Inhibition of protein synthesis by vaccinia virus I. Characterization of an inhibited cell-free protein-synthesizing system from infected Cells

VIROLOGY

99, 319-328 (1979)

Inhibition

of Protein

I. Characterization

Synthesis

by Vaccinia

Virus

of an Inhibited Cell-Free Protein-Synthesizing System from Infected Cells

M. SCHROM’l* AND R. BABLANIAN3 Department of Microbiology

and Immunology, State University of New York, Downstate Brooklyn, New York 11203

Medical

Center,

Accepted August 26, 1979

L-929 or HeLa cells infected with vaccinia virus in the presence of cycloheximide fail to resume protein synthesis upon removal of the drug 3.5 hr after infection. However, infectedtreated LLC-MK2 cells resume protein synthesis upon removal of the drug. Cell-free protein-synthesizing systems prepared from such vaccinia virus infected-cycloheximide treated L-929 or HeLa cells, 30 min after removal of the drug, fail to incorporate amino acids in vitro in response to endogenous mRNAs, while similar extracts of LLC-MK2 cells are functional in vitro. The inhibition of protein synthesis is also characterized by a failure to respond to exogenous mRNAs (Globin, L cell poly (A), or EMC RNA). Such inhibited extracts have been separated into supernatant, ribosome, and ribosomal salt wash fractions. The response of ribosomes from infected and uninfected cells to EMC RNA in the presence of homologous and heterologous supernatant fractions shows that defects can be found in both ribosomes and supernatant fractions of infected-treated cells. Readdition of salt wash fraction of infected and uninfected ribosomes to the ribosome and supernatant fractions of infected and uninfected cells shows an additional lesion in the salt wash fraction of infectedtreated cells. Addition of supernatant, ribosome, and salt wash fractions of infected and uninfected cells individually to reconstituted protein-synthesizing systems prepared from normal cells shows that the primary lesion in the infected-treated cells is the salt wash fraction of the infected ribosomes. This constitutes a crude initiation factor preparation and is consistent with previous observations that vaccinia virus-induced inhibition of protein synthesis is the result of a failure at the level of initiation. INTRODUCTION

One of the most prominent features of virus-cell interaction is the inhibition of cellular protein synthesis (reviewed by Bablanian, 1972, 1975). The inhibition of host protein synthesis has been extensively ’ Present address: Department of Microbiology and Immunology, Albany Medical College, Albany, N. Y., 12208. 2 Based on a portion of the dissertation which was submitted by M. Schrom to the School of Graduate Studies of the State University of New York in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 3 Author to whom reprint requests should be addressed.

studied in picornavirus-infected cells (Baltimore and Franklin, 1963; Willems and Penman, 1966; Hunt and Ehrenfeld, 1971). In this case preparation of cell-free amino acidincorporating systems from virus-infected cells has shown that these systems are capable of translating viral and cellular mRNAs equally well when tested separately. However when both are added simultaneously the translation of cellular mRNAs is substantially depressed (Lawrence and Thach, 1974; Abreu and Lucas-Lenard, 1976). This inhibition appears to be established at the level of a virus-specific initiation factor (Golini et al., 1976). In other systems, infected extracts are capable of translating viral but not cellular mRNAs. However, in

319

0042~6822/‘79/160319-10$02.00/O Copyright All rights

0 19’79 by Academic Press, Inc. of repmduction in any form reserved.

