Mechanism of interferon action Properties of an interferon-mediated ribonucleolytic activity from mouse L9z9 cells

89, 240-251

VIROLOGY

(1978)

Mechanism Properties

of an interferon-mediated

DEBORAH Section

of Biochemistry

of Interferon

Action

Ribonucleolytic

Activity

A. EPPSTEIN and Molecular California,

AND

CHARLES

from Mouse

E. SAMUEL

Biology, Department of Biological Santa Barbara, California 93106 Accepted

May

LSz9 Cells

Sciences,

University

of

10, 1978

The biochemical properties of an interferon-mediated ribonucleolytic activity isolated from mouse LcI, fibroblast cells were investigated. The following results were obtained: (1) The interferon-mediated nucleolytic activity was present in active form in interferontreated mouse LWX~ cells. Bio-Gel PI50 column fractions that contained the activated nuclease also contained a low molecular weight phosphorylated component (2 3509) labeled with ]‘“P]ATP (Eppstein and Samuel, 1977). A cryptic nucleolytic activity present in untreated LL)>:, cell-free extracts was activated by a low molecular weight effector molecule synthesized in the presence of double-stranded RNA and ATP by an enzyme preparation isolated from interferon-treated but not untreated cells. (2) The interferon-mediated nucleolytic activity was optimal at pH values between 6.0 and 6.5. Potassium chloride stimulated the enzymic reaction with maximal activity near 100 nuI4 KCl; high KC1 concentrations (2 300 mM) inhibited activity by 2 90%. Magnesium as Mg(OAch was inhibitory at all concentrations tested. (3) Reovirus messenger RNA (mRNA) was degraded more efficiently per fig RNA than either poly(A)-containing mouse cellular RNA or synthetic polyuridylate RNA by the interferon-mediated nucleolytic activity. Thus, the sensitivity of various single-stranded RNAs to degradation catalyzed by the interferonmediated nucleolytic activity correlates with the selectivity of translation inhibition characteristic of interferon-treated intact cells and some cell-free extracts. (4) The interferonmediated degradation of reovirus mRNA was impaired by bulk transfer RNA from mouse ascites cells. There was no detectable reduction in the 4 S size-class of the transfer RNA, but leucine-accepting activity of the tRNA was slightly impaired. (5) Leucine incorporation in response to exogenously added reovirus mRNA catalyzed by a cell-free protein synthesizing system prepared from mouse ascites tumor cells proceeds at comparable rates for 10 to 15 min in the presence and absence of the interferon-activated nuclease; thereafter, incorporation proceeds at a progressively lower rate in the presence of the activated nuclease. These results suggest that interferon treatment mediates the activation, through a low molecular weight effector molecule, of a nucleolytic activity. This nucleolytic activity may account, in part, for the reduction in accumulation of virus-specific RNA and the inhibition of protein synthesis observed in some interferon-treated cells and cell-free extracts. INTRODUCTION

Interferon treatment of many species of animal cells in culture greatly reduces their abi1it.y to support the multiplication of a wide range of unrelated DNA and RNA viruses (Finter, 1973; Ho and Armstrong, 1975). The interferon-mediated inhibition of viral multiplication requires both host cell RNA synthesis (Taylor, 1964) and protein synthesis (Levine, 1964; Friedman and Sonnabend, 1964), but the molecular mech0042~6822/78/0891-0240$02.00/0 Copyright All rights

0 1978 by Academic Pre.u, of reproduction in any form

Inc. reserved.

anism(s) by which the host-coded interferon-mediated gene product(s) function are not completely resolved. In reovirusinfected mouse fibroblast cells, the translation of early virus-specific mRNA is more sensitive to interferon than is the transcription of the mRNA (Wiebe and Joklik, 1975). However, the accumulation of virus-specific mRNA transcribed from parental genome templates is also reduced in reovirus-infected mouse cells by interferon treatment (Guantt, 1972;Wiebe and Joklik, 1975). The

