Selective inhibition of viral protein accumulation in interferon-treated cells; nondiscriminate inhibition of the translation of added viral and cellular messenger RNAs in their extracts

VIROLOGY

67, 49-63

Selective

(1974)

Inhibition

of Viral

Treated

Cells;

Nondiscriminate

Added

Viral

and

S. L. GUPTA, Department

Cellular

W. D. GRAZIADEI

of Moleculnr

Biophysics

Protein

Accumulation

Inhibition Messenger

III, H. WEIDELI,

Accepted

of the RNAs

Translation

in their

M. L. SOPORI,

Yale Utiversily,

and Biochemistry,

in Interferon-

Mew

AND

Haven,

of

Extracts P. LENGYEL Co~~necticul

06520

Septelnber 24, 1973

Treatment of monolayers of mouse L cells with 7.5 vesicular stomatitis virus plaque reduction units/ml of mouse interferon (specific activity 2 X IO7 NIH mouse reference standard unit,s/mg protein) results in a 95y0 decrease in the reovirus yield and, as shown earlier, an about 80% decrease in double-stranded and an about, 607; decrease in single-stranded reo RNA accumulation in cells infected with 10 plaqueforming units of virus/cell. Interferon at this concentration does not affect the rate of accumulation of host proteins in uninfected or reovirus-infected cells. It inhibits reo virion protein accumulation in infected cells. In control cells the rate of reo virion protein accumulation increases about 2-fold between 10 and 14 hr after infection. In cells treated with interferon it remains at the same low inhibited level. Reo virion proteins were assayed in cell extracts by immunoprecipitation with antisera specific for various size classes or by gel electrophoresis. These results reveal the selective nature of the inhibition of protein accumulation in interferon-treated, virus-infected cells. The effect of treating L cells with interferon was also tested on the capacity of their extracts to translate various mRNAs. The translation of endogenous mRNA, of mRNA in L cell polysomes (added as such) and of polyuridylic acid is at most onl~ slightly inhibited whereas that of added natural mRNAs (including reovirus and encephalomyocarditis virus mRNA, rabbit globin mRNA, and a mixture of L ccl1 mRNAs) is strongly inhibited. The dominant nature of the inhibitor(s) is indicated by the fact that translation of added mRNA is impaired in a mixture of extracts from inkrferorl-treated and from control cells. Thus an effect of interferon treatment, of cells is manifested in subcellular systems. However. the selectivity of the effect in clion. is not well reflected i/t vilro. INTRODUCTION

Interferons are macromolecules, presumably glycoproteins, which are synthesized in a variety of cells of vertebrates upon viral infection or some other stimuli. They are excreted, are bound to other cells and make these inefficient in supporting the growth of a broad range of viruses. No interferon has been purified to homogeneity, and their mechanism of action has not been definitely established (for a review see Ng and VilEek, 1972). Experiments are underway in many laboratories to identify the strp (or steps) in virus replication which is (or are) blocked in interferon-treated cells. 49 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

Studies on various types of intact cells which were treated with interferon and infected with a variety of viruses resulted in reports according to which viral mRNA synt’hesis is a step blocked (Taylor, 196.5; Oxman and Levin, 1971; Marcus et al., 1971; Bialy and Colby, 1972; Mandcrs et al., 1972; Gauntt, 1972; Gravel1 and Cromeans, 1972) and other reports according to which viral protein synthesis is blocked but viral mRNA synthesis is not affected (,Joklik and Merigan, 1966; XI& and Est’eban, 1972; Jungwirth et al., 1972). The effect of interferon treatment, of cells was also tested on protein synthesis in

50

GUPTA ET AL.

their cell extracts. According to some reports the capacity to translate added mRNA, except poly (U), is impaired in these, but only if the extract was prepared from cells which had been infected with a virus after treatment with interferon and the impairment is due to a labile factor which is inactivated by incubating the extract at 37” (Friedman et al., 1972). According to other reports, treatment of cells with interferon results in an impairment of the translating capacity of the extract even if the cells were not infected with the virus, ma this impairment persists in extracts incubated at 37” (Falcoff et al., 1972). Most of our studies on the interferon defense mechanism were performed with reovirus-infected mouse L fibroblasts. The choice of this system was based on the following considerations. The replication of reovirus in L cells is inhibited by interferon (Oie and Leh, 1968; Gauntt, 1972; Vassef et al., 1973) and the life cycle of reovirus is known at least as well as that of any other virus. Reovirus has a double-stranded RNA genome consisting of 10 segments. The virion contains a transcriptase which transcribes each of t,hese segments apparently end-toend into a different species of mRNA. The size of these mRNAs and those of known reovirus capsid proteins (XI, X2, ccl, 4% Ul, -3 and us) and noncapsid proteins be, and u2za)are consistent with the possibility that each mRNA specifies the synthesis of a single protein species. Both the mRNAs and the proteins specified by reovirus fall into three size classes: large (known proteins X1 and X2; mRNlis 11, 12, and 15), medium (proteins po, ~11,p2; mRNAs ml, m2, and m3), and small (proteins (~1, U% Us,, and a3; mRNAs sl, sz, s3, and ~4). The capsid protein pcLzis not a primary translation product, it is apparently formed by cleavage of ~1. The transcriptase in the virion can be activated in vitro by treating the virus with chymotrypsin. The resulting particles can synthesize the ten reovirus mRNAs if incubated with four ribonucleoside triphosphates in the presence of salts (for recent re+ws on reovirus, see Joklik, 1971; Millward and Graham, 1971; Shatkin, 1971). To enable us to study the manifestation of interferon treatment on viral

and host protein synthesis in vitro we developed a cell-free system in which reo mRNAs synthesized in vitro can be translated (Graziadei and Lengyel, 1972; McDowell et al., 1972). The translation products of the reo mRNAs in this system are identical in size with the authentic reo virion proteins. For a further test of the similarity of these translation products to virion proteins we prepared antisera against the large, medium, and small size classes of reo virion proteins. From the proteins whose synthesis was directed in the cellfree system by reo mRNAs each antiserum precipitates only those which correspond in size to the &ion proteins against which the antiserum was prepared (Graziadei et al., 1973). The results to be presented in this communication reveal a strong impairment in the capacity of extracts of interferontreated cells to translate various viral mRNAs including that of reovirus. Thus, the effect of interferon treatment is manifested in subcellular systems. The impairment of translation is, however, not restricted to those of viral mRNAs but is almost equally pronounced for host mRNAs. Indeed, the only messageswhose translation is not (or is only slightly) affected by interferon treatment of the cells are endogenous mRNAs present in not preincubated extracts of cells, mRNAs in L cell polysomes (added as such to the cellfree system), as well as polyuridylic acid, a messengerwhich can be translated without proper peptide chain initiation. In a mixture of two cell extracts, one prepared from interferon-treated cells and the other from untreated cells, the capacity of translating added mRNA is impaired. This indicates that the inhibitor is dominant. We also studied the effect of interferon treatment on protein synthesis in intact cells. The results to be presented reveal that in interferon-treated, reovirus-infected L cells the accumulation of reovirus specific proteins is blocked whereas that of host proteins is not affected. Thus, in intact cells the interferon effect is selective and this selectivity is apparently not well reflected in our experiments with the cellfree system in which protein synthesis is

