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VIROLOGY 58, 47@-491 (1974)

A Protein Synthesizing System From Interferon-Treated Cells ThatDiscriminates between Cellular and Viral Messenger RNAs

CHARLES E. SAMUEL AND WOLFGANG K. JOKLIK

Department of Microbiology and Immunology, Duke University Medical Center,Durham, North Carolina 97710

Accepted November 99, 1973

The effect of treating Krebs 11 ascites tumor cells with interferon on the abilityof extracts prepared from them to catalyze the translation of a variety of messengerRNAs was investigated. The following results were obtained : (1) Extracts of un-treated cells translated endogenous mRNA, as well as exogenously added syntheticmRNA [poly(U)l, cellular mRNA (Krebs cell and L cell), and viral mRNA (reovirusand vaceinia virus) . By contrast, extracts of Krebs cells treated in the ascitic formwith interferon translated endogenous mRNA, exogenously added cellular mRNAand poly(U) as efficiently as extracts of untreated cells, but they translated viralmlLNAs very poorly (less than 10% as efficiently as extracts of untreated cells) . Thus,the ability to discriminate between cellular and viral mRNAs that is characteristicof whole cells was also exhibited by these cell-free extracts . (2) Virus infection was notrequired for the development of the interferon-induced inhibition of viral mRNAtranslation . (3) Mixing experiments indicated that the inability of extracts of inter-feron-treated cells to cat alyze the translation of viral mRNAs was due to the presenceof an inhibitory factor(q) rather than to the absence of a required factor(s) . (4) Theinhibitory factor(s) was associated with ribosomes, and could be dissociated fromthem by washing with KC1 . (5) Polyacrylamide gel electrophoresis revealed the pres-ence of a 48,000 dalton polypeptide in the 0.3-0 .6 M KCl wash fraction of ribosmmesprepared from interferon-treated cells that was not detectable in the correspondingwash of ribosomes prepared from untreated cells . This fraction inhibited the trans-lation of viral mRNAs in cell-free extracts of untreated cells more than any othersalt wash fraction .

These results suggest that the antiviral activity of interferon is mediated, atleast in part, by a ribosome-associated polypeptide that permits discrimination be-tween cellular and viral mRNAs .

INTRODUCTIONInterferons inhibit the multiplication of

both DNA and RNA viruses (VWek, 1969 ;DeClercq and Merigan, 1970 ; Colby andMorgan, 1971 ; Kleinschmidt, 1972). Sinceinterferon-mediated inhibition of virus mul-tiplication requires both host cell messengerRNA synthesis (Taylor, 1964) and proteinsynthesis (Levine, 1964 ; Friedman andSonnabend, 1964), the mode of action ofinterferon may involve a derepression of thehost genome that ultimately results in thebiosynthesis of specific protein(s) whicheither directly or indirectly inhibit virus

Copyright © 1974 by Academic r're , Lm .All rights of reproduction in any form reserved .

476

growth. This protein has so far remainedelusive .

Interferon does not prevent virus adsorp-tion to cells, penetration into cells, uncoatingwithin cells, or release from cells (Finter,1966 ; DeClercq and Merigan, 1970) . Fromtheoretical considerations, it was suggestedsome time ago that the primary biochemicalprocess affected by interferon would be thefirst anabolic stage common to the growthcycles of both DNA and RNA viruses,namely the translation into protein of thegenetic information contained in the parentalviral genorne (Joklik, 1965) . For vaccinia

virus (Joklik and Merigan, 1966 ; Metz andEsteban, 1972 ; Jungwirth et al ., 1972; Bodoet al ., 1972; Esteban and Metz, 1973), cow-pox virus (Jungwirth et al ., 1972; Bodo et al .,1972), mengovirus (Levy and Carter, 1968),and Semliki Forest virus (Friedman, 1968),viruses representative of both DNA andRNA viruses, it has indeed been shown thatvirus-directed protein synthesis is inhibitedin vivo in interferon-treated cells while viralRNA synthesis is unaffected ; however therehave also been several reports that viralRNA synthesis is the primary target (Bialyand Colby, 1972; Marcus et al ., 1971 ; Oxmanand Levin, 1971) . Inhibition of viral RNAsynthesis has not yet been demonstratedin vitro, but inhibition of viral mRNAtranslation has ; thus extracts of interferon-treated mouse L and ascites tumor cells havebeen shown to be incapable of translatingencephalomyocarditis (EMC) RNA, mengo-virus RNA and rcovirus RNA (Friedmanet al ., 1972a, b; Falcoff et al ., 1972, 1973 ;Gupta et al ., 1973). However, these extractsalso failed to translate added cellular RNAto any significant extent .

We report here the results of an investiga-tion of the effect that interferon treatmenthas on the ability of cell-free protein syn-thesizing systems prepared from Krebs IIascites tumor cells to catalyze the translationof viral (reovirus and vaccinia virus), cellu-lar (ascites and L cell), and synthetic [poly(U)] messenger RNAs . 'lie reasons forchoosing this system were primarily the factthat vaccinia virus and especially reovirusmRNAs can readily be prepared in vitro inlarge amounts and are well characterized(Joklik, 1973 ; Shatkin, 1971), that cell-freeextracts of Krebs II ascites cells catalyzeefficiently the translation of reovirus mRNAinto known viral polypeptides (McDowellet al., 1972), and that Krebs II ascites cellsare available in large quantities and aresensitive to interferon (Wagner and Huang,1966) .

MATERIALS AND METHODS

Materials. L-[G'H]Phenylalanne, 7 .79Ci/mmole, was purchased from New Eng-land Nuclear ; r.-[4,53Hz]leueine, 53 Ci/mmole, from Schwarz/Mann ; creature phos-

MECHANISM OF ACTION OF INTERFERON

477

phate, creatine phosphokinase, DTT' andpoly(U), from Sigma ; ATP, GTP, CTP, andUTP, from either Sigma or Schwarz/Mann;Sephadex G-25, from Pharmacia ; filter papercircles (25 mm), from Schleicher & Schuell ;protein (crystallized bovine albumin) stand-ard solution, from Armour ; Eagle's MEAT,Eagle's MEM (Joklik's modification), andfetal calf serum, from Grand Island Biologi-cal Co . ; and female albino mice (strain CDI ),from Charles River Breeding Labs. Rat livertRNA was either purchased from GeneralBiochemicals or prepared as previouslydescribed (Samuel el at., 1973) . Poly(U)-cellulose was prepared as described bySheldon et al . (1972) . Clostridium pasteuria-num mRNA, prepared as described byStallcup and Rabinowitz (1973), was thegift of Dr. J . C . Rabinowitz, Department ofBiochemistry, University of California,Berkeley. The original line of Krebs IIascites tumor cells was kindly provided byDr. A. T. H . Burners, Sloan-Kettering In-stitute for Cancer Research, New York .

