trans complementation by RNA of defective foot-and-mouth disease virus internal ribosome entry site elements.

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0022-538X/94/$04.00+0

trans

Complementation by RNA of

Defective Foot-and-Mouth

Disease Virus Internal Ribosome Entry Site Elements

JEFFDREWANDGRAHAM J. BELSHAM*

AFRC Institutefor Animal Health, Pirbright, Woking, Surrey GU24 ONF, United Kingdom Received 6 July 1993/Accepted 29 October 1993

A region of about 435 bases from the 5' noncoding region offoot-and-mouth disease virus RNA directs internal initiation of protein synthesis. This region, termed the internal ribosome entry site (IRES), is predicted to contain extensive

secondary

structure. Precise deletion of five predicted

secondary

structure features has beenperformed. The mutant IRES elements have been constructed into vectors which express bicistronic mRNAs and assayed within cells. Each of themodified IRES elements was defective in directing internal initiation when assayed alone. However, coexpression of an intactfoot-and-mouth disease virusIRES complemented four of these defective elements to an efficiency of up to 80o of

wild-type

activity. No complementationwasobservedwith thestructurally analogous element from encephalomyocarditis virus. The roleof RNA-RNA interactions in the function of thepicornavirus IRES is discussed.

The initiation of protein synthesis on picornavirus RNAs occursatAUG codons whichare600to 1300nucleotides (nt) downstream from the 5' termini of the molecules. No cap

structure is present at the 5' terminus of the viral RNA, in contrasttocellularmRNAs. Anumberof featuressuggestthat theinitiation ofproteinsynthesisontheseRNAsmustoccurby amechanism which is distinct fromthatdescribed for cellular

mRNAs (see reviews in references 11 and 16). The long 5' noncoding regions (NCRs) of picornavirus RNAs contain several unused AUGcodons, and these regions arepredicted to form extensive secondary structure. Translation of many

picornavirus RNAs also has to occur when cap-dependent

translation is abolished following the cleavage of the

cap-bindingcomplex(eIF-4F)componentp220. Inrepresentatives of each of the genera of these viruses, evidence has been obtained forthepresenceofanelementwhich directs internal

initiationofproteinsynthesis (2, 5, 12, 17, 26). This element is usually termedtheinternal ribosomeentrysite (IRES).

Thereisconsiderable conservation of thesestructureswithin certain groups of picornaviruses. The IRES elements from aphthoviruses (foot-and-mouth diseaseviruses[FMDV]) and cardioviruses (e.g., encephalomyocarditis virus [EMCV]) are

about50% identicalin sequence,and theirpredicted

second-ary structures arevery similar(29) (Fig. 1). These structures have been defined on the basis of phylogenetic comparisons andbiochemicalprobingof these RNAmolecules. The entero-virus(e.g.,poliovirus [PV])and rhinovirusIRES elements also show extensive similarity in their predicted secondary struc-tures (30, 33). However these elements are very different in

sequence and predicted structure from the aphthovirus and

cardiovirus elements. A conserved feature acrossall of these

elements is the presence of a pyrimidine tract about 25 nt

upstreamfromanAUGcodon. In thecaseof thecardioviruses and aphthoviruses, this AUG codon is an initiation site of

proteinsynthesis. In contrast,in the enterovirusesand rhino-viruses, aregionofupto 120nt ispresentbetween theAUG codon associated with the pyrimidine tract and the initiation codon. Mutagenesis of this cryptic AUG codon in PV has

*Correspondingauthor.Mailingaddress:Departmentof

Biochem-istry, McIntyre Medical Sciences Building, McGill University, 3655

Drummond St., Montreal, Quebec,Canada H3G 1Y6. Phone: (514)

398-7275. Fax:(514)398-1287.

shown that it is not essential for virus viability. However, uniquelyamongthe upstream AUGcodons,itsmodification is deleterious to the virus. The mutant has a small-plaque phenotype (25). InFMDV, two initiationcodons, some 84nt apart,are usedto initiateprotein synthesis. Ithasbeen shown thatsomeribosomesscanthrough the region following the first initiationcodon,presumablyfollowingrecognitionof theRNA at apoint upstream of the first initiation codon (1).

