Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

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0022-538X/87/062033-05$02.00/0

Society

for Microbiology

Effects of cDNA Hybridization

on

Translation of

Encephalomyocarditis Virus RNAt

DING S. SHIH,1* IN-WOO PARK,' CHRISTOPHER L. EVANS,' JESSE M. JAYNES,'

ANDANN C. PALMENBERG2

Departmentof Biochemistry andLouisiana Agricultural Experiment Station, Louisiana State UniversityAgricultural Center, Louisiana State University, Baton Rouge, Louisiana 70803,1andBiophysics Laboratory, University ofWisconsin,

Madison, Wisconsin 537062

Received 28 October 1986/Accepted 16 February 1987

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragmentsto different sites ofthe 5' noncoding region of the viral RNA. The following resultswereobtained. (i) The bindingof cDNA fragmentstothefirst41 nucleotides,tothepoly(C) tract(between nucleotides 149and263), and tothe sequencebetween nucleotides 309 and 338 didnotaffect translation of the viralRNA; (ii) the binding of cDNAfragmentstothesequencebetween nucleotides 420 and 449causedaslight inhibition;and(iii)the bindingof fragmentstoeight differentsites between nucleotides 450 and theinitiator AUG codon (nucleotide 834)causedhighdegreesofinhibition. The resultssuggestthat thefirst partof the 5' untranslated region, atleasttonucleotide 338, maynotbe requiredfor encephalomyocarditis viral RNA translation; however, the regionnearnucleotide 450 is important for translation of the viral RNA. The possibility that initiationoccursataninternal site is discussed.

Encephalomyocarditis (EMC) virus is a member of the

picornavirus family. Muchis known about the protein

syn-thesispathways ofpicornavirusesand about the products of

thesynthesisreactions. However, an important aspect of the

viral protein synthesis process, the initiation of translation,

isstillunclear at present.

For most eucaryotic mRNAs, the ribosome-scanning model has been accepted to explain the translational

initia-tion process (7, 8). In this model, 40S ribosomal subunits

first bindtothe 5' end of a messenger and then migrate along

theRNAchainuntil they encounter the first favorable AUG

triplet, at whichpoint translation begins. An essential

fea-tureof this scanningmodelisthat the 40S subunitsrecognize

andbindonlytothe 5' endsof themRNAmolecules before

thestartof thescanningprocess.The 5' cap structure,which

is present in most eucaryotic mRNAs, is known to be

involved in the ribosome-binding reaction (forareview, see

reference3).

Unlike most eucaryotic mRNAs, picornaviral RNAs do

nothavea5' nucleotidecap structure. Instead, they havea

covalently linked protein (VPg) at their 5' ends (26). This

genome-linked protein is not required for translation (18).

Therefore, during initiation of picornaviralRNAtranslation,

it is possible that an alternate ribosome recognition and

binding mechanism is involved. Forexample, theribosome

subunits mayrecognizeand initially bindto aninternal site

ofthe viralRNAbeforethe scanningprocessbegins.

We have investigated this possibility by usingEMC virus

as amodel. EMC viralRNAisaparticularlyactive

messen-gerinseveralcell-freeprotein-synthesizing systems(16, 20,

24). In rabbit reticulocyte lysates, the viral RNA is

com-pletely and efficiently translated, and the translation

prod-ucts are accurately processedtoform matureviralproteins

(16, 20, 21,24). Recently,thecomplete nucleotidesequence

of EMC virus was determined (14, 25; A. C.

Palmenberg

*Corresponding author.

tPaper 86-12-0184 of the Louisiana

Agricultural

Experiment

Station, LouisianaState University,BatonRouge.

and G. M. Duke, manuscript in preparation). The RNA is 7,835 nucleotides long, and the translation initiated AUG

codon is located at nucleotide 834 (14). This codon is

followed by an open reading frame of 6,876 nucleotides,

which encodes the viral polyprotein. The initiator AUG is

precededby 10otherAUGtriplets scattered throughout the

5' noncoding region. The present study was designed to

investigatethemechanism by which ribosomes initiate

trans-lationatthe particularAUG triplet in position 834.