Growth of cells. Mouse L and clone L929fibroblast cells were grown in suspensionculture in Joklik's Modification of Eagle'sMEM containing 5 % fetal calf serum. Krebsascites tumor cells were maintained in vivoby injection of 0 .15 ml of ascitic fluid into theperitoneal cavity of female CD1 mice; thecells were passed every 7 days .

Interferon treatment . Krebs cells in theascitic form were treated with interferon byinjection of partially purified interferon intothe peritoneal cavity of mice 6 days afterpassage. About 8 hr later, an identical seconddose of interferon was administered, andcells were collected 18 hr later. Control micewere treated with a volume of phosphatebuffer equal to that of interferon adminis-tered. Unless otherwise stated, the dose ofinterferon administered was 1 X 10° unitsper animal per treatment .

Preparation of cell free extracts . Cell-freeextracts of Krebs cells were prepared by a

I The abbreviations and trivial names usedare : DTT, dithiothreitol ; IIEPES, V-2-hydroxy-ethylpiperazine \N'-2-ethanesulfonic acid ; poly(U), polyuridylic acid ; poly(A), polyadenylicacid ; SOS, sodium dodecyl sulfate ; TCA, tri-chloroacetic acid ; PFU, plaque-forming units ;EDTA, ethylenediaminetetraacetic acid .

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SAMUEL AND JORLIK

method based on that described by Mathewsand Korner (1970) . All operations, unlessotherwise stated, were carried out at 4' .Liquid tumors were collected from mice,filtered through cheesecloth into cold isotonicbuffer (Tris Cl buffer, 35 mM, pH 7 .5, con-taining 146 mM NaCI and 11 mM glucose),and washed five times by differential cen-trifugation at 65 g for 5 min in isotonicbuffer. After centrifugation at 700 g for 10min, the cells were suspended in threepacked-cell volumes of hypotonic buffer(HEPES, 10 mM, pH 7 .5, 15 rn11M KCI,1.5 mM Mg(OAc)2 , and 1 mM DTT),allowed to swell 10 min, and disrupted withabout 30 strokes of a tightly fitting Douncehomogenizer. The extract buffer was im-mediately adjusted to a final concentrationof 20 mM HEPES, pH 7 .5, 5 mM Mg(OAc)2 ,120 mJ1 KCI, and 1 mM DTT. The homoge-nate was centrifuged at 5000 g for 5 min,the pellet discarded, and the supernatantsolution centrifuged at 10,000 g for 20 min .For nonpreincubated extracts, the resultingsupernatant solution, the S10 fraction, wasstored at -80° in small polypropylene vialswithout further treatment. For preincubatedextracts, the 810 fraction was supplementedwith 1 mM ATP, 0.2 mill GTP, 10 mMcreatine phosphate, and 0 .2 mg/ml creatinephosphokinase, and incubated at 37 ° for40 min ; it was then centrifuged at 10,000 gfor 10 min, and stored in small aliquots at-80° . Where noted, the 810 fractions werepassed through Sephadex G-25 equilibratedwith 20 mM HEPES, pH 7.5, 5 mM Mg-(OAc)2 , 120 mill' KCI, and 1 mM DTT . Theprotein concentrations of these fractionsranged from 8 to 14 mg/rnl .

Preparation of ribosome and cell-sap frac-tions . Ribosome and cell-sap fractions wereseparated by centrifuging S10 fractions for2.5 hr at 100,000 g . For the construction ofprotein synthesizing systems consisting ofcrude ribosomes from interferon-treated cellsand cell-sap from control cells, and viceversa, the ribosomal pellets were resuspendedin heterologous cell-sap ; control systemswere prepared by resuspending ribosomalpellets in homologous cell sap . After resus-pension, the reconstituted systems wereclarified by low-speed centrifugation andstored in small portions at -80° .

For the preparation of (a) salt-washed,preincubated ribosomes, (b) ribosome-asso-ciated factors released by washing with highsalt, and (e) cell-sap fractions, nonpreincu-bated S10 fractions were centrifuged at100,000 g for 2 .5 hr. The supernatant solu-tion (the 5100 fraction) was removed, passedthrough a column of Sephadex G-25, andstored in small portions at -80° . The cruderibosome pellet was resuspended in 25 m117Tris CI, pH 7.5, 10 mM Mg(OAc) 2 , 1 .0 MKC1 and 1 mM DTT, and stirred slowlyovernight (10 hr) at 4° . The suspension wasthen centrifuged for 10 min at 10,000 g, thepellet was discarded, and the salt-washedribosomes were collected by centrifuging for5 br at 100,000 g. The supernatant solution,which contained released ribosomal com-ponents, was dialyzed against 25 mM TrisCl, pH 7.5, 1 .0 roil Mg(OAc)2 , 50 mM KCI,1 mM DTT and 5 % glycerol, and stored at-80° in small aliquots . The pellet containingthe salt-washed ribosomes was resuspendedin 25 mill Tris •CI, 1 .5 mM Mg(OAc),,50 mM KCl and 1 mM DTT, centrifuged at100,000 g for 3 .5 hr, resuspended in the samebuffer and clarified by low-speed centrifuga-tion . The salt-washed ribosomes were thenpreincubated in a reaction mixture contain-ing the same components as a normal proteinsynthesis assay mixture (see below) exceptfor the following modifications : unlabeledamino acids were substituted for radioac-tively labeled amino acids, and ribosomal-wash factors and exogenous mRNA wereomitted. The mixture was incubated at 37 °for 30 min, then chilled and clarified by low-speed centrifugation. The salt-washed, pre-incubated ribosomes were then recovered bylayering them over 10 mM Tris-Cl, pH 7 .5,26% sucrose, 4 .5 mM Mg(OAc),, 50 mMKCl and 1 mM DTT, and centrifuging over-night at 85,000 g . The pellet was resuspendedin the above buffer minus sucrose, clarified,and stored in small aliquots at -80° .