INTERFERON

AND

PROTEIN

SYNTHESIS

51

of Vassef et al. (19%). Polyuridylic acid and goat antirabbit IgG serum were obtained from Miles Laboratories, Triton X-100 from Packard, 35S-methionine (spec. act. 60-130 Ci/mmole) and 3H-uridine (spec. act. 27.2 Ci/mmole) from New England Nuclear, *4C-phenylalanine (spec. act. 455 MATERIALS AND METHODS mCi/mmole), and 3H-valine (spec. act. 7 from Schwarz/Mann. OligoThe preparation of L cells (mouse L 929 Ci/mmole) (oligo dT-cellufibroblasts), the growth of reovirus (type 3, deoxythymidylate-cellulose the Dearing strain), the purification of t’he lose) was prepared according to Gilham virus, the synthesis of reo mRNAs in vitro, (1964). Growth medium for L cells was the preparation of the cell-free protein syn- Eagle’s MEM supplemented with 7 % fetal calf serum. Growth medium B was Eagle’s thesizing systems (530 extract and preincubated S30 extract) from L cells, the con- MEM containing 5% fetal calf serum and ditions for protein synthesis directed by g of the usual amount of amino acids. added viral mRNAs in the S30 extracts PBS solution (used to wash cells) was 10 and the processing of the 530 extract were mM potassium phosphate (pH 7.4) conas described by Graziadei and Lengyel taining 137 m&f NaCl and 2.7 mM KCl. (1972). The synthesis of radioactively RSBT was 10 mM Tris-Cl (pH 7.4), 10 labeled reovirus proteins in reovirus-inmd4 NaCl, 1.5 MgCl2 and 0.5 % Triton fected L cells, the preparation of antisera X-100. The multiplicity of infection (m.o.i.1 from rabbits against t’he various size classes is the ratio of the number of plaque-formof reo virion proteins (anti X specific for ing units of virus to the number of cells X1 and X2, anti p specific for p2 and possibly exposed to virus. Whenever cells were ~1, and anti u specific for ~1, a2 and u3) treatled with interferon, or infected wit,h were as described by Graziadei et cd. (1973). reovirus, the control cells were treated Encephalomyocarditis (EMC) virus was with the solution, not containing interferon grown, purified and EMC RNA was ex- or reovirus in which the interferon or reotracted according to Aviv et al. (1971). virus was administered. ,411 incubations Rabbit globin mRNA was prepared accord- were at 37” unless otherwise stated. ing to Evans and Lingrel (1969). Mouse Preparation of L cell mRNA. L cells interferon from L cells (treated with New- grown in a suspension culture at 37” (see castle disease virus) was purified according Graziadei and Lengyel, 1972) were fed to the procedure of Weideli and Lengyel with fresh medium and diluted to a density (manuscript in preparation). The interferon of 34 X lo5 cells/ml. Five hours lat>er t)he preparation used contained at least 2 X culture was harvested and polysomes were 10’ NIH mouse reference interferon units/ prepared according to the procedure of mg. The interferon was assayed by vesic- Schochetman and Perry (1972). ular stomatitis virus (VSV) plaque number The RNA from the polysomcs was cxreduction according to Vassef et al. (1973). tracted according to the phenol-chloroform One unit of interferon (throughout this procedure of Perry et al. (1972). The ixopaper) is that amount with which a monolated RNA was fractionated into a fraction layer of L cells has t’o be treated to reduce of RNA molecules with poly(A) segmems the VSV plaque number by 50 % compared (mRNA) and a fraction of RNA molecules to that in a control plate. One of our units without poly(A) segments on an oligo-dT corresponds to about 12 NIH mouse refer- cellulose column according to the proceence interferon units (Vassef et al., 1973). dure of Swan et al. (1972). About 3-4% The effect of trrating L cells with various of the total RNA was retained on the colconcentrations of interferon prior to infec- umn, i.e., contained poly(A) segments. The tion with reovirux on the yield of reovirus fractionation of R.NA on t$he oligo dTin a single replicative cycle (yield reduction) cellulose column was facilitat’ed by using was determined according to the procedure radioactivelv labclrd marker RNA and promoted by exogenous mRNAs. Nevertheless the fact that the effect of interferon treatment of cells is manifested in their cell-free extracts should be of use in attempt’s to understand the molecular basis of the interferon defense mechanism.

52

GUPTA

following the fractionation by determining the radioactivity in the fractions. The marker RNA was obtained by labeling an diquot (100 ml) of the L cell suspension with 3H-uridine (5 &Ji/ml) for 5 hr and mixing this solution into the suspension of unlabeled L cells before harvesting the cells. Preparation of L cell polysomes. An L cell extract (530) was prepared according to Graziadei and Lengyel (1972) except that the buffer B contained 120 mlM KCl. The S30 extract was treated with 0.5 % sodium deoxycholate and centrifuged through a linear sucrose gradient (15 to 40% sucrose w/v in 25 mM Tris.HCl (pH 7.5), 50 mM KCI, 5 m&f MgCh and 2 mlM dithiothreitol (DTT) in the SW 25.2 rotor at 24,000 rpm (70,000 g) for 1.5 hr. The gradient was fractionated and the optical density of the fractions was monitored. The fractions containing the polysomes were pooled and the polysomes were sedimented by centrifugat-ion at 150,000 g for 10 hr at 2’. The pellet containing the polysomes was suspended in 25 mM TrisaHCl (pH 7.5), 120 mM KCl, 4 mM MgCh, 1 m&f DTT at 150 Azao units/ml. This suspensionpromotes protein synthesis when added to cell extracts (Table 1). Assay of the e$ect of interferon treatment of L cells on the capacity of their extracts to translate added poly(U), viral and host mRNAs as well as endogenous mRNA (Table 1). A suspension culture of L cells was divided into two parts: one was treated with 37.5 units/ml of interferon in growth medium for 18 hr, the other served as a control. S30 extracts were prepared from each part. The translation was performed in preincubated S30 extracts except when L cell endogenous mRNA was translated. In this case the 530 extract was not preincubated. The total volume of each reaction mixture was 30 ~1. The incubation time was 1 hr. The Mg2+ was 10 mM in the case of poly(U)-directed amino acid incorporation; in all other cases it was 4 mM. The final concentration of KC1 was 120 m&f in the translation of rabbit globin mRNA, EMC RNA, and of L cell polysomes; in all other cases it was 80 mM. The other conditions were identical with those described for amino acid incorporation in