Considerable attention has been focusedondeterminingthe mechanism by which thepicornavirusIRES elementsfunction. Several studies have identified cellularproteinswhich interact with them. The IRES elements of FMDV and EMCV cross-linkprincipallyto aproteinof 57or58kDa. Twobindingsites havebeen mapped for thisproteinonthe FMDV IRES(18). Onlyasinglesite has beenidentified in EMCV(13).The latter is analogous to the 5' proximal site in FMDV. A distinct proteinof 52 kDa has been showntocross-linktothe PV IRES (21), althoughsome evidence for interaction with the 57-kDa protein has also beenpresented (28). Recently evidence has been obtained(3) that the 58-kDa protein is the polypyrimi-dine tract-binding protein (PTB), which has been previously assigned a function within the nucleus in the process of pre-mRNA splicing (24). The 52-kDa protein has been re-cently identified as La, a protein involved in maturation of RNA polymerase III transcripts, also located within the nu-cleus(20).Theroleof both of theseproteinsinIRES function isnotyetclear.Mutagenesisof the 58-kDaprotein bindingsite in theEMCV IRES indicated that loss ofprotein bindingwas associated with loss of ability to direct internal initiation of protein synthesis (13). However, other studies (7) suggested that this region of the IRES is notessential for the IRES to

function, and hence theimportance of this interaction is not certain.

Extensiveanalysisof thePVIRES has beenperformed.The sequence from nt 140 to 630 was initially defined as being necessary to direct internal initiation of protein synthesis. However, more recent studies (22, 27) have shown thattwo

predicted stem-loop structures within this sequence are not essential for IRES activity. Indeed, the3'-terminal structure, containing the cryptic AUG, can be deleted from

full-length

virion RNA without abolishing infectivity. However, the vi-ruses recovered have

regenerated

an AUG codon in an appropriate position

(31).

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FIG. 1. Secondary structure prediction for theFMDVIRES. The nomenclature used for the different predicted elements within the structure is indicated. Thestructure wasgenerated and presentedby using the FOLD and SQUIGGLES programs (6). The precise

se-quencesremovedby each ofthe indicated four domain deletions and the hammerheadstructure(deletion 5)areindicated in Table 1.The 5'

terminus of the IRES is indicated by the filled circle, and the 3'

terminus isrepresented by the filled oval.

Surprisingly, studies of the PV IRES showed that coexpres-sion offull-length PV RNA within cells containing defective PVIRES elements partiallyrestored activity tothe defective elements(27). Furthermore, these studiesrecently have been extended to show that merelycoexpression of the 5' NCR of PV with the defective IRES elements efficiently restored function (35). These studies suggested that complementation intrans between IRES elementsoccurred.

Inthisstudy,wehaveinvestigated therequirement foreach of the major predicted stem-loop structures in the FMDV IRES for efficient internal initiation of protein synthesis. Furthermore,wehavesought further evidence for theability of an intact IRES element to complement defective IRES ele-ments.Sincethe FMDVIRES isverydifferentin sequence and predicted structure from the PV IRES, equivalent results would strengthen the view that such complementation may have a role in thebiology ofpicornaviruses. We now showthat four of five defective FMDV IRES elements tested can be efficiently complemented in trans by an intact FMDV IRES. Thelossofthelargest motifrenders theIRES defectivewhen assayed alone or inthe presence ofan intact IRES.

