Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation.

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0022-538X/92/031602-08$02.00/0

Sequence

and Structural Elements That Contribute to Efficient

Encephalomyocarditis

Virus RNA Translation

GREGORY M. DUKE, MICHAEL A. HOFFMAN,AND ANN C. PALMENBERG*

Institutefor Molecular Virology and Department of Veterinary Science, University of Wisconsin,

Madison, Wisconsin53706

Received 23August1991/Accepted25November 1991

The nucleotide sequence of the5' nontranslatedregionofencephalomyocarditisvirus(EMCV-Rueckert)was

determined, and a consensus RNA structural model for this sequence (850 bases) and three other

poly(C)-containing cardioviruses (mengovirus, EMCV-B, and EMCV-D) was created through reiterative use of a

minimum-free-energyfolding algorithm.The RNA elements within thisregion which contribute to translation

ofEMCVproteinsweremappedin cell-free reactionsprogrammed with cDNA-derived RNAtranscripts. The data provide evidence that stem-loop motifs I, J and K, formed by viral bases 451 to 785, are important components of cap-independent translation. In contrast to other reports, a minimal role for stem-loop H (bases 406 to444) in translational activity is indicated. Small 5' nontranslated region fragments (bases 667 to 797) containing the J and K motifs proved strong competitive inhibitors when added to cell-free reactions

programmedwithexogenous capped or uncapped mRNAs. Theputativesequestering of required translational factors by this segment clearly contributes to translational activity, but also suggests a possiblecompetitive

mechanism for the downregulation of host proteinsynthesis duringviral infection.

Unlike most eucaryotic mRNAs, the positive-strand ge-nomesof picornavirusesareuncapped andhave verylong5'

nontranslated regions (600 to 1,300 bases), which contain extensive secondary structure and multiple noninitiating AUG triplets(9, 36). These uncommonfeatures, combined with the knowledge thatpoliovirus infection leads to inhibi-tion ofcap-dependenttranslationof normalcellularmRNAs, have long warrantedsuspicion about alternate translational mechanisms (9, 36). Ourrecent reports and those of others

have indeed confirmed that 5' non-translated regions (5'

NTRs) ofpoliovirus, encephalomyocarditis virus (EMCV) and foot-and-mouth disease viruspromote cap-independent translation initiation, whereby ribosome bindingoccurs via internalentry ontothe 5'NTRwithout scanning fromthe 5'

terminus.Internalentry,relativetothe 5'terminus,hasbeen clearly demonstrated with monocistronic and dicistronic cDNA-derived transcript RNAs (5, 10-12, 15, 16, 30), as

well as with virion RNA (36). The binding site has been

variously describedas aribosomal landingpad, translational

enhancer, orinternalribosomeentry site.

During poliovirus infection, cap-independent translation occursinconjunction with viral2Aprotease-induced

degra-dation of cellular proteinp220, a componentofthe

eucary-otic initiation factor 4F (eIF-4F) cap-binding complex. The

synergistic processes presumably promote viral translation while down regulating that of the host (18). Foot-and-mouth

disease virus infection also induces degradation of p220,

althoughadifferent viralprotease (Lrather than 2A) may be

responsible (3). With EMCV, the translational shift from host toviralmRNAislikewisevery proficient, but seems to resultfrom ahighly effective competition for cellular

trans-lational components rather than selective degradation of

p220(17, 21, 39).

The translational efficiency of EMCV RNA is especially

*Correspondingauthor.

tPresent address: Department of Microbiology/Immunology, StanfordUniversity, Stanford, CA 94305-5402.

apparent incell-freeextracts,inwhich 8to10copiesof viral

polyprotein aretypically obtained from eachtemplate (37).

We have shown thattheactivenature of these templates is

notuniqueto expression of viral codingsequences, but is a property that canbe qualitatively transferred to nearly any

eucaryotic cistron through ligation with viral fragments containing the internal ribosome entrysite element (12,27).

The method has been widely used to obtain high-level protein expression in a variety of eucaryotic cells (5, 41). The transferred EMCV element usually includes bases 335 to 837 (translation begins at base 834) (5, 12), although deletionanalyses with dicistronic chimericmRNAs suggest that bases 403 to 811 may provide the core fragment for efficient translation (6, 14).

In this report, EMCV enhancer sequences are further

defined through nested monocistronic RNAtranscripts de-rived from cDNA clones and expressed in reticulocyte lysates. Linker insertion and deletion mapping show that

translational functionality is achieved apparently through concertedcontributions of severalstructuresandsequences

within the required region. Moreover, the potent inhibitory effects ofoneparticular stem-loop element (theJ-Kdomain, bases680 to785)onexogenously translating capped mRNAs

suggest further biological involvement in putative

host-protein shutoff mechanisms during naturalviral infection.

