Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

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Vol. 56, No. 3 JOURNALOFVIROLOGY,Dec. 1985,p. 683-690

0022-538X/85/120683-08$02.00/0

Characterization of

Ribosome

Binding

on

Rous Sarcoma Virus

RNA

In

Vitro

ROBERTB. PETERSEN ANDPERRY B. HACKETT*

Department of GeneticsandCellBiology, University of Minnesota, St. Paul, Minnesota 55108 Received 15April 1985/Accepted 23 July 1985

Wedetermined the sitesatwhich ribosomes form initiationcomplexesonRoussarcomavirusRNA inorder todeterminehow initiation ofPr769'9 synthesisatthe fourth AUG codon from the 5' end of Roussarcomavirus strain SR-A RNAoccurs. Ribosomes bind almostexclusivelyatthe5'-proximal AUG codon when chlorideis presentasthe major anionadded to the translational system.However,when chloride isreplacedwith acetate,

ribosomes bindatthe two5'-proximal AUGcodons,aswellasatthe initiation site forpFr76ga.We confirmed that the 5'-proximal AUG codon is part ofa functional initiation site by identifying the seven-amino acid peptideencoded there. Our results suggest that(i)translation in vitro of RoussarcomavirusvirionRNAresults inthesynthesisof at least twopolypeptides; (ii) the pattern of ribosomebindingobserved for Roussarcoma

virus RNAcanbe accounted for bythe modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunitsor80S ribosomalcomplexesis strongeratthe5'-proximalAUGcodon than at sites farther

downstream, includingthe initiation site for themajorviralproteins.

The factors determining the site(s) at which ribosomes

initiate protein synthesis on eucaryotic mRNAs have not

beenfully elucidated. Although theoriginal scanning

hypo-thesis(15) explained initiation site selectiononthemajority

ofeucaryotic mRNAs, asignificant number ofmRNAs that

did notconformto this model remained. These exceptions,

inwhich proteinsynthesis is initiated downstreamfromthe

5'-proximal AUG codon, include poliovirus (8), Rous

sar-coma virus (RSV) (26), and mouse

ac-amylase

(11), which

initiate protein synthesis at the ninth, fourth, and second

AUGcodons fromthe 5' ends oftheirRNAs, respectively.

Such exceptions led to the formulation of the modified

scanning hypothesis,which rankspotentialinitiation sitesby

the strengthofthe sequencesflankingtheAUG codon (16).

The modified scanning hypothesis explains initiation of

protein synthesis at sites downstream from 5'-proximal

AUG codons by postulating that some 40S ribosomal

subunitscanbypass weakupstreaminitiation sitestoinitiate

protein synthesis atthefunctional AUG codon.

Ribosome-binding studies with RSV RNA have failed to

detectribosome bindingatthe AUGcodonknown to initiate

synthesis of thegag geneproduct, Pr76gag; rather,binding of

ribosomesunderconditionsprecludingpeptide bond

forma-tionhasbeendetected onlywithin the5'-proximal100bases

of RSV RNA with all of the strains tested (4, 23). This

observation ledtotheideathatspatial scanningmay occur,

inwhich40Sribosomalsubunits that bind at the 5' end of the

RNA can interact with downstream sequences via the

sec-ondary structure within the RNA molecule (5, 23). This

would allow the 40S ribosomal subunit to bypass upstream

AUG codons.

Anotherexplanation for initiation of protein synthesis at

downstreamAUG codons isthat acomplete 80S ribosome or its 40S subunit can reinitiate protein synthesis at

down-stream AUG codons (18, 20) after initiation of protein

synthesisat a5'-proximal AUG codon and synthesis of the

encoded peptide. Inthismodel,asin the modified scanning

hypothesis, sequencesflankingAUG codons may modulate

thelevel of initiation at agiven AUG codon.

*Corresponding author.

In apreviousreport (23)weidentifiedaribosome-binding

siteatthe5'-proximalAUG codononRSV RNA rather than

atthe initiationcodonforPr769'9. This resultwas obtained

by usinga rabbitreticulocyte translational system in which

chloridewastheprincipalanion added.However,the results

ofother studies suggested that initiation by 40S ribosomal subunits is sensitive to chloride and that replacement of

chloride withacetate results inenhanced initiationcomplex

formation (27). Furthermore, chloride differentially affects

initiation ofprotein synthesis on somemRNAs; e.g.,

trans-lation ofa-globinmRNAis more sensitiveto chloride than

translation of ,-globin mRNA is (6). We have found that

replacement of chloride with acetate as the major anion

added to the translational system directed by RSV RNA

results in(i) increased synthesis ofPr769ag and(ii) ribosome

bindingatthefirst, second,andfourthAUGcodonsfromthe

5'endof RSVRNArather thanatonlythe5'-proximal AUG

codon. In addition, the seven-amino acid peptide encoded

behind the5'-proximal AUG codonis synthesizedin

trans-lations ofRSV RNA,as predicted by theribosome-binding

studies.

