Inhibition of Protein Synthesis in Cell-Free Systems from Interferon-Treated, Infected Cells: Further Characterization and Effect of Formylmethionyl-tRNAF

Copyright01974 AmericanSocietyforMicriobiology Printed in U.SA.

Inhibition

of Protein

Synthesis

in Cell-Free

Systems

from

Interferon-Treated,

Infected Cells: Further Characterization

and Effect of

Formylmethionyl-tRNAF

IAN M. KERR, R. M. FRIEDMAN, R. E. BROWN, L. A. BALL, AND J. C. BROWN

National InstituteforMedicalResearch,MillHill, London NW71AA, andDepartment of Microbiology, University of Virginia, Charlottesville, Virginia, 22901

Received forpublication3August 1973

The translation of

encephalomyocarditis

virus (EMC) RNA is markedly

inhibited in cell-free systems from

interferon-treated,

vaccinia virus-infected

L-cells (10, 11). The polypeptide products synthesized inresponse toEMC RNA

incell-free systemsfromthese and

untreated

infected cells have been

analyzed

by electrophoresison

polyacrylamide

gels. Qualitatively,

thesame

EMC-specific

polypeptides were

synthesized throughout.

In

experiments using

preincubated

microsomes from normal Krebs cells to assay cell sap from L-cells which had

been

exposed

to interferon

prior

to

infection,

only

the amount of the

EMC-specific

polypeptide

products

was reduced. This resultsuggeststhat there isan

inhibitionvery

early

intranslation in

interferon-treated,

infectedcells. Initiation

seems a

priori

the more attractive site for this

inhibition,

butan effect

shortly

after initiation cannot be excluded. With unfractionated cell-freesystemsfrom

interferon-treated infected

L-cells, however,

there

appeared

tobe anadditional

minor inhibitory effect on

polypeptide

chain elongation, in that the

EMC-specific polypeptides

synthesized

showednot

only

areductionin amountbut also

abias towardslower molecular

weight.

The

formylated

methionyl initiator tRNA

(Fmet-tRNAF) was usedasafurther

probe

intotheapparent effect onintiation.

With thisreagent wehaveconfirmed that there isonemajor initiation site for the

translation of EMC RNA in these cell-freesystems.In

addition,

theresults have

shown that

EMC-specific

polypeptide

chains initiated with Fmet escape the

major

interferon-mediated inhibition

at or

shortly

after initiation.

In the

interferon-treated,

vaccinia

virus-infectedL-cell, vaccinia virus mRNA is

synthe-sized but not translated (16,

21).

Recently,

we

have shown that the translation of

enceph-alomyocarditis virus

(EMC)

RNA is inhibited

incell-freesystemsfromsuch cells (10, 11). It is

nowwell established fromour ownwork(6, 11,

18) and that of others (1, 2, 8, 23, 27) that the

EMC RNAgenomeistranslated withfidelityin

cell-free systems from mouse L- and Krebs 2

ascitestumourcells. The

products

are aseriesof

high-molecular-weight

EMC-specific

polypep-tides. The majority of these ariseas aresult of

prematuretermination atpreferred sites during

the translation of this large mRNA (molecular

weight 2.7 x 10') (2, 18). Here we have made

useof ourknowledge ofthesepolypeptide prod-ucts to analyze the nature of the inhibitory

event(s) in the translation of EMC RNA in

cell-free systems from interferon-treated,

vac-cinia virus-infected L-cells. In addition, we have used the formylated methionyl initiator 9

tRNA (Fmet-tRNAF) (14, 28) to confirm (23,

27)

that initiation oftranslation ofEMC RNA

inthe cell-free system is at aunique site as in

the intact cell. With this established for the

cell-freesystemswith whichweareworking,we

have used the Fmet-tRNAF as a probe in our

investigation ofthe translationof EMC RNA in

thesesystems.Theresults ofthe electrophoretic

analysis

oftheproducts suggestthat the major

inhibition in the translation of EMC RNA in

cell-free systems from interferon-treated,

in-fected cells isat orshortlyafter initiation, with

a minor secondary effect on chain elongation.

With EMC RNAas messenger, however,

poly-peptidechains initiated with Fmet from

Fmet-tRNAF appear to escape the major inhibition

occurring early intranslation.

MATERIALS AND METHODS

Materials. Chemicals for use in the cell-free system, for the isolation of cell fractions and viral RNA and forthepreparation of tryptic digests were as

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KERR ET AL.

described previously (6, 19). Allradiochemicals were supplied by the Radiochemical Centre, Amersham, Buckinghamshire, England. The calcium leucovorin was from Lederle Laboratories Division, American Cyanamid Company, Pearle River, N.Y. Some of the EMC virus was obtained from Products for Research, G.D. Searle and Co. Ltd., High Wycombe, Bucking-hamshire,England.

Methods. The Krebs 2 mouse ascites tumour cells and the preparation of cytoplasmic extracts from them (6, 19), the preparation andpurification of EMC virus(20) and the extraction of EMC RNA (19), the labeling of polypeptide products in thecell-free sys-tem, theperformate oxidation and tryptic digestion of theseproducts, and the analysis of the tryptic digests byelectrophoresis and chromatography on thin-layer silica gel plates (6) have already been described. Qualitative and quantitative analysis of the polypep-tide products by electrophoresis on 5 or7.5% polya-crylamide gels after treatment with sodium dodecyl sulphate (SDS) was as described previously (18). Furtherdigestion of the tryptic digests with Pronase wasovernight at 37 Cin0.2 Mammonium bicarbon-ate, 10mM CaCl2.

Preparation of L-cellextracts. Nonpreincubated post-mitochondrial supernatant-S10-fractions were prepared as before (11) from interferon-treated (50 to 100 reference units/ml) and control, vaccinia virus-infected L-cells. Centrifugationofthis material at100,000xg for90 minyielded the supernatant cell sap fractions used in the mixed system assays with Krebs cell microsomes.