Protein determination . Protein was esti-mated either by a modification of the Lowryphenol reagent method as described bySutherland et al . (1949) or spectrophoto-metrically (Layne, 19.57) .

Preparation of inlerferon . Mouse inter-feron was prepared in confluent aged mono-layer cultures of 1,929 cells (Lai and Joklik,

1973) infected with Newcastle disease virus(10 PFU/cell) . After a 1-hr adsorptionperiod the virus inoculum was removed andthe cells were incubated for 20 hr at 37° inserum-free Eagle's MEM. The medium wasthen collected, centrifuged at 15,000 g for8 hr and the interferon was precipitated with0.02M zinc acetate at pH 6 (Lampson et al .,1963) . The precipitate was dissolved by theaddition of 0 .2 N HCl to pH 2.5, and thesolution then extensively dialyzed firstagainst 0.01 M phosphate, pH 7.2, 0 .1 b1NaCl and 0.01 3 EDTA, and then against0.01 M phosphate, pH 7 .2. The interferonpreparation was then irradiated with UV-light for 5 min at a distance of 30 cm withtwo 15 W G . E . germicidal lamps, and thenstored at -80° in polypropylene tubes . Ithad a specific activity of about 2 X 10" units(equivalent to about 6 X 10 5 IU) per milli-gram of protein as determined by a 50 %plaque reduction assay on L929 cell mono-layers infected with vesicular stomatitis virus(Lai and Joklik, 1973) .

Preparations of messenger RNA . (a) ViralmRNAs. Reovirus mRNA was prepared asthe in vitro transcription product of cores ofDearing strain virions (Skchel and Joklik,1969 ; McDowell et al ., 1972). After incuba-tion at 37°, reovirus cores were removed fromthe reaction mixture by centrifuging at30,000 g for 30 min ; the supernatant solutionwas extracted with 1 volume of redistilledphenol, and then with freshly opened ether,and the RNA was precipitated by the addi-tion of 0.2 volume of 10 M LiCI . After stand-ing at 4 ° , the RNA precipitate was collectedby centrifugation and further purified byLiCl and ethanol precipitations as previouslydescribed . Electrophoresis in polyacryl-amide-agarose-urea gels (Floyd et al ., 1974)revealed that, the final mRNA preparationcontained approximately equal amounts ofthe 1, in, and s molecular species ; the relativemolar ratios of the 1, m, and s mRNA classeswere therefore about 1 :2 :4 .

For the preparation of vaccinia mRNA,the WE strain of vaccinia virus was grownas described by Becker and Joklik (1964)and purified as described by Joklik (1962) .The preparation of vaccinia virus cores andsynthesis in vitro of vaccinia virus mRNAwas essentially as described by Kates and

MECHANISM OF ACTION OF INTERFERON 179

Beeson (1970) ; vaccinia virus mRNA syn-thesized in vitro was purified from the reac-tion mixture essentially as described abovefor reovirus mRNA .

(b) Cellular mRNAs . Cellular mRNA wasprepared from polyribosomes of mouse Lfibroblasts or Krebs ascites tumor cells andpurified by chromatography on poly(U)-cellulose. Cells were rapidly chilled by theaddition of frozen crushed MEM, collectedby centrifugation, washed with isotonicbuffer (see above), suspended in hypotonicbuffer (see above) to a concentration of about5 X 10' cells per ml, allowed to swell 5 rainat 4°, and disrupted with 10 strokes in aDounce homogenizer. After centrifuging for3 min at 1000 g and 10 min at 10,000 g,sodium dcoxycholate was added to theresultant S10 fraction to a final concentrationof 0.5 % ; polyribosomes were then isolatedby centrifuging it through 40 % sucrose in20 mM Tris • CI, pH 7.6, 50 mM KCI, and2 mM MgC12. The polyribosomel pellet wasresuspended in 10 mM Tris . CI, pH 7.4,0.1 M NaCl and 1 mM EDTA, and extractedwith 1.5 volumes of chloroform : phenol (1 :1v/v) saturated with 10 mM sodium acetate,pH 6.0, 0.1 M NaCl and 1 mlM EDTA(Perry et al ., 1972) . After low-speed cen-trifugation, the aqueous phase was removedand extracted again with chloroform : phenol .RNA recovered from the aqueous phase byethanol precipitation was applied to a col-umn of poly (U) -cellulose equilibrated at 4 °with 10 mM Tris Cl, pH 7.4, 0.1 M NaCl .After washing with this buffer at 4 ° , hy-bridized poly(A)-containing RNA was elutedat 37° with 10 mM Tris • Cl, pH 7.4 .

Assay of in vitro protein synthesis . In vitroprotein synthesis was measured by determin-ing the incorporation of radioactively labeledamino acid, either ['H]leucine or ['H]phenyl-alanine, into acid-insoluble material . Thestandard reaction mixture (50 µl or 100 µlunless otherwise indicated) contained thefollowing ingredients : 30 mM Tris • Cl orHEPES, pH 7 .5; 96 mM KC1; Mg(OAc)2 ,3-4.5 mM for endogenous and exogenousviral mRNAs, and 9-11 mM for poly(U) ;1 mM ATP ; 0.2 mM GTP ; 0.6 mM CTP ;1 mM DTT ; 10 mM creatine phosphate ;0.2 mg/ml creatine phosphokinase ; 0.4mg/ml rat liver stripped tRNA ; 19 unlabeled

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SAMUEL AND JOKLIK

amino acids, 0 .1 trill each ; either 60 gCi/mlL-[4 .5 'H,]leucine or 40 pCi/mil L-[G 'H]-phenylalanine as indicated ; either no exoge-nously added mRNA, or 100 pg/ml of reo-virus mRNA, or 50 #g/ml of vacciniamRNA, or 200 pg/ml of poly(U) as indi-cated; and varying amounts of either S10fraction or salt-washed preincubated ribo-somes, ribosomal salt wash and cell sap asindicated. The assay reaction mixture waskept on ice during its assembly. After incuba-tion at 37 ° fir the amount of time indicated,aliquots of 20-80 pl were removed and ad-sorbed into filter paper disks, which weredropped into cold 10 % TCA and subse-quently treated with 5 % TCA at 90° for 15min followed by two washes in 5 % TCA at 4°,one gash in et}lanol :eLiter (1 :1), and one

wash in ether (Moll, 1969) . The dried filterdisks were then placed into toluene contain-ing 2 , 5-diphenyloxazole (0 .45c) and 1, 4-bis[2 - (5 - phenyloxazolyl)]benzene (0 .01 %)and the radioactivity on them was measuredin a Beckman LS-250 liquid scintillationspectrometer .