MATERIALS ANDMETHODS

The RNase Ti used for digestion of ribosome initiation

complexes was obtained from Calbiochem-Behring.

Se-quencing grade RNase

Ti,

dinucleotide cap m7G5'ppp5'A,

and T4 polynucleotide kinase were obtained from P-L

Biochemicals, Inc. Leupeptin was obtained from Vega

Biochemicals. RSV virion RNA was isolated from chicken

embryo fibroblasts infected with RSV strain SR-A as

previously described (10). Anisomycin was obtained from

Sigma

Chemical

Co.,

and sparsomycin was agenerous gift

from Marilyn Kozak and Matthew Suffness of the National Cancer Institute.

[-y-32P]ATP

was obtained from ICN Pharmaceuticals Inc., and

[5'-32P]pCp

was obtained from

NewEngland NuclearCorp. High-pressure liquid

chroma-tography (HPLC) grade acetonitrile, trifluoroacetic acid

(TFA), and methanol were obtained from PierceChemical

Co. n-Butanol was from Eastman-Kodak Co. The C18

Syncropak column resin used wasfrom Synspec, Inc. The

seven-amino acidmarkerpeptidewaspreparedbyFernando

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684 PETERSEN AND HACKETT

B

1

2

3

4 Pr180

Pr76

.. ..,. P,.

i :.

::: :'::

["'.

6 _.

._.

,.

X

1

.

::::i

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... ... i .:

o.r

.

F :

i

_._.

_

W

4 _

_r

i _;

wF

...

FIG. 1. Translation in the presence of eitheracetate orchloride

salts. (A) Translation of RNA isolated from RSV-infected chicken

embryofibroblasts. Lanes 1 and 2, Translation in the presence of

chloride salts with(lane 1)and without(lane 2)addedRNA;lanes 3

and4, translation in the presence ofacetate salts with(lane 3) and

without (lane 4)added RNA. (B)Translation of RSV virion RNA.

Lanes 1 and2,Translation in thepresenceofacetatesalts with(lane

1)and without(lane 2)addedRNA;lanes 3 and4,translation in the

presence ofchloride salts with (lane 3)and without (lane 4) added

RNA.Themajorvirion RNA-encodedprotein products,Pr8ga-( and ]?6(~ are indicated.

Albreccio and Sam Gunderson(Hackett etal., submittedfor

publication). The RSV DNA clones used, pSRA5' and

pSRA5' subclones through 6, have been described

previously (23).

Translation in vitro. The rabbit reticulocyte translation

system usedwasessentiallythe systemdescribed byPelham andJackson (22), with some modifications (3) (in addition,

noexogenous amino acids wereadded tothe system). RSV

RNA was added to a final concentration of 16 to 33 p.Lg/ml after heat denaturation for 2min at80'C in 0.2mM EDTA. In some experiments potassium acetate and magnesium

acetate replaced the chloride salts at finalconcentrations of

135 mM and 135 p.M, respectively. The experiments in whichleupeptinwasusedaredescribed in thefigure legends.

Forinhibition studies thecapanalog m7G5'ppp5'Awasused

at afinal concentration of 0.8 mM. Products of the in vitro translation reactions were separated on9% polyacrylamide gels with 3% stacking gels by using the procedure of Laemmli (19).

Isolation and analysis of ribosome-protected RNA

frag-meats. Isolation and analysis of the ribosome-protected

RNA fragments were performed essentially as described

previously (23), with the modifications described below. RNase Ti from Calbiochem-Behring was used to digest

ribosome initiation complexes formed in the presence of 3

mM anisomycin or 0.2 mM sparsomycin. Because of the difference in the unit definitions of the manufacturers, using RNase Ti from Calbiochem-Behring resulted in approxi-mately 60-fold-greater digestion than using RNase Ti from Boehringer Mannheim Biochemicals, as demonstrated by the smaller sizes of the oligoribonucleotides recovered after digestion. In addition to using RNA fragments labeled at their 5' ends, RNA fragments were labeled at their 3' ends with 32P after removal of the 3' phosphate left by RNase