ET AL,

vitro by Graziadei and Lengyel (1972) except that no tRNA was added. The rate of incorporation was nearly constant for the duration of the experiment in extracts of control cells to which mRNA from reovirus, from EMC virus, or from L cells was added. In all other casesit leveled off earlier. The effect of interferon treatment was also tested on the rate of protein synthesis in intact cells. For this purpose aliquots were taken from the same cultures of interferontreated and control cells from which the 530 extracts were also prepared. The rate of 35S-methionine incorporation into hot trichloroacetic acid-insoluble products in the aliquot from the cell culture treated with interferon was at most 10 % lower t,han in the aliquot from the control culture during the 5 hr of the assay (i.e., between 20 and 25 hr after the administration of interferon to the test culture). Xtudy of the dependenceof the fraction of L cells infected on the multiplicity of infection with reovirus (Fig. lb). Confluent monolayers of L cells on 60-mm diameter plates in growth medium were infected with reovirus in 0.5 ml growth medium at the m.o.i. shown in Fig. lb using two plates for each m.o.i. The plates were incubated at 37” for 90 min with an occasional tilting of the plates to allow adsorption of the virus. Subsequently the virus-containing solution was removed, the monolayers were washed twice with PBS, supplemented with 4 ml of growth medium, and incubated at 37’. Three hours later the growth medium was removed, the monolayers were washed twice with PBS and dissociated into single cells by incubation with 3 ml of a 0.05 % trypsin solution. After 10 min the action of trypsin was stopped by adding 6 ml of growth medium, and the cell suspension was centrifuged at 1500 g at room temperature for 5 min. The cells in the pellet were suspended in growth medium at a density of 8 X lo5 cells/ml. Approximately 240 cells (as determined by counting in the hemacytometer) in 3 ml of growth medium were plated in 60-mm diameter plates (in duplicates for each m.o.i. tested) and allowed to attach at 37”. Two hours later approximately 8 X lo6 noninfected L cells in 2 ml of growth medium were added to each plate and allowed

INTERFERON

AND

PROTEIN

TABLE EFFECT

ok INTERFERON ADDED posy, mRNA (I.4

Reovirus , 3 .O EMC virus, 2.6 Rabbit globin, 0.8 L cell, 1.0 Poly (U), 5.0 L cell endogenou@ L cell polysomese

TREATMENT VIRAL,

53

SYNTHESIS

1

OF L CELLS ON THE CAPACITY AND HOST mRNAs, AS WELL

OF THEIR EXTRACTS TO TRANSLATF: AS ENDOGENOUS mRNAa __-. Inhibition of amino acid Aminoacyl residues incorporated (pmoles) into protein in S30 extracts from incorporation in S30 extracts by treating cells with interferon” Control cells Cells treated with (%) interferon 4.5 9.6 4.6 4.4 18.5 IG

(l.O)b (1.0) (0.52) (1.1) (0.81) (6.3) (0.60)

1.4 1.6 1.2 2.1 20

(0.93)b (0.93) (0.50) (1.0) (0.81) (5.1) 12 (0.60)

87 92 8% 6a None 20 26

0 The incubations were carried out in preincubated S30 extracts from either control cells treated with 37.5 units/ml of interferon in growth medium for 18 hr. The labeled amino acid valine in all cases except when poly(U) served as mRNA, in which case it was “C-phenyIalanine. * The numbers in parentheses are picomoles of labeled aminoacyl residues incorporated mRNA was added to the reaction mixture. c The percent inhibition resulting from treating the cells with interferon was calculated in (except that for L ~11 endogenous mRNA) for the amino acid incorporation in the presence mRNA from which that occurring without added mR.NA was subtracted. d The S30 extracts were not preincubated. e The amount of L cell polysomes in the reaction mixture was 0.3 A LEOunits. For details see vant parts of the Materials and Methods section.

attach at 37” for 2 hr. By this time the cells formed a confluent layer attached to t’he plate. Thereafter the medium was removed, the plates were washed twice with PBS and overlaid with agar for assaying plaques according to the procedure of Vassef et al. (1973). Study of the e$ect of interferon treatment on the accumulation of various size classes of reo virion proteins and of host protein in infected II cells: Examin,ation by immwnoprecipitation with antisera against the various size classes of reo virion proteins (Fig. 3). Monolayers of L cells were grown on 60-mm diameter plates in growth medium in an atmosphere of 5% CO2 in air. About 1 day before reaching confluency the medium was removed and the monolayers were supplemented with 2.5 ml of growth medium (containing no interferon for monolayers in one group, 3 units/ml interferon for those in a second group, and 7.5 units/ml for those in a third group, as indicated in the figure) and incubated. After 18 hr the medium was removed and some of the monolayers in each group were infected with to

or cells was 8Hwhen

no.

all cases of added

the

rele-

reovirus at an m.0.i. of 10 in 0.5 ml growth medium B and incubated. Ninety minutes later the medium was removed and the monolayers were washed with 2.5 ml of PBS to remove the unadsorbed virus. One monolayer of each group was supplemented with 3 ml of growth medium B and further incubated. (This was used for determining the virus yield.) The other monolayers in each group were supplemented with 2 ml of growth medium B and further incubat,ed (0 time). At 7, 10, and 13 hr after infection the medium from one monolayer of each group was removed and each of these monolayers was supplemented with 2 ml of growth medium B containing 2 &i /ml of 35Smethionine (spec. act. 60 - 130 Ci/mmole) and incubated for 1 hr. Thereafter the medium was removed and the plates were frozen and stored (if necessary) at -50”. a. Extraction of proteins for immuno-precipitation. The plates were thawed at; 4’ and 0.2 ml of RSBT was spread evenlyon each plate. After 10 min at 4’ the cells were scraped off with a polyethylene scraper and the suspension was transferred int,o a

54

GUPTA

conical test tube. The plate was rinsed with 0.1 ml of RSBT and the rinsing solution was added to the‘ suspension. The suspension was gently agitated for 5 min and then centrifuged at 600 g for 10 min. The supernatant fraction was collected and the pellet fraction (containing nuclei and cell debris) was washed by suspension in 0.1 ml of RSBT and centrifuged in the same way. The supernatant fractions were pooled, supplemented with sodium deoxycholate to a final concentration of 0.3 % and centrifuged at 10,000 g for 10 min. The resulting supernatant fraction was supplemented with sodium dodecyl sulfate to a final concentration of 0.5 %, heated at 95” for 5 min, cooled and centrifuged at 3000 g for 10 min. The resulting supernatant fraction was used in immunoprecipitation experiments. b. Immunoprecipitation of various size classes of reo virion proteins. Aliquots of various amounts of the supernatant fractions (10, 20, and 40 ~1from the 7- and lo-hr samples, 2.5-, 5-, and lo-y1 fractions from the 13-hr samples) were made up to 40 pl with RSBT and supplemented with 5 ~1 of buffer T (100 rnlll sodium phosphate, pH 7.5; 150 mM NaCl), and 5 ~1of either anti X, anti p, or anti u serum. After 1 hr incubation at room temperature, 50 ctl of goat antirabbit IgG serum was added together with 6 ~1 of buffer T and 4 ~1 of 10% (w/v) Triton X-100 to each incubation mixture and the incubation was continued at room temperature to allow for the formation of the immunoprecipitates. After 45 min the suspension was diluted, with 1.5 ml of washing buffer (10 mM sodium phosphate (pH 7.5) containing 150 n&? NaCl, 1 mih! methionine, 1% sodium deoxycholate, and 1% Triton X-100) and centrifuged at 3000 g for 5 min. The pellet fractions containing the immunoprecipitates were washed by suspension in the washing buffer and sedimentation by centrifugation two more times. Subsequently the immunoprecipitates were filtered through nitrocellulose filters, the filters were washed with washing buffer and counted in a toluene based scintillator. The amount of labeled material in the immunoprecipitates was approxiL mately proportional ‘to the volume of the supernatant fraction used for at least two