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SCHROM

AND BABLANIAN

this case the inactivation of an initiation factor (eIF-4B) also appears to be the cause of the inhibition (Rose et aE., 1978). One of the cytopathic effects accompanying vaccinia virus infection is the inhibition of cellular protein synthesis (Kit and Dubbs, 1962; Shatkin, 1963; Salzman and Sebring, 1967; Holowczak and Joklik, 1967). Infection of cells in the presence of inhibitors of protein synthesis (cycloheximide, puromycin, or streptovitacin A) allows primary but not secondary uncoating (Joklik, 1964; Dales, 1965) and allows the development of an irreversible inhibition of both viral and cellular protein synthesis in HeLa (Moss, 1968; Moss and Filler, 1970) or L cells (Bablanian, 1975; Bablanian et al., 1978). In the absence of inhibitors or when inhibitors of protein synthesis are added later in the infection cycle (Moss and Filler, 1970) cellular, but not viral, protein synthesis remains inhibited upon removal of the drugs. However, LLC-MK2 cells infected in the presence of cycloheximide resume protein synthesis when cycloheximide is removed (Bablanian et al., 1978). This led to the suggestion that the inhibition of protein synthesis results, not from the virion itself, but from the synthesis, in the presence of cycloheximide, of a virus-induced product(s). Since cycloheximide allows, and indeed accelerates (Kates and McAuslan, 1967), the synthesis of viral RNA, the relationship of viral RNA to inhibition of protein synthesis was examined. It was found that a strong correlation exists between the inhibition of protein synthesis and the rate of virusinduced RNA synthesis (Bablanian et al., 19’78;Schrom and Bablanian, 1979). The complete inhibition of protein synthesis observed when L or HeLa cells are infected with vaccinia virus in the presence of cycloheximide provides a useful system in which to examine the inhibition of protein synthesis in vitro. In this communication we describe a cell-free amino acid-incorporating system from vaccinia virus-infected, cycloheximide-treated cells which reproduces the inhibition of protein synthesis in vivo. Evidence is presented which is consistent with the hypothesis that the inhibition of protein synthesis takes place at the level

of initiation of new polypeptide chains and is the result of a defect in the ribosomal salt wash fraction of such infected cells. MATERIALS

AND METHODS

Virus. The WR strain of vaccinia virus was propagated in HeLa cells grown in suspension and purified essentially according to Joklik (1962). The virus particle concentration was determined spectrophotometrically, 4.5 x log particles/ml having an A,,, of 1. This number was determined by counting representative virus preparations electron microscopically by the technique of Sharp (1949). Multiplicity of infection is expressed in terms of particles per cell and, although not routinely measured, the particle/PFU ratio was generally in the range of 2O:l. Cell cultures. Monolayer cultures of HeLa, L-929, and LLC-MK2 cells were grown in reinforced Eagle’s medium (REM: Bablanian et al., 1965) supplemented with 10% fetal bovine serum (FBS) and maintained in the same medium with 2% FBS. HeLa and L cells were grown in suspension in Eagle’s spinner medium supplemented with 5% FBS. Measurement of protein synthesis in vivo. Protein synthesis in infected cells was measured as described previously (Bablanian et al., 1978). Protein determinations were carried out according to Bramhall et al. (1969), and hot trichloroacetic acid (TCA)insoluble incorporation was measured according to Mans and Novelli (1961). Preparation of cell-free amino acidincorporating systems. Monolayer cultures were grown in 150 x 15-mm tissue culture dishes (Lux Plastics) and infected with purified vaccinia virus at a multiplicity of 300 particles/cell in the presence of 300 wg/ml of cycloheximide or mock infected and treated with the drug alone. Generally five such dishes containing a total of l-3 x lo* cells were used for each cell-free system. At the indicated time after infection and treatment with cycloheximide, cell-free amino acid-incorporating systems were prepared as follows: the cells were washed three times with cold Tris-saline (30 mM TrisHCl pH 7.4, 140 mM NaCl) and once with