Amino acid incorporation assays. For the Krebs andL-cell systems the assaysof aminoacid incorpora-tion inresponsetosaturatingamountsofEMC RNA (20to 40Mg/ml)werecarriedout aspreviously (6, 11). Assays in the mixed system in which L-cell sapwas assayed with preincubated microsomes from normal Krebs cellswere incubated at 30C under the same conditionsasfor theL-cell system(11) butusing200

jtgof Krebsmicrosomes and 10 ulitersof L-cellsap (approximately 150jtgofprotein) per 50-Mliter assay. Assays involving the use of35S-Fmet- or met-tRNAF and/orAUG(U)n,wereroutinelycarriedout inafinal volume of25Mliters.Tosaturatethese systems2pmol (35,000 to 250,000 counts/min per pmol for methio-nineat aspecific activityof 20to150Ci/mmolat80% efficiency of counting) of 36S-met- or Fmet-tRNAF were added per

25-Aliter

sample. Unlabeled methio-nine(50

AM)

wasincludedinthe amino acid mixture used in all such assays. AUG(U)ri was added at a saturating concentrationof 80Mg/ml(1.6OD260units/ ml) and was assayed at the same magnesium ion concentration (4.35to4.85mM) asforEMCRNAin

theL-cell system. It was prepared according to the methodofSundararajan and Thach(30).

Interferon preparation andassay. Mouse L-cell interferon (> 106 reference units per milligram of protein) was purified bythe method ofPaucker (25) andassayedaspreviously described (11).

Charging and formylation of Krebs

tRNANet.

The enzyme fraction used for the charging and formylation ofthe tRNA was prepared from Esche-richia coli (MRE 600) accordingtoMuenchandBerg (22).Thedialyzed andconcentrated DEAE-cellulose

fraction in 50% glycerol was employed throughout. Theactivity of the enzymes was monitored by using pure E. coli

tRNAFM,t

(thegenerous gift of Kellmers and Novelli, Oak Ridge National Laboratory). 100% charging and >80% formylation of this tRNA was routinely observed. E. coli Fmet-tRNAF has been reported (14) to be active at a low level in the rabbit reticulocyte system, but with the Krebs and L-cell systems employed here, although there was some incorporation of label from the E. coli

36S-Fmet-tRNA,

inresponse to EMC RNA, analysis of theFriiet trypticpeptides showed it to be nonspecific. Accord-ingly, the E. coli enzymes were used to charge and formylate Krebs celltRNAFMel.The Krebs cell tRNA was obtained by phenol extraction of whole cells followed by salt fractionation (31) and chromatogra-phy onDEAE-cellulose (32). Initially, it was fraction-ated on BD-cellulose to yield the tRNAFMet and tRNAFM"t species (27). Having confirmed (12) that the E.colienzymepreparation was inactive with the mammalian noninitiating

tRNAlNIet

species, how-ever,unfractionated tRNAwasused in all of the work described here. Charging and formylation were car-ried outsimultaneouslyin areaction mixture contain-ingthe followingcomponents: 50 mM HEPES buffer, pH 7.5; 50 mM KCI; 5 mM Mg acetate; 2 mM dithiothreitol, 4 mM ATP, 1 mM CTP; 10 MAM Ca leucovorin;200

Mg

ofKrebs cell tRNA per ml (approx-imately 7,000 pmol of total tRNAequivalent to 140 pmoloftRNAFMet at 2%)and 1 MM1-35S-methionine (40 to 150 Ci/mmol). Incubation was for 15 min at 30 C. For the preparation of nonformylated met-tRNAF the formyl donor Ca leucovorinwas omitted. TheCa leucovorinwasmade up and storedin0.25N HCI. On additiontothe cell-freesystem, the change in pH converts it to theunstable active form (26). The amount of E. coli enzyme required for optimum charging and formylation varied considerably from preparation topreparation and with storage andwas determinedon a smallscalepriortopreparativeuse. The charged and formylated tRNA was extracted withphenol before purification at pH4.5 on asmall DEAE-cellulose column (32). Afterethanol precipita-tion the RNAwasdissolvedin waterat2x 105counts perminper

Mlliter.

Toassaytheextentofformylation a sample of the 3`S-Fmet-tRNAF was precipitated with 5% trichloroacetic acidat 0C, washed with cold acid on a cellulose nitrate filter (13-mm diameter, Sartorius Membranfilter GMBH, Gottingen, Ger-many),rinsed withethanol,and theaminoacyl-tRNA wasdechargedby incubation of the filter for 30 minat

37Cin 0.4 NNH40H.The eluted Fmet andmetwere fractionated by electrophoresis for 1 h at 400 V on

Whatman 3 MM paperinpyridineacetatebuffer, pH 6.5. The amounts of Fmet and metweredetermined by usingastripscannerorbycuttingthedriedpaper into0.5-cmstripsandcountingthese inascintillation counter. Charging was to a level of2 to 2.4pmol of Met-tRNA, per 100 pmol of total tRNA. Only70 to

80%formylationoftheKrebsmet-tRNAFwasusually

obtained. However, the residual (20to 30%) nonfor-mylated methionine in these preparations was not

incorporatedintoacid-insolubleproductsinresponse toEMCRNA.AnynonformylatedN-terminal methi-onine is rapidly cleaved (14, 15) from the nascent

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polypeptide chain. Inaddition,althoughwhen work-ing with completely nonformylated initiator met-tRNAF there is a significantincorporationof methio-nine intointernal sites in thepolypeptidechain(7,13, and our own unpublished results), no significant incorporation of this type occurred from the nonfor-mylated fraction (20 to 30%) of the Fmet-tRNAF preparation used here. Forexample, complete diges-tion ofthe EMC RNA-stimulatedpolypeptide prod-ucts with trypsin and Pronase consistently yielded >95% Fmetand <5% methionine. It couldbe, there-fore, that the 20to30%ofapparently nonformylatable met-tRNAF wasartefactual orrepresented damaged nonfunctional RNA.

RESULTS

Throughout

the

experiments

tobe

described,

the L-cell extracts

employed

in the cell-free

systems were neither

preincubated

nor

sub-jected to

Sephadex

chromatography.