ET AL.

amounts of supernatant fraction in each case. The data derived (amount of immunoprecipitable material per volume) were calculated from the values in various tubes containing different amounts of supernatant fraction. RESULTS

Effect of Interferon Treatment of L Cells on the Capacity of Their Extracts to Translate Various mRNAs One of the processesin viral replication that may conceivably be impaired in interferon-treated cells is the translation of viral niRNA. The data shown in Table 1 reveal that this is indeed-affected, at least in the extracts of interferon-treated cells. The 530 extracts for these experiments were prepared either from L cells treated with interferon (in an amount resulting in a 90% decrease in the virus yield in cells infected with reovirus at an m.o.i. of 5), or from L cells not treated with interferon. The extracts were incubated before the experiment to lower the incorporation due to the translation of endogenous mRNA. The data shown are taken from one of several experiments which gave similar results. It can be seen that treatment of cells with interferon greatly impairs protein synthesis in their extracts as directed by the mRNAs of either of two interferon sensitive viruses: reovirus and EMC virus. Moreover protein synthesis in the presence of a heterologous host messenger (rabbit globin mRNA) is inhibited to almost the same extent as that in the presence of viral mRNA. Protein synthesis in the presence of a mixture of homologous host messengers(from L cells) is also inhibited though perhaps to a somewhat lesser extent. There is, however, no inhibition of amino acid incorporation directed by polyuridylic acid. The inhibition is very slight in the translation of endogenous mRNA (in a cell extract not preincubated) and is similarly small when homologous polysomes from L cells are added to the extract. These results seem to indicate that in the extracts of interferon-treated cells protein synthesis is greatly inhibited when promoted by any added natural mRNA but is less impaired

INTERFERON

AND

PROTEIN

SYNTHESIS

Fi.5

when it occurs on either endogenous or the effect of interferon c3n protein accumulation in intact cells, we performed t,he folexogenous polysomes. Mixing an extract from interferonlowing experiments. We ,followed the rat*e of labeled amino acids treat’ed cells with that, from control cells of the incorporation (in a volume ratio of 1: 1) results in an F into proteins of control cells and of cells treated for 18 hr with an amount of interabout 70% impairment of protein synthesis promoted by added EMC RNA in a 60- feron (7.5 units/ml) which causes about $53, inhibition of virus replication under min incubation (Table 2). This indicates that there is an inhibitor present in the the conditions of the experiment. During the 24 hr of the experime’nt no significant int.erferon-treated extract. difference was noted between protein synthesis in control and interferon-treated cells Effect of Interferon-Treatment on Viraland Host-Protein Accumulation in Reo- (Fig. la, curve C and INT). This indicates that interferon-t,reatment in our conditions virus-Infected and Uninfected L Cells does not affect host protein accumulation. Assay of total protein accumulation. To We also tested the effect of interferonestablish whether the results with extracts treatment on the accumulation of proteins of interferon-treated cells reflect faithfully in L cells which were infected with reovirus. The multiplicity of infection of cells with TABLE 2 the virus was 10 and in t’hese conditions EFIWCT OF MIXING OF .4~ S30 EXTRACT FROM L over 80% of the cells are infected (Fig. CELLS TREATED WITH INTEFGFERON WITH THAT lb). The rate of protein accumulation in FROM CONTROL CELLS ON PROTEIN SYNTHXSIS reovirus-infected cells increases above that DIRXCT~D BY EMC RNA: PRESENCE OF AN of control cells between 8 and 14 hr after INHIBITOR IN THE EXTRACT FROM CELLS infection, thereafter it decreases. Twent?;TRE:ATI.:D WITH INTERFERON~ four hr after infection, it is only half as .Volume of S30 ex3H-Val incorporaInhibition fast, as in uninfected cells (Fig. la, curve tract (~1) from ted (pmoles )into of aH-Val REO). In interferon-treated, infect’ed cells ___ protein in S30 incorporathe ratz of protein accumulation never Control Cells extract tion” treated cells (%I exceeds t’hat’ in control cells. Furthermore, with inbetween 14 and 24 hr after infection the terferon rate is even lower than in infected cells which are not treated with interferon (Fig. 10 -9.8 (0.36)b la, curve INT, REO). These rasults may 10 0.50 (0.33) 98 10 10 3.4 (0.41) 69 indicate that interferon treatment blocks 20 12.4 (0.34) 0 virus-specific protein accumulat8ion without affecting host protein accumulation in virusG The incubations were carried out in preininfected cells and does not overcome the cubated S30 extracts from control cells and cells inhibition of host protein accumulation treated with 37.5 units/ml of interferon in growth caused by reovirus infection. medium for 18 hr. The total volume of the reaction Assay by gel electrophoresis. To provide mixture was 30 ~1. The concentration of KC1 was further support.for these tentative conclu120 mM, the amount of EMC RNA added was 2 pg, the incubation was performed at 37” for 60 sions we analyzed polyacrylamide gel elecmin. trophrrograms of proteins accumulated in h The numbers in parentheses are pmoles of reovirus-infected interferon-t’reated, and 3H-valine incorporated when no EMC RNA was reovirus- infected not interferon-treated, added to the reaction mixture. cells. The electropherogram of reovirusc The percent inhibition was calculated from specific proteins accumulated in infcrted the 3H-valine incorporation in the presence of L cells is shown in Fig. 2a (REO Marker EMC RNA from which that occurring without curve). This was obtained by incubating added mRNA was subtracted. The incorporation with labeled amino acids reovirus-infert,ed into 10 ~1 of 530 extract from control cells was L cells in which host RNA (and consequently taken as 100. For details see the relevant parts of the Materials and Methods section. host protein) sgnt,hesiswas blocked by ac-

56

GIJPTA

z ::

ET AL.

-. *.