MECHANISM

OF

INTEKFEHON

reduction in reovirus mRNA accumulation in llilbo is interferon-specific; inhibitors of protein synthesis such as pactamycin and emetine do not mimic the effect of interferon. ’ The interferon-mediated inhibition of viral mRNA translation observed in ciao has also been demonstrated in h-o. Cellfree extracts prepared from interferontreated murine cells catalyze the translation of a variety of exogenously added mRNAs much less efficiently than extracts prepared from untreated cells (Carter and Levy, 1968; Friedman et al., 1972; Falcoff et al., 1973; Gupta et al., 1973; Samuel and Joklik, 1974). In several systems, the interferon-mediated inhibition of viral mRNA translation is caused by ribosome-associated inhibitor(s) (Falcoff et al., 1973; Gupta et al., 1973; Samuel and Joklik, 1974). Even though certain interferons are relatively species specific (Finter, 1973), the interferon-mediated inhibitor(s) of translation do not appear to be speciesspecific (Samuel and Farris, 1977; Cooper and Farrell, 1977). In the absence of exogenously added transfer RNA, the interferon-mediated inhibition of viral translation in oitro appears to be primarily exerted at a step subsequent to formation of the first peptide bond (Content et al., 1975; Samuel, 1976; Sen et al., 1976a). The apparent activity of the inhibitor(s) in oitro is reduced by exogenously added tRNA (Colby et al., 1976; Hiller et a Z.,1976; Sen et al., 1976a; Zilberstein et al., 1976a). Recent studies indicate that interferon treatment also mediates several additional changes in murine cell-free extracts that are dependent, at least in part, upon double-stranded RNA. These interferonmediated changes include an enhanced phosphorylation of at least three ribosomeassociated components (Lebleu et al., 1976; FZoberts et al., 1976; Zilberstein et al., 1976b; Samuel et al., 1977); an increased nucleolytic degradation of viral messenger FiNA (Brown et al., 1976; Kerr et al., 1976; Samuel et al., 1977; Eppstein and Samuel, 1977); and an activated synthesis of a low molecular weight inhibitor of mRNA translation (Hovanessian et al., 1977; Kerr and ’ K. Melamed preparation.

and

C. E. Samuel,

manuscript

in

241

ACTION

Brown, 1978). We recently reported the isolation of an interferon-mediated translational inhibitor that possessednucleolytic activity; associated with this nucleolytic activity was a low molecular weight interferon-mediated [“‘PIATP-labeled component (Eppstein and Samuel, 1977). In this paper we describe some of the reaction conditions and the substrate selectivity, as well as the activation by a low molecular weight effector, of the interferon-mediated nucleolytic activity. MATERIALS

AND

METHODS

Materials. L-[4,5-“H]Leucine (57 Ci/ mmol), [y-‘l’P]ATP (17.9 Ci/mmol). and Aquasolwere obtained from New England Nuclear; [5-“Hlpolyuridylate (40 $.X/~mol P) was from Miles; [5-“H]UTP ( 19 Ci/mmol) , [5-“Hluridine (21 Ci/mmol), and L-[14C]valine (260 mCi/mmol) were from Schwartz/Mann. L-‘“C-Amino acids, S-adenosyl-L-methionine, creatine phosphate, creatine phosphokinase, ATP, CTP, and UTP were obtained from Sigma; GTP and poly(1) poly( C)-Sepharose were from P. L. Biochemicals; pancreatic ribonuclease was from Worthington; oligo(dT)-cellulose (type T-2) was from Collaborative Research; and Bio-Gel P-150 was obtained from Bio-Rad. Eagle’s minimal essential medium (MEM) and Joklik-modified Eagle’s MEM were from GIBCO, and fetal calf serum was from Irvine Scientific. Pactamycin and sparsomycin were generously provided by Dr. J. D. Dourus, Drug Development Branch, National Cancer Institute, Bethesda. Cells. Mouse L fibroblast cells, clone LRic, cells, and Krebs ascites tumor cells were cultured as previously described (Samuel et al., 1977.) Interferon

treatment. Mouse L:,z:, interferon induced by Newcastle disease virus (Herts strain) was prepared as previously described (Samuel and Joklik, 1974). The specific activity was 5 X 10” units interferon/mg of protein as determined by a 50% plaque reduction assay on LF29cells monolayers infected with vesicular stomatitis virus (Lai and Joklik, 1973). Confluent Le,!, roller cultures were treated with 300 units