With such

nonpreincubated

material,

amino acid

incorpo-ration inresponse toEMC RNA inthe cell-free

system is not reduced

by

prior infection ofthe

cells and interferon treatment alone has

only

a

small inhibitory effect (11). A clear-cut 50 to

90% inhibition of the translation of the added

EMC RNA isseen,

however,

insystems

employ-ingmaterial ofthistypefromL-cellsexposedto

both interferon treatmentand infection

(10,

11,

I. M. Kerr et

al.,

Advances in the

Biosciences,

vol. 11, in press). It is with the further

charac-terization ofthis enhanced inhibitionof

transla-tion triggered

by

infection that we will be

con-cerned here.

Characterization

of the

polypeptide

prod-uctssynthesized in responsetoEMC RNAin

cell-freesystemsfrom

interferon-treated,

in-fected cells. An analysis by

electrophoresis

on

SDS

polyacrylamide

gels of the EMC-specific

polypeptide

products

synthesizedinresponse to

EMC RNA in cell-free systems from

vaccinia-virus-infected and

interferon-treated,

vaccinia-infected cells is shown in Fig. 1. A qualitative

autoradiographic analysis

ofthe

products

syn-thesized after incubation for 30, 60, 120, and

240

min in the presence ofEMC RNA yielded the

results shown inFig. 1A,

whereas

aquantitative

analysis

ofthe

distribution

ofthe products at

120 min is shown in Fig. 1B. It

should

be

emphasized that in this experiment

pretreat-ment ofthe cells with interferon prior to

infec-tion resulted in a 70% inhibition of

incorpora-tion in response to EMC RNA in the cell-free

system (Fig. 1C). To compensate for this the

autoradiographs of gels 6 to 9 (Fig. 1A)

(inter-ferontreated) were exposedfor longerthanthe

corresponding controls (gels 1 to 4, Fig. 1A).

Similarly,

for ease of comparison, the data in

Fig. 1B areexpressedaspercentageofrecovered

radioactivity rather than in terms ofabsolute

counts. From thisdata it canbc seen that the sameseries

(A

to

G, Fig. 1A)

of

high-molecular-weight EMC-specific polypeptides

was

synthe-sized in both systems. These are

apparently

identical in molecular weight to those

synthe-sized incell-free systems fromuninfected Krebs

and L-cells. The nomenclature A to G is that

which was used to

identify

the

EMC-specific

polypeptides

inourprevious work(Fig.3,

refer-ence

18).

In

addition,

theresults for the earlier 30-min

(gels

1 and

6)

and60-min

(gels

2 and

7)

time

points

indicate that there is no

striking

differencein the timeat which the

polypeptide

chains first reachasize of130,000in molecular

weight,

(polypeptide G), for example. This in

turn suggests that there is no gross overall

reduction in the rate of polypeptide chain

elongation

in the inhibited systems.

Neverthe-less,

there does appearto be aslight

inhibitory

effectonchaingrowthinthattheamountof the

major

polypeptideGaccumulatingatlater time

points (gels

3 and

4)

is significantly reduced

(gels

8 and

9).

This is shownquantitatively for

the120-mintimepointinFig. 1B, inwhich the

bias in the distribution of the

polypeptide

products towards lower molecular weight is

obvious in theinhibited system..Thenature of

this

relatively

smalleffect isuncertain, butitis

clearly insufficient to accountfor the 70%

inhi-bition of total incorporation (Fig. 1C).

Accord-ingly,

the results show that themajorinhibition

mustoccurpriortotheproducts reachingasize

detectableonthesegels, i.e., mostprobably at,

orpossibly shortly after, initiation.

Characterization of the

polypeptide

prod-uctssynthesizedinresponsetoEMC RNA in

mixed cell-free systems. In general, on

frac-tionation ofthe L-cell extracts, the amount of

the interferon-mediated inhibitory effect

re-coveredwith the cellsap (S100) andmicrosome

fractions has proved variable. It may be that

this variability iscorrelated, forexample, with

the degree ofpolysome

disaggregation

or

ribo-some dissociation in the system. The results

obtained in preliminary assay of the crude

microsome fractionwith normal Krebs cellsap

were qualitatively similar to those for the

un-fractionated system (Fig. 1). It should be

em-phasized that thismaymerely reflectthe

crude-ness of the fractionation procedure. Different

resultswereobtained, however, onassay of cell

sap from

interferon-treated,

vaccinia

virus-infected cells with preincubated microsomes

from normal Krebs cells. Withthissystem only

the majorinhibition at or shortly after initiation

was observed, with no secondary effect on

elongation. This is shown in Fig. 2. In this

experiment interferontreatment prior to

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KERR ET AL.

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FIG. 1. Characterization ofthe polypeptideproductssynthesized in response toEMC RNA with time in cell-free systems from interferon-treated, vaccinia virus-infected cells. Unfractionated postmitochondrial supernatant (S10) fractions were used throughout. Incubation in the cell-free system in the presence of 35S-methionine(100MCi/ml,100Ci/mmol)andEMC RNA (40

,g/ml)

was asalreadydescribed(Materialsand Methods). A,Analysis ofthepolypeptide products by electrophoresisinSDS-polyacrylamide gels. Gels6to9 werewithinterferon-treated,vaccinia-infected cell extracts; Gels1 to 4 werewithcorrespondingextractsfrom infectedcells withoutinterferonpretreatment.Afterincubationofthecell-freesystemsfor30min(gels1and6), 60 min(gels2and7),120min(gels3and8)and240min(gels4and9),50ulitersamplesweretreatedwithSDS andsubjectedtoelectrophoresison SDS-polyacrylamide gels. Thefigureshowsautoradiographs ofthedried gelsand,as anindexof molecular weight,aphotographofastainedgelonwhich thepolypeptides of purified reoviruswereelectrophoresedinparallel.Thefigurestotherightrepresent the molecularweightinthousandsof themajor reoviruspolypeptides Xl, A2, p2,and q3(29). Theautoradiogr:aphsofgels 1 to4 wereexposed for4 days; those for gels 6 to 9for20days. B, Quantitative analysis ofthe molecularweight distribution ofthe products at 120 min. Gels corresponding to3 and 8 (Fig. 1A) werefixed, sliced, prepared for radioactive counting, and countedaspreviously (18). Theabscissa shows the distance migrated fromthe top ofthegel