Conditions for electrophoresis . Standardconditions for discontinuous SDS-slab poly-acrylamide gel electrophoresis were essen-tially as described by Laemmli (1970) andmodified by Studier (1973) . The running gelwas routinely 12.5 % acrylamide, and thestacking gel 5 .0 % acrylamide . Electrophore-sis was carried out at 15 mA per slab forabout 5 hr. After electrophoresis gels werefixed in 107o TCA containing 25% isopro-panol for 1 hr at morn Lemperature, stained

100

200

300

5

2

30.a PROTEIN

MINUTESFro. 1 . Effect of interferon treatment on the ability of nonpreincubated 510 fractions of uninfected

and infected Krebs ascites cells to translate endogenous mRNA and added reovirus mRNA . (A) and (C)Dependence on the amount of extract . (B) and (D) kinetics of ['H]lcucine incorporation . Reaction timefor (A) and (C), 45 min at 37° ; reaction volume 50 At ; 40-v1 aliquots were processed . Amount of extractin (B) and (D), 200 pg protein per 50 µl reaction mixture ; 20-Al aliquots were processed . Round symbols,untreated cells ; triangles, interferon-treated cells; open symbols, no mRNA added (endogenous activ-ity) ; filled symbols, reovirus mRNA (100 µg/ml) added . (A) and (B) Uninfected cells ; (C) and (D),cells infected with reovirus intraperitoncally for 4 hr .

in 10 % TCA containing 25 % isopropanoland 0.1 % Coomassie Brilliant Blue for 1 hrat room temperature, and destained in 7 `!acetic acid .

RESULTSEffect of Interferon Treatment on Protein

Synthesis in S10 Fractions

S10 Fractions of Krebs ascites tmnor cellsprepared either from untreated cells or from

fa50

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axM


M0

a 2C-

INFECTED,INTERFERON

p

1waU- 15-aaOuz,,, 10

pg PROTEIN

481

cells treated in the ascitic form with inter-feron were examined for their ability tocatalyze the translation of various types ofmRNA.

Nonpreincubated 810 Fractions

The effect of interferon treatment on thetranslation of endogenous host cell mRNAwas measured by determining the extent of[aH]leucine incorporation catalyzed by S10

Fia . 2 . Effect of interferon treatment on the ability of preincubated S10 fractions of Krebs ascitescells to translate poly(U) . (A) Dependence on the amount of extract ; (B) kinetics of [aH]phenylalanineincorporation. Poly(U), 200 pg/ml . Reaction time for (A), 45 min at 37° ; reaction volume, 50 pl ; 40-p1

aliquots were processed . Amount of extract in (B), 90 pg protein per 50 id reaction mixture ; 20-p1 ali-quots were processed. Round symbols, uninfected cells ; triangles, cells infected with reovirus for 4 hr .Open symbols, untreated cells ; filled symbols, cells treated with interferon intraperitoneally . Fullcurves, poly(U) present ; broken curve, poly(U) absent .

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S.A-IUEL AND JOKLIK

fractions that had not been preincubated . Asshown in Fig. 1, interferon treatment hadlittle or no effect on the translation in vitro ofendogenous cellular mRNA in extracts ofuninfected cells or of cells infected for 4 hrwith reovirus (in which most of the endoge-nous mRNA was still cellular) . The. additionof reovirus mRNA to these nonpreincubated

FIG . 3 . Effect of interferon treatment on theability of preincubated 810 fractions of unin-fected Krebs ascites cells to translate reovirusm11NA. (A) Dependence on the amount of S10fraction . Reovirus mRNA, 100 pg/ml ; reactiontime, 45 min at 37 ° ; reaction volume, 50 pl ; 40-p1aliquots were processed . Round symbols, un-treated cells ; triangles, interferon-treated cells .Open symbols, reovirus mRNA absent ; filledsymbols, reovirus mRNA present . (B) Kinetics of['Rlleucine incorporation . Conditions and sym-bols as for (A) ; concentration of extract, 350 Mg

protein per 50 pl reaction mixture ; 2O-µ1 aliquotswere processed .

extracts failed to stimulate incorporation toany significant extent (Fig . 1) .

Preincubated SbO FractionsPreincubation of S10 fractions at 37 ° for

40 min reduced the endogenous activity bymore than 90% . Such preincubated extractswere then tested for their ability to translateexogenously added synthetic, viral andcellular mRNAs .

Translation of poly(U) . Interferon treat-ment of either uninfected or reovirus-infectedKrebs cells had no significant effect on theability of SiO fractions to catalyze the trans-lation of poly(U) (Fig. 2) . The optimum

a eau0~ 6aa0a0 4uZwZ

2J

u 0

Wrn0a0 z

z_Z

Wz

0 tWJ

zMJ

eIIIIII10 20 30 45 60

MINUTES

FIG. 4 . Effect of interferon treatment on theability of preincubated SlO fractions of Krebsascites cells infected with reovirus for 4 hr totranslate reovirus mRNA . (A) Dependence onthe amount of S10 fraction ; (B) kinetics of ['IIl-leucine incorporation. Conditions and symbolsas for Fig . 3 .

A

0

0

B

100

200

300PROTEIN (pq)

75I90

400

Mgg+ concentration for the translation ofpoly(F) in S10 fractions of both control andinterferon-treated cells was 9-11 mM .