Oligodeoxyribonucleotides were synthesized on an

Ap-plied Biosystems380A DNA synthesizer. After completion

ofthesynthesizing cycles, theoligonucleotide solution from

the synthesizer was incubated with 1 ml of concentrated

NH40H for 4 to 12 h at 50°C to completely remove the

blockinggroups. The solution was thenevaporatedto

dry-nessinarotary evaporator.Thedry residuewasdissolvedin 1 ml of 25 mM triethylammonium bicarbonate (TEAB), pH 7.6,and the solutionwasevaporated. ThisTEABtreatment

stepwasrepeatedtwice. The residuewasdissolvedin 3 ml

of TEAB, and the oligonucleotide fragments were isolated

by using a Sep-pak cartridge (11), whichserved to remove

prematurely terminated sequencesfromthecomplete

oligo-nucleotide chains. The oligonucleotide fractioneluted from

the cartridge was dried, and the 5'-protecting groups were

removedby dissolvingthenucleotidesin1 mlof80% acetic

acidandincubatingthe solutionat roomtemperaturefor25

min. Afterevaporation, the sample was dissolved inwater and dried. Thefully unprotected productwasfurther

purified

by fractionation on a 20%

polyacrylamide gel

under

dena-turing conditions. The conditions forelectrophoresisandfor

oligonucleotide

isolation fromthe

gel

have been described

elsewhere(11).

All hybridizations weredone in reaction mixtures

(8 ,ul)

containing20 mMHEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid)

(pH 7.6)-0.1 M NaCl-1 mM EDTA.

The RNA concentration was 0.5

mg/ml,

and cDNA was added inafivefold molarexcess.Thereaction mixtureswere heatedat

70°C

for10minfollowed

by

incubationat

45°C

for 2033

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Oigo(dG)

z2w

1170011

(Fig. 2A)wasthe result ofhybridizationtotheA10andA12

oligonucleotidefragments.

The effectsontranslation ofhybridizationofseven other

fragments(A4 through A9andAll) areshown(Fig. 2C,2D,

[image:2.612.66.280.69.271.2]

and3). The data indicate that fragments A4 through A7 and All caused more than 80% inhibition of translation; frag-ment A8 caused 65% inhibition, and fragment A9 caused 75% inhibition.

Figure 3depictsamino acid incorporation data for A4- and A3-hybridized RNA. As described above, hybridization of A4 caused a strong inhibition of translation. On the other hand, hybridization ofA3, which correspondstotheregion

immediately 5' tothe A4-hybridized region, caused only a low level of inhibition. The translational activity of the A3-hybridized viral RNA was about 70% of that of the control(unhybridized) RNA.

Itwaspossible that the lowdegree of inhibition observed with A3 wasdueto weak orincomplete hybridization with viral RNA. However, this possibility was ruled out by primer extension experiments. Theelectrophoresis patterns FIG. 1. 5'-Untranslatedregion of EMC viral RNA.Symbols:-,

5'-noncoding sequence; _, location of initiator AUG codon; , locations of other AUG triplets; -_ , positions of termination codons;L.J, cDNA fragments. For the indicated fragments, the lengths and theexactnucleotide locations(given inparentheses) of thesites of hybridization were:Al,41(1to41);A2,30(309to338); A3, 30 (420to449); A4,30(450to479);AS,30(480,to509);A6,30 (510to539);A7,30(590to619); A8,33(667to699); A9,34(710to 743); A10, 40 (744to783);All, 30 (784to813); A12, 41(814to854).

3 h.Nonhybridized control samplesweretreatedidentically,

exceptthat cDNA was omitted from the reactionmixtures.

Thehybridization procedure described abovewas

devel-oped by combining the procedures of Hastie and Held (5)

and Liebhaber et al. (10). The first method (5) involved

heating mRNA and single-stranded cDNA in an aqueous

solutionat65°Cfor30min. The second method(10) involved

heating mRNA anddouble-stranded DNA in a

formamide-containing buffer for10min at70°Cfollowedby incubation

at 45°C for 3 h. Each ofthe methods produces complete

hybridization.