MATERIALS AND METHODS

Plasmid constructions. Standard methods (32) were used for themanipulationandgrowth ofplasmidDNAs, whichwere purified by CsCl density gradient centrifugation priorto usein the transient expression assay. Plasmid pKSGC (Fig. 2) ex-presses abicistronic mRNAtranscript, encoding

3-glucuroni-dase (GUS) (14) and chloramphenicol acetyltransferase (CAT) (10) reporter proteins, under the control of the T7 promoter. The FMDV IRES, as an EcoRI-Clal fragment derived frompKSMRHMRI.Cla (2),wasinserted into pKS+ (Stratagene)toproducepKSRClaand used as the template for deletion analysis. The same fragment was also inserted into EcoRI-and Clal-digestedpSK+ to producepSKRCla,so that anantisensetranscriptis expressedfromthe T7 promoter (Fig. 2). The SmaI-Clal fragment from pKSRCla containing the wild-type (wt)FMDVIRESwasblunt ended andinserted into thevectorpKSGC, previously digested withHindlIl andblunt ended, toproduce pKSGSCC (Fig. 2).

The PCRwasperformed essentiallyasdescribed previously

GUS CAT

pKSGC=

(Smal) IRES

pKSGSCC

OEZIME

EcoRI (Clal) Hindlll

pKSGA1-5C5C

(EcoRI) (Clal)

pKSRCIa E _l

EcoRI Cla Hindlil

pSKRCIa

Clal EcoRI

pSKEMCRB

[image:2.612.353.519.69.278.2]

EcoRl BamHI

FIG. 2. Structure of the plasmids expressing bicistronic mRNAs and the FMDV and EMCV IRES elements. The T7 promoter is indicatedby filled circles. Note theoppositeorientation of the FMDV IRES(black rectangles) inpKSRCla (sense orientation)andpSKRCIa (antisense orientation).

(32). The EMCV IRES element was produced by using primers GTGGCCATATAAGGATCCTG7'FF'ITTC and GG GAGACCGGAATTCCGCC, which create BamHI and EcoRI restriction sites nearthe termini of the PCRfragment (about 550bases in length), using plasmid pE5LVPO (23) as the template. The BamHI site is at the position ofAUG10, some8 nt upstreamof the initiation site. The EcoRI-BamHI-digested PCR product was ligated into similarly digested pSK+ toproduce pSKEMCRB (Fig. 2). This EMCVelement functions efficiently to direct internal initiation of protein synthesis when assayed within cells in the context ofa bicis-tronicmRNA transcript (datanotshown).

Theprecise deletion of regions within the FMDVIRESwas performed by thePCR in two stages. One setof reactionswas performed with the forward sequencing primer (GTAAAAC GACGGCCAGT) together with specific primers 1 (Table 1), which introduced the required deletions. Separate reactions using the complementary mutagenic primers (primers 2;Table 1) togetherwith the reverse sequencing primer (AACAGCT ATGACCATG)werealsoperformed. The half-reaction prod-ucts were purified by agarose gel electrophoresis and mixed appropriately, and furtherPCRs wereperformed withonlythe forward and reverseprimers.Thefragments obtainedweregel purified, digested with EcoRI and Clal, and ligated into EcoRI- and Clal-cut pSK+ or pKS+. Modified FMDV ele-ments were constructed into the pKSGCvector as described abovefor thewtelement except thatblunt-endedEcoRI-ClaI fragments were used. All mutants were sequenced by direct double-stranded DNA sequencing using a T7 DNA poly-merase-basedsequencingsystem (Amersham).

Transient expression assays. Plasmids were assayed by transfection, using Lipofectin (Life Technologies), into HTK-143 cellsinfected with the recombinant vaccinia virusvTF7-3 (8), which expresses the T7 RNA polymerase as described previously (2). After 20 h, cell extracts were prepared and assayed for GUS activityin afluorescence assay (14) and for CATprotein ina quantitative enzyme-linked immunosorbent assay(ELISA) (Boehringer Mannheim).