IN VITRO INHIBITION

OF PROTEIN

BY VACCINIA

321

hypotonic medium (10 mM Tris-HCl pH tionated extracts were assayed with the 7.4, 10 mM KCl, 1.5 m&I magnesium following modifications. Ribosomes were acetate, ‘7mM 2-mercaptoethanol). The cells added at 0.45 A,,,, per assay, supernatant were drained nearly dry and scraped into fractions, where indicated, at 10 ~1 per what remained of the buffer (approximately assay (usually 80-100 Fg protein) and salt 1.5 cell volumes). After 5 min the cells were wash fractions, where indicated, at 5 ~1 homogenized by 20 strokes of a glass per assay (lo-15 pg protein). Salt concenDounce homogenizer. The homogenate was trations were usually 120 m&Z K+ (as brought to 30 n&f Tris-HCI (pH 7.4), potassium acetate) and 3.5 m&f Mg2+ (as 90 mlM KU, 3.5 r&I magnesium acetate, magnesium acetate) and were adjusted to and 7 n&I 2-mercaptoethanol and centrifuged account for the salts present in the volumes at 10,000 g for 10 min. The resulting super- of the various fractions added. natant (SlO) was stored at -70” in 100-~.~1 The reaction was carried out at 30” for up aliquots. Where indicated the SlO was de- to 120 min for exogenous mRNAs and 60 salted by passagethrough Sephadex columns min for endogenous mRNAs. At the approprior to storage and assay. Sephadex G-25 priate times lo-p1 aliquots were removed columns were prepared by equilibration of and placed on filter paper disks (Whatman the gel in 30 mJ4 Tris-HCl (pH 7.4), No. 42, 2.54 cm) which had previously been 100 mM KCl, 5 mZI4 magnesium acetate, spotted with 1% (w/v> casamino acids. When 7 miI4 2-mercaptoethanol, and 5% (v:v) all of the aliquots had been collected the glycerine. The SlO was passed through the papers were dried and hot TCA-insolcolumn and the fractions containing the uble radioactivity was measured as degreatest amount of protein (monitored scribed above. Fractionation of cell-free systems. The visually by turbidity), corresponding to the void volume, were pooled and stored at extracts, prepared as described above, -70” in 100~~1aliquots. Extracts had an were centrifuged at 40,000 rpm for 90 min in A 260 of 60-100 units/ml before passage the Beckman type 65 rotor and the superthrough Sephadex. All operations, unless natant was removed and dialyzed against otherwise indicated, were performed at 4”. 100 vol of buffer F (10 m&I HEPES, pH 7.5; 80 m&I KCl; 3.3 m&f magnesium Assay for amino acid incorporation in vitro. The reaction mixture contained, in acetate; 7 m&I 2-mercaptoethanol, and 10% a total volume of 50 ~1, the given concen- (v/v) glycerine) overnight with one change. trations of the following: Tris-HCl (pH 7.4), The pelleted ribosomes were resuspended 35 m&Z; 2-mercaptoethanol, 9 mM; ATP, 1 with a Dounce homogenizer in buffer F in m&I; GTP, 0.1 mM; CTP, 0.6 n-&I; creatine three-fourths of the original volume of SlO phosphate, 10 m&f; creatine kinase, 0.16 and washed with 0.5 M KC1 by addition of mg/ml; [35S]methionine (400-1000 Wmmol; one-seventh volume of 4 M KCL dropwise Amersham) 100 $X/ml; 40 ,uM each of the with gentle stirring. The ribosomes were other 19 amino acids; 10 ~1 of the appropriate stirred for 1 hr at 4” and clarified by centrifuSlO, and, where indicated, appropriate gation at 10,000 g for 10 min to remove unmRNAs. KC1 and magnesium acetate were dispersed clumps. The ribosomes were colincluded at optimum concentrations for the lected by centrifugation at 50,000 rpm for particular mRNA used. For endogenous 90 min in the type 65 rotor and the supermRNA KC1 was usually 110 n-&f and mag- natant (salt wash) was dialyzed overnight nesium acetate 4.5 mM, unless otherwise against buffer F with one change. The indicated. L cell mRNA and globin mRNA washed ribosomes were resuspended in were translated at 50 mA4 KC1 and 3.5 m&I puromycin-KC1 (0.5 M KU; 50 mM Trismagnesium acetate. EMC RNA was trans- HCl, pH 7.5; 2 n&I MgCI,; 1 n&I dithiolated at 120 mM KC1 and 3.5 mM mag- threitol; 1 m&I puromycin) and incubated nesium acetate. Later assays employed at 37” for 10 min (Blobel, 1972). After clarifipotassium acetate which was found to result cation as above, the puromycin-KCl-washed in greater incorporation than KCI. Frac- ribosomes were collected by centrifugation