Translation ofthehybridizedRNAsampleswasexamined

by using the mRNA-dependent rabbit reticulocyte system

(17). Reticulocyte lysates were obtained from Green

Hect-are; Oregon, Wis. The conditions fortranslationhave been

describedpreviously (20, 21).

Figure 1depicts schematically the5' region of EMCviral

RNA and the locations of cDNA hybridization sites. The

cDNAfragments, designatedAl through A12, are 30 to 41

nucleotideslong, and their positionsare indicated.

Translation of cDNA-mRNA hybrids. EMC viral RNA was testedforits abilitytodirect translation afterhybridization

to various cDNA fragments. The effects with two of the

fragments, A10 and A12, are shown (Fig. 2A). A12

hybrid-izes to the genomic region encompassing the polyprotein

initiation codon, including 18 upstreamand 20downstream

nucleotides.A10 hybridizes to a region that is 30 nucleotides

5' tothe A12-hybridized region. The data indicate thatA10

andA12caused 93 and77%inhibition of normalEMC viral

polyprotein

translation, respectively.The results of a control

experiment in which theA12-mRNA complex was

dissoci-atedby heatingat97°Cbefore translation are given (Fig. 2B).

Heating ofthe complexeliminated most of the inhibition. We

therefore concludethatthe decrease in translation observed

0

15 30 45 60 0 IS 30 45 60

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6-6-

4-x~~~~

0 153045 60 0 1530 45 60

Time (Min)

FIG. 2. InhibitorycDNAfragments.Theconditions for hybrid-ization and for translationwere asdescribed in thetext.The control RNAsampleswereheated andincubated in thesame manner asthe hybridizedRNAsamples,except that cDNAfragmentswere omit-ted.(A)Effectofhybridization ofA10andA12.Symbols:0,minus RNAcontrol;A, A10;x,A12;0, minuscDNA control. (B) Effect ofdehybridization of theannealedcomplexes ofA12andEMC viral RNA. The hybridization solution containing the annealed com-plexes wasdivided intotwoaliquots. Onewastranslated directly, andtheotherwasheatedat97°C for3min,quickly frozenin adry ice-ethanolbath, thawed,and translated.Symbols: 0,minusRNA control; A, A12-mRNAcomplexes; x,dehybridized mixture; 0, minuscDNAcontrol.(C) Effects of A8, A9, andAll. Symbols:0, minus RNA control; O, All; A, A9; x, A8; 0, minus cDNA control.(D) Effects of AStoA7.Symbols:0,minusRNAcontrol; O, AS; A, A6; X, A7;0,minus cDNA control.

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A4.A5

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ofprimer extension products synthesized from RNA tem-plates primed with A3 (nucleotides 420 to 449) or with All (nucleotides 784 to 813) are shown (Fig. 4). The formation of

only one major product was seen in each case. The chain

length was approximately 350 nucleotides for the major

A3-extension product and 750nucleotidesfor themajor All

extensionproduct. Densitometertracings (notshown) of the

gelprofiles confirmed that equal molar amounts of product

DNAweresynthesizedinreactions with the two primers (A3

andAll),indicatingthat the extent ofhybridizationof A3 to RNA wassimilar to that of All. Therefore, although the two

cDNAs differed significantly in the ability to inhibit EMC

viral polyprotein translation, these properties were not

causedby differences in hybridization efficiencies.

Thehybridization of Al,which corresponds to the first 41

nucleotides,causedno detectable inhibition of translation of

the viral polyprotein (Fig. SA). The same result was also

obtained when the hybridization reaction was done by

heating the solution to a much higher temperature (97°C)

beforetheannealing step (Fig.SB). Similarly, the

hybridiza-tion of oligo(dG) or of A2, which corresponds to the

se-quence from nucleotides 309 to 338, had no effect on

translation (Fig. 5C andD).

