Proc. NatI. Acad. Sci. USA Vol. 75, No. 1, pp. 175-179, January 1978
Biochemistry
In vitro inhibition of protein synthesis by purified cores from vaccinia virus (reticulocyte lysate/host cell shutoff/polypeptide chain initiation block)
F. BEN-HAMIDA AND G. BEAUD Laboratoire de Biochimie du Developpement, Institut de Recherche en Biologie Mol6culaire du Centre National de la Recherche Scientifique et de l'Universit6 Paris VII, 2 Place Jussieu, Tour 43, 75221 Paris Cedex 05, France
Communicated by J. Brachet, October 21, 1977
ABSTRACT The mechanism of the shutoff of cellular protein synthesis in vaccinia virus-infected cells has been investigated by using in vitro systems. Purified vaccinia cores cause inhibition of endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems. Translation of viral mRNA from turnip yellow mosaic virus is also impaired in wheat germ cell-free extracts. The block induced by vaccinia cores in protein synthesis is not due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. No nucleolytic activity able of digesting mRNA could be detected in purified vaccinia cores with three sensitive tests. There is a lack of inhibition in the poly(Phe)poly(U) system, which bypasses the normal initiation process. An almost complete disaggregation of polyribosomes in the reticulocyte lysate appears when vaccinia cores are present. These results indicate that mRNA translation in a cell-free system is affected predominantly at the level of polypeptide chain initiation.
The infection of animal cells with many viruses results in an inhibition of host protein synthesis (for review, see ref. 1). In the case of vaccinia (2), the inhibition of cell protein synthesis is rapid and extensive at high multiplicity of infection. It occurs in the presence of actinomycin D (3-5), cycloheximide (4), or cordycepin (6). This implies that inhibition of host protein synthesis does not require de novo viral RNA or protein synthesis and appears to be associated with the virions. Moreover, the time required for the establishment of the shutoff corresponds to the first stage of uncoating-i.e., the release of cores into the cytoplasm (7, 8). Which level of protein synthesis is affected by the shutoff of the cell in the vaccinia system? This question can be answered by using cell-free systems in -which the effect of vaccinia cores on mRNA translation can be directly studied. Here we report experimental results with purified vaccinia cores in cell-free systems. Endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems was inhibited by the vaccinia core preparations. The translation of exogenous mRNA such as the RNA of turnip yellow mosaic virus (TYMV) in wheat germ extracts also was impaired. The lack of inhibition of the poly(Phe)-poly(U)dependent system which bypasses the normal initiation process and the almost complete loss of polyribosomes in the reticulocyte lysate when vaccinia cores were present suggest that polypeptide chain initiation is the major site of action of the vaccinia virus core-mediated inhibition. MATERIALS AND METHODS Preparation of Purified Cores. The vaccinia virions (Lister strain) produced by infection of KB cells were purified by suThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
crose density gradient centrifugation as described (9) except that an additional cycle of sucrose cushion followed by sucrose gradient centrifugation was performed. Cores were prepared by incubation of purified virus (previously sonicated for 40 sec at 4 ,u on an MSE sonicator) at 370 for 30 min in 30 mM TrisHCl, pH 7.5/0.2% Triton X-100 (Koch-Light)/50 mM 2-mercaptoethanol (10). At the end of this incubation, the Triton X-100 was removed from the sonicated cores by adsorption on Bio-Beads SM2 (Bio-Rad) (11) by shaking at 40 for 3 hr. The supernatant was then layered onto 3 ml of a 36% (wt/vol) sucrose cushion containing 10 mM Tris-HCI, pH 7.5/1 mM dithiothreitol and centrifuged in a Beckman SW 50-1 rotor at 25,000 rpm for 30 min. The pellet of viral cores was suspended in 10 mM Tris-HCI (pH 7.5) and, after brief sonication (twice for 10 sec), was layered onto a 20-40% (wt/vol) linear sucrose gradient prepared in 2 mM Tris-HCI (pH 9) in a Beckman SW-25.1 rotor at 15,500 rpm for 55 min. The band of viral cores thus obtained was collected and concentrated by sedimentation in a Beckman 30 rotor at 28,000 rpm for 90 min. The purified cores thus obtained were suspended in 2 mM Tris-HCl (pH 9) and stored in small aliquots in liquid nitrogen. The vaccinia core preparations were adjusted to 20 A26o units/ml (corresponding to 1.5 mg of protein per ml). Cell-Free Systems. Reticulocyte lysate system. Rabbit reticulocyte lysates were prepared according to Housman et al. (12). Protein synthesis was assayed by the incorporation of L[35S]methionine into protein. Reaction mixtures (50 ,i) contained 10 ul of reticulocyte lysate, 30 mM Tris-HCI (pH 7.5), 3 mM Mg acetate, 60 mM KCI, 2 mM dithiothreitol, 1 mM ATP, 0.5 mM GTP, 16 mM K phosphoenolpyruvate (PePyr), PePyr kinase at 200 ,g/ml, 19 unlabeled amino acids at 40,uM each, 1 MM unlabeled methionine, and 4 ,gCi of L-[35S]methi. onine (300 Ci/mmol, Amersham). Incubation mixtures were supplemented with 35 ,M hemin (Fluka). Usually, in order to enhance the vaccinia core-mediated inhibition of the endogenous [in a reticulocyte lysate or an Ehrlich ascites tumor (EAT) cell-free extract] or exogenous (in a wheat germ cell-free extract programmed by TYMV RNA) mRNA translation (see below), preincubation of the cell-free extract with the vaccinia cores was performed for 10 min under suitable ionic conditions, as described with the results of each experiment. Similar results were obtained when hemin was added during preincubation with the reticulocyte lysate. Incubation for protein synthesis (reticulocyte lysate) was at 30°. Aliquots (5 ,l) were applied to Whatman 3MM paper discs, immersed for 10 min into 10% cold trichloroacetic acid containing 4 mM unlabeled methionine, transferred to 5% trichloroacetic acid, brought to 900 for 15 min, and rinsed twice in 5% cold trichloroacetic acid for 5 min, Abbreviations: TYMV, turnip yellow mosaic virus; PePyr, phosphoenolpyruvate; EAT, Ehrlich ascites tumor.