242

EPPSTEIN

AN11

interferon/ml for 22-24 hr before harvesting (Eppstein and Samuel, 1977). Isolation of interferon-mediated nuclease. The isolation and partial purification of activated interferon-mediated nuclease from ribosomal salt-wash fractions prepared from non-preincubated S 10 extracts of interferon-treated mouse LcYL9cells was as previously described (Eppstein and Samuel, 1977). Synthesis of low molecular weight effector. Postribosomal cell-sap fractions were isolated from preincubated cell-free S 10 extracts prepared from untreated and interferon-treated mouse Ls29 cells essentially as described by Hovanessian et al. (1977). The postribosomal cell-sap fractions were applied to columns of poly (I) .poly(C)-Sepharose which were then used to catalyze the synthesis, in the presence of ATP, of the low molecular weight oligonucleotide inhibitor of protein synthesis (Hovanessian et al., 1977; Kerr and Brown, 1978). Preparation of RNAs. Reovirus mRNA was prepared as the in vitro transcription product of the virion-associated doublestranded RNA-dependent single-stranded RNA polymerase (Skehel and Joklik, 1969) in the presence of 10 fl S-adenosyl-L-methionine, and was purified as previously described (Samuel and Joklik, 1974). Radioactively labeled mRNA was transcribed with 20 @i/ml [SH]UTP added to the reaction mixture. Mouse L cell poly(A)-containing RNA was prepared from L cells labeled with [3H]uridine (15 &i/ml) at 37”. The RNA was extracted with phenol-chloroform (1:l) at 60” (Penman, 1966) and fractionated on a column of oligo(dT)-cellulose (Hirsch and Penman, 1974). Ascites tRNA was prepared from untreated ascites cells as previously described (Colby et al., 1976). Assay of nucleolytic activity. Ribonucleolytic activity was determined by measuring the formation of cold perchloric acid (6.25%)-uranyl acetate (0.19%)-soluble radioactively labeled oligonucleotides (Kalnitsky et al., 1959) from 3H-labeled RNA substrates. Unless otherwise stated, the standard reaction mixture (25 ~1) contained 40 mM 2-( N-morpholino)ethane sulfonate

SAMlJEL

(MES) (pH 6.2), 120 m&Z KCl, 1 mM dithiothreitol (DTT), 1 pg of methylated reovirus [“H]mRNA (8400 cpm/pg), and, as indicated, partially purified activated nuclease prepared from interferon-treated Lsjg cells, unactivated cell-free extracts prepared from untreated Ly29 cells, and interferon-mediated low molecular weight effector molecule. Incubation was at 25” for 60 min, after which the reaction mixture was processed as previously described (Samuel et al., 1977). Unless indicated otherwise, all reactions were carried out under conditions such that the rate of degradation of the substrate RNA was measured, not the extent of degradation. Assay of in vitro protein synthesis. In vitro protein synthesis catalyzed by preincubated ascites cell-free extracts was determined as previously described (Samuel and Joklik, 1974), measuring the incorporation of [“Hlleucine into hot trichloroacetic acid (TCA)-insoluble material. The standard reaction mixture (50 ~1) contained 30 m&f HEPES (pH 7.5); 120 n&f KCl; 3.4 mM Mg(OAc),; 1 mM ATP; 0.2 m&f GTP; 0.6 n&f CTP; 1 mM DTT; 10 m&f creatine phosphate; approximately 0.2 mg/ml creatine phosphokinase; 19 unlabeled L-amino acids, 50 fl each; 50 @i/ml L-[“Hlleucine; 50 pg/ml reovirus mRNA; and 0.3 ml of ascites cell-free extract/ml. The reaction mixture was incubated at 25” for the amount of time indicated and IO-$ aliquots were processed. Assay of tRNA aminoacylation. An enzyme fraction containing leucyl-tRNA synthetase activity was prepared from the S 100 supernatant solution of untreated Ls,, cells, and endogenous tRNA was removed by chromatography on DEAE-cellulose as previously described (Samuel et al., 1973). The aminoacylation reaction mixture (25 ~1) contained 100 mM HEPES (pH 7.5), 1 miV DTT, 4 m&Y ATP, 8 m&f Mg(OA&, 20 fl [“Hlleucine at 10 Ci/mmol, 20 fl unlabeled amino acids minus leucine, enzyme, and tRNA as indicated. After 20 min of incubation at 37’, the reaction mixture was processed as previously described (Samuel et al., 1973). Electrophoresis of tRNA. Electrophoresis of tRNA was performed on 20% acryl-

MECHANISM

OF

INTERFERON

amide-0.65% bisacrylamide gels at pH 8.0 (DeWachter and Fiers, 1971). Electrophoresis on l-m&f thick slab gels was at 20 mA for 4 hr. Gels were stained with methylene blue, and scanned at 260 nm with a Gilford model 2520 gel scanner. RESULTS