digestionwithcalf intestinal phosphatase (CIP), as described

by England and Uhlenbeck (9), except that glycerol was omitted fromthereaction mixture (7). The hybridization of 32P-labeled, ribosome-protected RNA to cloned RSV DNAs was performed at 34°C, and elution was accomplished by heating the preparation twice at 80°C for 10 min in 0.2 mM EDTA. The eluted RNAs were suspended in a solution containing 7Murea, 20mM sodium citrate (pH 5.0), 1 mM EDTA, 0.05% (wt/vol) bromphenol blue, 0.05% (wt/vol) and xylene cyanol and heated for 10 min at 50°C before loading

onto a 16.5% polyacrylamide gel. The gel buffer used, elution ofspecific RNA fragments, and analysis by partial RNaseTi digestion have been described previously (23).

HPLC analysis of translational products. RSV RNA or water wasadded to atranslation mixture containing acetate salts (see above) and 50

[Lg

of leupeptin per ml. After incubation for 4 min at 29°C, a portion of the translation mixture wasremoved and added to a 0.5-ml microcentrifuge tubecontaininganequal volume of methanol. After vortex-ing for 15 s, a volume of n-butanol equal to the volume of

methanol wasadded, and the mixture was vortexed for 15 s.

Morethan99% of theproteinsinthelysate were precipitated

by cooling at -80°C for ca. 5 min. The supernatant was

cleared of the precipitated proteins by centrifugation for 5 min in a0.5-ml microcentrifuge (Fisher Scientific Co.) and transferredto afreshmicrocentrifuge tube. Then 20 nmol of

a synthetic marker peptide,

H-Met-Ala-Gly-Pro-Leu-Ile-Pro-OH (Hackett et al., submitted), was added, and the samplesweredried inaSpeed Vac concentrator. Each dried sample was suspended in 30

plA

of water, and 20 pJ was

applied to a C18 column and eluted on a linear 0 to 30%

acetonitrile gradient in 0.1% TFA by using a Beckman

HPLC apparatus. The HPLC run conditions used were as

follows: 0.1% TFA 5 min; a gradient from 0 to 30%

acetonitrile in0.1% TFA for 40 min; and 30% acetonitrile in

0.1%TFA for 5 min. The flow rate was 1 ml/min, and the

absorbancewasmonitored at 210nmwithan LKBUvicord

II detector. [35S]methionine incorporation was detectedby

collecting 1-ml fractions directly into minivials and adding

Tritosol, followed by counting with a Beckman model

LS-100C scintillationcounter.

RESULTS

Translation of cellular mRNA in rabbit reticulocyte

ly-sates is usually performed by using chloride as the major

monovalent anion. Indeed, chloride is more efficient at

promoting translation of intracellular mRNA isolated from

RSV-infected chicken embryo fibroblasts than acetate is

(Fig. 1A). However, since we were unable to detect

ribo-somebindingattheinitiationsite forPr%6ga onRSVRNA,

we replaced chloride with acetate in the translational

sys-tem.The useofacetate saltsinsteadof chloridesalts in the translational system resulted in enhanced translation of RSV

RNA(Fig. 1B); the increasewas threefold, asmeasured

by

the incorporation of

[35S]methionine

into trichloroacetic

acid-insoluble protein.

A

1

2

3

4

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CHARACTERIZATION OF RIBOSOME BINDING ON RSV RNA

The increased synthesis of viral proteins when acetate

saltswere used in the translational system could have been dueto an increase in ribosome

binding (i)

at all ribosome-binding sites or(ii) at specific

ribosome-binding

sites.

Con-sequently, weexamined thesites of ribosomebindingunder

the new salt conditions by using anisomycin to inhibit

peptide

bondformation inordertodetermine whether there

wasdetectablebindingattheinitiationsitefor

Pr76gag,

AUG

4 (Fig. 2A). RNA fragments which had been isolated from

RNase

T1-digested

monosomes werelabeledattheir 5' ends

with 32P and hybridized to pSRA5' subclones 1 through 6

(Fig.

2A) (23). After

elution, the RSV-specific

RNA

frag-ments wereseparatedon a16.5%

denaturing polyacrylamide

gel (Fig.

2B). The RSV RNA

fragments ranged

in size from

26bases (band E) to 35 bases

(band

D). In contrast to our

previous results, which demonstrated ribosomebinding only

at AUG 1, replacement of chloride with acetate resulted in

ribosome

bindirng

at AUG 1 (bands A, C, and D), AUG 2

(band

B), and AUG 4 (bands E, F, and G). There was no

detectable

binding

to AUG 3; the RNA

fragments

that

hybridized to

pSRA5'

subclone 3 bound to the region

up-stream from AUG3, as

reported

previously

(23). As shown

in

Fig. 2C,

an AUG codon is

located

at the 5' end of the

seven

major ribosome-protected

RNA

fragments.