Translation of viral mRNAs . ReovirusmRNA stimulated the incorporation of ['H]-leucine into protein severalfold above theendogenous level when added to preincu-bated S10 fractions prepared from untreated,but not from interferon-treated cells . Reo-virus mRNA-stimulated protein synthesiswas dependent both upon the amount of ex-tract used to catalyze the reaction (Fig . 3A)and upon the duration of incubation (Fig .3B). In similar manner, extracts preparedfrom cells infected intraperitoneally with

TABLE 1

EFFECT OF INTERFERON DOSAGE ON THE ABILITYof PREINCUBATED 810 EXTRACTS TO CATALYZE

THE TRANSLATION OF REOVIELS mRNA

° Injected intraperitoneally .Concentration of reovirus mRNA, 100 mg/ml .

Reaction time, 60 min at 37° . The backgroundincorporation due to the translation of residualendogenous message was subtracted .

m0-20


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AREOVIRUS

0 40 60RNA (pg/ml)

e0

reovirus for 4 hr were much more active incatalyzing reovirus mRNA-stimulated ['H]-leucine incorporation than extracts of suchinfected cells which had been treated withinterferon (Fig. 4) . The optimum concentra-tion of Mg''* for the translation of reovirusmRNA was 3-5 mM, that is, about one-third of that for the translation of poly(U) .The diminished ability of preincubated S10fractions prepared from interferon-treatedcells to catalyze the translation of reovirusmRNA was observed at all [Mg' ] tested .

The effect of interferon dosage on the abil-ity of preincubated S10 fractions to catalyzethe translation of reovirus mRNA is shownin Table 1 . Leucine incorporation in responseto exogenously added reovirus mRNA wasreduced to less than 10 /o of the normal levelby the administration of 1 X 101 units ofinterferon per animal . With this dose of inter-feron neither endogenous mRNA norpoly(U) translation was signifcantly in-hibited (,Figs . 1 and 2) .

Vaccinia virus mRNA stimulated leucineincorporation as efficiently as reovirusToRNA in preincubated S10 fractions fromuntreated cells, but, like reovirus mRNA, itfailed to do so in S10 fractions from inter-feron-treated cells. Fig . 5 compares thetranslation of various concentrations ofthese two viral niRNAs in preincuhated S10fractions of untreated and interferon-treatedKrebs cells : the efficiency of translation ofboth reovirus (Fig. 5A) and vaccinia virus

a40- VACC.NIA

-INTERFERON

+INTERFERON

100 0

20

40

60

e0

100RNA (go/ml)

FIG . 5 . The translation of reovirus and vaccinia virus mRNA by preincubated 810 fractions of un-treated and interferon-treated Krebs cells. 320 pg protein per 50 µI reaction mixture ; reaction time, 45

min at 37° . Background incorporation due to residual endogenous mRNA was subtracted . Round syno-hols, untreated cells ; triangles, interferon-treated cells. (A) Reovirus mRNA ; (B) vaccinia mRNA.

Amount of interferonadministered°

(units)

[3H]Leucine incorporatedin response to reovirus

mRNAM (cpm)

0 653110 61 ,15

100 57381,000 1129

10,000 466

484

(Fig. 5B) mRNA in the latter was onlyabout 10 % of that in the former .

Translation of cellular mRNAs . L cell andKrebs cell mRNAs were isolated by passingRNA associated with polyribosomes throughpoly (U)-cellulose columns and isolating thoseRNA species that were retained . SuchmRNA preparations, both from L and Krebscells, were translated in S10 fractions of in-terferon-treated Krebs cells nearly as effi-ciently as in S10 fractions of untreated Krebscells (Fig . 6). These results with cellularmRNAs should be compared with those con-cerning viral mRNAs (see above) . The S10fractions clearly possessed the ability todiscriminate between viral and cellularmRNAs :S10 fractions of untreated Krebscells translated both added cellular and viraltnRNAs, while S10 fractions of interferon-treated cells only translated cellular mRNAs .Messenger RNA from the bacterium, C .

pasteurianum, was not translated to anysignificant extent by preincubated S10 frac-tions from either untreated or interferon-treated Krebs cells .

Cellular Localization of the Interferon-InducedInhibitor of Viral mRA'A Translation

Crude reconstituted 810 fractions . In orderto determine whether the interferon-inducedinability to translate viral mRNA was asso-ciated with the ribosome fraction or the solu-ble fraction of the protein synthesizing sys-

SAMUEL AND JORLIK

tcm, hetcrologous systems consisting of crudeunwashed ribosomes from one type of cell(untreated or interferon-treated) and 5100fractions from the other were constructedand examined for their ability to stimulateleucinc incorporation in response to reovirusmRNA and phenylalanine incorporation inresponse to poly(U) . As shown in Fig. 7, thecontrol homologous reconstituted S10 frac-tion prepared by combining the ribosomeand 5100 fractions from an untreated S10fraction was active in translating reovirusmRNA. The heterologous reconstituted S10fraction consisting of the ribosome fractionfrom untreated cells and the 5100 fractionfrom interferon-treated cells was about two-thirds as efficient, while the heterologousreconstituted S10 fraction consisting of theribosome fraction from interferon-treatedcells and the 5100 fraction from untreatedcells was only about one-third as efficient . Bycontrast, the ability of all these systems tobe stimulated by poly(U) was identical (notshown) . These results suggested that thefailure to translate reovirus mRNA was as-sociated predominantly with the ribosomefraction of interferon-treated cells .

Reconstituted systems consisting of washedribosames, ribosomal salt wash, and 8100 frac-tion. 810 fractions of interferon-treated andcontrol cells were fractionated into salt-washed preincubated ribosomes, factors dis-sociated from ribosonies by washing with

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50RNA (/i0/ml)

RNA ('p/m1)

Fra. 6 . The translation of L cell and Krebs cell mRNAs by preincubated S10 fractions of untreatedand interferon-treated Krebs cells . For preparation of the cellular ruRNAs see Materials and Methods .320 pg protein per 50 pl reaction mixture ; reaction time, 45 min at 37' . Background incorporation due toresidual endogenous mRNA was subtracted . Round symbols, untreated cells ; triangles, interferon-treated cells. (A) L cell mRNA ; (B) Krebs cell mRNA .

s


FIG. 7 . Kinetics of reovirus mRNA-directedstimulation of ['H]leucine incorporation catalyzedby reconstituted 810 fractions containingribosomes and 5100 fractions derived from eitheruntreated or interferon-treated Krebs cells . Forexperimental details see -Materials and Methods .270 µg protein per 50 Al reaction mixture ; con-centration of reovirus mRNA, 100,ug/ml . Extractreconstituted by combining : crude ribosomesfrom untreated cells and 8100 fraction from un-treated cells (0-0); crude ribosomes fromuntreated cells and 5100 fraction from interferon-treated cells (D-D) ; crude ribosomes frominterferon-treated cells and 5100 fraction fromuntreated cells Broken curve : noreovirus mRNA added .