(1-mm slices)with theorigintotheleft; themajor peak(fractions8to10)correspondstoG in thegels3and8. Theordinategivestheradioactivityincorporatedintopolypeptiderecoveredineachfractionas apercentageof

the totalrecoveredfrom thecompletegel.Symbols:0, nointerferonpretreatment ofthecells; 0, interferon

pretreated. The total recoveries from the gels (100% values) were 53,000 counts/min without and 15,000 counts/min with interferon pretreatment. C, Direct quantitative assay of83S-methionine incorporation in responsetoEMC RNA with timeinthe cell-freesystemsused in IA andIB above.Afterincubationforthe indicated time

5-pgliter

sampleswereassayed forincorporation of radioactivity intoproteinin the usual way. Symbols:*,vaccinia-infected cellextracts; 0, interferon-treated, vaccinia-infected cellextracts.

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INTERFERON

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FIG. 2. Characterizationofthepolypeptide products synthesizedinresponseto EMC RNA with timeinthe

cell-free systems in the presence of cell sap fractions from vaccinia virus-infected and interferon-treated vaccinia virus-infectedcells. The cell sapfractionswereassayedwithpreincubated microsomesfrom Krebs

ascites tumourcells. Incubation in the cell-free systemin the presence of3"S-methionine (100pCi/ml, 100

Ci/mmol) and EMC RNA (40ug/ml) was asalreadydescribed.A,Analysis ofthepolypeptide products by electrophoresis inSDS-polyacrylamidegels. Gels 1 to 3employedcellsapfrom vacciniavirus-infectedcells.

Gels 4to 6were with cellsapfrom interferon-treated, vacciniavirus-infected cells.Afterincubationofthe

cell-freesystemsfor30min (gels1 and4),60min (gels2 and5),and 120min (gels3 and6),40-plitersamples weretreatedwith SDS andsubjectedtoelectrophoresisonSDS-polyacrylamidegels inparallelwith reovirus polypeptidesasdescribedinFig.1.Thefigurestotheright representthe molecularweightsinthousandsofthe

major reoviruspolypeptides (Fig. 1).Theautoradiographswereexposed for4days. B, Quantitative analysis of the molecularweightdistributionoftheproductsat 120min.Thiswascarried out withgels correspondingto 3

and6 in A aboveasdescribedinFig. lB. The dataareexpressedasinFig. lB.Symbols: 0,Nointerferon;0,

interferon pretreated. The total recoveriesfrom the gels (100%/o values) were 75,000counts/minwithout and

21,000 counts/minwithinterferonpretreatment.C,Adirectquantitativeassayof"'S-methionineincorporation inresponsetoEMCRNA withtime in thecell-freesystems usedinA and BabovewascarriedoutasinFig.1C. Symbols: 0, vaccinia virus-infected cellextracts; 0, interferon treated, vaccinia virus-infected cellextracts.

tion reduced the level of translation of EMC RNA to 50% of the control value (Fig. 2C) without apparently affecting the nature ofthe EMC-specific polypeptides formed (Fig. 2A and B). In the inhibited system identical products

were synthesized (Fig. 2B) at apparently the

same rate ofpolypeptide chain growth (gels 1 and 4, Fig. 2A) but in reduced amounts (Fig. 2C).

Taken together these results are consistent

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KERR ET AL.

with a major block at orshortlyafter

initiation,

with a minor inhibition of polypeptide chain

elongation, the latter being seen only in the

unfractionated system.

IncorporationofFmet fromFmet-tRNAFin

response to EMC RNA. The initiation of

polypeptide chain synthesis in eukaryotes is

thought to occur exclusively with methionine

fromoneof themethioninetRNAs: the initiator

tRNAF\et.

Normally, this N-terminal methio-nine is cleaved from the nascent polypeptide

chain when it isstillquite short (14, 15). Thus,

35S-methionine from labeled initiator

35S-met-tRNAF is removed from the N-terminus of the

nascentpolypeptide chains almost immediately

after it is incorporated and is not normally

detected. If, however, the methionine on the

initiator tRNA is formylated, the Fmet once

incorporatedN-terminally onthe nascent chain

is not cleaved and is readily detected (14, 27).

Here, therefore, wehave used Krebs cell

initia-tor

tRNA,NIet

loaded and formylated with en-zymesfromE. coli as a probe in our studieson

initiation.The E. colienzymesdonotrecognize

the noninitiator mammalian methionine tRNA species (tRNAlet )responsiblefor thedonation

ofmethionineintointernal sites(12,and

Mate-rials and Methods). Moreover, the presence of

the formyl group both prevents the proteolytic

cleavageofthe N-terminal (formyl) methionine

and removes any possibility ofincorporation of

the methionine into internal sites in the

poly-peptide chain. Incubation of

35S-methionine-labeled Fmet-tRNAF with EMC RNA in the

Krebs cell-freesystemprovidesaproductwhich

on digestion with trypsin yields a single major

EMC-specific Fmet-peptide (23, 27). Further

analysis of this peptide

by

Smith has shown it

tobe identical (with the additionof the

formyl

group) totheunique methionineinitiation

pep-tide formedinthe translationoftheentireEMC

RNA genome in the intact EMC-infected cell

(27). Thus, initiation ofprotein synthesiswith

Fmet-tRNAF in response to EMC RNA in the

Krebs cell-freesystem occursatthesamesiteas

does initiationofEMCpolypeptide synthesisin the intact cell.