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TABLE 1. Deletions introduced into the FMDV IRES

Plasmid Mutagenic oligonucleotide Sequencedeleted"

pKSGSCC None

pKSGllC 1,ACGAATTCCGCTGTGCTTGA -432to -379

2,CGAGCGTGGAGTCAAGCACAGCGGAATTCGT

pKSGA2C 1, CACTTTGTACTGCTGGTGACAGGC -375to -164

2,CATCCTTAGCCTGTCACCAGCAGTACAAAGTG

pKSGA3C 1, AGCACTGGTGACGAATAGGTGACC -155 to -48

2,GCCTCCGGTCACCTATTCGTCACCAGTGCT

pKSGA4C 1, TACGCCTGAATATTTCTTTTATA -42to -23

2,GTGGTTATAAAAGGAAATATTCAGGCGTA

pKSGA5C 1, GGAGCATGGTGGCGGCATGCCCATCATGTGTG -302 to -269,

2, CACACATGATGGGCATGCCGCCACCATGCTCC -266 to -258, and -253 to -244 "Thenumbering system used positions the A of the first initiation codon as nucleotide 1.

RESULTS

Determination of secondary structure motifs required for FMDVIRESfunction.The secondarystructurepredictionfor the FMDV IRES, essentially as derived previously (29), has been usedasthe basis for precisely deleting predicted domains within theIRES (Fig. 1).Verysimilarmodels were proposed for the FMDV IRES and for the element from EMCV. Different laboratories have used alternativenomenclatures (7, 13) for the various predicted secondary structure elements within the cardiovirus and aphthovirusIRES. Asimplersystem is shown in Fig. 1 and was used throughout this study. An alternative model for the EMCV IRES(7) is similar inmany respects.However,atthe 3' terminus, several short stem-loop structures(termedL, M, andN)whichare notcompatible with theFMDV sequence arepredicted by the latter model. Within thisregion, the latter model (7) also includes the polypyrimi-dine tract in secondary structure, in contrast to the models predicted forFMDVand EMCV previously (29). Alternative foldings of shortsequenceswithin thelargest domain(domain 2)remain unresolved butdo notaffect theexperiments under-taken in thisstudy.

a)

pKSGGIC 1

pKSG.62C 100

pKSGG3C 39

pKSGtbC pKSGSCC pKSGC

C)

pKSGAlC

pKSGQ2C

pKSGA3C

pKSGLAC pKSG.5C pKSGSCC

0 20 40 60 81 _ 102

_100 - 125

pKSGC 5

0 20 40 60 80 100 120 140

Each of the deletions indicated Fig. 1 and Table 1 was constructed by PCR as described in Materials and Methods. Each deletion was designed to precisely delete a predicted secondarystructurefeature.Notethat deletion5 removesonly the hammerhead structure at the endofdomain2. The PCR fragmentsgeneratedwerecloned,sequenced, and constructed into vectors expressing bicistronic mRNAs transcribed from the T7 RNA polymerase promoter. The vectors contain the GUS gene as an indicator ofcap-dependent translation, and this activity alsoverifies the expression of the plasmid within the cells. The efficient expression of CAT requires internal initiation ofprotein synthesis and is dependent on an active FMDVIRES.

Each of theplasmidsexpressingbicistronicmRNAs contain-ing thewt or mutantIRES elementsefficientlyexpressed GUS activity (Fig. 3a). However, marked differences inthe level of CAT expression were observed (Fig. 4a). The expression of CATdirectedby thewtIRES (plasmidpKSGSCC) servedas a positive control, whereas the lack of expression from a plasmid (pKSGC) containing the GUS and CAT genes but lacking anyIRES element (Fig.2)servedas acontrol forany

b)

pKSG61C

pKSGL22C

pKSGA3C 7

pKSGA4C _5

pKSGA5C 6

pKSGSCC pKSGC _

0 20 40 60

d)

pKSG 1C

pKSG62C

pKSG,5C pKSGA5C pKSGSCC pKSGC

I*+pKSRCla

0 20 40 60 80 100 120 140

FIG. 3. Effects of coexpression of IRES elements on the expression of GUS activity. The indicated plasmids were transfected into

vTF7-3-infectedcells alone(a)orwithpKSRCla (wtFMDVIRES; b),pSKRCla (antisenseFMDVIRES; c)orpSKEMCRB (EMCVIRES;d).