MATERIALS AND METHODS

Sequence determination. EMCV Rueckert strain

(EMCV-R) was passaged in HeLa cells, and vRNA was

purified as described previously (33). Comprehensive se-quence data for the 5' noncoding region of EMCV-Rwere

generatedbythreeseparatetechniques: (i) primerextension

dideoxynucleotide reactions withreverse transcriptase,

us-ing EMCV vRNA templates and 5'-labeled

(32p

or

35S)

oligonucleotide primers; (ii) Maxam-Gilbert determinations ofcDNAssynthesized from EMCVvRNA[poly(C) region];

and(iii) primer extension dideoxynucleotide reactions with Klenow polymerase, using EMCV cDNA-containing plas-1602

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mids as templates (20, 25, 38). All nucleotides were

con-firmed by multiple determinations.

Aconsensus RNAsecondary structure for the 5'

noncod-ing regions of poly(C)-containnoncod-ingcardioviruses was created

through reiterativeuseof the folding algorithm of Zuker and

Stiegler (42), modified to accept large (e.g., >2,500

nucleo-tides) data fields. The minimum-free-energy structures for

the 5'-most 2,500 nucleotides fromEMCV-R, mengovirus-M

(24), EMCV-D, and EMCV-B (1, 2), were calculated

sepa-rately and then compared. Thefolding energies were those

defined by Freier et al. (7) and averaged to about -1,180

kcal/mol (ca. -4,937 kJ/mol) for each sequence. Elements

commontoalldeterminationswereretained, and intervening

segments wererefolded untilaconsensus structure maximiz-ing total paired nucleotides in analogous configurations for all strains was achieved. The model was adjusted until the

calculated minimum freeenergy didnotdiffer by more than

5%fromanyof the optimum individual sequencesolutions. Construction of cDNA. Standard DNA cloning reactions (34)wereused, and Escherichia coli JM101wasthebacterial hostfor propagation of recombinantDNA.Thetranscription

vector Bluescribe M13+ was purchased from Stratagene. Plasmid pEA1, a subclone of pE13 (4), contained EMCV bases 335 to 4229 inserted between the EcoRI and BamHI sites of Bluescribe M13+. This construction isvery similar

to plasmid pESA1 (28), except for the choice of vector sequences. The pBalAl plasmids (numbered 1 to 14) were

derived frompEAl andcontainedanested series of

sequen-tial deletions (fromprogressive Bal 31 reactions)within the 5' noncoding segments of the viral cDNA. Insertion

muta-tionscontaining EcoRI restriction sites were made at natu-rallyoccurring MaeII restriction sites in the viral5' noncod-ing region ofplasmidpEAL.The sequence at theinsertion is

ACGCGCGCGGAATTCGT (inserted sequences are under-lined). Additional 4-base insertionswerecreatedbyfillingin AvrII and HindIII restriction sites within the viral 5'

non-coding regionofpEAlwith Klenowfragment. An insertion-deletion plasmid was made byjoining (with EcoRI linkers)

sequencescleavedatKpnI(base701) and MaeII(base 776).

The construction of all plasmids was confirmed by nucleo-tide sequencing.

Transcription ofplasmid cDNA. Plasmid DNAs were lin-earizedbydigestionwithBamHIandusedastemplatesin T7

polymerase runoff transcription reactions containing

[a-32P]CTP

(13 mCi/mmol). Reactions were terminated by

addition of EDTA (to 10 mM), and the samples were extracted withphenol-chloroform, precipitatedwithethanol,

and then resuspended in water. Reaction efficiency was monitored by scintillationcounting afterabsorptionof sam-ples to Whatman DE81 filters. To determine the transcript

size andpurity, sample aliquotswere denaturedby incuba-tionat50°Cfor1hin thepresenceof1 Mdeionizedglyoxal

and 10 mM Na2HPO4 (pH 6.5) and then loaded onto 1%

agarose gels. Afterelectrophoresis (1 V/cm for11 h), dried

gelswerescanned forradioactivitywith anAmbis radiation detector. The radioactivity in full-length

transcript

bands relative tothatfrom the total samplewas used toestimate

the percentage offull-length RNA synthesized in the

reac-tion.