This

ex-plains

the observation that the RNA

fragments generated by

protection

at AUG 1

hybridized

to pSRA5' subclone 2,

which contains RSV DNA sequences starting two bases'

downstream from AUG 1. A

comparison

ofthe patternsof

ribosome

binding

obtained with chloride andacetate in the

translational system suggested that initiation complex

for-mation at AUG 4 is more sensitive to the anion used than

initiation complex formationatAUG 1 is.

We repeated the ribosome protection studies by usinga

different inhibitor of protein synthesis, sparsomycin, to

establishthatthenewribosome-bindingpatternwas notdue

to the inhibitor but rather to the anion in the translational

system. The patterns of ribosome protection when

sparsomycin

was used in the presenceof either chloride or acetate salts are shown in Fig. 3. The presence ofchloride

salts (Fig. 3A) nearly abolished the formation of initiation

complexes

downstream from AUG 1 (Fig. 3C). The RNA

fragments generated by ribosome bindingatAUG4couldbe

seen

only

in long exposures of the gels. In contrast, when

acetatesaltswereusedin the translational system(Fig. 3B),

easily

identifiable initiation complexeswereformedatAUGs

2and 4 inadditiontoAUG 1(Fig.3D),as wasthecasewhen

anisomycin

was used asthe inhibitorofprotein synthesis.

When acetate was used in the translational system with

either

anisomycin (Fig.

2) or sparsomycin (Fig. 3),ca. 25%

of the

ribosome-protected

RNA

fragments

hybridized tothe

region

aroundAUG4.Themajor differencein the pattern of

ribosome-protected fragments

thatresulted fromthe use of

sparsomycin

was the intense 55-base RSV RNA fragment

that

hybridized

to pSRA5' subclone 1 (Fig. 3A andB, lane

1);therewasnocomparable protectionof thisregion ofRSV RNAwhen anisomycin was used (Fig. 2B, lane 1, arrow).

However,

the procedure which we usedto label the RNA

fragmentswouldnothave labeled thosefragmentsthatwere

terminatedbya5' cap structure.

Todetermine whether any 5'cappedRSV RNAfragments

were protected by ribosomes, we labeled RNA fragments

from the samples used in the experiments shown in Fig. 2 and 3 with 32P at their 3' ends. The 3'-terminal phosphate groups on the RNA fragments generated by RNase

Ti

digestion

were removed with CIP before labeling. To

facili-tate comparison with our previous results and to

demon-strate that the phosphatase reaction had not caused degra-dation of the RNA, phosphatase-treated and untreated RNAs were labeled at their 5' ends as in our previous experiments. The 32P-labeled RNA fragments were hybrid-ized to pSRA5' subclone 1, and the RSV RNA fragments were analyzed on 16.5% polyacrylamide gels after elution

(Fig.4). Figure4shows theproducts of 5' labeling reactions

performed with untreated (lane 1) and CIP-treated (lane 2) RNAfragments. The patterns of labeling of the fragments in the lanes were not changed, indicating that the CIP

treat-mentdid notdegradethe RNAs. Lane 3 shows theproducts of the 3' labeling reaction. Note that the 3' labeling resulted in band shifts due to the addition of one base to the 3'

termini; 5' labelingonly added a phosphate groupto the 5'

termini.Acomparisonof the RSV RNAfragments in lanes 4

and5, in which fragmentswerelabeledattheir 5' ends, with thefragments in lane 6, in which fragments were labeled at

their3'ends,showed that whenanisomycinwasused in the

translational system, a 3'-end-labeled capped RSV RNA

fragment of 25 bases was protected (lane 6, arrow). This

fragmentcontainednoAUG codons andmostlikelywasthe result of a 40S ribosomal subunit stalled at the cap. In contrast, lanes 8 through 10 show a 55-base oligoribo-nucleotide which could be labeled atboth its 5' (lanes 8 and 9)and 3'(lane10)ends, These resultsdemonstratethat there

were no detectable capped RSV RNAfragments protected byribosomes whensparsomycinwasusedastheinhibitorof

protein synthesis. It is interesting that the capped RNA

fragment recovered from reactions containing anisomycin

(lane 6) and the 55-base RNA fragment recovered when

sparsomycin was used (lane 7) were present in

stoi-chiometric amounts. In both cases these protected RNA

fragments probably resulted from preinitiation complexes.