After 20h, cell extractswere prepared and assayed for GUS activity. GUS activity from pKSGSCC assayed alone wasset at 100% in each experiment,andother valueswererelatedtoit. Mean values from fourindependentdeterminationsareshownexceptforpaneld,in whichvalues

arefromasingle experiment repeatedtwicewithsimilarresults.

* 100 120 140

f17 |*+pSKRCIa

4

*19

in2 *1

15i

80 100 120 140

-38 *+pSKEMCRB

_48

2

N2

-34

_8

ND

r-6

pKSGu,n3G

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a)

pKSG A1 C

pKSGt62C

pKSGA3C pKSGA4C

pKSGt65C

pKSGSCC 100

pKSGC

C)

pKSGA1C pKSG 02C pKSGA3C pKSG 64C

pKSGt65C

pKSGSCC pKSGC

b)

pKSGA1 C m

pKSG62C

pKSGA3C pKSGt64C

pKSG&5C

pKSGSCC 1

0

pKSGC

d)

pKSGA1C

pKSG&lC pKSGb3C pKSGMnC pKSG65C pKSGSCC pKSGC

0 20 40 60 80 100 120 0 20 40

[image:4.612.155.464.75.273.2]

0O

FIG. 4. Complementationof defective FMDV IRES elementsby coexpressionof thewtFMDVIRES. Cellextractspreparedasdescribedfor Fig. 3werealsoassayedfor CATexpression by usingaquantitative ELISA. CATexpressionfromplasmidpKSGSCC (containingthe wt FMDV

IRES) assayed alonewassetat100%,and CATexpressionfrom otherconstructswascomparedwith this value in eachexperiment. Appropriate dilutionsof cellextractswereusedsothattheassays wereperformedwithin therangeof thestandardcurve.Theresultspresentedarethemean

valuesobtained from fourindependent determinationsexceptforpanel d,in whichvaluesarefromasingle experiment repeatedtwice with similar

results.

expression resulting from reinitiation. Essentially no CAT

expressionwasdetected from thisconstruct(Fig. 4a). Individ-ual deletion of domains 2, 3, and 5 from the FMDV IRES reduced CAT expression to almost the background level. However,significantbutlow-levelexpression (ca.3% of thewt

level) of CAT was observed when domains 1 and 4 were

individuallydeleted(Fig. 4a).

Complementation of mutant FMDV IRES elements. To determinewhether the defects in the ability ofthe defective IRES elements were complementable in trans, each of the plasmids expressingthe bicistronic mRNAswascotransfected

withaplasmid (pKSRCla) expressingthe intactFMDV IRES.

Theresultsobtained fortheIRES-directedexpression of CAT

are presented in Fig. 4. The results, mean values from four

determinations, are presented as a percentage of the CAT expression obtained fromplasmid pKSGSCC, which contains thewt FMDV IRES. The coexpression of the FMDVIRES, unlinkedto any open reading frame, fromplasmid pKSRCla enhancedexpressionof CAT frommutantswithdomains 1, 3, 4, and 5 individually deleted (Fig. 4b). The level of CAT expressionobservedwasbetween 12 and81% ofthat observed

from thewt IRES. Thedeletion of domain 1wasparticularly

efficientlycomplemented. Noenhanced CATactivitywas ever

observed from the construct lacking the large domain 2 or

lacking allIRES sequences. Itisnoteworthy thatthe plasmid containing deletion 5(deletion ofonly partofdomain 2[the hammerhead]) was very efficiently complemented. It may be

thatdeletion of all ofdomain 2interferes with theability of the

restof the IREStoadopt its native conformation.