An interferon-mediated nucleolytic activity (IF-nuclease)” was isolated by BioGel P150 column chromatography from the 0.5 M KC1 ribosomal salt-wash fraction prepared from ribosomes derived from nonpreincubated extracts of interferon-treated Lr,,!, cells. This nucleolytic activity was not detectable in the corresponding fractions obtained by chromatography of preparations from untreated control cells (Eppstein and Samuel, 1977). The Bio-Gel column fractions possessing the IF-nuclease contained a small phosphorylated component, previously designated Pq, that was labeled with [Y-““P]ATP (Eppstein and Samuel, 1977). The molecular weight of P,$ estimated electrophoretically is I 3500.” Other investigators (Horvanessian et al., 1977; Kerr and Brown, 1978) have recently described an interferon-mediated LMW oligonucleotide inhibitor of encephalomyocarditis virus RNA translation; the LMW inhibitor was synthesized by mouse L cellfree preparations on poly(1) poly(C)-Sepharose columns in the presence of ATP.

243

ACTION

As shown in Fig. 1, activation of nucleolytic activity in untreated L929 cell-free extracts was obtained with LMW effector molecule(s) synthesized from ATP on a column of poly(1). poly(C)-Sepharose by enzyme(s) from interferon-treated L%z, cells. Synthesis of the positive effector of ribonucleolytic activity was not catalyzed by the corresponding poly( I) poly (C) Sepharose-supported enzyme fraction from untreated control cells, or by the poly(1). poly(C)-Sepharose-supported enzyme fraction from interferon-treated cells in the absence of ATP (Fig. 1). I

‘-1-T

10 c

“t

4c 2c

4’ IF

Activution of Nucleolytic Activity in Untreated Cells by an Interferon-mediated LMW Effector To test the possibility that the LMW oligonucleotide inhibitor of protein synthesis observed by Kerr and co-workers (Horvanessian et al., 1977) may be related functionally to the LMW phosphorylated component Ph that we observed associated with activated IF-nuclease (Eppstein and Samuel, 1977), we synthesized the LMW inhibitor of protein synthesis and examined its effect on nucleolytic activity. ’ Abbreviations used: IF-nuclease, the nucleolytic activity observed as a result of those components activated or synthesized in response to interferon treatment: LMW, low molecular weight; dsKNA and ssRNA. double-stranded and single-stranded RNA. ’ 11. A. E:lqrstein and C. Is. Samuel. unpublished (observation

0

.----------------------. 0

10

ATPI

-1 20

30

U

1

FIN. 1. Activation of nucleolytic activity in cellfree S 10 extracts prepared from untreated cells by an interferon-mediated low molecular weight effector. Nucleolytic activity of preincubated S IO extract (‘7.5 al) from untreated Lrlc, cells was measured as described under Materials and Methods, except the reaction was carried out under protein synthesis conditions. The reaction mixture contained reovirus [‘H]mRNA (29; 000 cpm/g) and the product (7.5 ~1 eluate) of poly(I) poly(C)-Sepharose column-supported enzyme fractions as indicated. Product obtained by incubation of the column-supported S 100 supernatant enzyme fraction from interferon-treated (IF) cells (@, 0) or from untreated (C) cells (A, a) with either buffer tr‘:. a) or with buffer containing 1 mM ATP (0, A). Column buffer before incubation with poly(1) polytC)Sepharose supported enzymes (Cl); (O----O) is the same as (-1. except broken line is in the absence of s 10.

244

EPPSTEIN

AND

SAMUKL

Figure 2 compares the effect of relative concentration of the interferon-mediated LMW effector on the activation of the nucleolytic activity in untreated cell-free extracts and on the activity of the IF-nuclease isolated by chromatography from interferon-treated cells. Concentrations of the effector that yielded maximal activation of the nuclease present in untreated murine extracts did not enhance the activity of the activated IF-nuclease isolated directly from interferon-treated cells (Fig. 2). Reaction Conditions of the Interferon-mediated Nucleolytic Activity pH optimum. IF-nuclease activity was optimal at pH values between 6.0 and 6.5 at 25” with reovirus r3H]mRNA as a substrate (Fig. 3). Effects of potassium. Potassium ion, tested as KCl, stimulated the IF-nuclease