Invitroproteinsynthesis. Cell-free translation reactions in reticulocyte extracts were performed as described

previ-ously (29). Typically,vRNA(1to3,ug)or

transcript

RNA(1

to3 ,ug)wasusedtodirectprotein synthesisinreactions(15

to30,ul) radiolabeledwith

[35S]methionine

(1 ,uCi/u). After

60to90 minof incubationat30°C,reactionswere

stopped by

theaddition ofcycloheximide and

pancreatic

RNase(to0.3

61

121

181

241

301 361

421

481

541

601

661

721

781

841

901

UUGAAAGCCGGGGGUGGGAGAUCCGGAUUGCCAGUCUGCUCGAUAUCGCAGGCUGGGUCC GUGACUACCCACUCCCCCUWUCAACGUGAAGGCUACGAUAGUGCCAGGGCGGGUACUGCC

GUAAGUGCCACCCCAAAAUAACAACAGACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

cCCCCCCCCcCCCCCCCcCCCCCCCCCCCCCCCCCCCCCCCCCCCCCcCcCCCCCCCCCC CCCCCCCCCCCCCCCCCCCCCCCUCUCCCUCCCCCCCCCCUAACGUUACUGGCCGAAGCC GCUUGGAAUAAGGCCGGUGUGCGUUUGUCUAUAUGUUAUWWUCCACCAUAUUGCCGUCUU

UUGGCAAUGUGAGGGCCCGGAAACCUGGCCCUGUCUUCUUGACGAGCAUUCCUAGGGGUC UUUCCCCUCUCGCCAAAGGAAUGCAAGGUCUGUUGAAUGUCGUGAAGGAAGCAGUUCCUC

UGGAAGCUUCUUGAAGACAAACAACGUCUGUAGCGACCCUWUGCAGGCAGCGGAACCCCC

CACCUGGCGACAGGUGCCUCUGCGGCCAAAAGCCACGUGUAUAAGAUACACCUGCAAAGG

CGGCACAACCCCAGUGCCACGUUGUGAGUWGGAUAGUUGUGGAAAGAGUCAAAUGGCUCU

CCUCAAGCGUAUUCAACAAGGGGCUGAAGGAUGCCCAGAAGGUACCCCAWUGUAUGGGAU CUGAUCUGGGGCCUCGGUGCACAUGCUUUACAUGUGUUUAGUCGAGGUUAAAAAACGUCU

AGGCCCCCCGAACCACGGGGACGUGGUUUUCCUUUGAAAAACACGAUGAUAAUAUGCCA M A T

CAACCAUGGAACAAGAGACWUGCGCGCACUCUCUCACUUWUGAGGAAUGCCCAAAAUGCU

T M E Q E T C A H S L T F E E C P K C S

CUGCUCUACAAUACCGUAAUGGAUUUUACCUGCUAAAGUAUGAUGAAGAAUGGUACCCAG

AL Q Y R N G F Y L L K Y D E E W Y P E

96AGAUA.AUAGAGGA UUUUAC 1000

60

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

9 61 AGGAGUUAW~GACUGAUGG'AGAGGAUGAUGUCUUUGAUCC 100 0

E L L T D G E D D V F D

FIG. 1. Nucleotide sequence of the5'-most1,000nucleotides of EMCV-R. Thepolyproteinreadingframebeginsatbase834,and the poly(C)tractcontains 132pyrimidines(C115UCUCCCUC10). mg/ml

each).

Samples (3

RI)

were

analyzed by

sodium

dodecyl

sulfate-polyacrylamide

(Maizel) gel

electrophoresis

(SDS-PAGE)

(26), and labeled

proteins

were visualized

by

autoradiography

of the dried

gel.

Incorporation

of

[35S]me-thionine into acid-insoluble

products

was

performed

as de-scribed

previously

(22).

Nucleotide sequence accession number. The GenBank/

EMBL accession number for this sequence

(complete

EMCV-R genome)is M81861. RESULTS

Sequence and structure model for EMCV-R. The nucleo-tidesequenceof the 5'-most

1,000

nucleotides of EMCV-R is

presented

in

Fig.

1. Thedatacorrectminor

discrepancies

in the

poly(C) length

differences are

discounted,

the 5' NTRof EMCV-R shares95.2%nucleotide

identity with those of EMCV-B and EMCV-D

(1,

2)

and 92.7%

identity

with the same

region

from

mengovirus-M

(24).

AconsensusRNA

secondary

structurefor the 5' ends of four cardiovirus

RNAs,

highlighting

strain

differences,

is

presented in Fig. 2. The model contains many elements similartothose

suggested

from

partial

sequence

alignments

(13, 31), but it also differs in three

regards.

First,

these

dominant

patterns

were derived from

minimum-free-energy

calculations which considered

simultaneously

all

possible

pairing

options

for

large

segments of thegenome

(the

5'-most

2,500

nucleotides).