322

SCHROM

AND BABLANIAN

min at 2”, to remove traces of bentonite. at 50,000 rpm for 90 min in the type 65 rotor and resuspended in one-fourth starting vol- Two percent potassium acetate and 2 vol of ethanol (-20”) were added and the RNA ume of buffer F. The ribosomes were finally passaged through Sephadex G-25 equili- was stored at -20” until required. brated with buffer F to remove any traces RESULTS of puromycin. All fractions were stored in small volumes at -70”. Amino Acid Incorporation in Extracts of Preparation of cellular mRNA. Poly (A)Vaccinia Virus-Infected, Cyclohexicontaining RNA from polyribosomes was mide-Treated L-929, HeLa, and LLCisolated from log L cells grown in suspenMK2 Cells sion by the method of Krystosek et al. (1975). Previous results (Bablanian et al., 1978) By measuring the incorporation over a range of K+ and Mg2+concentrations, it was have shown that while protein synthesis determined to have translation optima for does not resume in infected cycloheximideK+ of 50 mM and for Mg2+ of 3.5 mM when treated L or HeLa cells upon removal of added at 40 pi/ml to a preincubated cell- cycloheximide, infected-treated LLC-MK2 free system from normal L cells. Rabbit cells resume protein synthesis upon removal globin mRNA, obtained from Searle Diag- of the drug. Essential to the examination nostics, was determined to have similar of the inhibition of protein synthesis in cellsalt optima and was included at 14 pg/ml. free systems is the demonstration that such an inhibition may be faithfully reproPurijkation of encephalomyocarditis (EMC) virus. EMC was grown in L cells duced in vitro. Cultures of HeLa, L-929, and LLC-MK2 and purified from the infected culture fluid by the method of Kerr and Martin (1972) cells were infected with vaccinia virus except that the precipitation with protamine (300 particles/cell) in the presence of cyclosulfate was omitted. heximide (300 pg/ml) or mock infected and Extraction of RNA from purijkd EMC treated with the drug. At 3.5 hr after infecvirus. The virus was thawed, and bentonite tion the drug was removed by washing and (20 mgiml) and EDTA (0.1 M, pH 7.2) were the cells were incubated for an additional added to final concentrations of 0.5 mgiml 30 min. Table 1 shows that cell-free extracts and 0.001 M, respectively. Sodium dodecyl prepared in this manner from infected cyclosulfate (SDS) (5% w/v) was added to 0.5% heximide-treated HeLa or L-929 cells, after and an equal volume of distilled phenol allowing completion of the nascent polypep(saturated with 0.05 M Tris, pH 7.4 and tides initiated prior to the addition of the 0.001 M EDTA) was added. After 10 min drug, fail to incorporate amino acids in vitro. at 44” and room temperature for 20 min, Uninfected-treated extracts under similar with occasional mixing, the mixture was conditions synthesize proteins in vitro, centrifuged at 3000 rpm for 10 min at 2”, and suggesting that during the 30-min incubathe aqueous layer was collected. The intertion in vivo the infected-treated cells comface and phenol layer were washed with 0.5 plete their nascent polypeptides but fail to vol of sterile distilled water, then centri- initiate new polypeptide chains. Furtherfuged. The aqueous layer was pooled with more, preparation of cell-free amino acidthe first aqueous extract. To the combined incorporating systems after similar infecaqueous layers, 2% potassium acetate (pH tion and treatment from LLC-MKZ cells in 5.0) and 2 vol of ethanol (precooled to -20”) which protein synthesis is not inhibited were added. The RNA was left at -20 in vivo results in extracts which are funcovernight and centrifuged at 3000 rpm for tional in vitro (Table 1). This indicates that 10 min. The pellet was washed three times the preparations described here are repreby centrifugation at 0” with 4-ml portions sentative models for the inhibition of proof ethanol (2 parts): 0.15 M NaCl (1 part). tein synthesis in vivo. The inhibition of The RNA was dissolved in sterile distilled protein synthesis in extracts of infectedwater and centrifuged at 13,000 g for 20 treated HeLa or L-929 cells is still evident