I f 1 [POLY

,

1

I

3

10

30

1,POLY

C

SEPHAROSE

I 100 ELUATEI

FIG. 2. Effect of poly(I) poly(C)-Sepharose column eluate concentration on the activation ofnuclease in untreated S 10 cell-free extracts and on the activity of the purified IF-nuclease isolated from interferontreated cells. Solid lines: nucleolytic activity in S 10 extracts from untreated cells; reaction conditions and symbols as for Fig. 1. except varying concentrations of poly(I).poly(C)-Sephadrose eluate (7.5 ~1 = 100 on abscissa) were added to the reaction mixture. Results are normalized to maximal nucleolytic activity obtained in presence of LMW effector (8600 cpm) minus the activity due to endogenous nucleolytic activity of the untreated S 10 extract (3550 cpm). Broken line: activity of IF-nuclease purified from interferon-treated cells by Bio-Gel chromatography, assayed in the presence of the LMW effector synthesized from ATP by poly(1) poly(C)-supported enzyme fraction from interferon-treated cells (2400 cpm = 100).

FIG. 3. Effect of pH on the degradation of reovirus [‘H]mKNA catalyzed by IF-nuclease isolated from interferon-treated cells. The standard incubation mw ture for assa,v of nucleolytic activity was as described under Materials and Methods. except that the buffer was either 20 m&f MES (pH 6.0 to 7.0) or 20 n&f HEPES (pH ii.5 to 8.5). Broken line Indicates harkground radioactivity observed in the absence of added IF-nuclease.

with maximal activity obtained between 75 and 125 mM KC1 (Fig. 4). However, high KC1 concentrations strongly inhibited the nucleolytic activity. At 300 m.iV KCl, the degradation of reovirus mRNA was inhibited by 90% and at 500 mM KCl, essentially no degradation of the viral mRNA into acid-soluble oligonucleotides was observed (Fig. 4). Effect of magnesium. Magnesium ion, tested as Mg(OAc)Z, inhibited the degradation of reovirus mRNA catalyzed by the activated IF-nuclease (Fig. 5). The synthetic RNA, poly(U), was not degraded as efficiently as reovirus mRNA; however, the limited degradation of poly(U) was slightly reduced with increasing magnesium ion concentration (Fig. 5). Sensitivity of Viral, CeLLular, and Synthetic Single-stranded RNAs to Degradation Catalyzed by the IF-NW clease The relative ability of the IF-nuclease to catalyze the degradation of three classes of

MECHANISM

I 0

“1

L._ 03

02 [KCI]

I-b 04

OF

INTERFERON

05

M

FIG. 4. Effect of potassium chloride on the degradation of reovirus [‘H]mRNA catalyzed by IF-nuclease isolated from interferon-treated cells. The standard incubation mixture for assay of the nucleolytic activity was used as described under Materials and Methods, except that the concentration of KC1 was varied as indicated. Immediately before addition of the cold perchloric acid-many1 acetate, the reaction mixtures were diluted to a final volume of 125 (11 containing 0.1 M KCl. Broken line indicates assay blank value.

LL I

----e .'

J [1”19’

6 11 111IL1

FIG. 5. Effect of magnesium on the degradation of viral and synthetic RNA catalyzed by IF-nuclease isolated from interferon-treated cells, Conditions were a.< described under Materials and Methods, except that the reaction mixture contained either 1 pg of reovirus (,‘H]mRNA to----O) or 1 wg of r’H]polyuridylate (a----Al and Mg(OAc)> as indicated.

ACTION

245

mRNA that differ in their sensitivity to translation inhibition in interferon-treated cell-free protein synthesizing extracts was examined. The rate of degradation of reovirus mRNA, poly(A)-containing mouse cellular RNA, and synthetic polyuridylate catalyzed by the IF-nuclease is summarized in Fig. 6. Reovirus mRNA, which is the most sensitive of these three RNAs to interferon-mediated inhibition of translation (Samuel and Ioklik, 1974), was degraded approximately 4 to 6 times more rapidly than either cellular RNA or poly(IJ) at a concentration of 1 pg RNA per 25 ~1 of reaction mixture (Fig. 6). As shown inTable 1, double-stranded RNA tested in the form of reovirus genome RNA did not significantly affect the degradation of any of the ssRNA substrates, reovirus mRNA, L-cell mRNA or poly(U), catalyzed by the activated IF-nuclease.