Second,

the

poly(C)

tracts and their discontinuitieswerenotexcludedfromconsideration

by

the

algorithm,

but were allowed to

(potentially)

participate

in base

pairing,

as

long

asthese interactions couldcontributeto the

lowest-energy

solutions.

Third,

calculations from the

finalconsensusmodel allowedamaximumdeviation of

only

5% from the

optimum folding

solution foreach of thefour

participating

(2,500-base)

sequences. This model is

consis-tent with most available data

concerning

enzyme-sensitive

sites,

exposed loops,

etc.,for relatedisolates of EMCV

(6,

13, 31,40).

The structure suggests a linear series of 5'

noncoding

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G AUA

C U

40U C

(a)G--C

(C)U--A(G)

C--G U--G

G--C

(0)A--U(C) C--G GC--G U)_U--G A--U G--C

C--G

( UC -G

C--G U--A

A)

G2-C

A--U

(A

G--C

G--C G--C U--A G--C G--U G--C

G-C

cG--C(u)

C C (a)

G--c pseudo-knot

A--U A--U G--C

U--A A G A

(U--A AAC&

G-U U t U-A C

A C-G C-G A A

G--C G-c U (A) A

ACAG(G--CU)AG

0 (c)A

A C C

C C

(C)U C C

pseudo-knot 140 (-.)(A C C

(-)A C C

(-(C C C

(C)A C C

A C C

C C C

A C C

G(U)C0 C (+A)A C C

c C C

C C C

C C

EMCV-R (C

Mengo (C

EMCV-B (C

EMCV-D (C c c c

c

c c c

c

c c c c c c c c

c

c c c

rC

()A AA

C A

C-G

G-C

560 G-CAC 580 (G) A

(a)GA9AGGUG-I; 9U-riiG9; GLT~TAU

Mu) G6 AC GCG,AC,G,G-C AG

CAC .C(U.:CG A

U-A

-GCGG AC c

C-GCACC GU&ACC A-U

G-U62

C-G

G--UG

A GAPC

G-5U

U-G

C-G

U-A G-U

( A

C-G

A-U

A-U C-G

(G.g)A-UGA

SOA AGA

C-GG A-U

A G--C

A A

GU--A

U-G

C-G

G-U (Ac

c 480A--U{)