1 M KCI, and S100 fractions (see Materialsand Methods) . Heterologous combinations ofthese fractions derived from untreated andinterferon-treated cells were then tested fortheir ability to translate reovirus and vac-cinia virus mRNA . As shown in Table 2, theinability of extracts of interferon-treatedcells to translate viral mRNA was traced tothe fraction that could be dissociated fromribosomes by washing with 1 M KCI .

Demonstration of an inhibitory factor(s) .The above results suggested that a factorseparable from ribosomes by washing withI M KC1 is either absent in extracts of inter-feron-treated cells or that ribosomes in inter-feron-treated cells were associated with afactor(s) that inhibited protein synthesis . Inorder to discriminate between these two al-ternatives, mixing experiments were per-formed : to reaction mixtures containing con-stant amounts of salt-washed preincubatedribosomes and 5100 fractions from untreated

485

cells, mixtures of varying amounts of ribo-somal salt wash fractions from untreated andinterferon-treated Krebs cells were added,and the ability of these reaction mixtures totranslate reovirus mRNA was determined(Fig . 8) . The results indicated not only thatthe amount of ['H]leucine incorporated was afunction of the concentration of the ribo-somal salt wash fraction from untreated cells(with an optimum at about 500 µg protein/ml), but that the salt wash of ribosomes frominterferon-treated cells strongly inhibitedreovirus mRNA translation . The inability ofribosomes from interferon-treated cells totranslate viral mRN A is thus not due to thedeficiency of a factor(s), but to the presenceof an inhibitory factor(s) . This conclusion isin accord with that reached by Gupta el al.(1973) using Ehrlich ascites tumor cells andFalcoff et al. (1973) using L cells .Effect of ribosomal salt wash concentration

from interferon-treated cells on the translationof cellular and viral tsRNA . The results de-scribed so far indicate that the ribosomal saltwash fraction from interferon-treated cellssupports the translation of cellular mRNAs,but not only does not support that of viralmRNAs, but actually inhibits it . The con-centration dependence of these effects wasinvestigated next. The results (Fig. 9) indi-cated that the optimal concentration of thisfraction for the translation of Krebs cellmRNA was about 700 ug protein/ml, when[$H]leucine incorporation was stimulatedsome 400-500 %. By contrast, this fractionwas unable to stimulate [VH]leucine incor-poration in response to reovirus mRNA atany concentration ; the small increase thatwas observed was also obtained in the ah-sence of any added reovirus mRNA .

Identification of the difference in the proteincomposition of ribosomal salt wash fractionsfront untreated and interferon-treated Krebscells. Experiments were carried out to iden-tify differences in the protein complementsof the ribosomal salt wash fractions from un-treated and interferon-treated Krebs cells bymeans of discontinuous SDS-polyacrylamidegel electrophoresis . S10 fractions were pre-pared in 0.05 M KCl (instead of the usual0.12 M KC1), and the ribosomes were col-lected by centrifugation . After washing in0.05 M KU, the ribosomes were washed

480

TABLE 2

EFFECT OF INTERFERON TREATMENT ON THE ABILITY OF SALT-WASIIED PREINCUBATED RIBOSOMAS,RIBOSOMAL SALT WASH, AND 5100 FRACTIONS TO TRANSLATE REOVIRUS AND VACCINIA VIRUS

,nRNAs'

The assay reaction conditions were as described in Materials and Methods for the standard proteinsynthesis assay . Reaction mixtures contained in a total volume of 100 µl :3 .0 At6o units of salt-washedpreincubated ribosomes, 50 pg of ribosomal salt wash protein, and 200 pg of high-speed 8100 fractionprotein. The concentrations of reovirus and vaceinia m U.NA were 65 and 35 pg/ml, respectively .

0XEa

~ 6

U 4

w

o .-IIIIIIJ 0

20

40

60

s0AMOUNT OF R1sOSOMAL SALT WASH PROTEIN

FROM UNTREATED CELLS(,ug /oJ MI)

FIG . 8 . Effect of varying the eonecntlations ofribosomal salt-wash fractions from untreatedand interferon-treated Krebs cells on the transla-tion of reovirus mRNA . Salt-washed preincubatedribosomes and 8100 fractions from untreatedcells were used . Except for the amount ofribosomal salt wash fractions added, conditionswere as fin Table 2 . 40-Ml aliquots were processed .The number above each curve indicates theamount (in pg protein) of ribosomal salt washfraction from interferon-treated cells ; the amountof ribosomal salt wash protein from untreatedcells present in each reaction mixture is indicatedon the abscissa .

successively for 30 nun at 4° in buffer (seeMaterials and Methods) containing 0 .07,0.17, 0 .3, 0 .6, and 1 M KC1 . The polypeptidecomplement of each wash fraction was then

SAMUEL AND JOKLiK

NONE

u

_111l110

20

40

60

so

100CONCENTRATION OF RIBOSOMAL SALT WASH FRACTION

FROM INTERFERON-TREATED CELLS(Ag PROTEIN /Q1 r1)

FIG. 9. Concentration dependence of the ri-bosomal salt wash fraction from interferon-treated Krebs cells for the translation of Krebscell mRNA and reovirus mRNA . The reactionmixture was the same as for the experiment de-scribed in Table 2, using salt-washed preincubatedribosomes and 5100 fractions from interferon-treated cells . The concentrations of mRNA addedwere : 0, none (endogenous activity ; ; •, Krebscell mRNA, 35 pg/mi ; A, reovirus roRNA, 100pg/oil .