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Proc. Nati. Acad. Sci. USA 75 (1978)
B
x
x
B
E
E0.
30_
04
20 _ 10
30 20 Time, min FIG. 1. Effect of preincubation of reticulocyte lysates with vaccinia cores on protein synthesis. (A) Without preincubation. (B) Preincubation of reticulocyte lysate with vaccinia cores at 200 for 10 min in 3 mM Mg acetate/60 mM KCl/40 mM Tris-HCl, pH 7.5. Both samples were then incubated at 300 with the components required for protein synthesis. Aliquots (5 Il) were spotted onto Whatman 3MM discs and analyzed as above. 0, Control; 0, with cores.
0
10
once in ethanol for 5 min, in ethanol/diethyl ether, 1:1 (vol/vol), and finally in diethyl ether. The radioactivity was measured by using a toluene-based scintillator. EAT cell-free system. Extracts were prepared from EAT cells as described (9). The endogenous EAT cell mRNA translation activity was measured in nonpreincubated extracts. The assay system for measurement of protein synthesis by the incorporation of L-[3sS]methionine consisted of 50 ,l of the reaction mixture containing 3.5 mM Mg acetate, 120 mM KC1, and the other components required for protein synthesis as above. As in the case of the reticulocyte lysate, preincubation was performed before addition of the radioactive amino acid and the components needed for protein synthesis. The experimental procedure was as above. When poly(U) was used, 0.4 ,UCi of L-['4C]phenylalanine (260 mCi/mmol) was added instead of the amino acids and the Mg2+ concentration was increased to 5 mM. Wheat germ cell-free system. The wheat germ cell-free extract was prepared essentially according to the procedure of Davies and Kaesberg (13). The standard in vitro reaction mixture for amino acid incorporation directed by TYMV RNA (80 ,g/ml) consisted of 100 ,ul of a reaction mixture containing 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) (pH 7.6), 3 mM Mg acetate, 140 mM K acetate, 1 mM ATP, 0.2 mM GTP, 8 mM PePyr, 19 unlabeled amino acids at 25 MuM each, and L-[s5S]methionine (2 ,Ci, 300 Ci/mmol). Incubation was at 300, and samples were analyzed as above. Radioactive Vaccinia mRNA Preparation. The vaccinia [3H]mRNA used as substrate to detect residual nuclease activity in the vaccinia cores was made from [3H]UTP and purified as
described (9). Detection of Residual Nuclease Activity in Vaccinia Cores by Breakdown of [l4C]Phenylalanine-tRNAPhe. Purified tRNAPhe from rat liver was a gift from G. Petrissant (Paris). Charging with [14C]phenylalanine was carried out with an EAT cell-free extract (4 A260 units/ml) in a reaction mixture containing 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.6), 5 mM Mg acetate, 100 mM KC1, 2 mM
0
20
40 60 Time, min FIG. 2. Effect of vaccinia cores on globin synthesis in reticulocyte lysates. In this experiment, both preincubation and incubation for protein synthesis were performed at 30°. Hemin was added in the second incubation. At the indicated times, 5-gl samples were applied Whatman 3MM discs and analyzed for protein incorporation. Curve A, control; curve B, with 10 ttl of vaccinia cores; curve C, with 20 gl of vaccinia cores.
dithiothreitol, 2 mM ATP, 16 mM PePyr, Pepyr kinase at 200 Mg/ml, cycloheximide at 300 ,g/ml, and [14C]phenylalanine (10 ,Ci/ml, 370 mCi/mmol). Incubation was at 300 for 50 min. To detect residual nuclease activity in vaccinia cores, the preparation was assayed by adding vaccinia cores at 50 min when charging of tRNA with [14C]phenylalanine had reached a plateau. For this, to 50 Mul of the above mixture were added, in a final volume of 100 Mul, 0.8 A260 unit of vaccinia cores (or the corresponding buffer in control samples) and 20 mM phosphate buffer (pH 7.5). Incubation was continued at 300. The reaction was stopped by addition of 0.2 mg of yeast RNA, immediately followed by 1 ml of cold 5% trichloroacetic acid; the insoluble radioactivity was measured as above.
RESULTS Vaccinia Core-Mediated Translational Inhibition in Cell-Free Systems. When a reticulocyte lysate was incubated in the presence of vaccinia cores, protein synthesis was inhibited by approximately 30% (Fig. 1). Kinetic analysis showed a lag period of 10-20 min before inhibition of protein synthesis. Preincubation, for 10 min, of the reticulocyte lysate with the vaccinia cores resulted in a greater inhibition of incorporation, reaching 60-70% of the control. The level of this inhibition depended on the concentration of the vaccinia cores. A 50% decrease of protein synthesis (relative to the control) was obtained with 10 ,l of cores, corresponding to 300 Mug of viral protein per ml (Fig. 2). In the experiments described in Figs. 1 and 2, hemin was not added in the preincubation step; however, the addition of hemin (35 or 75 MM) at this step did not change the magnitude of the protein synthesis inhibition. Impairment of translation, although to a lesser extent, also was obtained with the EAT cell-free system (data not shown). Vaccinia cores were also assayed for exogenous mRNA (TYMV RNA) translation in a wheat germ cell-free extract. The results (Fig. 3) show that vaccinia cores cause the inhibition of not only endogenous or cellular mRNA but also of viral mRNA trans-
lation.
Biochernistr : Ben-Hamida and Beaud
Proc. Natl. Acad. Sci. USA 75 (1978)
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Table 1. Absence of residual nuclease activity in vaccinia cores (breakdown of [l4C]phenylalanine-tRNAPhe)
Incubation time, min 0 10 30 50 70
Insoluble radioactivity,* cpm/10 ,l With Control vaccinia cores 3800 3691 3409 3311 3134
3703 3465 3310 3144 3180
* Insoluble in cold trichloroacetic acid.