G U 8GGC661

300C-G A UUCC-UG A

GOGAU°U G-U G)G s(AU G

A-- A(-9 CGG- G-C G

Au u~~~~~00 A4

AA c u 382 U-~AAG

-G - U A -U (GaAC, G AU G

C UC--G(GucUCU UA)(GA- Mc)aCAU CU- - C GC-C U A C-Geu G-- AM- U--G

CGCC U UC-G(A) G C-GuA G--C

UC-GCCa)G--C(u)u C G--Croa) AA

'M'C(QU--

C--G U--A

C--GG A~~~~~~~~~--M2

GC-CCCU20C G-C

C C (C.C)U-G G--C U-A

CACG U () 32 U-A c

c AA ) u (UCU-()()A. GCA 9 C

C-- UC U AU(OAA GG

CC--G--C UGC UA C-G-- AG--CAQU--GCA --C C 70 (-

-(C (CU } (A)-Cu C U.-(Ge) (A) G

C-G G c).A(I C-- A--U A A

A--U C-jCU-G

Ag)C--GUAG-c ccc UU(CAA.CG-G--CA

c (c) A UG-C A

c CCC GC-..-- G-CAAu

(

C) _ccU U- (U)_{G--~~~~~C-IUlG-}(A),CAU u

'C C C -CUGA

C C C U-AC440GUGCC

C c C UCU -G

C C C C-GAG

CC C c

C2

u G A-jUA

C C CCA-C A--UlA

C C C C ~~AU(

--Cccc~ ~ ~ ~ ~ G- GO

C

C-C C CUG-C

C C C C G-U720

C,CC

~~~~~~~~~~~~~~AU-A_

115 UCUCCCUC10)

50 UC10)

27 ),(C141)* 130), (C144)*

E)cCG C-G

(C)U-I(C)

U (G)

G

* independentdeterminationsofsequence

FIG. 2. Consensus secondary structurefor 5' NTRsoffourrelatedcardioviruses. The sequence shown is for EMCV-R. Changes in

mengovirus and other EMCV strains (EMCV-B and EMCV-D) aregiven parentheticallyas capital and lowercase letters, respectively.

Stem-loopfeatures arelettered consecutively, and AUG sequences(EMCV-R)areboxed.

Initiating AUG, base834

L_0c U_UA82 CA84_c _A

- U--A

C U--A (~C A U--A~a PCAC A

i--C G A(Cx) G-C

-G G--C GC(u)

-G U--A A

-G G-C U

,-G C--G C (U--GO

A u Au) A G 0

kA> AUA A..(3.)

(G.g)

763AUU (C) cA

;AGCUG UG IGU AICU

: : U(C)

UCGGUGC A U

Z3

(G)

)~

,

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T7 RNA Transcripts.

E-Al p3alAl -14

4tk

-3 kb

-6kb

FIG. 3. RNA transcript integrity. T7 transcript samples from pBalAl-1 to pBalAl-14 wereprepared, denatured, fractionatedby electrophoresis, and detected asdescribed in Materialsand Meth-ods.

stem-loop motifs (Fig. 2, letters A to N), within which at least65% of the nucleotides [exclusive of the poly(C) tract] participate in base pairing. Surprisingly, the poly(C) region did not make a major contribution to the most favorable configuration ofany sequence (5'-most 2,500 bases), even

whenallowed topair with distant regions. The pseudoknot motifs in B and C differ from apublished structure of this region (40), but thisarrangementis energeticallyvery

favor-able and has been independently suggested by algorithms sensitivetopseudoknot configurations (31a).

Among the considered viral strains, transitions (e.g., U-*CorA--G)accountfor60% of thesequence variation.

More than two-thirds of theseexchanges occurinstemsand

cause little (if any) perturbation of the common structure. The model has 10 examples of double substitutions within paired bases, althoughsomeof thesecompensating changes are separated by more than 40 to50 nucleotides within the

sequence(seestemsA, F, H, I, and K). Transversions (e.g., U-iAorG-*C), insertions,anddeletionsaremore common

totheunpaired regions (77%) andaretypicallyatthetopsof stemsorinbulge-loops between pairedstems.

Translation of RNAs containing 5' NTR mutations. To defineimportant translational elements within theEMCV5' NTR, weconstructedaseries of cDNAplasmids containing progressive 5'--3' deletions. The constructions, designated pBalAl-1 topBalAl-14, each includedcodingsequencesfor the amino-terminal half of the polyprotein open reading frame (1,131 codons). RNA transcripts from these cDNAs

wereprepared in parallel and assayed by gel electrophoresis

(Fig. 3) to ensure minimum variation in the amount of full-length material (ca. 70%). Thetranscripts wereused to

program reticulocyte lysate reactions, takingcarethateach sample received the same amount of RNA (2.6 ,ug), at a

concentration (86 ,ug/ml) previously determined to give optimal, saturating protein synthesis.

The pEAl transcripts which began at viral base 335

A. d

LI|1AI|

1B

1C

I|

1D

I2AI12B

Al

virlon RNA pEAl

pBalAl

1-14

B.

400

c 3007

x

v-2

200-21

CIO

"6

- 363 387

406 416

443 454 466 485

505 513 546

620

776-L

802-CPM Incorporated

perul of lysate

UG(834) (x 103) 302 (1 00%)

1 253 (84%)

^, 251 (83%)

* 254 (84%h)

.4 230 (76%)

"= 95 (31%)

.= 64 (21%°h)

.= 82 (27%)

,= 169 (56%)

,= 76 (25%)

,~ 14 (5%)

,= 18 (6%)

.= 21 (7%)

,= 23 (8%)

.= 45 (15%)

.= 105 (35%)

-\&-_406

l-_466

416 1485 ----802

AUG (base 834)

200 460 600 800 1000

5' most viral nucleotide

FIG. 4. 5' deletion mutations. (A)EMCV virion RNA(1 ,ug)or T7 RNAtranscripts (2.6 ,g)asshown inFig.3[pEA1andpBalAl-1

topBalAl-14]wereusedtoprogramtranslation reactions (30RI)in rabbit reticulocyte extracts containing [35S]methionine (1 ,uCi/,ul). After 90 min at 30°C, triplicate sampleswere removed(2 ,ul)and quantitated for trichloroacetic acid-insoluble radioactivity. Aver-agedresults(standard deviation, <2%)arepresentedascountsper minute incorporated per microliter oflysate and also as percent