IN VITRO INHIBITION

OF PROTEIN BY VACCINIA

after the extracts have been passed through Sephadex G-25, and subsequent experiments have been performed with extracts which have been so treated. Because of their resistance to the toxic effects of cycloheximide, vaccinia-infected, cycloheximidetreated HeLa cells have been selected for further studies and in order to consistently produce inhibition of protein synthesis comparable to that observed in vivo and with L-929 cells, a multiplicity of infection of 900 particles per cell has been used. Translation of Exogenous mRNAs in Extracts of Infected -Treated and Uninfected Treated HeLa Cells Since the incorporation of amino acids in response to endogenous mRNAs consists primarily of completion of nascent polypeptides, the translation of which was initiated in vivo (Weber et al., 1975; Reichman and Penman, 1973), an analysis of the inhibition of protien synthesis should be carried out in a system capable of initiation in vitro. HeLa cells were infected, SlOs were prepared as described previously, and the TABLE 1 AMINO ACID INCORPORATION IN INFECTED-~YCLOHEXIMIDE TREATED AND CYCLOHEXIMIDE-TREATED EXTRACTS OF L-929, HeLa, AND LLC-MK2 CELLS”

Extract

Cells L-929 HeLa LLC-MK2

Uninfectedtreated (cpm) 10,054 5,651 15,703

Infectedtreated (cpm) 1,357 1,937 17,317

Percentage of control 13 34 110

u Cells were infected (300 particles/cell) or mock infected in the presence of cycloheximide (300 pg/ml). Three and one half hours after infection the drug was removed by washing and 30 min later cell-free protein-synthesizing systems were prepared as described under Materials and Methods. The extracts were assayed for [Wlmethionine incorporation for 60 min at 30”. Incorporation at 0 time has been subtracted.

323

TABLE 2 TRANSLATIONOFEXCGENOIJS mRNAs IN INFE(;TEDCYCLOHEXIMIDE

TREATED

AND CYCLOHEXIMIDE-

TREATED HeLa CELL EXTRACTS” mRNA (cpm) Extracts Infected-treated Uninfected-treated

None

Globin

L cell

EMC

1,531 1,947 3,502 4,849 14,710 28,943 50,556 43,327

(1Extracts were passed through Sephadex G-25 and treated with 10 pgiml micrococcal nuclease in the presence of 1 mM CaClp for 10 min at 18” to reduce incorporation due to endogenous mRNAs. EGTA was added to 2 mM to stop the action of the nuclease before addition of the exogenous mRNAs. The respective mRNAs were added under conditions found to be optimal for their translation in vitro as described under Materials and Methods. Incubation was for 120 min at 30” and incorporation at 0 time has been subtracted.

resulting extracts were treated with micrococcal n&ease as described by Pelham and Jackson (1975) to reduce the incorporation due to endogenous mRNA. L cell poly (A)containing RNA, rabbit globin mRNA, and EMC viral RNA were added under conditions found to be optimal for their translation in vitro. Table 2 shows that extracts of infected-treated HeLa cells are severely reduced in their ability to translate all of the RNAs added. There does not appear to be any discrimination between viral and cellular RNAs of the type observed by others (Samuel and Joklik, 1974; Golini et al., 1976). This result is consistent with our. previous in vivo (Bablanian et al., 1978) and in vitro (Table 1) observations that the synthesis of both cellular and viral proteins in inhibited in vaccinia-infected, cycloheximide-treated HeLa cells and that the inhibition appears to result from a failure of the infectedtreated cells to initiate new polypeptides. Further information on the reason for this failure to initiate new polypeptides can be obtained by fractionating the infectedtreated extracts and mixing their various fractions until the defective component is isolated. Such an approach is described in the following section.