52

, 4

I 6

I 8

I 10 Hours

I 12 After

I 14 Infection

I 16

1 t8

I 20

% D INT, REO

, 22

I 24

FIG. 1. Effect of interferon treatment on protein accumulation in L cells and reovirus-infected L cells. (a) Monolayers of L cells were grown on 35 mm diameter plates in growth medium in an atmosphere of 570 COS in air. About 1 day before reaching confluency the medium was removed and the monolayers were supplemented with 2 ml of growth medium containing either no interferon or 7.5 units/ml and incubated. After 18 hr the medium was removed and some of the monolayers in each group were infected with reovirus at an m.o.i. of 10 in 0.3 ml of growth medium and incubated. Ninety minutes later the cells were washed with 2.5 ml of PBS, and 2 ml of growth medium was added to each plate (0 time). For labeling, the medium was replaced by 1 ml of growth medium containing 0.5 &i a%-methionine (spec. act. 62 Ci/mmole) for 1 hr at 37”. The midpoint of the various labeling periods is given in the figure. After labeling, the medium was removed, the cells in the plate were washed twice with 5 ml of PBS and solubilized by incubation in 0.5 ml of 0.1 N NaOH at room temperature. Five minutes later the resulting suspension was transferred to a test tube, the plate was washed with 0.5 ml of H20, and the washing solution was also transferred to the same test tube. The suspension was supplemented with 1 ml of 20% trichloroacetic acid containing O.Sa/, casamino acids, incubated at 95” for 15 min to precipitate the proteins and filtered through a Whatman GFA glass fiber filter. The filter with the protein precipitate was washed first with 5y0 trichloroacetic acid, subsequently with 70y0 ethanol, finally with ether, and dried and counted in a toluene based scintillator. C stands for control cells; INT for cells treated with interferon; REO for cells infected with reovirus; and INT,REO for cells first treated with interferon and subsequently infected with reovirus. (b) Dependence of the fraction of L cells infected on the m.o.i. with reovirus. For details see the relevant parts of the Materials and Methods section.

tinomycin D (see Zweerink and Joklik, 1970). The designation of the reovirus protein size class in the various peaks is indicated in the figure. The electropherogram designated REO in Fig. 2b is that of proteins accumulated in reovirus-infected cells between 7 and 14 hr after infection. (In this and the subsequent experiments no actinomycin was added to avoid complicating effects: cf., e.g., Singer and Penman, 1972.) Some of the peaks in the REO curve coincide with those of the REO Marker curve in Fig. 2a. This indicates that much of the protein synthesized in these conditions is reovirus-specific. As a control the electropherogram of proteins in uninfected cells (Fig. 2b, curve C) and in uninfected interferon-treated cells (Fig. 2b, curve

INT) are also shown. The curve in Fig. 2c designated A (REO - C) is a plot of the difference in the amount of label between the REO curve and the C curve in Fig. 2b. This difference curve is supposed to reflect the electropherogram of reovirus-specific proteins. It is consistent with this expectation that the correlation is good between the peaks in this difference curve and those of the REO Marker curve in Fig. 2a. The curve designated A (INT, REO - INT) is a plot of the difference in the amount of label between the INT, REO curve (from interferon-treated, reovirus-infected cells; not shown) and the INT curve from Fig. 2b. The calculated number of counts are scattered around the 0 value in this difference curve. This seemsto indicate that in inter-

INTERFERON

10

AND

20

30 Gel

PROTEIN

40 Slice

50 No.

57

SYNTHESIS

60

70

80

+

FIG. 2. Effect of interferon treatment on reovirus protein accumulation in infected L cells: examination by gel electrophoresis. (a) Reovirus specific proteins (in the figure REO Marker) synthesized in infected L cells in which host specific RNA (and consequently protein) synthesis was blocked with 0.5 &ml actinomycin D. The cells were labeled between 7 and 14 hr after infection, and addition of actinoQ (small) in the three regions mycin D. The designation of the protein size class X (large), p (medium), of the electropherogram is indicated in the figure. For details see Graziadei et al. (1973). The mixture of labeled proteins (2700 cpm) was fractionated by gel electrophoresis as described in (b). L cells (INT), reovirus-infected L (b) Protein synthesis in control L cells (C), interferon-treated cells (REO), and int.erferon-treated and reovirus-infected L cells (INT,REO). For details of the procedure followed until 0 time see the legend to Fig. la except that 60 mm diameter plates were used and the monolayers were infected with reovirus suspended in 0.5 ml of growth medium and at 0 time 3 ml of growth medium was added to each plate. The cells were incubated from 0 time to 7 hr. Thereafter the medium was removed, each plate was supplemented with 3 ml of growth medium (however, containing only 1% fetal calf serum and x of the usual amount of amino acids) as well as 5 &i/ml 3%-methionine (spec. act. 60 Ci/mmole) and incubated further. At 14 hr after 0 time the plates were washed and protein was extracted from the cells in each monolayer as described by Graziadei et aZ. (1973). The extracted proteins were fractionated by electrophoresis at pH 7.2 in sodium dodecyl sulfate, urea, polyacrylamide gels and the gels were sliced and the slices were counted as described by Graziadei et al. (1973). The following amount of 36S-methionine labeled material was applied to each gel: to (C) 10,000 cpm, to (INT) 9000 cpm, to (REO) 13,000 cpm, to (INT,REO) 9600 cpm. The difference between the labeling of the slices of the (INT) sample and the (INT, REO) sample was small (see c). (c) The curve A(REO-C) is a plot of the difference in the amount’ of label between the (REO) curve and the (C) curve (in b). The curve A(INT,REO - INT) is a plot of the difference in the amount of label between the (INT,REO) curve (not shown) and the (INT) curve (shown in b).

feron-treated, reovirus-infected cells little if ( any) detectable reovirus-specific protein is synthesized. Assay by ir?zmunoprecipitation. A sensitive

assay for virus proteins in cell extracts is based on immunoprecipitation. This was used as a third approach in testing the selectivity of the effect of interferon treatment

58

ET AL.