N-terminal incorporation of 35S-methionine

labeled Fmet from

Fmet-tRNAF,

as a measure

ofinitiation, and a mixture of free "C-amino

acidsor

3H-leucine,

as anindex of total

synthe-sis, werecompared forthesystems under

study

here. With systems using material from

inter-feron-treated, infected cells there wasthe usual

inhibition of "C-amino acid or 3H-leucine in-corporation in response to EMC

RNA,

but no such inhibitionintheincorporationof35S-Fmet

was observed(Fig.3). Onoccasion,with

unfrac-tionated systems showing a profound effect

some inhibition (<50%) of Fmet incorporation

did occur, but this was always much less than

thatobservedwiththefree amino acids (> 80%).

Accepting that EMC-specific polypeptide

chains initiated with Fmet-tRNAF do escape the major inhibition one might, a priori, have expected to see a decrease in the inhibition of

3H-leucine incorporation in those systems

re-ceiving Fmet-tRNA as in the experiments

shown in Fig. 3B. However, the added Fmet

tRNAF is in competition with endogenous met

tRNA,

andonly 0.01 to 0.05 pmol of Fmet are

incorporated per 0.34 pmol of added EMC

RNA. No accuratefigureforthe total number of

EMC-specific polypeptide chains initiated in

response totheadded RNA is yetavailable(not all EMC RNAmoleculesneed be active inthis

respect), but it is clearly possible that Fmet

from the added Fmet-tRNAF is only used in a small fraction of the initiation events (our

calculations suggest a figure of no more than

15%). Acceptingthis, no significant decrease in the inhibition of3H-leucineincorporation would be expected in the presence ofFmet-tRNAF.

There are a number of possible explanations

why incorporation of Fmet from

Fmet-tRNAF

might escape inhibition. It could be, for

exam-ple, that the major inhibition operates at the

level of charging or breakdown of the initiator

tRNAF

et. Thus, by adding 35S-Fmet-tRNAF

one mightalready be beyond the site of inhibi-tion in the sequence of events involved in initiation. Indeed, we have some preliminary

evidence for a reduced ability to charge the methionine tRNAs in these systems, but on fractionation this does not appear to be respon-sible for the major inhibition observed (L. A. Ball, unpublished data). Nevertheless a re-duced charging activity could result in a re-duced pool of endogenous met-tRNAF in the cell-free system. This, in turn, would result in an increase in the effective specific activity of the added 35S-Fmet-tRNAF in the inhibited system and a spuriously increased level of incorporation. The results obtained with

AUG(U)yg

as message, however, argue against

any such explanation.

Incorporation from

Fmet-tRNAF

and

met-tRNAF in response to AUG(U)n-. When

as-sayed with

AUG(U)r,

as mRNA, incorporation

of label from initiator tRNAF "let was inhibited under conditions where incorporation of Fmet in response to EMC RNA escaped inhibition

(Table 1). This was true with

AUG(U)3n,

as

message both for the

formylated

Fmet-tRNAF

and thenonformylated

met-tRNAF

species,

al-though in someexperimentsthe inhibition with

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INTERFERON MEDIATED INHIBITIONOFTRANSLATION

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~

~

CM

3

-5e

51 3SP

20

40

60

120

TIME

(mins).

FIG. 3. Incorporation of "S-Fmet from 'S-Fmet-tRNAF in the cell-free system: absence of inhibition in extractsfrominterferon-treated, vacciniavirus-infectedcells. A,Unfractionated extracts. The graph on the left shows theincorporationof a mixture of14C-aminoacids, that on the right the incorporation in parallel assays of

35S-Fmet from 85S-Fmet tRNAF with time at 30 C in the cell-free system. Interferon-treated, vaccinia

virus-infected cellextractsplus O--Oandminus 0--* EMC RNA; vaccinia virus-infected cell extracts plusO-Oand minus0-* EMC RNA. Thesymbols are the same for both graphs. B, Assays of L-cell sap withpreincubatedmicrosomesfrom normal Krebs cells. Here the incorporation in response to EMC RNA of free 3H-leucine andof3'S-Fmetfrom3"S-Fmet-tRNAFincluded in the sameassay mix was followed with time at 30 C. 3H-leucine incorporation with vaccinia virus-infected cell extracts (0) and interferon-treated, vaccinia virus-infected cell extracts (A). 3"S-Fmet incorporation with vaccinia virus-infected cell extracts (0) and with interferon-treated, vaccinia virus-infected cell extracts (A). The assays were carried out as described in Materials and Methods with 0.25

,gCi

of

"4C-amino

acid mix (50 mCi/mAtom of carbon)or5

5gCi

of3H-leucine (52 Ci/mmol) plus 35S-FmettRNAF equivalent to 3 x 101 to 4 x10&counts per min (4 pmol at 45 to 50 Ci/mmol). VOL.13, 1974

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[image:7.495.123.398.82.549.2]

TABLE 1. Incorporation of 35S-methionine from the initiator tRNAFMe' species in response to AUG(LT)n in cell-free systems frominterferon-treated, infected cells"

Incorporation in response toAUG(U)a-with Incorporation in response to EMC RNA with Cell-free systems from 35S-met-tRNAF

35S-Fmet-tRNAF

35S-Fmet-tRNAF

3H-phenylalanine

(counts/min) (counts/min) (counts/min) (counts/min) Vacciniavirus-infected

cells ... 15,600 6,500 2,500 11,500

Interferon-treated,vaccinia

virus-infectedcells ... 4,400 (28%)b 2,200 (30%)b 2,000(80%)b 2,050(18%)b

aUnfractionated postmitochondrial supernatant (S10) fractions were used throughout. Met-tRNA and Fmet-tRNA were from the same preparation and had essentially the same specific activity. Two

Atliters

of one or other of the35S-met-labeled initiatortRNAFMet species (4 x 105counts/min,2.2 pmol) wereadded to each assay (25 ,liters). All assays were carried out in duplicate under the conditions already described (Materials and Methods). Incorporation from the abnormal Fmet-tRNA was apparently less efficient than from the normal met-tRNAF in accord with the results of Brown and Smith (3).

b%ofincorporation in the vaccinia-infected system.

the formylated species was marginally less.