324

SCHROM AND BABLANIAN TABLE 3

EFFECT OF RIBOSOMALSALT WASH FRACTIONSFROMINFECTED-CYCLOHEXIMIDETREATED AND CYCLOHEXIMIDE-TREATED HeLa CELL EXTRACTS ON EMC RNA TRANSLATION BY RECONSTITUTEDPROTEINSYNTHESIZINGSYSTEMS* Ribosomes Infected-treated

Supernatant I I I I C C C C

salt wash I C I C

EMC RNA

cpm

+ + + + + f

0 2,440 6,315 10,194 10,431 3,177 14,291 18,583 22,635

Percentage maximumb

11 23 24 33 45 57

Uninfected-treated

cpm 0 11,095 13,968 18,512 18,233 12,437 34,089 25,824 46,438

Percentage maximum

8 22 21 64 39 100

(1Extracts of infected-treated and uninfected-treated cells were fractionated and protein synthesizing systems were reconstituted as described under Materials and Methods. EMC RNA was added as indicated and the assay mixtures were incubated for 120 min at 30”. Incorporation at 0 time has been subtracted. The salt washes have no effect in the absence of added mRNA. I = fractions from infected cycloheximide treated cells; C = fractions from uninfected cycloheximide treated cells. b Response to EMC RNA was estimated by subtracting the incorporation in the assay mixtures without EMC RNA from the same ribosome-supernatant mixtures with the mRNA. The response is expressed as a percentage of the maximum observed response.

their homologous supernatant (64%) but function poorly with the supernatant from the infected-treated cells (8%). From this we can conclude that the function which is lacking in the infected-treated cells is deficient in both the ribosomes and superThe approach in this fractionation scheme natant of those cells. If the activity responwas to obtain the following components: (1) sible for the incorporation in these assays is ribosomes freed of their endogenous the initiation factors, then the majority of mRNAs, (2) soluble supernatant factors, this activity should be present in the salt and (3) crude initiation factors. In order to wash fraction. Therefore this fraction was analyze the inhibition of protein synthesis, added to the ribosome and supernatant incorporation in vitro should be dependent fractions in order to determine whether upon the presence of all three fractions and function could be restored. an exogenous mRNA for maximal activity. It can be seen (Table 3) that the salt Table 3 shows that ribosomes from in- washes of both infected-treated and uninfected-treated HeLa cells respond poorly fected-treated ribosomes show little stimto EMC RNA when assayed with homol- ulation with either ribosomes in the presence ogous supernatant fraction (11%) and of the infected SlOO fraction (21 and 24%, respond only slightly better in the presence respectively) or in the presence of the inof the uninfected supernatant (33%). The fected ribosomes (45 and 5’7%,respectively). ribosomes from uninfected-treated cells The only substantial stimulation is observed will translate EMC RNA efficiently with when the salt wash of the uninfectedTranslation of EMC RNA by ProteinSynthesizing Systems Reconstituted from Fractions of Infected -Cycloheximide-Treated and Uninfected -CycEoheximide-Treated HeLa Cells

IN VZTRO INHIBITION

OF PROTEIN TABLE

BY VACCINIA

325

4

AMINO ACID INCOWORATIONBY FRACTIONSOF INFECTED-TREATED AND UNINFECTED-TREATED

CELLS MIXED WITH FRACTIONSOF NORMAL CELLS” Ribosomes

SlOO

Salt wash

U

U

U

I C U U U U

u U I C U U

U U U U I C

Stimulation by EMC RNA (cpm)

Percentage control

5,388 5,160 5,063 7,698 13,497 2,700 5,304

96 94 143 251 50 98

u Protein-synthesizing systems were reconstituted from the indicated fractions and the stimulation of amino acid incorporation by EMC RNA was measured by subtracting the endogenous incorporation in the absence of EMC RNA. Incorporation in the presence of EMC RNA was generally 1.5 to 2-fold that in its absence. U = fractions from normal cells, I = fractions from infected-cycloheximide-treated cells, C = fractions from uninfected-cycloheximide-treated cells.