0
20 40 Time, min FIG. 3. Kinetics of protein synthesis directed by TYMV RNA in wheat germ cell-free extracts. The wheat germ extract (40 Ml) was first incubated at 200 for 20 min in the presence of vaccinia cores (50 Ml) or the corresponding buffer for controls, in a final volume of 100 Ml containing 3 mM Mg acetate, 140 mM K acetate, and 20 mM N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.5). The vaccinia cores were then removed by centrifugation in the cold at 14,000 rpm for 10 min in a Beckman Microfuge. The supernatant (75 Ml) was then incubated at 300 in the presence of TYMV RNA (80 Mg/ml) in a final volume of 100 Ml containing 1 mM ATP, 0.2 mM GTP, 8 mM PePyr, 19 unlabeled amino acids at 25 MM each, and L-[35S]methionine (2 MCi, 300 Ci/mmol, Amersham). Samples (20 Mul) were removed at various times, applied to Whatman 3MM filters, and analyzed for methionine incorporation into protein. 0, Control; 0,
with vaccinia cores.
The decrease in protein synthesis induced by vaccinia cores does not seem to be due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. Indeed, the same extent of inhibition was obtained when the cores were removed by centrifugation after preincubation with the wheat germ extract and then TYMV RNA was added as mRNA for translation, as shown in Fig. 3. When the effect of vaccinia cores was assayed on poly(U) translation in an EAT cell-free system, there was no shutoff (Fig. 4). The failure of vaccinia cores to inhibit poly(Phe) synthesis under the direction of poly(U) indicates that the vaccinia core-mediated inhibition does not predominantly affect peptide chain elongation.
S
3Table 2. Assay of breakdown of vaccinia [3H]mRNA Insoluble radioactivity, cpm/5 ,l With Incubation Control vaccinia cores time, min
0
x
EQ
Lack of Residual Ribonuclease Activity in Vaccinia Core Preparations. A critical point to be established is the level of the nuclease activity that may be a contaminant of the viral preparation and may interfere with the observed inhibition. The earliest preparations of vaccinia cores used for in vitro studies of the inhibition of host cell protein synthesis showed some breakdown of [l4C]phenylalanine-tRNAPhe and, to a lesser extent, a nuclease activity of vaccinia [3H]mRNA (14). The present technique of purification allowed us to decrease the nuclease activity in the vaccinia core preparations to a level not detectable by the following three tests. (i) Possible breakdown of [14C]phenylalanine-tRNAPhe was followed as cold trichloroacetic acid-soluble material and compared to samples containing vaccinia cores. There was no increase in the degradation of [l4C]phenylalanine-tRNAPhe in the presence of vaccinia cores, even after 70 min of incubation at 30' (Table 1). (ii) 3H-Labeled vaccinia mRNA was also used as substrate for possible nuclease contaminating the vaccinia core preparations. The acid-insoluble radioactivity from 3H-labeled vaccinia mRNA was followed, and no breakdown occurred on incubation for 60 min (Table 2). (iii) A third highly sensitive control to check the absence of endonuclease activity in vaccinia core preparations used for in vitro studies was the absence of any significant change of the polyribosome distribution pattern from reticulocyte lysates (15) when incubated in the presence of vaccinia cores, under conditions that do not allow protein synthesis. The profiles of absorbance at 260 nm of polyribosomes were identical in the absence and in the presence of vaccinia cores when incubated for 5 min after 10 min of preincubation (Fig. 5 A and B). Core-Mediated Inhibition Is Not Due to Nucleoside Triphosphate Phosphohydrolase. The translation inhibitory activity present in vaccinia cores could be attributed to the nucleoside triphosphate phosphohydrolases I or II, two enzymes present in the cores (16-19). In fact, under our conditions, the
2I -I
20
.I I
40
Time, min
FIG. 4. Kinetics of poly(Phe) synthesis in response to poly(U) (200 Mug/ml) in a nonpreincubated EAT cell-free extract. Reaction mixtures were first preincubated at 300 for 10 min before addition of L-[14C]phenylalanine at zero time. A 50-Ml mixture was used, and 10-Mul samples were removed at various times and applied to Whatman 3MM discs; the synthesis of [14C]poly(Phe) was measured as hot trichloroacetic acid-insoluble material. *, Control; 0, with vaccinia cores (20 Ml).
405 409 366 427
386 321 378 379 The reaction mixture (final volume, 50 ,l) contained 40 mM N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.5), 3 mM Mg acetate, 80 mM KCl, 1 mM dithiothreitol, 3 Mg of vaccinia [3HJmRNA (100 cpm/Mg), and 15 Ml of vaccinia cores. Incubation at 300 was stopped at the times indicated by the addition, to 5-,ul samples, of 5% trichloroacetic acid and 200 gg of yeast RNA. 0 20 40 60
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Biochemistry: Ben-Hamida and Beaud
This effect on the polyribosome profiles indicates that polypeptide chain initiation is the major site of action of vaccinia cores in the inhibition of in vitro protein synthesis.
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FIG. 5. Effect of vaccinia core extracts on the polyribosome profile of reticulocyte lysate. A reticulocyte lysate (30 p1) was first preincubated at 200 for 10 min in the absence (mixture I) or in the presence (mixture II) of vaccinia cores (40 pl) in a final volume of 85 Ml containing 25 mM Tris.HCl, pH 7.5/80 mM KCl/3 mM Mg acetate/30 MM hemin. Both mixtures were then duplicated in A and C and in B and D, respectively. C and D received 1 mM ATP, 16 mM PePyr, PePyr kinase at 200 Mg/ml, and a mixture of 20 amino acids (40 MM each) for protein synthesis. A and B lacked on exogenous energy-generating system and amino acids. All samples (in a final volume of 50 Ml) were incubated at 30°. After 5 min, the samples were quickly cooled to 0° and 3 volumes of cold gradient buffer (20 mM Tris.HCl, pH 7.5/100 mM KCl/5 mM Mg acetate) containing cycloheximide at 400 ,g/ml was added. Each sample thus diluted (total volume, 200 Ml) was layered onto a linear (15-30%) sucrose gradient in the above buffer. After centrifugation at 20 for 85 min at 40,000 rpm in a Beckman SW 41 rotor, the contents of the tubes were pumped from the bottom through the flow-cell of a Gilson spectrophotometer recording optical density at 260 nm. (A and C) No vaccinia cores; (B and D) vaccinia cores present; (A and B) no protein synthesis; (C and D) with amino acids and an energy-generating system. The direction of sedimentation was from left to right.