GUPTA

on virus protein accumulation. For this assay we prepared three antisera against the three size classesof reo virion proteins: one specific for the large (A), one for the medium (P), and one for the small (u) size class. Using the antisera we tested the effect of treating cells with interferon on the accumulation of reo virion proteins of various size classesbetween 2 and 3, 4 and 5, 7 and 8, 10 and 11, as well as 13 and 14 hr after infection. The cells were grown in monolayers and were treated with either no interferon or with 3 or 7.5 units/ml for 20 hr before infection. In t’hesecondit’ions 3 units/ ml of interferon caused an 87 % decrease and 7.5 units/ml a 95 % decrease in the yield of reovirus (m.o.i. 10) formed by 24 hr after infection. Between 2 and 3 as well

Hours

After

as 4 and 5 hr after infection no virion protein accumulation was detected either in interferon-treated or control cells. The curves in Fig. 3a, b, and c, reveal (1) a linear increase in the rate of accumulation of virion proteins of each size class between 7 and 14 hr after infection and (2) a strong and about equal inhibition of the accumulation of reo virion proteins of each size class in cells treated with interferon. The extent of this inhibition increases with the concentration of interferon. It is remarkable that in control cells the rate of virion protein accumulation about doubles between 10 and 14 hr after infection whereas in interferon-treated cells it barely changes. The sum of the accumulation of reo virion proteins of all three size classesand the effect

Infection

FIG. 3. Effect of interferon treatment on the accumulation of various size classes of reo virion proteins and of host protein in infected L cells: Examination by immunoprecipitation with antisera against the various size classes of reo virion proteins. The points on the curves represent amounts of labeled methionine incorporated into various size classes of reo virion proteins (A in a, p in b, (r in c), between 7 and 8, 10 and 11, and 13 and 14 hr after infection with reovirus of L cells which had been treated for 20 hr preceding infection with either no interferon (0) or 3 or 7.5 units/ml of interferon. The immunoprecipitates of the reo virion proteins were obtained by treating extracts of cells with either anti X (in a), anti fi (in b), or anti (r serum (in c) and counting the radioactivity in the precipitate. The data were calculated by subtracting from the amount of label in the immunoprecipitate of an extract of reovirus-infected cells that from uninfected cells (2 to 3 X lo3 cpm) which had been treated identically with the infected cells. The data in (d) were calculated by adding the amounts of label incorporated into x , p, and ,Y size class virion proteins (i.e., represent amounts of label in all virion proteins). The data in (e) were calculated by subtracting from the total amount of label incorporated (i.e., label in material insoluble after heating in 57, trichloroacetic acid containing 0.2 mM methionine at 95” for 15 min) the amount of label in all virion proteins. These data reflect upon the amount of host specific proteins synthesized. This is, however, only an approximation since some of the label is present in reovirus specified nonvirion protein. Such nonvirion proteins (e.g., w, oao) are not precipitated by our antisera (Graziadei et al., 1973) and therefore the label in these was not subtracted from the total amount. The amount of label shown is that present in cells of an entire monolayer in a 60-mm diameter plate. For details see the relevant parts of the Materials and Methods section.

INTERFERON

AN11

PROTEIN

thereon of interferon treatment is shown in the curves in panel d. The data for these were calculated by adding those in panels a, b, and c. An approximation of the amount of host proteins accumulated in reovirusinfected cells is shown in panel e. The data for this panel were calculated by subtracting from the total amount of label incorporated into hot acid-insoluble product the amount of label incorporated into immunoprecipitated reo virion proteins. It can be seen that host protein accumulation decreases slightly between 7 and 11 hr and strongly between 11 and 14 hr after the infection of the cells with reovirus, and that interferon treatment does not overcome the inhibition of host protein synthesis in reovirus-infected cells. The results shown in Figs. 1, 2, and 3 indicate that interferon treatment blocks virion protein accumulation without affecting the extent of total host protein accumulation in reovirusinfrrt’ed cells. We did not test, however, if interferon treatment alters the rate of turnover of host proteins, or the proportion in which t’he various host proteins are accumulated. Dependence oJ’ the Inhibition of Reovirus Replication and Reovirus Specific Protein Accumulation, on the Interferon Concentration The dependence on interferon concentration of the inhibition of reovirus replication, and c size class reo virion protein accumulation are compared in Fig. 4. Virus yield was determined 24 hr after infection (i.e., after a single replicative cycle) and u virion protein accumulation was determined between 7 and 8 hr after infection. At all concentrations of interferon tested the inhibition of virus replication was more extensive t,han that of c reo virion protein accumulation early in infection. (It was shown in Fig. 3 that the accumulation of all thrcar size classesof reo virion proteins follow the same pattern and interferon trt>atment affects all three similarly. j The difference in the concentration depcndencc of the inhibition of virus yield and virion protein accumulation is greatly diminished late in infection. This is so b~ausc~ thr rate of virion protein accumu-

59

SYNTHESIS

INT

Concentration

(U/ml1

FIG. 4. Effect of treating L cells with various concentrations of interferon prior to infection with reovirus on the yield of reovirus and on the accumulation of (r size class reo virion proteins. The yield of reovirus and the amount of methionine incorporated into c size class reo virion proteins in L cells in monolayers not treated with interferon were taken as 100. The corresponding values obtained in cells in monolayers treated with various amounts of interferon for 20 hr preceding infection are given as per cent of the former. The yield of reovirus in a single replicative cycle (25 hr after infection) was determined according to the procedure of Vassef et al. (1973) for assaying single cycle yield reduction. The cells were exposed to 35%methionine for 1 hr between 7 and 8 hr after infection with reovirus at an m.o.i. of 10 in conditions otherwise identical to those described for the experiment shown in Fig. 3. For details of the determination of the amount of methionine incorporated into (r size class reovirion proteins see the relevant parts of t#he Materials and Methods section.

lation increases more than twice as fast in cells not treated with interferon than in those treated with interferon from 7 t,o 14 hr after infection (see Fig. 3). DISCUSSION

The following arc the main conclusions that can be drawn from our experiments on the effect of interferon-treatment on virus and host metabolism in rtovirus-infected I, cells: Interferon (at a concentration at’ which it blocks reovirus replication in L cells by 95 ‘35) (1) does not affect prot,cin

60

GUPTA ET AL.

accumulation in uninfected L cells, (2) does not affect total protein accumulation early after infection in reovirus-infected cells, (3) does block reovirus protein accumulation (early and even more late) after infection. [For other reports on the inhibition of viral protein accumulation in interferon-treated cells see, e.g., Friedman (1968) ; Yamazaki and Wagner (1970).] (4) Interferon does not overcome two effects of reovirus infection on L cells, i.e., the inhibition of host protein accumulation late in infection and cell lysis. Ten times more interferon (75 units/ml) than needed for causing 95 % inhibition of reovirus yield does not protect the cells from lysis either (see also Levy, 1964; Gauntt and Lockart, 1966; Haase et al., 1969; Yamazaki and Wagner, 1970). The fact that reovirus protein accumulation is impaired in interferon-treated L cells at a time (7 and 11 hr after infection, see Figs. 1 and 3) when total protein accumulation is not yet, or is at most only slightly, affected indicates that the inhibition of reovirus replication in interferon-treated L cells is not (or at least not only) a consequence of the shutdown of all macromolecular synthesis i.e., the “suicide” of the cells. Earlier we established that penetration and uncoating of reovirus is not impaired in interferon-treated L cells (R. Galster, unpublished data) but synthesis of single stranded and double stranded reo RNA is (Vassef et al., 1973; see also Gaunt& 1972). The extracts of cells treated with our interferon preparation have an impaired capacity for translating various added mRNAs including viral and host mRNAs. The following findings are consistent with the assumption that this impairment is due to interferon and not to another component of the interferon preparation: (1) Virus replication is not blocked in cells exposed to interferon in the presence of actinomycin D (an agent blocking RNA synthesis) (Taylor, 1964; Lockart, 1964); and we find that in these conditions (i.e., exposure of cells to interferon in the presence of actinomycin D) the capacity of the cell extract to translate exogenous mRNA is not impaired by the interferon treatment. We derived this conclusion from an experiment with