incorporation relative to virion RNA. (B) Data from panel A displayed graphically.

directedprotein synthesistoabout 84% of the level obtained withvirion RNA. However, this is probably nota

substan-tivedifference intranslational efficiency (6), sincethe trun-catedcoding regions of the transcripts containonly halfas manymethionines(24versus51)asintactpolyproteins.The pEAl incorporationrepresents about threetofive copiesof protein product from each transcript template (pEAl), a

significant activity consideringthe saturatingconcentrations ofRNA,andprovidesthetruebaseline forcomparisonwith othertranscripts.

Engineered deletionof thenext52 viral bases(tobase387)

gave transcript activities similar to those of pEAl and confirmedpreviousobservations thatstem-loops A to G do notcontributesignificantlytoEMCV translational function-ality (12, 36).Furthertruncations, however,whichimpinged

on structure H, showed significantdecline in incorporation (Fig. 4B). Computer-generated structural models suggest thattranscripts shortened within this region (i.e., beginning

pEAl (1131 codons)

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J n--rc: .ee et -.. 'T ..

--2IA

P'-FIG. 5. Proteinproducedin cellextractsis viral. Samplesfrom thetranslation extracts inFig.4wereprocessedwithexogenous3C proteaseandanalyzed by SDS-PAGE andautoradiography.

with bases 406to450)may assumestablepairing

configura-tionsquitedissimilartotheconsensus structure(notshown).

The predictedalterations would disrupt stem-loopI as well

as remaining H fragment, but it is also anticipated that

slightly larger deletions (i.e., to bases 451 to 500) might

restore most of the I configuration (see also reference 6).

Indeed, complete removal ofthe H domain (to base 466)

from thepBalAl-7transcript partiallyreestablished transla-tion in areproducible manner. This level ofincorporation

was alwayslower(e.g., 56 versus 84%)than thatachieved with pEA1, but clearly indicates thatintactHsequencesare not anabsolute requirementfortranslationalactivity.

Tran-scriptsbeginningwith base505,513, 546,or620werepoorly

translated in the reactions, although very shorttranscripts

with only 32 5' NTR bases had somewhat better activity.

These highly abbreviated RNAs (e.g., beginningwith base

802) have been suggested to initiate synthesis in a

5'-end-dependentmannerunrelatedtointernalribosome entrysite function (15).

The translation data presented in Fig. 4 were obtained withoptimal concentrations of added templateRNA.

How-ever, the differential levels of incorporation were clearly

dependent on template sequence, not concentration,

be-causesimilar resultswerealsoobtained undersubsaturating

RNA transcript conditions (e.g., 50 ,ug/ml), as long as

compared samples contained similar molar equivalents of

template(multiple determinations notshown).

To confirmthatprotein synthesis bythe transcript series

was uniformly initiating at the same AUG (base 834), we

processed selected product samples with exogenous,

unla-beled 3C protease and then analyzed them by SDS-PAGE (Fig. 5). Although thepatternsvaried in intensity according

to incorporation, all lanes showed bands characteristic of L-P1-2AB* sequences and there was no clear evidence for

significantlevels ofinitiationatalternate AUG triplets.Even

the shortest transcripts, whicharemissingmajor portions of

the internal ribosome entry site, seemed to direct most

residual synthesis towards the correct, in-frame AUG and

hence reinforcefindingsthat sequencesand/orstructures in the immediate vicinity of this codon probably play strong

selectiveroles in synthesis initiation(15).

pEAI (1131codons)

A 1B IC

I

1D I 2A

126B|

Relati' inco

(p

nomutation UG834)

335 ₄₁₅

Hin tillin

335 488

EcoRIlinker

ln"rfit

335 5

EcoRIlinker

insertion

335

EcoRIlinker

insertion

335 77

ive35-Met wrporation

ercent)

100

88

90

85

15

8

777 EcoRIlinker

insertion/deletlon

335

A4

701-776 2

FIG. 6. Localinsertion and deletion mutations. Mutationswere

created in pEAl through restriction enzyme digest (AvrII at base 415;HindIl atbase488;MaeIIatbases506, 577, and777;orKpnI at base 701) followed by EcoRI linker insertion, filling in with Klenowpolymerase, orsequentialdigestion (with KpnIandMaeII) and EcoRI linker insertion. T7 RNAtranscriptssynthesized from these cDNAs(1.6to1.7 jig)weretranslated inreticulocyte lysates (15 iul) as in Fig. 4, and protein synthesis was quantitated by trichloroacetic acid-insoluble radioactivity. Presented values are relative to unmutated pEAl transcript incorporation (ca. 250,000 cpm/,ul),whichwas set at100o.

The deletion experiments placed the 5' boundary for minimum translational activity within the lower regions of

stem-loop I (roughly between bases 466 and 505). Local