Coexpressionof theintact FMDVIRES hadsomeinhibitory

effect on theexpression of GUS from the plasmids (compare

Fig.3aandb),butthiseffectwas verysimilarineachcaseand

probably reflectscompetitionfor cellularcomponents. The high efficiency of the complementation and also the inability tocomplement thedeletion of domain 2 bothargue

strongly against theeffectresultingfromsomeformof

recom-bination event between the plasmid DNAs. As a further

control against this possibility, cotransfection with a plasmid

(pSKRCla) containing the FMDV IRES in the opposite orientation to the T7 promoter was also performed. Under

these conditions, no complementation, as indicated by

en-hanced CAT expression, of the defective mutants lacking domain 1, 3, 4,or5wasobserved(Fig. 4c). Very surprisingly,

however, a consistent enhancement of CAT expression from

the domain 2 deletionconstructwas observed. The antisense IRES also inhibited GUS expression (Fig. 3c). The latter findingwasobservedpreviously(35)in analogousstudies with

the PV IRES. The inhibition of GUSactivity bythe antisense IRES was most marked on the RNAs which contained the FMDV IRES. A substantially less severe inhibition of GUS

expression from pKSGCwasobserved. This findingindicates

that the interaction with the antisense RNA reduces the activity of the bicistronic mRNA in translation, perhaps by reducing its stability.

Todeterminewhether thestructurally related EMCVIRES

wasabletocomplementthedefectiveFMDVIRESelements,

weconstructedaplasmid (pSKEMCRB; Fig. 2) containingthis element unlinked to any open reading frame. The element

used extends from the poly(C) tract to AUG10 just 8 bases upstream from the initiation codon. This EMCV IRES was

unable to complement any of the defective FMDV IRES elements(Fig. 4d)butcouldcomplement deletions withinthe EMCV IRES when assayed with analogousplasmids (2a). It had aninhibitoryeffectsimilartothat of theFMDV IRESon

the expression ofGUS activity (Fig. 3d). DISCUSSION

The FMDV and EMCV IRES are strikingly similar in

predicted secondary structure. There is considerable

agree-mentamongseveral laboratoriesthatasegmentof about 450 bases issufficienttodirectefficientinternalinitiationofprotein synthesis. However,somecontradictorydataontheprecise5'

terminus of the elementhave been presented. Studiesonthe FMDV element (2) and the EMCV element (13) suggested that loss of sequence within domain 1 grossly impaired the

0.1

0.2 *12 *13

_J1

*+pKSRCla

0.2 20 40 60 80 100 1

1.3 0 .8

1.2 _27

1.3 |*+pSKRCIa

0 20 40 60 80 100 121

, .4 .3 .3 .5 :0.3

I; 9

:ND |*+pSKEMCRB d)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

11 -A A . .11nrs 4on1

60 80 100 120I'll

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activity of the element. However, other studies (17) reported thatpartial loss of domain1 of FMDVonly hadamodest effect (50% loss) on translational efficiency. Similarly, it has been reported that partial loss of sequence from this terminal stem-loop structure in EMCV impaired activity by about 70%, while complete loss of the element (and loss of a few bases from the next stem) resulted in a deficiency of only about 35% compared with the wt element (7). These results are of importance since this terminal structure is reported to be the major site of binding of the 58-kDa protein (PTB) which interacts with each of these elements. Resolution of these various results is not entirely clear. The data suggesting a minimal role for domain1 have been obtained by using in vitro translation reactions on monocistronic RNAs, whereas the evidence forarequirement for this structure has been obtained by using bicistronic mRNAs in cell culture. It has been suggested that partial motifs may interfere with other struc-tures and hence give false answers (7). The presence of the upstream open reading frame in the bicistronic mRNAs may alsoperturb the structure of partial motifs. The precise dele-tion of this element in this study, without anypredicted effect onthe neighboringstructures, severely inhibited the abilityof the FMDV IRES to direct internal initiation, although some low-level (ca. 3% of the wtlevel) activity persisted. This result was consistent with our previousobservations that the partial loss of this element made itseverely defective (2).