mouse Ehrlich ascites tumor cells (Gupta et al., 1973). Treatment of these cells with 60 units/ml of our interferon preparation diminished the capacity of their extract to translate EMC RNA by 88%, whereas treating the cells in the presence of 1.2 pg/ml of actinomycin D with 60 units/ml of interferon had no such effect. The same conclusion was reached earlier by Falcoff et aE. (1972) from an experiment with L cells and a less highly purified interferon preparation. (2) Treating of cells in suspension culture with interferon (37.5 units/ml) inhibits the virus yield in intact infected cells and the capacity of the cell extract to translate added viral mRNAs to a comparable extent (by about 90%). The manifestation of the interferon effect in the cell extract does not require that the cells from which the extract is prepared be infected with a virus and the inhibition persists even after incubating the extract at 37” for 45 min. These results are in agreement with those of Falcoff et aZ. (1972; 1973). The in vitro manifestation of the interferon effect should make it possible to identify the component responsible for the impairment of translation. The fact that in a mixture of two cell extracts: one from control cells and one from interferon-treated cells the inhibition is dominant indicates that the block is not due to a lack of a component but to an inhibitor. Falcoff et al. (1973) reached the same conclusion though they did not find an inhibition upon mixing a crude extract from interferon-treated cells with that from control cells. There is a discrepancy between the selective block of viral protein accumulation in interferon-treated cells on the one hand and the general block of translation of exogenous viral and host mRNAs in the cell extracts of interferon-treated cells on the other hand. It is conceivable, however, that this discrepancy may be more apparent than real. The translation of endogenous mRNA or mRNAs present in polysomes (and added as such) is not inhibited in extracts of interferon-treated cells. It is the translation of exogenous “naked” mRNAs only which is blocked. It is possible that host messengers are processed differently from viral messengersin vivo and that the outcome of

INTERFERON

AND

this different processing determines whether the translation of an mRNA will or will not be impaired in cells treated with interferon. [Such differences in processing may, e.g., consist of differences in cleavage, or modification of the RNAs, or attachment of different proteins to them; for recent references see Williamson et al. (1973).] The translation of viral (i.e., EMC or reo) mRNA is slightly more inhibited than that of host (i.e., L cell) mRNA (Table 1). This difference may perhaps indicate that the translation of different natural mRNAs is impaired to a different extent in interferon-treated cells and that in turn the interferon defense mechanism is based on differences in sensitivity between various mRNAs. [For various mRNA-specific translation initiation factors see, e.g., Heywood (1969), Revel et al. (1970), Lee-Huang and Ochoa (1971), Wigle and Smith (1973), Nude1 et al. (1973).] The available data are, however, clearly insufficient for substantiating this possibility. The results with intact cells and with cell extracts taken together still do not allow the identification of the step (or steps) in virus replication which is (or are) blocked in interferon-treated cells. However, if we assume that only one step is inhibited (which does not have to be the case) and base our considerations in part on the impairment of mRNA translation in the extracts of interferon-treated cells, then the following tentative conclusion may be drawn: The block may occur after the synthesis of viral mRNA (at least in the case of reovirus) but before the synthesis of viral protein. This leaves open as possible causes of the inhibition of virus replication in interferon-treated cells a block in the translation proper of the viral mRNA or an accelerated inactivation and degradation of the viral mRNA. This latter possibility could account’ for the decreased amount of viral mRNA and viral protein accumulated in interferon-treated cells as well as the impairment of the translation of added mRNA in extracts of interferon-treated cells.

PROTEIN

SYNTHESIS

61

ACKNOWLEDGMENTS This study haa been supported by a grant (GB 30700x) from the National Science Foundation. W.D.G. has been a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. H.W. haa been a postdoctoral fellow of the Swiss National Fund. We thank R. P. Perry and L. Schaefer for preparations of oligo dT-cellulose. and M. Revef for preprints. REFERENCES AVIV, H., BOIME, I., and LEDER, P. (1971). Protein synthesis directed by encephalomyocarditis virus RNA: Properties of a transfer RNAdependent system. Proc. Nat. Acad. Sci. U.S. 68,2303-2307. BIALY, H. S., and COLBY, C. (1972). Inhibition of early vaccinia virus RNA synthesis in interferon-treated chicken embryo fibroblasts. J. Viral. 9,286289. DIANZANI, F., BUCKLER, C. E., and BARON, S. (1968). Kinetics of the development of factors responsible for interferon-induced resistance. PTOC. Sot. Exp. Biol. Med. 129, 535-538. EVANS, M. J., and LINGREL, J. B. (1969). Hemoglobin messenger ribonucleic acid. Synthesis of 9 S and ribosomal ribonucleic acid during erythroid cell development. Biochemistry 8, 300&3005. FALCOFF, E., FALCOFF, R., LEBLEU, B., and RYVNL, M. (1972). Interferon treatment inhibits mengo RNA and hemoglobin mRNA translation in cell-free extracts of L cells. Nature (London.) New Biol. 240, 145-147. FALCOFB, E., FALCOFF, R., LXBLNJ, B., and REVEL, M. (1973). Correlation between the antiviral effect of interferon treatment and the inhibition in vitro of messenger RNA translation in non-infected L cells. J. Viral. 12,421-430. FRIEDMAN, 1~. M. (1968). Inhibition of arbovirus protein synthesis by interferon. J. Viral. 2, 1081-1085. FRIEDMAN, Ii. M., MICTZ, D. H., ESTEBAN, R. M., TOWLL, D. It., BALL, L. A., and KERR, 1. M. (1972). Mechanism of interferon action: Inhibition of viral messenger ribonucleic acid translation in L cell extracts. J. Viral. 10, 11841198. GAUNTT, C. J. (1972). Effect of interferon on synthesis of ssRNA in reovirus type 3 infected 1, cell cultures. Biochem. Biophys. Res. Commun. Pi, 1228-1236. GAUNTT, C. J., and LOCKART, R. Z., JR. (1966). Inhibition of mengovirus by interferon. J. Bacterial. 91, 176-182. GILHAM, P. T. (1964). The synthesis of polynucleotide-celluloses and their use in the fractiona-

62

GUPTA

tion of polynucleotides. J. Amer. Chem. Sot. 86,49824985. GRAVELL, M., and CROMEANS, T. L. (1972); Inhibition of early viral RNA synthesis in interferon treated cells infected with frog polyhedral cytoplasmic deoxyribo virus. virology 50, 916919. GRAZIADEI, W. D., and LENGYEL, P. (1972). Translation of in vitro synthesized reovirus messenger HSAs into proteins of the size of reovirus capsid proteins in a mouse L cell extract. Biochem. Biophya. Res. Commun. 46, 1816-1823. GRAZIADEI, W. D., ROY, D., KOSIQSBERG, W., and LICNGYEL, P.’ (1973). Translation of reovirus messenger ribobuclcic acids synthesized in c&o into reovirus proteins in a mouse L cell extract. Arch. Biochem. Biophys. 158, 21X-275. GUPTA, S. L., SOPORI, M. L., and LRNGYEL, P. (1973). Inhibition of protein synthesis directed by added viral and cellular messenger RNAs in extracts of interferon-treated Ehrlich ascites tumor cells. Location and dominance of the inhibitor(s). Biochem. Biophys. Res. Commun. 54, 777-783. HAASE:, A. T., BARON, S., LEVY, II., and KASEL, J. A. (1969). ?uIengovirus-induced cytopathic effect in I, cells: Protective effect of interferon. .I. Viral.