FIG. 6. Sensitivity of viral, cellular, and synthetic single-stranded HNAs to degradation catalyzed by IFnuclease isolated from interferon-treated cells. Con ditions were as described under Materials and Methods, except that the reaction mixture contained: reovirus [ ‘H]mRNA. 8400 clnn/~~g: t-1. [ ‘H lpolyuridylate, “5.ooo CM), CpWpg; to----0). polytA)-containing mouse L cell [ ‘HIRNA, IfiOn cpm/iig. Immediately prior to the addition of the perchloric acid-uranyl acetate the total concentration of RNA in the reaction mixtures was adjusted to 1.5 bg (0. 0) or 1 pg (0) in a total volume of 35 ~1 by addition of the respective unlabeled RNA.

246

EPPSTEIN

AND

SAMUEL

Effect of Transfer RNA on the Interferonmediated Nucleolytic Activity The degradation of reovirus [“H]mRNA by the IF-nuclease was reduced by transfer RNA prepared from untreated mouse ascites tumor cells (Fig. 7). However, the amount of tRNA migrating as 4 S material on polyacrylamide gels was not reduced by the IF-nuclease under conditions where the apparent degradation of reovirus mRNA was inhibited by 70-90% (Fig. 8, A and B) . Under comparable conditions, the degradation of reovirus [3H]mRNA catalyzed by pancreatic RNase was inhibited much less by added tRNA (results not shown); in addition, the added tRNA was significantly degraded by the pancreatic RNase as measured by the reduction in 4 S material (Fig. 8, A and C). Although the apparent size of the ascites tRNA was not detectably reduced by the IF-nuclease (Fig. 8, A and B), the capacity to aminoacylate the tRNA with leucine was slightly reduced following treatment with the IF-nuclease (Table 2). Effect of Interferon-mediated Nucleolytic Activity on the Kinetics of Reovirus mRNA Translation in Vitro The effect of the IF-nuclease on the kinetics of reovirus mRNA translation as a function of the time of addition to the protein synthesis reaction mixture is shown in Fig. 9. When the IF-nuclease was added to the reaction mixture at the initial time of TABLE

1

EFFECT OF dsRNA ON THE DEGRADATION CATALYZED BY THF. INTERFERON-MEDIATED NUCLEOLYTIC

Substrate

(I

ACTIVITY

RNA

degraded

-dsRNA Reovirus [‘H]mRNA L cell [“HlmRNA [‘H]Polyuridylate

OF ssRNA

0.263 k 0.008 0.073 5 0.016 0.083 f 0.005

(kg)’ +dsRNA

0.261 + 0.002 0.084 f 0.003 0.077 f 0.004

n Reovirus and L cell mRNAs were tested at a concentration of 1.0 Kg/‘25 ~1; polyuridylate was tested at 0.5 pg/25 ~1. ’ RNA degradation was performed as described for Fig. 6, either in the absence or presence of 1.0 pg/ml reovirus genome dsRNA. Values given are the averages of three independent reactions.

0L

0

+,,,’

2

3

4

5

FIG. 7. Effect of transfer RNA on the degradation of reovirus mRNA catalyzed by the IF-nuclease isolated from interferon-treated cells. Conditions were as described under Materials and Methods, except that the reaction mixture contained the indicated amount of tRNA. Incubation was at 25” for 78 min. Immediately before addition of the perchloric acid-uranyl acetate reagent tRNA was added to a final concentration of 5 @g/30 ~1.

incubation, viral mRNA translation was not significantly affected during the first 10 min; however, after 10 min of incubation, a reduction in both the rate and the ultimate extent of [“Hlleucine incorporation was observed (Fig. 9A). Addition of the IF-nuclease at 10 min after initiation of reovirus mRNA translation also resulted in significant inhibition of [“Hlleucine incorporation, although by 60 min the relative inhibition was slightly less than that obtained when the IF-nuclease was added at time zero (Fig. 9B). Pactamycin, an inhibitor of initiation of translation, reduced reovirus mRNA-directed leucine incorporation more than 90% when added at time zero (Fig. 9A); whereas, addition of pactamycin 10 minutes after initiation of incubation resulted in only 30% reduction of incorporation. Incorporation was completely blocked when both pactamycin and sparsomycin, an inhibitor of elongation, were added after 10 min (Fig. 9B). When both