ascertained by electrophoresis in SDS-poly-acrylarnide gels : each fraction was also as-sayed for its ability to inhibit the translationof reovirus mRNA using the test systemdescribed in Table 2 . As shown in Fig . 10, apolypeptide is clearly detectable in the 0.3-0.6 M KCI and 0.6-1 .0 M KCI salt washfractions of ribosomes from interferon-

Origin of

8100 Frac on

[3H]T .cucine incorporated inresponse to

Salt-washedribosomes

Ribosomal saltwash fraction

Reovirus mRNA Vaccinia mRNA

Cpm % Cpm %

Untreated cells Untreated cells Untreated cells 19,346 100 10,178 100Interferon-treated Untreated Untreated 16,841 87 7,649 75Untreated Interferon-treated Untreated 5,126 27 1,704 17Untreated Untreated Interferon-treated 17,765 92 6,489 64Interferon-treated Interferon-treated Interferon-treated 5,857 30 1,073 11Interferon-treated Untreated Interferon-treated 13,139 68 5,170 51

150 ----

72

34 -~

1

2

3

4

5

6

7FIG . 10. SDS-polyacrylamide gel patterns of ribosomal salt-wash fractions prepared from ribosomes

of untreated and interferon-treated cells . Ribosomes were washed as described in Materials and Methodsexcept for the following modifications : the wash buffer contained increasing concentrations of KCI asindicated ; the ribosomes were washed successively at the indicated salt concentrations for 30 min withstirring at 4°, then isolated by centrifuging for 1 .5 hr at 150,000 g ; each supernatant was then recen-trifuged at 150,000 g for 4 .5 hr before dialysis . Electrophoresis was carried out as described in Materialsand Methods; the direction of migration was from top to bottom in a 12.5% running gel with a 5.0%stacking gel . Gel A : (1) 6 .3 pg reovirus polypeptides ; (2) through ( 13), ribosomal salt-wash fractionsfrom untreated (even numbers) and interferon-treated (odd numbers) cells, respectively : (2) 12 .9 pg,and (3) 15 .3 pg, of 0 .05 M KCl wash ; (4) 8 .8 pg, and (5) 7 .6 , g, of 0 .05-0 .07 M KCl wash ; (6) 7 .4 pg, and(7) 7 .0 pg, of 0 .07-0 .17 M KC1 wash ; (8) 10 .0 pg, and (9) 8 .0 pg, of 0 .17 to 0 .30 M KCI wash ; (10) 12 .6mg, and (11) 12 .3 mg, of 0.30 to 0 .60 M KCI wash; (12) 10 .2 pg, and (13) 9 .8 mg, of 0 .60 to 1 .0 bf XCIwash. Gel B : (1) 6 .3 pg reovirus, (2) 8 .4 pg, (4) 16 .8 pg, and (6) 25 .2 pg of 0 .3-0 .6 M KCl wash fractionof ribosomes from untreated cells ; (3) 8 .2 pg, (5) 16 .4 pg, and (7) 24 .6 pg of 0.3-0 .6 M KCI wash fractionof ribosomes from interferon-treated cells . In gels Al and 131 only the major reovirus eapsid polypeptidesare visible (since only 6 .3 µg total polypeptide was electrophoresed) ; the three bands correspond topolypeptides a1 + A2, p2, and o3, and their molecular weights (in thousands) are indicated . The 48,000Dalton polypeptide, which is found only in interferon-treated cells, is indicated by the arrows .

487

488

SAMUEL AND JOKLIK

treated cells that is not discernible in theanalogous salt washes of ribosomes from un-treated cells (arrow) . The apparent molecu-lar weight of this polypeptide was about48,000 .

Samples of these ribosomal salt wash frac-tions were then tested for their effect on thetranslation of reovirus mRNA in the stand-ard protein synthesizing system. It wasfound that the salt wash fractions from inter-feron-treated ribosomes that contained thelargest amounts of the additional componentas judged by SDS-polyacrylamide gel elec-trophoresis (the 0.3-0.6 M KCl wash frac-tions) also possessed the greatest ability toinhibit the translation of reovirus mRNA(Fig . 11) .

5 005 007 017 030 060to to to to to007

0.17

o,w 060

1 .0RIBOSOMAL SAL' WASH FRACTION (M KCI)

Frc. 11. Effect of ribosomal salt wash fractionsfrom untreated and interferon-treated cells on thetranslation of reovirus mIINA. The assay con-ditions were as described in Materials and Meth-ods for the standard protein synthesis assay .Reaction mixtures contained in a total volume of100 µl:3.6 Am units of salt-washed preincubatedribosomes, 50 pg of 1 M KCI ribosomal salt wash,and 200 µg of S100 fraction, all prepared fromuntreated cells . In addition, the following amountsof ribosomal salt wash fraction from untreated(cross-hatched bar) or interferon-treated (hatchedbar) cells, respectively, were added (these weresamples from the same ribosomal salt wash frac-tion as were electrophoresed in Fig . 10) : 0.05 MKCI, 20 .4 gg and 17 .2µg ; 0 .05-0 .07 M KCI wash, 8 .8µg and 6 .4 Mg ; 0 .07-0 .17 M KCI wash, 7 .4 µg and 8 .4µg ; 0.17-0 .30 M KCI wash, 4 .0 pg and 3 .2 pg ; 0 .30-0.60 M KCI wash, 16 .8 pg and 16 .4 µg ; and 0 .60-1 .031 KCI wash, 8 .2 pg and 7 .8 pg . The concentrationof reovirus mRNA was 100 pg/ml ; 5927 epm of['H]leucine were incorporated in the absence ofadded ribosomal salt wash fraction from ribosomesof either untreated or interferon-treated cells .