4, 490-495.

H~~woou, S. hf. (1969). Synthesis of myosin on heterologous ribosomes. Cold Spring Harbor Symp. Quant. Biol. 34, 799-803. JOKLIK, W. K. (1971). The molecular biology of rcovirus. J. Cell Physiol. 76,289-302. JOKLIK, W. K., and MERIOAN, T. C. (1966). Concerning the mechanism of action of interferon. Proc. Nat. Acad. Sci. U.S. 56, 558-565. C.,

JUNGWIRTH,

HORAI(,

I.,

BODO,

G.,

LINDNER,

J., and SCHULTZE, B. (1972). The synthesis of pox virus-specific RNA in interferon-treated cells. Virology 48, 59-70. LEE;-HUANG, S., and OCHOA, S. (1971). Messenger discriminating species of initiation factor F3. Nature

(London)

New

Biol.

234.

236-239.

H. B. (1964). Studies on the mechanism of interferon action. IT. The effect of interferon on some early events in mengo virus infection in I, cells. Virology 22, 575-579. LOCKART, It. %. (1964). The necessity for cellular RNA and protein synthesis for viral inhibition resulting from interferon. Biochem. Biophys. Res. Commun. 15. 513-518. i\Icl~ow~~r., hf. J., JOKLIK, W. K., \rILLA-KohlAROFF, L., and LODISH, H. F. (1972). Translation of reovirus messenger RNAs synthesized in vitro into reovirus polypeptides by several mammalian cell-free extracts. Proc. Nat. LWY,

Acad.

Sci.

c’.S. 69, 2649-2653.

ET

AL.

E’K., TI.LLES, J. C;., and ILL.I\Nc;, A. d. (1972).. Interferon-r+iated inhibition of viriondirected transcription. V&OZO~~ 49, 573-581. MARCUS, P. I., ENOELHARDT, 1). L., HUNT, J. JI., and SEKEL~ICK, M. J. (1971). Interferon action: Inhibition .of vesicular stomatitis virus RNA synthesis induced by virion-bound polpinerase. Science 174, 593-598. METZ, I). H., and ESTEBAN, J. (1972). Interferon inhibits viral protein synthesis in L:cells infected with vaccinia virus. Xalure (London) 238, 385-388. MILLWARD, S., and GRAHAM, A. E. (1971). Structure and transcription of the genomes of doublestranded RNA viruses. “Comparative Virology.” (K. Maramorosch and E. Kurstak, cds.), pp. 387406. Academic Press, New York. Nc, M. H., and VILEEK, J. (1972). Interferons: Physicochemical properties and control of cellular synthesis. Aduan. Protein Chem. 26, 173-241. NUDEL, U., LEBLEU, B., and REVEL, M. (1973). Discrimination between mRNAs by a mammalian translation initiation factor. Proc. Nat. Acad. Sci. U. S. 70. 2139-2144. OII:, II. K., and LEH, P. C. (1968). Reovirus type 2: Production of and sensitivity to interferon in human amnion cells. Proc. Sot. Ezp. Biol. Med. 127, 1210-1213. OXMAN, M. N., and LEVIS, %I. J. (1971). Interferon and transcription of early virus-specific RNA in cells infected with simian virus 40. Proc. Nat. MAXDERS,

Acad.

Sci.

U.S.

68, 299-302.

R. P., LA TORRP, J., KELLEY, D. E., and GREENBERG, J. R. (1972). On the lability of poly (A) sequences during extraction of messenger RNA from polyribosomes. Biochim. Biophys. Acta 262, 220-226. REVEL, M., Avxv, II., GRONER, Y., and POI.LACK, Y. (1970). Fractionation of translation initiation factor B (Fz) into cistron-specific species.

PERRY,

FEBS

Lett.

9,213-217.

G., and PERRY, It. I’. (1972). Characterization of the messenger RNA released from L cell polgribosomes as a result of temperature shock. .J. Mol. Biol. 63. 577-590. SHATKIN, A. J. (1971). Viruses with segmented ribonucleic acid genomes: Multiplication of influenza versus reovirus. Racleriol. Rev. 35, 250-266. SINGICR, R. H., and PISHMAN, S. (1972). Stability of IleLa cell mBNA in actinomycin. Nature (London) 240, 100-102. SWAN, D., Av~v, H., and LEDI;R, I’. (1972). Purification and properties of biologically active messenger RNA for a myeloma light chain. Proc. Nat. Acad. Sci. I,‘.S. 69, 1967-1971. TAYLOR, J. (1964). Inhibition of interferon action SCHOCHXTMAS,

INTERFEIION

AND

by actinomycin. Biochenz. Biophys. Res. Commun. 14, 447451. TAYLOR, J. (1965). Studies on the mechanism of action of interferon. I. Interferon action and RNB synthesis in chick embryo fibroblasts infected with Semliki Forest virus. Virology

25,34&349. VASSEF,

A., BEAUD, G., PAUCKER, K., and LENGYEL, P. (1973). Interferon assay based on the inhibition of double-stranded reovirus RNA accumulation in mouse L cells. J. Gen. Viral. 19, 81-87. WIGLE, D. T., and SMITH, A. E. (1973). Specificity in initiation of protein synthesis in a fraction-

PROTEIN ated

63

SYNTHEsIS mammalian

cell-free

system.

~VU~ZITP (I,orr--

don) New Bid. 242, 13G-140. WILLIAMSON, R., I~REWIENKII~ZVICZ, C. E., and PAUL, J., (1973). Globin messenger sequenccx in high molecular weight RNA from embryonic mouse liver. Nature (London) :Vcw Bid. 2&l,

06-68. YAM~ZAKI, S., and WAGNER, R. P. (1970). -Yetion of interferon: Kinetics and differential ri'fects on viral functions. .1. Vi&. 6, 421~-129. ZWXI:RISK, H. J., and JOPLIK, W. K. (1970). Studies on the intracellular synthesis of reovirus-specified proteins. Virology -41, 501 -UNIX.