core-mediated inhibition of mRNA translation is not due to a depletion in the level of ATP or GTP. Indeed, by adding either 3H-labeled ATP or GTP to the reaction mixtures and measuring the amount of radioactivity present in the nucleoside mono-, di-, and triphosphates at different times, the levels of ATP or GTP were shown to remain unchanged when vaccinia cores were present (data not shown). The Block Induced by Vaccinia Cores Affects Polypeptide Chain Initiation. The fact that the poly(U) system, which bypasses the normal initiation process, is unaffected by vaccinia cores (see above and Fig. 4) is consistent with an inhibitory site of action at the initiation level in protein synthesis. To confirm this, an analysis of the change of the polyribosome profiles of reticulocytes lysates was carried out in the presence and in the absence of vaccinia cores under conditions of protein synthesis. As illustrated in Fig. 5, vaccinia cores caused an extensive disagreggation of polyribosomes within 5 min when incubated with a reticulocyte lysate and amino acids plus an energy source (Fig. 5 C and D). At the same time, there was a corresponding increase of monosomes. In the absence of amino acids and an energy source, no breakdown was observed (Fig. 5 A and B).
DISCUSSION The results presented in this report demonstrate that a component causing inhibition of protein synthesis in cell-free systems is associated with vaccinia cores. Based on the viral core concentration used (600 ,tg/ml) and on the ribosome content of rabbit reticulocyte lysates (1 iug of ribosomes per ,l of undiluted lysate), one can calculate that the inhibition in this system occurs at the low ratio of 1 viral core to 100 ribosomes. Moreover, the block affects translation of both endogenous and cellular mRNA (in nonpreincubated reticulocyte lysates and EAT cell-free extracts) and viral mRNA (in wheat germ extracts). This possible lack of discrimination between cellular and viral RNA translation is in agreement with earlier findings (4) that some viral protein synthesis is apparently required to allow extensive early viral mRNA translation. The kinetics of protein synthesis in reticulocyte lysates incubated in the presence of vaccinia cores shows that the initial rate of amino acid incorporation is not greatly reduced during the first minutes of incubation. This early resistance to inhibition could reflect the gradual inactivation of a component(s) that is essential for translation of mRNA. This is in agreement with the finding that prior incubation of the cell-free extract with the cores (in the absence of protein synthesis) increases translational inhibition
(Fig. 1).
It is possible that the protein shutoff observed is due to the effect of double-stranded RNA (20) possibly present in the cores. The fact that inhibition of TYMV RNA translation occurs in wheat germ extracts (Fig. 3) excludes this possibility. Indeed, as shown by Grill et al. (21), inhibition of protein synthesis by double-stranded RNA is a phenomenon limited to the mammalian system and does not occur in wheat germ extracts. The block of protein synthesis mediated by vaccinia cores is not due to a decrease in the availability of mRNA but rather to a lowering of the rate of polypeptide chain initiation. (i) No nucleolytic activity able to digest mRNA could be detected in purified vaccinia cores. (ii) Poly(U) translation, which does not use the initiation mechanisms characteristic of natural mRNA, is not affected. (iii) Incubation of vaccinia cores with reticulocyte lysates under conditions of protein synthesis leads to an almost complete disaggregation of polyribosomes. These results indicate that the level at which mRNA translation in cell-free systems is affected is predominantly polypeptide chain initiation. A similar conclusion has been obtained from in vivo experiments showing that initiation is also the major site of inhibition of protein synthesis in vaccinia virusinfected cells exposed to actinomycin D (4, 5) or cordycepin (22). It can be suggested that inhibition of protein synthesis might be related to the phosphorylation of a component essential for initiation of translation. One plausible hypothesis is that the protein kinase, one of the enzymes located in the vaccinia cores (23, 24), may be involved in this inactivation by phosphorylation of the eIF-2 initiation factor. Preliminary experiments have not allowed clear proof of a significant phosphorylating activity of vaccinia cores when purified reticulocyte initiation factor eIF-2 was used as substrate. On the other hand, in order to support the same hypothesis, we also tested the ability of eIF-2 to reverse the protein synthesis inhibition induced by the vaccinia cores. However, only a partial reversal of this inhibition was obtained with purified eIF-2 under the conditions of protein synthesis inhibition in this work. [The highly purified reticulocyte initiation factors used in these
Biochemistry: Ben-Hamida and Beaud experiments were provided by T. Staehelin and B. Ei.( e Institute for Immunology).] Another explanation of the vaccinia core-mediated translational inhibition might be related to the specific phosphorylation of 40S ribosomal proteins which occur in the vaccinia virus-infected HeLa cells (25). Fractionation of the vaccinia core proteins and solubilization of the inhibitor should lead to a better understanding of the mechanism of action of the core-mediated shutoff of host cell protein synthesis. We thank Prof. F. Chapeville for encouragement and helpful advice during this work. We are grateful to Alfred Dru for his excellent technical assistance in preparing and purifying vaccinia virus cores. We thank Dr. Petrissant for his generous gift of tRNAPhe. The wheat germ extract and TYMV RNA were donated by Drs. A. L. Haenni and Cl. Benicourt. Thanks also are due to Drs. Schapira and C. Vaquero for supplying the rabbit reticulocytes. We are grateful to Dr. A. L. Haenni for reading of this manuscript. The work was supported in part by a grant from the Centre National de la Recherche Scientifique (ATP 2114) and from the C.E.A. (Saclay, France). 1. Bablanian, R. (1975) Prog. Med. Virol. 19,40-83. 2. Moss, B. (1974) in Comprehensive Virology, eds. FraenkelConrat, H. & Wagner, R. R. Vol. 3, pp. 405-474. 3. Shatkin, A. J. (1963) Nature 199,357-358. 4. Moss, B. (1968) J. Virol. 2, 1028-1037. 5. Rosemond-Hornbeak, H. & Moss, B. (1975) J. Virol. 16, 3442. 6. Esteban, M. & Metz, D. H. (1973) J. Gen. Virol. 19,201-216.