Only limited mutagenesis of domains 2 and 3 has been performed previously. Linker insertions into the hammerhead structure atthe end of domain 2 (as deleted in the domain 5 deletion) and into domain 3 completely abolished the function ofthe EMCV IRES(7). The results presented here show that precise deletion of the hammerhead structure and domain 3 within theFMDV IRES also totallyabolished function.

Wehave used thesecondarystructuremodel for the3' end of the IRESproposed byPilipenkoetal. (29), since theL, M, and N motifs suggested by Duke et al. (7) for the EMCV sequence are notcompatiblewith the FMDV sequences. The stem-loopstructure proposed by Pilipenko et al.

(29)

is com-patiblewith bothFMDVandEMCV sequences and leaves the polypyrimidine tract unstructured. Precise deletion of the predicted 3'-terminal motif(domain 4), leavingthe

polypyri-midine tract intact, also severelyinhibited the activity of the IRES, although the activitywasabove thebackgroundlevel.

Complementation of defectiveIRES elements. The FMDV IRES is substantiallydifferent from that ofPV.The sequence has little similarity, and the secondary structure

prediction

is completelydifferent. However, theydo have thesame

biolog-ical activity. In each case, not surprisingly, it has been shown that precise deletion ofpredicted secondary structure motifs can abolish the activity of the element when assayed alone. Deletion of domain IV from the PV IRES produces an element which retains significant activity, but none of

major

features of theFMDVIRES appeardispensable. More

signif-icantly, uponcoexpression withanintacthomologous element, unlinkedtoanyother gene, enhancedactivityisobserved from thedefective elements within bicistronicmRNAs. Recombina-tion between theplasmidDNAsis ruledout

by

three lines of evidence. First, theefficiency of this unselected event is very high; up to 80% of the wt activity is observed from the defective elements when

coexpressed

with the intact IRES. Second, no enhanced CAT

expression

is observed when a plasmid containingthe identicalFMDV cDNA sequence,but in the opposite sense to the T7 promoter, is used

(the

one

exceptionisdiscussedbelow). This

plasmid

should be ableto recombine with the defective IRES elementsin an

equivalent

manner totheconstruct containing the IRES in the

opposite

orientation.

Third,

the construct

lacking

domain 2 did not exhibit enhanced CAT

expression

when the wt IRES was

coexpressed.

Ifrecombinationwas

responsible

for the restora-tion of CAT

expression

from the defective

elements,

one would expectthat this defect would also be corrected.

It may be

significant

that the deletion of domain 1 is the most

efficiently complemented (up

to81% ofwt IRES

activi-ty).

This is the

region

of the IRES towhich different labora-tories attribute very distinct

properties

(as

discussed

above)

andtowhich

p57/p58

(PTB)

binds. It may be that this terminal region has an

enhancing

function as

proposed

for the

stem-loop structure

only

on the 5' side of this domain in EMCV

(13),

which may alsoconstitutea

binding

site for

p57.

Thus,

its activity may be

fully

revealed

only

under

stringent

assay

conditions,

such aswhen the RNAis

assayed

in

competition

with other RNAs

(cf.

in vitro translation

assays). However,

since the EMCV IRES failedto

complement

eventhedeletion of this motif from the FMDV

IRES,

this element must do morethan

simply

bind PTB.

The

structurally

related EMCV IRESwas unable to

com-plement

any of the defective FMDV elements. This

closely

mirrors the

inability

of the rhinovirus 5' NCRto

complement

thedefectivePVIRES elements and the very limited

ability

of themore

closely

relatedcoxsackievirusB45' NCRto

perform

thesamefunction

(35).