The translation data presented above (Fig. 2, 3, and 5)

demonstrated asequential transition ofthe effect of cDNA

hybridization fromstronglyinhibitorytopartiallyinhibitory

to noninhibitory. The area of viralRNA thatis critical for

efficienttranslation seemedto centeraround nucleotide450,

which is the 5'-most nucleotide within a region, to which

cDNA fragmentA4 washybridized, leadingto strong

inhi-bition.

In addition to the hybridization-translation studies

de-scribed above, weexamined translation of EMC viral RNA

after the deletion of specific segments of the 5' noncoding

region byRNase Hdigestionof cDNA-mRNA hybrids. We

found that deletion of the 5' sequence to approximately

nucleotide 338 caused no discernible effect on translation,

but deletion of the sequence extending beyond nucleotide

450diminished the translation activity (datanotshown).

Analysis of translation products. The protein products

synthesized by various hybridized RNA sampleswere

ana-lyzedby sodium dodecyl sulfate-polyacrylamidegel

electro-phoresis. Figure 6, lanes a, b, and c, represents 45-min

translation reactions directed by A2-, oligo(dG)-, and

Al- 5-0

4-3~~~~~~~~

0L

o

2

0 15 30 45 60

Time

(Min)

FIG. 3. Activities ofviralRNAhybridized toA3 orA4. Sym-bols: 0, minus cDNA control; x, A3; A, A4;

0,

minus RNA control.

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163

D ;- W:

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FIG. 4. Gel analysis of the primer extension products from A3-and All-hybridized primers. A3 and All were hybridized sepa-rately to the viral RNA. Each hybridized sample (8 ,ug of RNA) was precipitated with ethanol and dried under vacuum. The residue was dissolved in 15 ,ul of water and added to a reverse transcription reaction mixture. The transcription reaction was done by the methodof Ahlquist et al. (1), based on the original procedures of Landet al. (9)and Kacian and Myers (6). The cDNA products were analyzed on2% alkaline agarose gels bytheprocedure of McDon-nellet al.(13).Lanes: a,products from A3-hybridized template;b, products from All-hybridized template; c, radioactively labeled standards. Thepositions of stained molecular weight markers are shownatthe left oflaneaandattheright of lanec.

hybridized RNA, respectively. Also shown are the products from intact virion RNA (Fig. 6, lane h). The protein bands

(Fig. 6, in lanes a, b, c, and h) were indistinguishable in

position, although there was some variation in band

inten-sity. This variation could beattributed tothe fact that the

translation of the different RNA samples was done at dif-ferent times. Variations in the conditions oflysate prepara-tion and in thecomponents of theprotein synthesisreaction mixture couldcertainlyhavecauseddifferencesin the rates of translation andprocessing,which woulddirectlyaffect the product pattern.

Theproducts from RNA hybridized to A8, A10 plus A12, A10, and A12 are shown(Fig. 6, lanes dthrough g, respec-tively). In agreement with the amino acid incorporation data (Fig. 2 and 3), much less of the viral proteinwasproduced in thesesampleswhencomparedwiththe controlsample.

Our data demonstrated thatbinding of cDNA fragmentsto

the beginning portion of EMC viralRNA (5' to nucleotide

338) hadnodetectableeffectonpolyprotein translation, but binding of cDNA fragments to sequences 3' to nucleotide

450 severely inhibited translation. Furthermore, we found

that removal of the first 338 nucleotides by RNase H

digestionhad nodiscernible effectonsubsequenttranslation

activity of the remaining RNA fragment, while removal of

nucleotide sequencesextending beyond nucleotide 450

vir-tuallyabolished the messengeractivity.

Our resultsare incomplete agreementwith experimental data from other studies.The Lfragment generated byRNase Hdigestion ofoligo(dG)-hybridizedfoot-and-mouthdisease virus RNA serves as very active mRNA in vitro and pro-duces the same

products

as

full-length

virion RNA (5).