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7. Joklik, W. K. (1964) J. Mol. Biol. 8, 263-276. 8. Dales, S. (1963) J. Cell. Biol. 18,51-72. 9. Jaureguiberry, G., Ben-Hamida, F., Chapeville, F. & Beaud, G. (1975) J. Virol. 15, 1467-1475. 10. Easterbrook, K. B. (1966) J. Ultrastruct. Res. 14, 484-496. 11. Holloway, P. W. (1973) Anal. Biochem. 53,304-308. 12. Housman, D., Jacobs-Lorena, M., RajBhandary, U. L. & Lodish, H. F. (1970) Nature 227,913-918. 13. Davies, J. W. & Kaesberg, P. (1973) J. Virol. 12, 1434-1441. 14. Ben-Hamida, F., Chapeville, F. & Beaud, G. (1975) "In vitro transcription and translation of viral genomes," INSERM Colloq. 47,355-361. 15. Clemens, M. J., Pain, V. M., Henshaw, E. C. & London, I. M. (1976) Biochem. Biophys. Res. Commun. 72, 768-775. 16. Munyon, W., Paoletti, E., Ospina, J. & Grace, J. T., Jr. (1968) J.
Virol. 2, 167-172. 17. Gold, P. & Dales, S. (1968) Proc. Natl. Acad. Sci. USA 60, 845-852. 18. Paoletti, E., Rosemond-Hornbeak, H. & Moss, B. (1974) J. Biol. Chem. 249, 3273-3280. 19. Paoletti, E. & Moss, B. (1974) J. Biol. Chem. 249,3281-3286. 20. Ehrenfeld, E. & Hunt, T. (1971) Proc. Natl. Acad. Sci. USA 68, 1075-1078. 21. Grill, L. K., Sun, J. D. & Kandel, J. (1976) Biochem. Biophys. Res. Commun. 73, 149-156. 22. Person, A. & Beaud, G. (1978) J. Virol., in press. 23. Paoletti, E. & Moss, B. (1972) J. Virol. 10, 417-424. 24. Kleiman, J. H. & Moss, B. (1974) J. Biol. Chem. 250, 24202437. 25. Kaerlein, M. & Horak, I. (1976) Nature 259, 150-151.
Biochemistry
In vitro inhibition of protein synthesis by purified cores from vaccinia virus (reticulocyte lysate/host cell shutoff/polypeptide chain initiation block)
F. BEN-HAMIDA AND G. BEAUD Laboratoire de Biochimie du Developpement, Institut de Recherche en Biologie Mol6culaire du Centre National de la Recherche Scientifique et de l'Universit6 Paris VII, 2 Place Jussieu, Tour 43, 75221 Paris Cedex 05, France
Communicated by J. Brachet, October 21, 1977
ABSTRACT The mechanism of the shutoff of cellular protein synthesis in vaccinia virus-infected cells has been investigated by using in vitro systems. Purified vaccinia cores cause inhibition of endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems. Translation of viral mRNA from turnip yellow mosaic virus is also impaired in wheat germ cell-free extracts. The block induced by vaccinia cores in protein synthesis is not due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. No nucleolytic activity able of digesting mRNA could be detected in purified vaccinia cores with three sensitive tests. There is a lack of inhibition in the poly(Phe)poly(U) system, which bypasses the normal initiation process. An almost complete disaggregation of polyribosomes in the reticulocyte lysate appears when vaccinia cores are present. These results indicate that mRNA translation in a cell-free system is affected predominantly at the level of polypeptide chain initiation.
The infection of animal cells with many viruses results in an inhibition of host protein synthesis (for review, see ref. 1). In the case of vaccinia (2), the inhibition of cell protein synthesis is rapid and extensive at high multiplicity of infection. It occurs in the presence of actinomycin D (3-5), cycloheximide (4), or cordycepin (6). This implies that inhibition of host protein synthesis does not require de novo viral RNA or protein synthesis and appears to be associated with the virions. Moreover, the time required for the establishment of the shutoff corresponds to the first stage of uncoating-i.e., the release of cores into the cytoplasm (7, 8). Which level of protein synthesis is affected by the shutoff of the cell in the vaccinia system? This question can be answered by using cell-free systems in -which the effect of vaccinia cores on mRNA translation can be directly studied. Here we report experimental results with purified vaccinia cores in cell-free systems. Endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems was inhibited by the vaccinia core preparations. The translation of exogenous mRNA such as the RNA of turnip yellow mosaic virus (TYMV) in wheat germ extracts also was impaired. The lack of inhibition of the poly(Phe)-poly(U)dependent system which bypasses the normal initiation process and the almost complete loss of polyribosomes in the reticulocyte lysate when vaccinia cores were present suggest that polypeptide chain initiation is the major site of action of the vaccinia virus core-mediated inhibition. MATERIALS AND METHODS Preparation of Purified Cores. The vaccinia virions (Lister strain) produced by infection of KB cells were purified by suThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
crose density gradient centrifugation as described (9) except that an additional cycle of sucrose cushion followed by sucrose gradient centrifugation was performed. Cores were prepared by incubation of purified virus (previously sonicated for 40 sec at 4 ,u on an MSE sonicator) at 370 for 30 min in 30 mM TrisHCl, pH 7.5/0.2% Triton X-100 (Koch-Light)/50 mM 2-mercaptoethanol (10). At the end of this incubation, the Triton X-100 was removed from the sonicated cores by adsorption on Bio-Beads SM2 (Bio-Rad) (11) by shaking at 40 for 3 hr. The supernatant was then layered onto 3 ml of a 36% (wt/vol) sucrose cushion containing 10 mM Tris-HCI, pH 7.5/1 mM dithiothreitol and centrifuged in a Beckman SW 50-1 rotor at 25,000 rpm for 30 min. The pellet of viral cores was suspended in 10 mM Tris-HCI (pH 7.5) and, after brief sonication (twice for 10 sec), was layered onto a 20-40% (wt/vol) linear sucrose gradient prepared in 2 mM Tris-HCI (pH 9) in a Beckman SW-25.1 rotor at 15,500 rpm for 55 min. The band of viral cores thus obtained was collected and concentrated by sedimentation