Thus, although

therewere

readily

apparentdifferences inthe

exact sites ofprotectionwhen the differentinhibitors were

used,

the overall distribution of ribosomes was essentially

thesame.

Synthesis of PrW6ga is sensitive toinhibitionby avariety

of capanalogsinreticulocyte lysates(2, 4; Petersen,

unpub-lished data). Therefore, to demonstrate that the ribosome

binding which we observed was the result ofa bona fide

initiation event, we repeated the ribosome-binding experi-mentsby using sparsomycin to inhibitpeptidebond forma-tion in the presence or absence of the dinucleotide cap

analog m7G5'ppp5'A. RNAfragments isolatedfromRNase

Ti-digested

monosomes were labeled at their 5' ends with

32p,andequalnumbersofcountsfromeachreactionnmixture

were hybridized to pSRA5' which contained the

EcoRI-BamHI fragment shown in Fig. 2A. The eluted RSV RNA

fragments were analyzed on a 16.5% polyacrylamide gel

(Fig. 5). Addition of the cap analog to the translational

system nearly eliminated ribosome binding to RSV RNA when either chloride or acetate salts were used, as deter-mined by the numbers of counts eluted from the pSRA5'

filters. This suggests that the ribosomeprotection shown in

Fig.2through4 wasthe resultofribosomebinding to normal

initiation sites onRSV RNA.

Although the results described above demonstrated

ribo-some binding at the initiation site for Pr76gag, ribosomes

bound at the 5'-proximal AUG codon under all conditions tested. Productive initiation ofprotein synthesis at this site would result in thesynthesis of a seven-amino acid peptide

designated leaderpeptide 1 (LP1). Consequently, we

initi-ated a search for this peptide in the translational system

containing RSV RNA. The results of an early experiment

indicatedthat adipeptidewasdegraded rapidly when itwas

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-50 1 100 200 300 400

ILwI _A I I _(I..,)

s5.

1

FE ---- "- -:--

2 ET3

EA ( H

AUGI AUG(2 A

*

WG(3) H

4

Pr769

H B

At

AUG(4)

40

0 1 2 3 4 5 6

a _

DO

a04

GO:.i

G

_0

as

a.

30

4...

df

A_

- ..

E -.