Deletion and recombination experiments with cloned

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0X 2- =

0 15 30 45 60 0 15 30 45 60

FIG5. DoihbtrcDAfamns0Afeto 1y

Th Cyrdzto 0ecinmxuewshaet9° nta fa

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3-

2-0 15 30 4560 01530 45 60

Time

(min)

FIG. 5. NoninhibitorycDNA fragments. (A) Effect ofAl. Hy-bridization was doneunder standard conditions. (B) Effect ofAl. Thehybridizationreaction mixturewasheatedat970C instead ofat thenormal 70°C. (C) Effect of oligo(dG). The oligonucleotide was added at afivefold molarexcess of the entire poly(C) region (115 nucleotides). (D) effect ofA2. Symbols: 0,minus RNAcontrol; x, hybridized samples; 0,minuscDNAcontrol.

cDNAs have shown that the translational properties of transcribed EMC viral mRNAs reside exclusively in the 5'

noncoding portion ofthe viral genome, specifically in the

segmentextending 3'from(approximately)nucleotide 460 to

.L-1-2A

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I-2A

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a

b

c

d

e

f

g

h

FIG. 6. Sodium dodecyl sulfate-polyacrylamide gel electropho-resispatternsof translationproducts from hybridizedRNA samples. Allsamples werewithdrawnafter 45 min of translation. Lanes: a

throughg,productssynthesized from viralRNAhybridizedtoA2, oligo(dG),Al, A8, A10 plus A12, A10, and A12, respectively; h, products from a control RNA sample. The viral proteins are

indicatedattherightof lane h.

theAUG codon thatbeginsthepolyprotein. The 5'-most 460 nucleotides, the intactpolyprotein reading frame,and the3' noncoding regions are not necessary for efficient mRNA

translation (15; Palmenbergetal., inpreparation).

Thus itis reasonabletoconclude thattheinitialsequence (about 400 bases) of theEMC viralRNAisnotnecessaryfor

translation; however, the region near nucleotide 450 is

important. On thebasis ofthisconclusion,wepropose that

for the initiation of EMC viral RNA translation the 40S ribosomal subunits may first bind at asite near orincluding

nucleotide 450 and that from this site the subunits migrate

toward the initiator AUG siteand beginprotein synthesis.

If our internalinitiation hypothesisis correct, an obvious questioniswhich factors are responsible for ribosome

rec-ognition and binding? The Shine-Dalgarno sequence is

thought to act as a ribosome-binding site in procaryotic

mRNAs (22, 23). This conserved, purine-rich sequence is

complementary to the 3' end of bacterial 16SrRNA. Since

the 3' ends of eucaryotic 18S rRNAs also contain a

con-served sequence (2, 4) which is very similar to the 3' sequence ofthe 16S rRNA, itisreasonable tosuggest that the3' endof18S rRNA maybe involvedin theinitiation of

translation ofsome eucaryotic mRNAs. In fact, the

hypo-thetical involvement of 18S rRNA in translation initiation

has already been proposed (2, 4, 19). For picornavirus

RNAs, the possibility of mRNA-rRNA interaction is

particularly relevant because of the lack of the 5' cap

structure ofthe viral RNA. Examination ofthe EMC viral RNA sequence reveals a partial complementarity

(GAAGCUUCUUGAAG [underscores indicate a

potential

basepairing])existingbetween EMC viralnucleotides483to

496 andthe 3' endof rabbit reticulocyte 18S rRNA (2,

4).

This limited

complementarity

may not be sufficient for

effective ribosome

binding.

However,the

possibility

of other

sequence

complementation

between the 5'

noncoding

re-gions of EMC virus and different parts of the 18S rRNA

cannotbe ruled out. Inthis

regard,

itis

interesting

to note that arecentstudyshowed thatpoliovirusRNA can

hybrid-ize(understringentconditions)to aspecific region ofthe18S

rRNA as well as to several regions of28S rRNAof

higher

eucaryotes (12).

The studies presented here provide new insight into the

mechanisms of translation of EMC viral RNA and suggest

avenues for future experimentation. In light of the high

translational efficiency of this particular mRNA, we hope

that clarification of the translational mechanism may serve

as amodel forotherexperimentalsystems.

We thank Simon Chang for assistance in the synthesis of the cDNAfragments.

This workwassupported in part by the Agency forInternational Development, the U.S. Department of Agriculture Competitive Research Grants program, and theLouisianaAgricultural Experi-mentStation.

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