(With

AUG[U]n

as message, nonformylated

met tRNAF can be used since the N-terminal

methionine is not cleaved from the relatively

shortnascent

met(phe)n oligopeptides

[3].)

Initi-ation with saturating amounts of

AUG(U)n-

is

over morerapidly (5to 10 min) inthese systems

than with EMC RNA (20 to 40 min, Fig. 3).

However, control experiments involving the

additionofthesemessengers progressivelywith

timehave shownnodifferenceinthestabilityof

functional Fmet and met-tRNAF species in

thesesystems. Itseemsunlikely,therefore, that

the apparent side-stepping by Fmet of the

inhibitory effect with EMCRNA (Fig. 3) simply

reflectsareductioninthe sizeofthe endogenous

met-tRNAF pool (Table 1), or anydifferencein

the relative stabilitiesofthenormal endogenous

(or added) initiator met-tRNAF as compared

with the formylated

Fmet-tRNAF

species.

Characterization of the Fmet-labeled

poly-peptide products synthesized in response to

EMC RNAincell-free systems from

interfer-on-treated,

infected cells. Anobviouspossible

alternative explanation for the escape of the

Fmet incorporation from the major inhibition

would be provided if, in these particular sys-tems, initiation with Fmet occurred at an

ab-normal site on the EMC RNA genome. The

Fmet-labeled

polypeptide products

synthesized

in response to EMC RNA were, therefore,

analyzed

by electrophoresis

on SDS

polyacryl-amide gels (Fig. 4). In

addition,

the

major

EMC-specific Fmet-labeled initiation

peptide

released ontryptic digestionofthe

polypeptide

products was

compared

(Fig.

5)

with that

formed in responsetoEMC RNA in theKrebs

cell-free system. This latter has been shown

by

Smithtobeidentical

(plus

the

formyl

group)

to

thatnormally involvedinthe initiationofEMC

polypeptide

synthesis inthe intactinfected cell

(27). Fromacomparisonofthe

autoradiographs

shown in Fig. 4 with those in Fig. 1 and 2and

previously (18), it is clear that

35S-Fmet

from

Fmet-tRNAF is

incorporated

predominantly

into the same series of

high-molecular-weight

EMC-specific

polypeptides

as is free

methio-nine. Double labeling experiments with

35S-Fmet-tRNAF

and 3H-leucine have confirmed

this and similar results have been

reported by

Oberg and Shatkin for the Krebs cell-free

sys-tem (23). The

relatively

greater amounts of

radioactivity in the lower molecular

weight

material with 35S-Fmetaslabel(compareFig.4

with Fig. 1 and2) isconsistent with

incorpora-tion ofFmet

exclusively

at the

N-terminus,

for

over the molecular

weight

range of

25,000

to

130,000 observed here, the ratio ofN-terminal

Fmet per molecule to

randomly

distributed

methionine or leucine would be expectedto

de-crease

by

afactor of5.Accurate

quantitation

in

these systems, whichseemto show less

prema-ture termination of translation of EMC RNA

thanisobservedinthe Krebssystem, is

compli-catedby thefact thatsome

cleavage

ofnascent

high-molecular-weight

EMCprecursor

polypep-tide may beoccurring

(R.

M. Esteban andI. M.

Kerr, manuscript in

preparation).

There

is,

in

addition, a significant (e.g.,

Fig.

3A)

back-ground of

endogenous

incorporation

in these

nonpreincubated

systems.

Nevertheless,

on

complete digestion ofthese Fmet-labeled

prod-uctswithPronase, >95% of thelabel

co-electro-phoresed with Fmetand<5% withmethionine,

confirming thatat least95%ofthelabel

incor-porated isN-terminal. On

tryptic

digestion

the

sameEMC-specificFmet initiation

peptide

(A,

Fig. 5A) wasobtained with all of thesesystems

16 KERR ET AL. J. VIROL.

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A

G-

1"

l 40

E t.

a_h*

i2

-72

1

2

3

4

5

6

7

8

REO

B . v

i

55

G_

_t

140

t

S3V** -34

*:..Sf..1

A-1

2

3

4

5

6

REO

FIG. 4. Characterizationofthe35S-Fmet-labeledpolypeptide productssynthesizedin responsetoEMC RNA with time in the cell-free system. A, Unfractionated extracts from vaccinia virus-infected (gels 1 to4) and interferon-treated,vacciniavirus-infected (gels5to8)cellswereincubated in the presenceof8x10J countsper minof35S-Fmet-tRNAF (equivalentto100pmol ofI'S-metat45Ci/mmol)and

20,ug

ofEMC RNA(7.4pmol) per 0.5-ml assay.At 30min(gels1and5),60min(gels2and6),120min(gels3and7),and240min(gels4and 8)0.1-mIsampleswere treated with SDS andsubjectedtoelectrophoresisonSDS-polyacrylamidegelsin the presenceof reoviruspolypeptidesasmolecularweightmarkers. Thefigurestotherightrepresentthe molecular weightsinthousandsofthemajorreoviruspolypeptidesdetailed in thelegendtoFig.1.Autoradiographs ofthe driedgels are shown.Incorporation ofFmet in the interferon-treated, infected cell system (gels5to8) was inhibited to some extent (25 to 40%) in this experiment, but much less so than was the incorporation of 3H-leucine andfree 5S-methionine(70to80%)assayedinparallel. B,L-cell sapfrom vaccinia-infected(gels1 to3) andinterferon-treated, vaccinia-infected (gels4 to6)cellswereincubatedwithpreincubatedmicrosomes fromnormalKrebscells in the presenceof4 x10J countsperminof3S-FmettRNAF (equivalentto50pmol of 3"S-metat 45Ci/mmol)and

20Mgg

ofEMC RNA (7.4pmoles) per 0.5-mI assay.At 30min(gels 1 and 4),60min (gels 2 and 5), and 120 min (gels 3 and 6) 0.125-mI samples were treated with SDS and subjected to electrophoresisasinA. Therewas nosignificantinhibition(<10%)of incorporation of Fmet in this experiment whereas therewas a