in a Beckman 30 rotor at 28,000 rpm for 90 min. The purified cores thus obtained were suspended in 2 mM Tris-HCl (pH 9) and stored in small aliquots in liquid nitrogen. The vaccinia core preparations were adjusted to 20 A26o units/ml (corresponding to 1.5 mg of protein per ml). Cell-Free Systems. Reticulocyte lysate system. Rabbit reticulocyte lysates were prepared according to Housman et al. (12). Protein synthesis was assayed by the incorporation of L[35S]methionine into protein. Reaction mixtures (50 ,i) contained 10 ul of reticulocyte lysate, 30 mM Tris-HCI (pH 7.5), 3 mM Mg acetate, 60 mM KCI, 2 mM dithiothreitol, 1 mM ATP, 0.5 mM GTP, 16 mM K phosphoenolpyruvate (PePyr), PePyr kinase at 200 ,g/ml, 19 unlabeled amino acids at 40,uM each, 1 MM unlabeled methionine, and 4 ,gCi of L-[35S]methi. onine (300 Ci/mmol, Amersham). Incubation mixtures were supplemented with 35 ,M hemin (Fluka). Usually, in order to enhance the vaccinia core-mediated inhibition of the endogenous [in a reticulocyte lysate or an Ehrlich ascites tumor (EAT) cell-free extract] or exogenous (in a wheat germ cell-free extract programmed by TYMV RNA) mRNA translation (see below), preincubation of the cell-free extract with the vaccinia cores was performed for 10 min under suitable ionic conditions, as described with the results of each experiment. Similar results were obtained when hemin was added during preincubation with the reticulocyte lysate. Incubation for protein synthesis (reticulocyte lysate) was at 30°. Aliquots (5 ,l) were applied to Whatman 3MM paper discs, immersed for 10 min into 10% cold trichloroacetic acid containing 4 mM unlabeled methionine, transferred to 5% trichloroacetic acid, brought to 900 for 15 min, and rinsed twice in 5% cold trichloroacetic acid for 5 min, Abbreviations: TYMV, turnip yellow mosaic virus; PePyr, phosphoenolpyruvate; EAT, Ehrlich ascites tumor.
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B
x
x
B
E
E0.
30_
04
20 _ 10
30 20 Time, min FIG. 1. Effect of preincubation of reticulocyte lysates with vaccinia cores on protein synthesis. (A) Without preincubation. (B) Preincubation of reticulocyte lysate with vaccinia cores at 200 for 10 min in 3 mM Mg acetate/60 mM KCl/40 mM Tris-HCl, pH 7.5. Both samples were then incubated at 300 with the components required for protein synthesis. Aliquots (5 Il) were spotted onto Whatman 3MM discs and analyzed as above. 0, Control; 0, with cores.
0
10
once in ethanol for 5 min, in ethanol/diethyl ether, 1:1 (vol/vol), and finally in diethyl ether. The radioactivity was measured by using a toluene-based scintillator. EAT cell-free system. Extracts were prepared from EAT cells as described (9). The endogenous EAT cell mRNA translation activity was measured in nonpreincubated extracts. The assay system for measurement of protein synthesis by the incorporation of L-[3sS]methionine consisted of 50 ,l of the reaction mixture containing 3.5 mM Mg acetate, 120 mM KC1, and the other components required for protein synthesis as above. As in the case of the reticulocyte lysate, preincubation was performed before addition of the radioactive amino acid and the components needed for protein synthesis. The experimental procedure was as above. When poly(U) was used, 0.4 ,UCi of L-['4C]phenylalanine (260 mCi/mmol) was added instead of the amino acids and the Mg2+ concentration was increased to 5 mM. Wheat germ cell-free system. The wheat germ cell-free extract was prepared essentially according to the procedure of Davies and Kaesberg (13). The standard in vitro reaction mixture for amino acid incorporation directed by TYMV RNA (80 ,g/ml) consisted of 100 ,ul of a reaction mixture containing 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) (pH 7.6), 3 mM Mg acetate, 140 mM K acetate, 1 mM ATP, 0.2 mM GTP, 8 mM PePyr, 19 unlabeled amino acids at 25 MuM each, and L-[s5S]methionine (2 ,Ci, 300 Ci/mmol). Incubation was at 300, and samples were analyzed as above. Radioactive Vaccinia mRNA Preparation. The vaccinia [3H]mRNA used as substrate to detect residual nuclease activity in the vaccinia cores was made from [3H]UTP and purified as
described (9). Detection of Residual Nuclease Activity in Vaccinia Cores by Breakdown of [l4C]Phenylalanine-tRNAPhe. Purified tRNAPhe from rat liver was a gift from G. Petrissant (Paris). Charging with [14C]phenylalanine was carried out with an EAT cell-free extract (4 A260 units/ml) in a reaction mixture containing 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.6), 5 mM Mg acetate, 100 mM KC1, 2 mM
0
20
40 60 Time, min FIG. 2. Effect of vaccinia cores on globin synthesis in reticulocyte lysates. In this experiment, both preincubation and incubation for protein synthesis were performed at 30°. Hemin was added in the second incubation. At the indicated times, 5-gl samples were applied Whatman 3MM discs and analyzed for protein incorporation. Curve A, control; curve B, with 10 ttl of vaccinia cores; curve C, with 20 gl of vaccinia cores.
dithiothreitol, 2 mM ATP, 16 mM PePyr, Pepyr kinase at 200 Mg/ml, cycloheximide at 300 ,g/ml, and [14C]phenylalanine (10 ,Ci/ml, 370 mCi/mmol). Incubation was at 300 for 50 min. To detect residual nuclease activity in vaccinia cores, the preparation was assayed by adding vaccinia cores at 50 min when charging of tRNA with [14C]phenylalanine had reached a plateau. For this, to 50 Mul of the above mixture were added, in a final volume of 100 Mul, 0.8 A260 unit of vaccinia cores (or the corresponding buffer in control samples) and 20 mM phosphate buffer (pH 7.5). Incubation was continued at 300. The reaction was stopped by addition of 0.2 mg of yeast RNA, immediately followed by 1 ml of cold 5% trichloroacetic acid; the insoluble radioactivity was measured as above.