MECHANISM

2.5pg

OF

INTERFERON

tRNA

I

AA A 20 addlllon

B + lhuclease 5.Opg

C

+ pant

RNase

tRNA

A;,

L

A no addition

B

C

- IF-nuclease

pant

RNase

FIG. 8. Effect of IF nuclease on the structural stability of ascites transfer RNA. Transfer RNA was analyzed by electrophoresis on 20% polyacrylamide slab gels following incubation with buffer, IF-nuclease, or pancreatic RNase; degradation of reovirus [“HImRNA was determined with identical reaction mixtures in the presence and absence of the tRNA. The 4 S region of each gel was scanned at 260 nm; electrophoresis was from left to right. The reaction mixtures contained either 2.5 pg (top) or 5.0 pg (bottom) of tRNA and: (A) buffer control; (B) IF-nuclease; (C) pancreatic RNase. The apparent inhibition of IF-nuclease activity by the tRNA was 70% (2.5 pg of tRNA) and 90% (5.0 pg of tRNA); pancreatic RNase was inhibited 20% (2.5 pg of tRNA) and 70% (5.0 pg of tRNA)

the IF-nuclease and pactamycin were added after 10 min, translation of reovirus mRNA was likewise completely inhibited within 10 min after the addition (Fig. 9B). These results suggest that the apparent level of inhibition of translation is the elongation of nascent polypeptide chains, and are consistent with the nucleolytic degradation of the message; however, they do not exclude an additional direct effect mediated by interferon on the elongation process per se. DISCUSSION

The results

reported

here demonstrate

247

ACTION

that a nuclease present in an inactive cryptic form in extracts of untreated mouse Lx9 cells can be activated by the LMW oligonucleotides synthesized from ATP by enzymes from interferon-treated cells (Figs. 1 and 2). The IF-nuclease isolated in active form from the interferon-treated Lz~ cells had associated with it a LMW component labeled with [32P]ATP (Eppstein and Samuel, 1977); this IF-nuclease was catalytically active in the absence of exogenously added LMW oligonucleotide effector (Fig. 2). Preliminary results suggest that the previously described interferon-mediated [32P]ATPlabeled LMW component (designated P4) associated with Bio-Gel P-150 fractions that possessed an in vivo interferon-mediated nucleolytic activity (Eppstein and Samuel, 1977), and the interferon-mediated class of LMW component(s) synthesized from ATP in vitro as described by Hovanessian et al. (1977) may, indeed, be identical. Thus, it appears that the inhibition of protein synthesis attributed to the interferon-mediated LMW oligonucleotides (Hovanessian et al., 1977; Ball and White, 1978) results from the degradation of the mRNA catalyzed by an activated nuclease rather than from a direct inhibitory effect on the host cell protein synthetic machinery (Figs. 1 and 2; Eppstein and Samuel (1977)). TABLE LECCINE TREATED

2

ACCEPTANCE BY AKITES WITH INTERFERON-MEDIATED

Conditions of treatmerit” Nuclease 0 min 60 min Buffer 0 min 60 min

Leucine

TRANSFER RNA NUCLEASE

accepted”

(cpm)

(‘J)

2132 -+ 121 1608 +- 118

100 75

2151 + 226 1993 t 40

106 93

I‘ Ascites tRNA (10 pg/25 ~1) was incubated at pH 6.2 with either interferon-mediated nuclease or buffer as described for Fig. 9. Incubation was at 25” for either 0 or 60 min. ‘Leucine acceptance determined with a IO-$ aliquot of the nuclease or buffer treatment reaction mixture. The standard reaction mixture for measuring aminoacylation with leucine was as described under Materials and Methods; values given are the average of three independent reactions.