perturbations affectingthisregionwereexamined with

engi-neeredlinker insertions orinternaldeletions (Fig.6).

Four-base insertionsatposition415(stem H)or488(lowerstemof I)decreased translation of the downstream cistron by only

10 to12%, consistent with the idea thatanintactHelement

and the lower portion of stem-loop I are not required for translational functionality. Since deletions past base 485 gave virtually inactive templates (Fig. 4), it was surprising

that insertions creatinganextended bulge loopatbase 506

were quite effective templates; this suggests that sizable alterationsin the middle ofstem I mayalso betolerable, if

stemintegrity is generally maintained.In contrast, insertion

at position 577, which disrupts the top of this stem (from bases527 to591), strongly decreased translationalactivity. TheJ-K domain also contributestoefficient translational function. Deletions within thisregion (e.g., bases701 to776)

or insertional augmentation near the A-rich bulge loop at

base 777 significantly decreased protein synthesis (Fig. 6). The detrimental effects occurredwithout alteration of

(pre-dicted) base pairs within adjacentstems (H, I, L, M, etc.) and are consistent with data implicating this region as a required translational element and possible protein-binding site(35).

Translation competition assays. Cardiovirus RNAs have

fj,

e.-2...

http://jvi.asm.org/

[image:5.612.341.532.80.347.2] [image:5.612.102.259.81.280.2]

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Re (p Pr Fh (a Competing RNA

Fragment

(none)

335 '848

elative Incorporation lesspronounced with thefragment frombases 335 to775or ercent) of 35S-Met in whenthe competitor includeda 13-base insertion at base 777 resence of Competing RNA (within J-K). The fragment from bases 335 to 619 had almost

Template RNA no inhibitory effect, which makes it unlikely that the ob-IVRNA B-globin pEAl served results were a nonspecific consequence of added

apped) (capped) (EMCV) RNA.Therelativeinhibitoryactivityof thetested fragments was also evident at higher and lower molar excess of 100 100 100

competitor

RNAs

(e.g.,

molar ratios of

1,

2.5,

and

10)

and confirma dependence on sequence, rather than

concentra-36 12 42 tion (all data not shown).

620-848 620-797

667-797

335 619

335 775

EcoRi hiker

whieron

335

%-7

848

EcoRt hiker

335 77

1.48

FIG. 7. Translation competition scripts containing the indicated El added toreticulocyte reactions (15 excess(oratwo- tothreefold molar overthetemplate RNA. The templat (FHV) RNA (1.2 ,ug), cellular 3-glo

T7 transcripts (1.8 ,ug). Synthesizt

trichloroacetic acid-insoluble radioa tion at 30°C. The results (average ( incorporation (percent) relative to s tor RNA(typically, for FHV300,00 125,000cpm/,ul=100%,andfor pEA results are reproducible to a stand done.

been demonstrated to be effit eucaryotic mRNAs in cell-fre Regions within the EMCV 5'

proteinswereinvestigated intra RNAtranscriptscontainingbase thereofwereaddedtoreticulocy

lation-competent mRNAs from

,B-globin,orintactpEAl (Fig.7)

sequences wereomitted from th interference fromsynthesizedv assays on initial translation evc

Since the molar ratios of the

deliberately kept low (molarral

proteinsynthesisto50% of thel1

offragment wasindicative ofst

Theassaysrevealed that intac and all smaller segments contai

reasonably effective competito withinthesefragmentscenters c

predictions suggest that these inhibitory RNAs and make it lik

teringofrequiredtranslational c

ble for this effect. Both capp

3-globin)

and uncapped(pEA1)

competition, although

1-globin

tory than pEAl and could typi

molarratios offragment. With

-37 13 42

cc 'OA AR

DISCUSSION

Do Am Translation is initiated on

picornavirus

RNA by

cap-nd 44 52 independent, internal entry ofribosomes onto the

5'

NTR.

Previous analyses of EMCV bases involved in efficient 88 95 114 translation were carried out predominantly with dicistronic mRNAs (14). We have further defined the required RNA 71 72 125 sequences by deletion and insertion analysis with

monocis-tronic mRNAs. Although our results generally agree with

13

<5

20 those of Jang and Wimmer (14), they do call into question the

role of stem-loopH. These authors suggestedthat integrity 68 62 105 of H correlates with translational efficiency of the down-streamcistron in dicistronicmRNAs and also with theability

MCV 5' NTR sequences were ofa57-kDa cellular

protein

tobindtothis

specific

structure.

RI)

ina four- to sixfold molar However, their tested deletions within this region were less excesswith

P-globin

reactions) comprehensive than our pEAlBal series, and we have Le RNAs were:flockhouse virus createdatleastonetranscriptin which this entire domainis obin mRNA(0.8

Rg),

andpEAl cleanly removed(deletiontobase466) and yet the RNAstill ed protein was quantitated by translates with reasonable efficiency (70% of that ofpEA1).