RESULTS Vaccinia Core-Mediated Translational Inhibition in Cell-Free Systems. When a reticulocyte lysate was incubated in the presence of vaccinia cores, protein synthesis was inhibited by approximately 30% (Fig. 1). Kinetic analysis showed a lag period of 10-20 min before inhibition of protein synthesis. Preincubation, for 10 min, of the reticulocyte lysate with the vaccinia cores resulted in a greater inhibition of incorporation, reaching 60-70% of the control. The level of this inhibition depended on the concentration of the vaccinia cores. A 50% decrease of protein synthesis (relative to the control) was obtained with 10 ,l of cores, corresponding to 300 Mug of viral protein per ml (Fig. 2). In the experiments described in Figs. 1 and 2, hemin was not added in the preincubation step; however, the addition of hemin (35 or 75 MM) at this step did not change the magnitude of the protein synthesis inhibition. Impairment of translation, although to a lesser extent, also was obtained with the EAT cell-free system (data not shown). Vaccinia cores were also assayed for exogenous mRNA (TYMV RNA) translation in a wheat germ cell-free extract. The results (Fig. 3) show that vaccinia cores cause the inhibition of not only endogenous or cellular mRNA but also of viral mRNA trans-
lation.
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Table 1. Absence of residual nuclease activity in vaccinia cores (breakdown of [l4C]phenylalanine-tRNAPhe)
Incubation time, min 0 10 30 50 70
Insoluble radioactivity,* cpm/10 ,l With Control vaccinia cores 3800 3691 3409 3311 3134
3703 3465 3310 3144 3180
* Insoluble in cold trichloroacetic acid.
0
20 40 Time, min FIG. 3. Kinetics of protein synthesis directed by TYMV RNA in wheat germ cell-free extracts. The wheat germ extract (40 Ml) was first incubated at 200 for 20 min in the presence of vaccinia cores (50 Ml) or the corresponding buffer for controls, in a final volume of 100 Ml containing 3 mM Mg acetate, 140 mM K acetate, and 20 mM N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.5). The vaccinia cores were then removed by centrifugation in the cold at 14,000 rpm for 10 min in a Beckman Microfuge. The supernatant (75 Ml) was then incubated at 300 in the presence of TYMV RNA (80 Mg/ml) in a final volume of 100 Ml containing 1 mM ATP, 0.2 mM GTP, 8 mM PePyr, 19 unlabeled amino acids at 25 MM each, and L-[35S]methionine (2 MCi, 300 Ci/mmol, Amersham). Samples (20 Mul) were removed at various times, applied to Whatman 3MM filters, and analyzed for methionine incorporation into protein. 0, Control; 0,
with vaccinia cores.
The decrease in protein synthesis induced by vaccinia cores does not seem to be due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. Indeed, the same extent of inhibition was obtained when the cores were removed by centrifugation after preincubation with the wheat germ extract and then TYMV RNA was added as mRNA for translation, as shown in Fig. 3. When the effect of vaccinia cores was assayed on poly(U) translation in an EAT cell-free system, there was no shutoff (Fig. 4). The failure of vaccinia cores to inhibit poly(Phe) synthesis under the direction of poly(U) indicates that the vaccinia core-mediated inhibition does not predominantly affect peptide chain elongation.
S
3Table 2. Assay of breakdown of vaccinia [3H]mRNA Insoluble radioactivity, cpm/5 ,l With Incubation Control vaccinia cores time, min
0
x
EQ
Lack of Residual Ribonuclease Activity in Vaccinia Core Preparations. A critical point to be established is the level of the nuclease activity that may be a contaminant of the viral preparation and may interfere with the observed inhibition. The earliest preparations of vaccinia cores used for in vitro studies of the inhibition of host cell protein synthesis showed some breakdown of [l4C]phenylalanine-tRNAPhe and, to a lesser extent, a nuclease activity of vaccinia [3H]mRNA (14). The present technique of purification allowed us to decrease the nuclease activity in the vaccinia core preparations to a level not detectable by the following three tests. (i) Possible breakdown of [14C]phenylalanine-tRNAPhe was followed as cold trichloroacetic acid-soluble material and compared to samples containing vaccinia cores. There was no increase in the degradation of [l4C]phenylalanine-tRNAPhe in the presence of vaccinia cores, even after 70 min of incubation at 30' (Table 1). (ii) 3H-Labeled vaccinia mRNA was also used as substrate for possible nuclease contaminating the vaccinia core preparations. The acid-insoluble radioactivity from 3H-labeled vaccinia mRNA was followed, and no breakdown occurred on incubation for 60 min (Table 2). (iii) A third highly sensitive control to check the absence of endonuclease activity in vaccinia core preparations used for in vitro studies was the absence of any significant change of the polyribosome distribution pattern from reticulocyte lysates (15) when incubated in the presence of vaccinia cores, under conditions that do not allow protein synthesis. The profiles of absorbance at 260 nm of polyribosomes were identical in the absence and in the presence of vaccinia cores when incubated for 5 min after 10 min of preincubation (Fig. 5 A and B). Core-Mediated Inhibition Is Not Due to Nucleoside Triphosphate Phosphohydrolase. The translation inhibitory activity present in vaccinia cores could be attributed to the nucleoside triphosphate phosphohydrolases I or II, two enzymes present in the cores (16-19). In fact, under our conditions, the
2I -I
20
.I I
40
Time, min
FIG. 4. Kinetics of poly(Phe) synthesis in response to poly(U) (200 Mug/ml) in a nonpreincubated EAT cell-free extract. Reaction mixtures were first preincubated at 300 for 10 min before addition of L-[14C]phenylalanine at zero time. A 50-Ml mixture was used, and 10-Mul samples were removed at various times and applied to Whatman 3MM discs; the synthesis of [14C]poly(Phe) was measured as hot trichloroacetic acid-insoluble material. *, Control; 0, with vaccinia cores (20 Ml).