treated ribosomes is added to the homologous ribosome and supernatant fractions (100%). Mixing of Fractions of Infected -Treated and Uninfected -Treated HeLa Cells with Fractions of Normal HeLa Cells In order to further define the inhibited component, puromycin-KC1 washed ribosomes, SlOO, and salt wash fractions were prepared from normal uninfected-untreated HeLa cells. Protein-synthesizing systems were reconstituted by adding one fraction of infected-treated or uninfected-treated cells to the remaining two fractions of normal cells. Amino acid incorporation of these systems in response to EMC RNA is shown in Table 4. The only mixture in which the response to EMC RNA is less than that in the reconstituted system from normal cells is that of the infected-treated salt wash with the normal ribosomes and SlOO. With any other combination the response to EMC RNA is as good or better than that of the normal system. This experiment demonstrates that there is a specific defect in the ribosomal salt wash of infected-treated cells. DISCUSSION

One observation made by Moss (1968) and subsequently confirmed by us (Bablan-

ian et al., 1978)is that when cells are infected with vaccinia virus in the presence of cycloheximide an almost total inhibition of protein synthesis is observed even after the removal of the drug. We feel that such a complete inhibition of protein synthesis could provide a suitable model system for studying the inhibition of protein synthesis in cell-free protein-synthesizing systems. Such an approach has been tried in extracts of picornavirus-infected cells. These infected cells demonstrate a complete inhibition of cellular protein synthesis to which viral protein synthesis is resistant. However, cell-free amino acid-incorporating systems prepared from such cells are capable of translating both viral and cellular mRNAs in vitro if added separately (Abreu and Lucas-Lenard, 1976, Lawrence and Thach, 1974; Golini et al., 1976). Protein synthesis in extracts prepared from vaccinia-infected, cycloheximidetreated L-929 or HeLa cells following removal of cycloheximide is inhibited by 6090% compared to extracts prepared from uninfected controls. Preparation of cellfree amino acid-incorporating systems from infected-treated LLC-MK2 cells under identical conditions results in extracts which are capable of amino acid incorporation in vitro (Table 1). These results indicate that the presence or absence of inhibition in cycloheximide-treated vaccinia-infected,

326

SCHROMANDBABLANIAN

cells may be reproduced faithfully in vitro. Further characterizing this inhibition of protein synthesis in vitro is the failure of the infected-treated extracts to respond to exogenous mRNAs (Table 2) regardless of whether they are cellular (globulin or L cell) or viral (EMC). When the inhibited extracts are fractionated the ribosomal salt wash appears to be the affected component. This fraction fails to stimulate protein synthesis by ribosomes from either infected-treated or uninfectedtreated cells (Table 3). The ribosomal wash fraction from uninfected-treated cells can partially restore function of the ribosomes from infected- treated cells in combination with the SlOOfrom uninfected cells (Table 3). Only the ribosomal salt wash of infectedtreated cells fails to function in combination with the ribosomal and SlOOfractions from normal cells (Table 4). The ribosomal salt wash constitutes a crude preparation of initiation factors and it is therefore possible that either an initiation factor(s) is inactivated or some virusspecific inhibitor is present in the salt wash fraction and is the cause of the inhibition. The process of initiation, and the initiation factors specifically, have appeared to be the control site for protein synthesis in several systems. The translational control by hemin in reticulocyte lysates has been shown to take place through phosphorylation of eIF-2 (Farrell et al., 1977). The inhibition of protein synthesis by double-stranded RNA has been shown by Kaempfer and Kaufman (1973) to be the result of inactivation of IF-3. Studies with picornavirus-infected cells have shown that the initiation is a likely site for the establishment of the inhibition of host protein synthesis in vivo (Leibowitz and Penman, 1971). When cell-free amino acid-incorporating systems are prepared from infected cells and fractionated extracts are assayed for translation in vitro, the inhibitory activity has been shown to reside in a ribosomal salt wash fraction (Kaufmann et al., 1976). The reason for the selective inhibition of cellular protein synthesis remains unclear, however, it is generally agreed that the inhibition resides at the level of the initiation factors, either