DISCUSSION1. The results reported here demonstrate

that treatment of uninfected Krebs 11 ascitestumor cells with interferon has no effect onthe ability of S10 fractions prepared fromthem to translate endogenous message, syn-thetic message or exogenously added cellularmessage, but greatly decreases their abilityto translate viral message . These effects wereindependent of the [M&], an importantpoint since the [Mg2+] is known to influencedrastically the specificity of protein synthe-sizing systems . These results may be relatedto several reports in the literature concerningthe ability of extracts of interferon-treated 1,cells, as well as of Ehrlich and Krebs II as-cites tumor cells, to translate exogenouslyadded mRNAs . We find, for example, inagreement with Faleoff et al . (1972, 1973),that the effect of interferon manifests itselfin uninfected cells as well as infected cells ;this is in contrast to the results of Friedmanet at. (1972a,b), who reported an absoluterequirement for viral infection in order forinterferon to exert its protein synthesis in-hibitory activity . We also find, as did Falcoffet al . (1972, 1973) and Gupta et al . (1973),that the interferon-induced inhibition of thetranslation of exogenous mRNA is discerni-ble in preincubated systems. In nonpreincu-bated Krebs cell systems exogenous reovirusmRNA was essentially inert (or even inhibi-tory) as measured by ability to stimulate['H]leucine incorporation ; similar results hadbeen obtained with nonprcincubated rabbitreticulocvte cell-free extracts (McDowellei al ., 1972) . Preincubation of S10 extracts at37° for 40 thin reduced endogenous activityby more than 90% and permitted subse-quently added viral mRNA to stimulatelcueinc incorporation some 5- to 10-fold. Bycontrast, Friedman el al . (1972a,b) reportedthat the interferon-induced inhibition ofEMC RNA translation in L cells was de-stroyed when (tell-free extracts were preincu-bated. The source of the apparent incon-sistency of the effect that preincubation haseither on reversing or conserving the inhibi-tory effect of interferon treatment in vitro isnot obvious .

The most striking difference between ourresults and those reported so far is that ourS10 fractions were able to translate addedcellular mRNAs . S10 fractions from un-

treated cells translated both cellular mRNAs(Krebs and L cell) and viral mRNAs (reo-virus and vaccinia) ; 810 fractions from inter-feron-treated cells translated the cellularmRNAs equally well, but failed to translatethe viral mRNAs. The ability of intact cellsto discriminate between cellular and viralmRNAs is therefore retained by the Krebscells S10 fractions . By contrast, Falcoff el al.(1972, 1973) and Gupta et al . (1973) foundthat in extracts of interferon-treated L cellsand Ehrlich ascites tumor cells, respectively,the translation of added cellular mRNAs wasinhibited as strongly, or almost as strongly,as that of viral mRNAs . Whether the sourceof this difference lies in the protein synthe-sizing systems or in the mRNA preparationsis not clear .

2. The difference between the protein syn-thesizing systems from untreated and inter-feron-treated Krebs cells is associated withfactor(s) that can be removed from ribo-somes by washing with KCI, since, otherconditions being equal, their capacity totranslate viral mRNA depended on whetherthe factor source was untreated or interferon-treated cells . This difference was not due tothe absence of factor(s) that are normallypresent in untreated cells, but rather to thepresence of additional factor(s) in interferon-treated cells that are inhibitory . This pointwas also established by Gupta et al . (1973)in an Ehrlich ascites tumor system . In oursystem and that of Falcoff el at . (1973) thesefactor(s) could be separated from ribosomesby washing with concentrated salt solutions ;in the system of Gupta et al . (1973) they werereleased by incubation, No doubt this differ-ence reflects a difference in the cells thatwere used .

3. The factor(s) released by KCI fromribosomes of interferon-treated Krebs cellswas capable of discriminating between cellu-lar and viral mRNAs : it permitted or sup-ported translation of the former, but not ofthe latter. By contrast, Gupta et al . (1973)found that the corresponding factors of inter-feron-treated Ehrlich ascites tumor cellswere unable to discriminate between cellularand viral mRNAs . As pointed out above,this difference may lie either in the nature ofthe cellular mRNAs used or in the compo-nents of the cell-free protein-synthesizingsystem .


489

4. Proof that the ribosomal salt wash frac-tions of ribosomes from untreated and inter-feron-treated cells actually contained differ-ent proteins was provided by analyzing thepolypeptide composition of the protein mix-tures that were removed from ribosomes bywashing with salt solutions of gradually in-creasing concentration . Using an assay sys-tem based on the ability of these washes toinhibit the translation of reovirus mRNA byextracts of untreated Krebs cells, we foundthat those washes that possessed the highesttranslation inhibitory activity also containeda polypeptide with an apparent molecularweight of 48,000 that was not present in com-parable washes of ribosomes from untreatedcells. It is conceivable that it is this proteinthat is induced by interferon and that inhib-its the translation of viral mRNAs in inter-feron-treated cells .

5. Finally, it should be pointed out that itis not at all certain that the function of theinhibitory protein is prevention of the trans-lation of viral mRNAs. There is much evi-dence that interferon profoundly affects avariety of host functions ranging from multi-plication (Macieira-Goelho et al ., 1971 ;Greaser and Bourali-Maury, 1972 ; Lee et al .,1972; Frayssinet et al., 1973), the expressionof surface antigens (Lindahl ei al ., 1973), andthe response to mitogenic stimuli (Lindahl-11lagnusson et al., 1972; Rozee et al., 1973) tomodification of the immune response (Lin-dahl and Gresser, 1972; Hirsch et at., 1973) .It is conceivable that all these functions aremediated by mRNAs that share a certainsequence and therefore structural similarity ;that the primary function of interferon is toregulate the translation of these mRNAs ;and that many viral mRNAs share thisstructural feature and are therefore nottranslated in cells that have been exposed tointerferon. Further, it is likely that mRNAspossess this structural feature to varyingdegrees, which would explain why the trans-lation of some is more sensitive to interferonthan that of others (as has been shown forcertain viral mRNAs) . It is therefore offundamental importance not only to eluci-date the function of the 48,000 dalton ribo-some-associated protein, but also to discoverwhich mRNAs it prevents from beingtranslated .

490

SAMUEL AND JOKLIK

ACKNOWLEDGMENTS

This work was supported by Research GrantAI-08909 and Health Sciences AdvancementAward 5 804 RR 06148 from the National In-stitutes of Health and by Research ContractAT-(40-1)-3857 from the Atomic Energy Com-mission . C . E . S. was a Damon Runyon CancerResearch Fellow (DRF-809) . The expert technicalassistance of Mrs . Jail Polk is gratefully acknowl-edged .

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