ictivity after 60 min of incuba- We can onlyspeculate about the reasons for these exper-ofduplicate samples) represent imentaldiscrepancies. However, it might be considered that

camples

withnoaddedcompeti- monocistronic and dicistronic contexts could place entirely

)0

cpm25

l = 10%, for

1 T-globin

different constraints

on the

ability

of the

5'

NTR

to

initiate

lard deviation of

10%.

nd, not translation. Indeed, Jang et al. showed previously (12) that some EMCV deletion mutants are moreeffective in

mono-cistronic than dimono-cistronic contexts. Moreover, it ispossible

thatengineered constructions which only partiallyremove a

localRNAmotif have unanticipated configuration effectson

cient competitors of other adjacent structural elements. Thus, certain transcripts,

ei-e synthesis reactions (32). thermonocistronic ordicistronic, could potentially exhibit NTR that might sequester altered activities independent of the deleted sequences.

,nslation competitionassays. Computer-aided RNA folding supports this idea and sug-s335to848orsubfragments geststhat thedeletions within stem-loopH (topositions421 (te extractscontainingtrans- and 425) performed by Jang and Wimmer (14) might have

i flock house virus, rabbit disturbed the natural folding of adjacentstemI, ina manner

.Except for14bases,coding analogousto ourpEAlBal constructions which beginatbase

etestedfragmentstoreduce 406, 416, or443. Insertions specific to stem-loop H (linker iralproteinand tofocus the insertion atbase415)orremoval ofHwithout disruption of

ents rather thanelongation. I(deletion ofbase 466) allowedus to independently assess

competing segments were these motifs and to map the 5'-most required translational tio of2 to 6), depression of elementtothelowerregionsofstemIrather thanstem-loop

evelachieved in the absence H. trongcompetition.

St 5' NTRs(bases 335to848)

ining bases 667 to 797were trs. The common sequence Dn the J-Kregion. Computer

stems are conserved in all ;ely thatJ-K-specific

seques-components (35)is

responsi-ted

(flock house virus and

templateswere sensitiveto RNA was much less refrac-ically be inhibited by lower all templates, inhibitionwas

Our dataare in agreement with those of Shih etal. (36), whofound that cDNAfragmentshybridizingtonucleotides

420 to 449 caused only a slight translational inhibition of EMCVvRNA, whereas cDNAshybridizingbetween

nucle-otides 450 and 834 caused a high degree of inhibition. Correlative results were also obtained with the related

foot-and-mouth disease virus, in which the RNA

region

controlling translation appears to maintain the same basic structuralpatternasthatof EMCV(31).Thefoot-and-mouth diseasevirusanalogof EMCVstemHwasmutatedsuch that the 57-kDaprotein boundvery weakly, yet translationwas

decreasedonly50% whencomparedwith thatofunmutated transcript RNA (16, 18a, 19). This lends support to our

http://jvi.asm.org/

[image:6.612.71.289.83.312.2]

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conclusion that theintegrityofstemHdoesnot seemcrucial forproper translationalfunction.

We suggest instead that the J-K element and the ever-present stem-loop I play more critical roles in translation initiation. Independent of other 5' NTR regions, the J-K sequences (bases 667 to 797) were remarkably active in

competitive inhibitionassays. It is notable that linker inser-tions instemsIand J-K(atbases 577and777,respectively)

both destroyed translational activity but showed quite dis-tinct effects when used as inhibitors in the competition

assays. The insertion at base 577 (stem I) was an active

competitor,whereas theinsertionatbase777(stem J-K)had lost inhibitory activity. The terminal loop of stem K

(CU-UUA,bases746to750)wasrecently reportedas anefficient

bindingsite foreucaryotic initiation factor eIF-2/2B (35), a

protein complex presumed to function in initiator AUG

recognition (8). Consistentwith ourdata, astrong

competi-tiveJ-K sequence-dependent sequesteringof these required translational elements may explain the overall

efficiency

of

EMCV

fragments

at

directing

protein synthesis. Moreover,

such interactions could provide a plausible mechanism by which EMCV vRNA may outcompete host mRNAs for ribosomesorinitiationfactors(eIF-2/2B?)andeffect shutoff ofcellularprotein synthesis during infection. We are

cur-rently examining the role ofpurified eIF-2 and eIF-2B on translational

activity

and

specifically

onJ-K-dependent inhi-bitionofexogenous mRNA translation.

ACKNOWLEDGMENTS

We thank DingShih for critical readingof the manuscript and CornelisPleijfor corroborative dataconcerningpseudoknot struc-turesBandC.

This workwassupportedbyPublic HealthService grant AI-17331 from the NIH.

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