by a message discriminatory IF-M3 (Golini et al., 1976) or by inactivation of eIF-4B (Rose et al., 1978). Thus the effect we have observed on initiation of protein synthesis in extracts of vaccinia-infected, cycloheximide-treated cells is consistent with the results with picornavirus-infected cells. It is also consistent with the indirect evidence presented by Person and Beaud (1978) that the inhibition of cellular protein synthesis observed by these authors in vacciniainfected cells in the presence of cordycepin takes place primarily at the level of initiation. Ben-Hamida and Beaud (1978) have also observed that translation of poly (U), which does not require normal initiation, can take place in reticulocyte lysates in which protein synthesis has been inhibited by the addition of vaccinia cores. If the inhibition is active at the level of the initiation factors, it also shows certain similarities with control of protein synthesis in other uninfected (Kaempfer and Kaufman, 1973; Datta et al., 1977) eucaryotic cells. What still remains unresolved is what virusinduced event is the cause of the inhibition of initiation. Two general hypotheses have been advanced to explain the inhibition of protein synthesis by vaccinia virus. Previous work in our laboratory has demonstrated a correlation between the synthesis of early virusinduced RNA and the inhibition of protein synthesis in infected cells (Bablanian, 1975; Bablanian et al., 1978). What this RNA represents is as yet unclear. It is known that double-stranded RNA is synthesized in vaccinia-infected cells (Colby and Duesberg, 1969) and also that such a molecule is capable of inhibiting protein synthesis at the level of initiation of new polypeptide chains (Ehrenfeld and Hunt, 1971; Kaempfer and Kaufmann, 1973). We have recently reported (Schrom and Bablanian, 1979) that the increase in cytoplasmic RNA synthesized in vaccinia-infected, cycloheximide-treated cells does not result in an increase in the percentage of nucleaseresistant RNA, although an increase in the absolute amount of nuclease-resistant RNA is observed. Thus if double-stranded RNA is responsible for the inhibition of protein

IN VZZ’RO INHIBITION

OF PROTEIN BY VACCINIA

synthesis, it could only be active in small amounts in a catalytic fashion, perhaps by activation of latent cellular enzymes as has been observed in interferon-treated cells (Roberts et al., 1976; Lebleu et al., 1976). However, we have not been able to detect any ribonuclease activity in infected-cycloheximide treated SlOs (Schrom and Bablanian, unpublished observations). The second alternative (Moss, 1968) holds that protein synthesis is inhibited by a component of the infecting virion. This has received recent support from the observations of Ben-Hamida and Beaud (19’78)that protein synthesis in reticulocyte lysates may be inhibited by the addition of vaccinia cores. When cores are added to coupled transcription-translation systems, protein synthesis is stimulated up to a point and then depressed (Cooper and Moss, 19’78) which can be the result of an increase in the amount of a viral component or an increase in the amount of viral RNA synthesized by the greater number of cores, or both. Thus the cause of the inhibition still remains unresolved. Our aim in these studies has been to fractionate infected cells and isolate the inhibited component. It should be noted that the present fractionation does not exclude an effect on the ribosome or soluble components, however it appears likely that the inhibited component resides primarily in the ribosomal salt wash fraction. It is also possible that there is an inhibitor in the salt wash fraction or that one of the initiation factors has been inactivated. It is now experimentally feasible to examine these possibilities directly. REFERENCES ABREU, S. L., and LUCAS-LENARD,J. (19’76).Cellular protein synthesis shutoff by mengovirus: Translation of nonviral and viral mRNAs in extracts from uninfected and infected Ehrlich ascites tumor cells. J. Viral. 18, 182-194. BABLANIAN, R. (1972). Mechanisms of virus cytopathic effects. Symp. Sot. Gen. Microbial. 22, 359-381. BABLANIAN, R. (1975). Structural and functional alterations in cultured cells infected with cytocidal viruses. Progr. Med. Viral. 19, 40-83. BABLANIAN, R., EEGGERS, H. J., and TAMM, I.

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