50%o

inhibitionof incorporation of 3H-leucine and of free3S-methionine assayed inparallel. InbothA (240min) and B (120 min) samples were taken and completely digested with trypsin then with Pronasefor further electrophoretic analysis which showed that essentially all (> 95%) of the radioactivity incorporated into polypeptideswasrecoverableasFmet.

on November 10, 2019 by guest

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[image:9.495.104.393.76.460.2]

!~~~~*

A

B~~~~~~~~~~~~~~~~B

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

l

2

3

4

5 6

7

8

9

10 11

[image:10.495.117.395.62.461.2]

PR

0

FIG. 5. Fmet-tryptic peptides formed in response to EMC RNA in Krebs and L-cell-free systems. The polypeptideproducts from the variouscell-freesystemslabeled with35S-Fmet-tRNAF or, in the case of band 3 in A,free3"S-methionine,weredigestedwithtrypsin.A,Electrophoresisonthin-layerplatesatpH 6.5.Samples (2to5uliters)wereplacedat0with theanodetothetopofthefigure.Krebscell-free system plus (1) andminus (2)EMC RNA. (3)Krebscell-freesystemplusEMC RNA withfree83S-methionine. Vaccinia-infected L-cell systemplus(4) andminus(5)EMC RNA. Interferon-treated, vaccinia-infected system plus (6) and minus(7) EMC RNA. Theremainingassayswerewithpreincubated microsomesfrom normal Krebs cells andvaccinia

virus-infected cellsapplus (8)and minus (9) EMC RNA and interferon-treated, vaccinia virus-infected cell

sapplus(10) and minus (11) EMC RNA. (B) Two-dimensional chromatography andelectrophoresis onthin layer plates (6). The digests were placed at the bottom center of the sheets (arrow) and subjected to

electrophoresisatpH6.5with the anodetotheleft.P.R. indicates thepositiontowhichaphenol red marker migrated during theelectrophoresis. Chromatography was towards thetopofthe sheet. The results withthe Fmet-trypticpeptides fromall ofthesystems inFig.5A wereessentially identical tothe examplefrom the Krebs cell system shown here. ThesamemajorFmetpeptidewasdetectedonanalysisoftheproduct after 5, 10, and120minofincubation in the Krebscell-freesystem.Experimentsin which thedigests labeled withFmet were runseparatelyand mixed withdigestslabeled withfreemethionine showed that themajor Fmetpeptide

migratedslightlyfasterin bothdimensions than the major peptideX(sample3 inFig. 5A) labeled withfree

methionine. The assays carriedoutasinMaterials andMethodswerescaled upto0.3mlforthe Krebscell systemand0.5mlforthe L-andKrebs microsome L-cellsapsystems.3BS-Fmet-tRNAFequivalentto4 x 10.

countsper min (22.6pmol ofmethionine at a specific activity of100 Ci/mmol) was added toeach assay. Incorporation in responsetoEMC RNA variedfrom 20,000 counts/min (0.11 pmol) forthe Krebssystemto

100,000counts/min (0.55pmol)forthe mixed system.

18 KERR ET AL. J. VIROL.

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(inhibited and control, unfractionatedand frac-tionated cell sap with Krebs microsomes) as

with the Krebs cell system(Fig. 5A). The minor Fmet-peptide (B, Fig. 5A) (usually<10%ofthe total radioactivity) is thought to be a further

breakdown product of the major (23, 27). It

seems reasonable to conclude that initiation with Fmet occurs at the normal site on EMC

RNAand that the Fmet is incorporatedintothe normalspectrumofEMC-specificpolypeptides in these systems. Thus, the ability of Fmet incorporation toescape the major inhibition is

not the result of its incorporation at an

abnor-mal site.

Comparing the resultsfor theanalysesofthe

Fmet N-terminally labeled polypeptide

prod-ucts synthesized at different times inthe cell-freesystemusing unfractionated (S10) material

frominterferon-treated and untreated, infected

cells (Fig. 4A), the same bias away from the

higher molecular weight products isseeninthe

interferon case as wasnoted for the

incorpora-tion of free amino acids (Fig. 1). Again, in agreementwith the results in Fig. 2, there isno

such bias in thesystemsinwhichcorresponding cell sap fractions were assayed with Krebs

microsomes (Fig. 4B). Thus, even though

poly-peptide chains initiated with Fmet escape the

major inhibition early in translation, they are

stillsubjecttothe minor inhibitionaffecting the accumulation ofhigh molecular weight material in the unfractionatedsystem.

DISCUSSION

It is impossible asyet to tell howthe inhibi-tion characterized here for systems from inter-feron-treated, vaccinia virus-infected cells, which apparently affects the translation of

EMC RNA, AUG(U)11 and mouse globin

mRNA (I. M. Kerr et al., in press; Friedman

unpublished data) but notofpoly U (11),

cor-relates withthe effectwe have seen in systems

from interferon-treated, EMC-infected L-cells,

which appears toshow somespecificity for

dif-ferent mRNAs (I. M. Kerr et al., in press; R.

M.Esteban, D. R. Tovel, and I. M. Kerr,

manu-script in preparation). Nor is it clear how this inhibition correlates with the much smaller effect of interferon treatment in the absence of infection that we have reported for these (11) and other systems (17), orwith the largeeffect

reported in the absence ofinfection by Falcoff et al. (9). The possibility exists that the inhibi-tion ofproteinsynthesis seenin the

interferon-treated, vaccinia-virus-infected cells, is a

sec-ondary effect of the abortive replicationof virus in these systems. Alternatively, it could

repre-sent the sum of a virus-mediated

inhibition-ofhost protein synthesis andan

interferon-me-diated inhibition ofvirus, rather than ablanket

interferon-mediated inhibition of both. On the

other hand, our preliminary analyses of these

different systemshave indicatedpoints in

com-mon as well as apparentdifferences, suggesting that a common underlying mechanism may be

involved. Moreover, the idea that

infection,

or

other insultto the cell, mayberequiredto

trig-gerthe fulldevelopment of the

interferon-medi-ated inhibition has its attractions.