Theseelementsareabout 60 and

70%,

respectively,

identical tothe PV IRESoverthis

region

of the genome. Thisis a

higher degree

of

identity

than between the EMCV andFMDV IRES

(about

50%).

Deletion of any sequence uptont528within thePV5' NCR (742nt

long)

has

proved

tobe

complementable,

but deletionof sequencesatthe 3'endof the

IRES, including

the

region

from nt528to

620,

werenot

complemented by

the cotransfection of theintactPVgenome

(27).

Four

independent

deletions of the FMDV IRES have been

complemented. However,

itwas not

possible

to

complement,

with theFMDV

IRES,

the deletion of the

largest element,

domain 2. It may be that the

tertiary

structure of the IRES is

severely

compromised by

this

large

deletion. It is of interest that a

single point

mutation at the base of this

large

domain enhanced the

activity

of thetype C FMDV IRES

(19), suggesting

that this domain is

key

to the structure

and/or

activity

of the IRES.

The strangestresult thatweobservedwastheenhancement of CAT

expression

directed

by

thedefective element

lacking

domain2when

coexpressed

with the antisense

transcript

of the FMDV IRES. We have no very

satisfactory

explanation

for this

phenomenon

atpresent. Antisense

oligonucleotides

have been shown

previously

to enhance the translation of masked mRNAs

(34).

Itwas

proposed

that this resulted from compe-tition with cellular

proteins

which were

binding

to the RNA and

inhibiting

its translation. It isdifficultto

adapt

thisconcept to the enhanced

activity

of the defective

IRES,

however. An alternative

possibility

is that the antisense

transcript,

when folded

by

itself,

forms a structure which allows the defective IRES to

partially

anneal to it and form a scaffold for the residual featuresof the defective IRESto function.

All of thedataso far obtained

indicating

complementation

of defective IRES elements

by

the

coexpression

of an intact

IRES havebeen obtained

by

using

the vaccinia virus-T7RNA

polymerase

expression

system

(this

report and reference

35).

The cellular environment is

clearly

modified

by

the vaccinia virus infection.

However,

thereisconsiderable accordbetween the results obtained in this assay system for otheraspects of IRES function and those obtained

by

other workers

using

alternative

strategies (2, 17,

22, 27,

35).

An

advantage

of the vaccinia virus system is that it

produces

high

levelsof

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mic transcripts asobtained with a natural picornavirus

infec-tion.

Two models for complementation of the PV IRES were

suggested (35). One modelsuggestsanRNA-RNA interaction inwhich the defective (and missing) element inone IRES is substitutedby that domain from the intact element sothat a

functional unit is re-formed. This would beanalogous to the regeneration in vitro ofa functional EMCV IRES from two

molecules by the melting and annealing ofoverlapping

tran-scripts (4).Thecosynthesisof the RNAtranscriptswithin cells in our studies could obviate the need for such a melting

procedure. The second model involves the transfer of a

functional initiation complex from the functional IRES ele-menttosomepointonthe defective IRESstructureintrans.In thecaseof FMDV andEMCV,thisseemsparticularly

attrac-tive sinceprevious studies (15) suggest that the viral RNA is recognizedwithrespect toitsabilitytoinitiateprotein synthe-sis fromapointwithin 8ntof the initiation codon itself.Thus, the region of the RNA in the immediate vicinity of the initiation codon may serve as anacceptorsite forapreformed

initiation complex. Very recently, it was shown (9) that a

nonlinear migration of ribosomeson cauliflower mosaic virus

RNA,whichwasdemonstratedonindividual RNAmolecules,

could also be achieved between two RNA molecules. These authors suggested analogous donor/acceptor regions for the ribosome shunt mechanism proposed.

Since thecomplementationof defective IRES elements has been observed for both majortypes of element found within the picornavirus family, it seems likely that this activity is important in the lifecycleof these viruses.

ACKNOWLEDGMENTS

We thank JuliaBrangwynfor excellent technical assistance. J.D. acknowledges receiptofanIAH studentship.

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