20

A~~~~~~~~~~~

(¢~ ~

~~~ ~

- -- 1 --8-

-rnt

©RSVRNA 20 40 60 s0 100

ProtectedAdBf

Fragments DC

L . . D

---;-;---I;-RSVRNA

~360

38 400 420

RSV RNA__=

ProtectedV E _

Fragmentsl GF

FIG. 2. Ribosomeprotectionof RSV RNAbyusing anisomycinandacetatesalts in the translational system.(A) Map of the5'endofRSV strain SR-ARNAandpSRA5' subclones1through6of the RSV leaderregion.The mapshowsthepositionsof the AUG codons in theleader region of RSVRNA. Thestippled regionsindicate the openreading frames behindtheAUGcodons, and the cross-hatchedareaindicatesthe gag gene coding region. The lower line indicates the subclones of the RSV RNA leader region. E, EcoRI; B, BamnHI; H, HaeIII; nt, nucleotide. (B) Distribution of ribosome-protected RSVRNAfragments determined whenusingacetatesaltsin thetranslational system.Lane 0, 32P-labeled RNAfragments before selection ofRSV-specific fragments; lanes1 through6, RNAfragmentsselectedby hybridization to pSRA5'subclones 1through6,respectively.Thearrowindicates thepositionof themajor55-base RNAfragmentwhichwasprotectedwhen sparsomycinwasused in the translational system (seeFig.3AandB, lane 1).(C)Map of ribosomeprotectiononRSV RNA in the presence ofacetatesalts. Theribosome-protectedRSVRNAfragments(designated byletters inpanel B)wereeluted andidentifiedby partialRNase

Tidigestion(23). The locations of thefragmentsareshownwith respecttotheAUG codons. ThehexagonsindicateAUG codons(thenumber in thehexagon refers to the position relative tothe 5' endofRSVRNA). The upper linesindicate the distances(in bases)from the 5' end of RSV RNA (note thediscontinuityin thescale).

added to the reticulocyte lysate (11), so we tested the ability of the lysate to translate RSV RNA in the presence of

leupeptin, a protease inhibitor. Translation of RSV RNA

was not measurably inhibited by the presence of leupeptin

up to a concentration of 300

[Lg/ml

(Petersen, unpublished data).

The seven-amino acid peptide predicted by the nucleic

acid sequence was manufactured by solid-phase synthesis

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1 2 3 4 5 6

(#

0

A %

50

1 2 3 4 5 6

A

40 40 _

E _

De

30 *

D

C.

H _

G

-C

B-

D'_

E _

20

co) 20 n

RSV RNA - i)74-- --

--Protected jA

______--Fragmentsl

E--360 380 400 420

RSVRNA'

20

RSV RNA

_-Protected A

Fragments- l

-IC-nt

360 380 400 420

RSV RNA.

Protected' FE-- ..

Fragments G H

FIG. 3. Ribosome protectionof RSV RNAdetermined whenusingsparsomycinand either chlorideoracetatein the translationalsystem. (A and B) Distribution of ribosome-protected RSV RNA fragments on 16.5% polyacrylamide gels when chloride and acetate salts.

respectively,wereused.(Cand D) MapsofribosomeprotectiononRSV RNA when chloride andacetatesalts, respectively,wereused.For additionalinformation see thelegend toFig. 2. nt, Nucleotide.

(1) foruse as amarkeronanalytical HPLC columns,which could separate LP1 from the other peptides in the rabbit

reticulocytetranslationalsystem(Hackettetal., submitted).

TheA210 profile of marker LP1 extractedfrom the

reticulo-cyte lysateshowed that the peptideelutedat 24min (Fig. 6)

under the gradient conditions described in Materials and

Methods. To demonstrate that LP1 was synthesized in translations of RSV RNA, we extracted alcohol-soluble peptides produced in rabbit reticulocyte lysates that had beenincubated for 4mininthepresenceofRSVvirion RNA or water. A peak of [35S]methionine activity which comi-grated with synthetic LP1 was synthesized in translational

mixtures containingRSV RNA but notin mixtures

contain-ing water (Fig. 6). From this we concluded that LP1 was synthesized in vitro from RSV RNA, as predicted by the

ribosome-binding data. On the basis of [35S]methionine

incorporation kinetics during 4-min translations, we

calcu-lated that the molar amount of LP1 synthesis was roughly equivalent to the molar amount of P-761"(' synthesis (Petersen, unpublished data). Because of the instability of

the peptide in the translational system (Hackett et al., submitted),LP1couldnotbedetectedintranslations ofRSV

RNAperformedwithout addedleupeptin(R.Petersen,P. B.

Hackett, and S. Gunderson, unpublished data).

DISCUSSION

Thecurrentmodelsexplaininginitiation ofprotein

synthe-sis on RSV virion RNA include (i) the modified scanning hypothesis, in which 40S ribosomal subunits can bypass

weakupstreamAUG codonstoinitiatefurtherdownstream,

(ii)thespatial scanninghypothesis,inwhich RNAsecondary structurejuxtaposes the initiation site with the 5' end of the

RNA, and (iii) the reinitiation hypothesis, in which the ribosome or 40S subunit reinitiates downstream after

syn-thesizinga shortpeptide. Previously, ribosome bindingwas reported within the5'-proximal 100 bases of RSVRNA, but there was noevidence ofbindingat the initiation codon for

Pr76c("'l (4, 23). Consequently,therewere nodatato support

any of the modelspresented above. We havenowidentified conditions under which binding at the initiation site for

Pr76"''i` occurs;thisallowsustoevaluatetheapplicabilityof the modelsto the problem oftranslation of RSV RNA.

The results of the ribosome-binding assays performed in

the presence ofacetate saltssupport the modified scanning hypothesis. Initiation complexes form predominantlyat the

5'-proximalAUGcodon,withsomeinitiationatdownstream sites. Ribosome binding at AUG 4 under conditions that preventpeptide bondformationdiscounts reinitiationasthe *

50 *

30

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1

2

3

4

5

6

7 L

8

9

10

~~~~~~~~~~~~~~~~' '' 0 v.

FIG. 4. Detection of capped RSVRNAfragments by3'32P endlabeling ofribosome-protected RNAs. The RSVRNAfragments labeled with 32Pat their3' endswere isolated by hybridizationtoand elution from pSRA5' subclone 1; thiswasfollowed byanalysison 16.5%

polyacrylamidegels. To facilitate comparison with the other experiments(Fig.2and3),parallel5'32P-labelingreactionswereperformed with CIP-treatedoruntreated RNAs. Lanes1through 3, Products of5' and3' 32P-labelingreactionsperformed with theRNAfragmentsisolated from RNase

TI-digested

initiation complexesbefore selection for RSV mRNAfragments (lane 1, 5' labeled, noCIPtreatment;lane 2,5'

labeled,CIPtreated;lane3,3'labeled);lanes4through 6,RSV-specificRNAsfromareaction in whichanisomycinwasusedastheinhibitor