According

to

onevariation onthis

hypothesis,

there could be

a switch-offofproteinsynthesison infection of

the interferon-treated cell

during

which time

either the events leadingtocell death are

initi-ated, i.e., the cell

dies,

thus

preventing

the

spread of infection, or the

input

viral nucleic

acid is inactivated so that, after a

period,

nor-mal host metabolismcan resume.Inmany cases

interferon pretreatment does not prevent cell

death afterinfection, it merely reduces the virus

yield and this hypothesis could

provide

the

basis for further work should it turn out that

there is no significant selection between host

andviral mRNAs in the effectupontranslation.

This,however, isonlyone

possibility;

theresults

in the interferon-treated

SV40-infected

cell

systems

(24)

strongly

suggest a specific

in-hibition of viral functions, and the absence of

selectivity

forhost and viralmRNA inthe cell-free system has

by

no means beenestablished.

Globin

mRNA,

particularly

intheformin which

it is presented tothe cell-free system may, for

example,

beatypical ofhost message inthe

in-tactcell. Amuchmoredetailedanalysis of these

cell-free systems and of the

possible

impor-tance of the different methods used in their

preparation and assay will be required before

the relationships between them and their

sig-nificancetointerferon actioncanberesolved.

Itis,

however,

withthe further

characteriza-tion of the enhanced inhibition of

translation,

triggered

by

infection inthe

interferon-treated,

vaccinia-virus-infected cellsystemthatwehave

been concerned here. Even if this inhibition is

secondary

to the primary event of interferon

action (an event perhaps capable of affecting

transcription and translation in different

cell-virussystems in differentways), the absence of

aneffect ofthetranslation ofpoly U (11) and on

the incorporation of Fmet from Fmet-tRNAF

(Fig. 3) clearly indicates that a highly specific

mechanismisinvolved which must beof

inter-est in the control of protein synthesis. The

resultsofthe analysis of thepolypeptide

prod-ucts synthesized in response to EMC RNA in

cell-freesystems of this type are consistent with

VOL. 13, 1974

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KERR ET AL.

amajorinterferon-mediatedinhibition of

trans-lation at, or shortly after initiation, with a

minorsecondary inhibition ofchain elongation

(Fig. 1and2).On the basis of this data alone it

is possible that the major inhibition occurs

shortly after initiation whenthe productis too

smalltobeseen onacrylamidegels. It would be

premature to exclude such a mechanism, but

for it to be unique to an early event in chain

elongation it seems likely that it would be

closely linked to initiation, as indeed is

sug-gested by the results obtainedwith theinitiator

Fmet-tRNAF.

Fmet from Fmet-tRNAF is incorporated

N-terminally into the normal spectrum of high

molecular weight polypeptides synthesized in

response toEMC RNAinthese systems (Fig. 4),

but ispresent as a commonN-terminalpeptide

(Fig. 5). This, therefore, provides further

evi-dence in favour ofthe model thatwe (18) and

others (2) have previously proposed for the

translation of EMC RNA in the cell-free

sys-tem. According tothis model, initiation in the

cell-free system occurs at a unique site on the

EMC RNA as in the intact infected cell, the

majority of the highmolecular weight

polypep-tidesbeing formed

by

prematureterminationof

translationatdifferent preferred sites (possibly

of

nucleolytic cleavage)

onthe

large EMC

RNA

message.

Theinterestingaspect oftheresultsobtained

with Fmet-tRNAF here, however, is that the

incorporation of Fmetfrom it escapesthe

inhi-bition of translationseeninthe

interferon-treat-ed, infected-cell system. The results with

AUG(U)"

as message

(Table

1) and those

concerning the

stability

ofthechargedinitiator

tRNAsargueagainstany

simple

explanation

for

this escape, such as would be involved ifthe

inhibitory eventaffected the chargingor

break-down of thenormal initiatormet-tRNAFsothat

the inhibition

preceded

the reactions afterthe

addition of the

precharged Fmet-tRNAF.

Nor

does the

side-stepping

ofthe inhibition

by

the

Fmet-tRNAF reflect initiation at an abnormal

site(Fig. 5). The most likely explanationseems

to be that the abnormal formyl group on the

Fmet-tRNA is not

recognized

by

the inhibitor.

But if this were thewholeanswer onewouldnot

have expected the incorporation of Fmet from

Fmet-tRNAF

tobeinhibitedwith

AUG(U)n

as message

(Table

1). Thereisevidence fromwork

with thereticulocytesystem, for

example,

that

both with Fmet-tRNAF (5) and with

synthetic

messages(4),the factorrequirementsfor

initia-tion aredifferent from those observedwith the

nonformylated initiator met-tRNAF or natural

messages. This emphasizes,

incidentally,

the

fact that the inhibition of incorporation in

response to

AUG(U)51

in these systems should

not be taken as proofthat all messages will be

affected: ifthere areadditional factors

specifi-cally required for the translation of host

mes-sages it is possible that they could provide the

specificity not apparent here. Returning to the

effect of Fmet-tRNAF, however, there are, in

fact, a numberofpossibleexplanations for this

intriguing phenomenon which are currently

under investigation. Meanwhile, taking these

results together, they suggest that the presence

of the abnormal formyl group on the initiator

Fmet-tRNAF allows a factor(s) involved in the

initiation oftranslationofEMCRNA, at least, to escape recognition at the site ofthe major

interferon-mediated inhibition oftranslation.

ACKNOWLEDGMENTS

Wethank D.Risby, A.Douglas,andSusan O'Connor for technical assistance and J. A. Sonnabend for much useful discussion.R.M.F., NationalCancerInstitute staffmember atthe National Institutes ofHealth,was avisiting scientist. The interferon was obtained from The Microbiological Re-search Establishment, Porton, Wiltshire, England (C. J.

Bradish)and from K.Paucker,andpurifiedreoviruswasthe generousgiftofJ. J.Skehel.

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