is small: FLC/REPis 408 bases or 5% smaller than poliovirus RNA. Kaplan and Racaniello (9) found that the replication rates of three subgenomic replicons were inversely proportional to their sizes, although this effect was not pronounced unless the deletions were large. The largest of their replicons had a deletion of 1,295 bases (17% of the genome) and yet replicated at a rate only 1.4 times greater than that of the full-length genome. Thus, FLC/REP is unlikely to interfere with the functions of the helper virus. Indeed, the observation that CAT activity does not significantly change over four passages supports this view, and preliminary hybridization data confirm that the numbers of FLC/REP and wild-type genomes are similar and remain constant through each of these passages (18a).

The genomes of naturally occurring DI poliovirus particles always contain the sequences coding for capsid protein VP4 (12). However, this region is not required for genome replication, since some RNAs constructed without VP4 coding sequences were able to act as replicons (9). Taken together, these observations have led to speculation that VP4 might contain the signal for encapsidation of poliovirus RNA. Our results show that a recombinant genome lacking VP4 coding sequences is efficiently packaged. Thus, the observations from the study of DI particles may reflect the mechanism of their generation rather than their specific packaging requirements, and the packaging for poliovirus RNA must lie outside the VP4 coding region. The CAT marker gene within FLC/REP provides an unambiguous way to distinguish itfromhelper virus RNA, and studies to locate the encapsidation signal are currently under way.

ACKNOWLEDGMENT

This work was supported by the Medical Research Council of GreatBritain.

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