(lane4,5'labeled,noCIP treatment;lane5, 5'labeled, CIPtreated; lane 6, 3' labeledafterCIP treatment); lanes 7through10,RSV-specific RNAsfrom areaction in which sparsomycin wasused as the inhibitor (lanes7and 9,5' labeled, CIP treated; lane8,5'-labeled, noCIP treatment;lane10, 3'labeled, CIP treated). Lane L was amarker lanecontaining5',32P-labeled,partiallyalkaline-hydrolyzed poly(A). The arrowindicates the position ofthecapped RSVRNAfragmentprotected whenanisomycinwasused.

only method forinitiationatdownstream sites on RSV virion

RNA. If reinitiationwerethemajor modeby whichinitiation

occurred at downstream sites, we would expect to see

ribosome binding mainly at the 5'-proximal AUG codon

since reinitiationrequires synthesis ofLP1before ribosome

movement downstream. In sum, reinitiation should be pre-ventedwhenpeptide bondformation is inhibited. Ribosome

binding at AUGs 1 and 2 would not be predicted by the

spatial scanningmodel (5);rather, we would expectbinding

predominantly atAUG 4. This interpretation must be

tem-pered since the secondary structure that an RNA molecule

assumes isundoubtedly dynamic. However, there is a pro-nounced difference in the pattern ofribosome protection

observed thatis dependentontheionic conditions used.

Protein synthesis directed by RNA isolated from

RSV-infected chicken embryo fibroblasts is more efficient when

chloride is themajormonovalentanion added to the invitro

translational system (Fig. 1A). However, protein synthesis

directed by RSV RNA is more efficient when acetate

re-places chlorideasthemajor monovalentanion added to the

translationalsystem, asdemonstratedbythe enhanced

syn-thesis of Pr769'9 (Fig. 1B) and ribosome binding at the initiationsiteforPr76Rag (Fig. 2 and3).These results canbe interpretedto meanthatchloride inhibitsinitiation complex formation at AUG codons that are not 5' proximal. An alternativeexplanationis that the useof acetate salts allows

nonspecific bindingtoRNAby ribosomes, thus resultingin

multiple initiation sites. The similarity of the translational

products shown in Fig. 1 and thespecificity of initiation on

RSV RNA demonstrated by the m7G5'ppp5'A inhibition experiment (Fig. 5) indicatethatacetatedoesnotreduce the

specificity of initiation. However, there is precedence for

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1 2 3 4

8

7

Ce)

0

i

E

0

Cf)

U.

a.

6

5

4

3

2

4.

[image:7.612.107.243.69.339.2]

0

FIG. 5. Effect of cap analog on ribosome protection when

sparsomycin and eitheracetate or chloride saltswere used in the

translationalsystem. Initiation complexeswereformed in the pres-ence orabsenceof 0.8 mMm7G5'ppp5'A. Isolated RNAwas5' end labeled, and the RSV-specific sequences were selected by hybrid-izationtopSRA5'; for each sample 2x 10i cpmwashybridized. The

RSV-specific RNA fragments were analyzed on 16.5%

polyacryl-amidegels. Lanes 1 and2, Initiation complexes formedinacetate salts; lanes 3and4, initiation complexes formed inchloride salts; lanes 2 and4,capanalog addedtothetranslational system.

inhibition of initiation complex formation by chloride inthe

rabbit reticulocyte translational system.

Weber et al. (27) reported that high concentrations of chloride inhibit initiation ofprotein synthesis.Theseauthors

hypothesizedthat the highelectronegativity ofthechloride

ion interferes with protein-protein interactions or protein-nucleic acid interactions or both, probably resulting in the dissociation of initiation factors from the 40S ribosomal

subunit. This may explain the sensitivity to chloride of

initiation complex formation at the initiation codon for Pr76gag. A 40S ribosomal subunit migrating downstream alongan RNAmolecule maylose initiation factors whilein

transit, andasaconsequencethe40S subunitwouldnotbe

competent to initiate protein synthesis upon reaching the downstream AUG codons. Alternatively, a 40S ribosomal subunit that reaches a downstream AUG codon in the translational system containing chloride may not form a

stablecomplex; beforethecomplete80S complex is formed,

the 40S subunitmay dissociate from the RNA. If the latter explanationis correct,there mustbe additional factors that

stabilizeinitiation complexes formedatAUG 1.