248

EPPSTEIN

AN11

The substrate selectivity displayed by the interferon-mediated nucleolytic activity in vitro correlates partially with the selectivity of the apparent interferon-mediated inhibition of translation observed both in uiuo (Finter, 1973) and in vitro (Carter and Levy, 1968; Falcoff et al., 1973; Samuel and Joklik, 1974; Sen et al., 1976a). Cellular poly(A)-containing RNA and synthetic poly(U) were degraded less efficiently than was reovirus mRNA (Fig. 6). The apparent selective degradation of reovirus mRNA as compared to cellular poly(A)-containing RNA was determined per pg rather than per mole of the respective RNA and, hence, does not unequivocally establish that the IF-nuclease is specific for viral RNAs. The reovirus mRNA preparation consisted of 10 mRNA species that do not contain detectable poly(A) at their 3’-termini (Stoltzfus et al., 1973; Joklik, 1974); by contrast, the cellular RNA preparation represents a large number of different poly(A)-rich RNA species. It is conceivable that the 3’-poly(A) affected the sensitivity of the mRNAs to nucleolytic degradation, or that some cellular mRNAs present at low molar concentration were selectively sensitive to the IFnuclease. The question of substrate selectivity of the IF-nuclease is currently under further investigation. The activity of the interferon-mediated nuclease was enhanced by KCl; however, at high concentrations of KCl, the nuclease activity was significantly reduced (Fig. 4). This result may be related to the recent finding that the in vitro accumulation of simian virus-40-specific RNA in nuclei isolated from interferon-treated, SV-40-infected Vero cells is reduced when measured at 100 mM KCl, but not at 300 m&f KC1 (Metz et al., 1977). The interferon-mediated inhibition of viral mRNA translation observed in vitro can be partially reversed in several cell-free protein synthesizing systems by addition of transfer RNA (Colby et al., 1976; Hiller et al., 1976; Sen et al., 1976a; Zilberstein et al., 1976a). The apparent ability of tRNA to inhibit the interferon-activated nuclease (Fig. 7) may be related to the observed reversal by tRNA of the interferon-mediated block in translation.

SAMLJEL B I,‘*(

,2

r/:t,,,

A T=O~mrl /

10

2 I: 61

.,’

0 0

10

1’

-~-. .-be20 30 40 TIME (mlnl

~~

~~_

50

_ FIG. 9. Effect of IF-nuclease, pactamycin and sparsomycin on the translation of reovirus mRNA catalyzed by the ascites in vitro protein synthesizing system. The standard reaction conditions were as described under Materials and Methods. Incubation was at 25’; IO-p1 aliquots were processed. (A) Additions at time zero: (D--U), 20 ~1 of buffer; (O---U), 20 ~1 of buffer, activity due to endogenous mRNA only; (M), 20 ~1 of IF-nuclease; (A-A), 2 x lOme M pactamycin. (B) Additions after incubation for 10 min: (0. ---O), 20 ~1 of buffer; (O----O), 20 al of IF-nuclease; (O-O), 20 pl of buffer plus 2 x lo-’ M pactamycin; (M), 20 aI of IF-nuclease plus 2 x 10 ’ M pactamycin; (n---A), 2 x 10 4 M sparsomycin plus 2 x 10 ’ M pactamycin.

There appear to be at least two mechanisms by which the translation of viral mRNA may be inhibited in cell-free protein synthesizing systems derived from interferon-treated cells. By one mechanism, the inhibition of viral mRNA translation by an interferon-mediated ribosome-associated inhibitor (Falcoff et al., 1973; Gupta et al., 1973; Samuel and Joklik, 1974) does not involve the degradation of viral mRNA (Joklik and Merigan, 1966; Gupta et al., 1974; Samuel, 1976; Ball and White, 1978). By the other mechanism, nucleolytic degradation of the viral message is apparently

MECHANISM

OF

INTERFERON

associated with the inhibition of viral mRNA translation (Sen et al., 1976b; Eppstein and Samuel, 1977; Clemens and Williams, 1978; L. A. Ball, personal communication). The particular mechanism(s) observed no doubt is (are) dependent upon the particular cell and virus combination investigated, as well as upon the conditions during the preparation of the cell-free protein synthesizing systems and the viral mRNAs. The presence in vim of a particular type and concentration of RNA with double-stranded character, for example, either dsRNA regions resulting from secondary structure of ssRNA, or dsRNAs per se such as replicative-intermediates or dsRNA genomes, may favor one mechanism over the other. Finally, it should be pointed out that interferons also appear to affect several host functions in addition to inhibiting the multiplication of animal viruses (Gresser, 1972; Finter, 1973; Ho and Armstrong, 1975; Metz, 1975; Stewart et al., 1976; Knight, 1976). It is conceivable that some of the biochemical changes observed in interferon-treated cells and cell-free extracts may be related to the antiviral action(s) of “interferon”; whereas, other of the changes may be more closely associated with the putative anticellular actions(s) of “interferon.” ACKNOWLEDGMENTS This work was supported, in part, by research grants from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service (AI12520). and from the American Cancer Society (VC192A). D. A. E. was a U. S. Public Health Service Postdoctoral Fellow (F32-AI-05472).

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ZILBERSTEIN, REVEL,