Initiation complex formation at the initiation site for Pr76909is more sensitive to the anion added to the

transla-tion mixturethaninitiationatthe 5'-proximalAUG codon is.

This result is somewhat surprising since analysis of the sequences flanking the two AUG codons (AUG 1,

UUGAUGG; AUG4, AGCAUGG) suggests thatAUG 4is

10 20 30 40

FRACTION NUMBER

FIG. 6. HPLC analysis of translation products from RSV-directedorcontroltranslations. Translation reaction mixtureswere extracted and analyzed with a reverse-phase C18 column as de-scribed in Materials and Methods. The line labeled LP1 shows the elution profile ofsynthetic LP1 monitored byA210. Theother lines show the elution profiles of incorporated [35S]methionine from RSV-directed(0)and control(0) translations.

thestrongerinitiation site based on the consensus sequence proposed in the modified scanning hypothesis (16). The

stronger binding of ribosomes at AUG 1 indicates that the ability ofanAUG codon to act as aninitiation site depends

on features of the RNA other than its immediate flanking

sequence. The distance betweenanAUG codon and the 5' terminus of the mRNA may be important (18). In this

context we note that AUG 1 is 41nucleotides fromthe 5' end of the RNA molecule; this is within the typical range of cap-to-initiation site distances found on most eucaryotic

mRNAs(17). AUG4is 331nucleotidesfarther downstream. Thus, the relative insensitivity of ribosomebindingat AUG

1to the anion present in the translation mixture mayresult

from the proximity of this site to the cap. Preliminary

ribosome-bindingstudies withuncapped RSV RNAs

synthe-sized in vitro showed areversal ofbinding strengths, with

AUG 4 being stronger than AUG 1 (R. Petersen and C.

Hensel, unpublisheddata). This suggests that the cap struc-ture at the 5' terminus of RSV RNA enhances ribosome bindingto the5'-proximal AUGcodonby conferring

stabil-itytothe initiationcomplex formed there.

The observation that ribosome binding occurs more

fre-quentlyat AUG 1 than atAUG 4 isdifficulttoexplain since AUG4isusedtoinitiatesynthesis of the major viral proteins

Pr76gag,

M809'9-Pl,

andgpgoenlu. One possible explanation

is that while the5'-proximal 374 bases of RSVRNA contains theinitiation site for viral proteinsynthesis, it also contains the sequences necessary to initiate viral replication (the

tRNATrPbindingsite[12,13, 25])and tofacilitate viralRNA

packaging (24). The relative instability of the interaction

between the 40Sribosomal subunitorthe80S ribosomeand

RSV RNAbeyond AUG 1 which was demonstrated in the

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chloride-versus-acetate experiments may reflect a means of regulating these different functions. Weak binding by 40S ribosomal subunits may facilitate cDNA synthesis by allow-ing the polymerase to displace the 40S subunit from the RNA.Alternatively, initiation complex formation at AUG 1 may preventviral proteins from associating with the RNA. A thirdpossibility is that initiation complex formation at AUG 1 may keep the RNA free of ribosomes and available for packaging or cDNA synthesis. Consequently, although we have demonstrated that the ribosome binding which we observe at AUG 1 is functional by isolating LP1 from translational reactions containing RSV RNA, LP1 may not be important in the viral life cycle; rather, initiation by ribosomes at AUG 1 may be a regulatory event in and of itself (Hackett et al., submitted).

Although our data suggest that the modified scanning hypothesis adequately explains initiation ofPr76gag

synthe-sis, thismodel does not accountforthesynthesis of all ofthe

proteins encoded by the RSV genome. After splicing to produce src mRNA (10), AUG 4 is followed by a termination codon, and an AUG codon downstream from the 3' splice site isused to initiate pp6Osrc synthesis. Two recent reports (14, 21) have indicated that multiple AUG codons are

probably functional on src mRNA. Consequently,

reinitia-tion mayplaya majorrole in thesynthesis ofpp6Osr,.

ACKNOWLEDGMENTS

We thank Fernando Albericio, Sam Gunderson, and George Baranyfor synthesizingLP1, Vicki Iwanij forthe use of the HPLC apparatus, Kris Kohn for the artwork, and GailErickson for typing the manuscript. Vicki Iwanij, Marilyn Kozak, LindaSabatini, and Chuck Hensel provided helpful suggestions.

This workwassupported by National Science Foundationgrant DMB 8405225 (P.B.H.) andby American Cancer Society Institu-tional Research grant IN-13-V-59 to the University of Minnesota (R.P.). R.P. was supported by Public Health Service predoctoral traininggrantGM07094 from the National Institutes ofHealth.

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