405 409 366 427
386 321 378 379 The reaction mixture (final volume, 50 ,l) contained 40 mM N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.5), 3 mM Mg acetate, 80 mM KCl, 1 mM dithiothreitol, 3 Mg of vaccinia [3HJmRNA (100 cpm/Mg), and 15 Ml of vaccinia cores. Incubation at 300 was stopped at the times indicated by the addition, to 5-,ul samples, of 5% trichloroacetic acid and 200 gg of yeast RNA. 0 20 40 60
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This effect on the polyribosome profiles indicates that polypeptide chain initiation is the major site of action of vaccinia cores in the inhibition of in vitro protein synthesis.
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FIG. 5. Effect of vaccinia core extracts on the polyribosome profile of reticulocyte lysate. A reticulocyte lysate (30 p1) was first preincubated at 200 for 10 min in the absence (mixture I) or in the presence (mixture II) of vaccinia cores (40 pl) in a final volume of 85 Ml containing 25 mM Tris.HCl, pH 7.5/80 mM KCl/3 mM Mg acetate/30 MM hemin. Both mixtures were then duplicated in A and C and in B and D, respectively. C and D received 1 mM ATP, 16 mM PePyr, PePyr kinase at 200 Mg/ml, and a mixture of 20 amino acids (40 MM each) for protein synthesis. A and B lacked on exogenous energy-generating system and amino acids. All samples (in a final volume of 50 Ml) were incubated at 30°. After 5 min, the samples were quickly cooled to 0° and 3 volumes of cold gradient buffer (20 mM Tris.HCl, pH 7.5/100 mM KCl/5 mM Mg acetate) containing cycloheximide at 400 ,g/ml was added. Each sample thus diluted (total volume, 200 Ml) was layered onto a linear (15-30%) sucrose gradient in the above buffer. After centrifugation at 20 for 85 min at 40,000 rpm in a Beckman SW 41 rotor, the contents of the tubes were pumped from the bottom through the flow-cell of a Gilson spectrophotometer recording optical density at 260 nm. (A and C) No vaccinia cores; (B and D) vaccinia cores present; (A and B) no protein synthesis; (C and D) with amino acids and an energy-generating system. The direction of sedimentation was from left to right.
core-mediated inhibition of mRNA translation is not due to a depletion in the level of ATP or GTP. Indeed, by adding either 3H-labeled ATP or GTP to the reaction mixtures and measuring the amount of radioactivity present in the nucleoside mono-, di-, and triphosphates at different times, the levels of ATP or GTP were shown to remain unchanged when vaccinia cores were present (data not shown). The Block Induced by Vaccinia Cores Affects Polypeptide Chain Initiation. The fact that the poly(U) system, which bypasses the normal initiation process, is unaffected by vaccinia cores (see above and Fig. 4) is consistent with an inhibitory site of action at the initiation level in protein synthesis. To confirm this, an analysis of the change of the polyribosome profiles of reticulocytes lysates was carried out in the presence and in the absence of vaccinia cores under conditions of protein synthesis. As illustrated in Fig. 5, vaccinia cores caused an extensive disagreggation of polyribosomes within 5 min when incubated with a reticulocyte lysate and amino acids plus an energy source (Fig. 5 C and D). At the same time, there was a corresponding increase of monosomes. In the absence of amino acids and an energy source, no breakdown was observed (Fig. 5 A and B).
DISCUSSION The results presented in this report demonstrate that a component causing inhibition of protein synthesis in cell-free systems is associated with vaccinia cores. Based on the viral core concentration used (600 ,tg/ml) and on the ribosome content of rabbit reticulocyte lysates (1 iug of ribosomes per ,l of undiluted lysate), one can calculate that the inhibition in this system occurs at the low ratio of 1 viral core to 100 ribosomes. Moreover, the block affects translation of both endogenous and cellular mRNA (in nonpreincubated reticulocyte lysates and EAT cell-free extracts) and viral mRNA (in wheat germ extracts). This possible lack of discrimination between cellular and viral RNA translation is in agreement with earlier findings (4) that some viral protein synthesis is apparently required to allow extensive early viral mRNA translation. The kinetics of protein synthesis in reticulocyte lysates incubated in the presence of vaccinia cores shows that the initial rate of amino acid incorporation is not greatly reduced during the first minutes of incubation. This early resistance to inhibition could reflect the gradual inactivation of a component(s) that is essential for translation of mRNA. This is in agreement with the finding that prior incubation of the cell-free extract with the cores (in the absence of protein synthesis) increases translational inhibition
(Fig. 1).
It is possible that the protein shutoff observed is due to the effect of double-stranded RNA (20) possibly present in the cores. The fact that inhibition of TYMV RNA translation occurs in wheat germ extracts (Fig. 3) excludes this possibility. Indeed, as shown by Grill et al. (21), inhibition of protein synthesis by double-stranded RNA is a phenomenon limited to the mammalian system and does not occur in wheat germ extracts. The block of protein synthesis mediated by vaccinia cores is not due to a decrease in the availability of mRNA but rather to a lowering of the rate of polypeptide chain initiation. (i) No nucleolytic activity able to digest mRNA could be detected in purified vaccinia cores. (ii) Poly(U) translation, which does not use the initiation mechanisms characteristic of natural mRNA, is not affected. (iii) Incubation of vaccinia cores with reticulocyte lysates under conditions of protein synthesis leads to an almost complete disaggregation of polyribosomes. These results indicate that the level at which mRNA translation in cell-free systems is affected is predominantly polypeptide chain initiation. A similar conclusion has been obtained from in vivo experiments showing that initiation is also the major site of inhibition of protein synthesis in vaccinia virusinfected cells exposed to actinomycin D (4, 5) or cordycepin (22). It can be suggested that inhibition of protein synthesis might be related to the phosphorylation of a component essential for initiation of translation. One plausible hypothesis is that the protein kinase, one of the enzymes located in the vaccinia cores (23, 24), may be involved in this inactivation by phosphorylation of the eIF-2 initiation factor. Preliminary experiments have not allowed clear proof of a significant phosphorylating activity of vaccinia cores when purified reticulocyte initiation factor eIF-2 was used as substrate. On the other hand, in order to support the same hypothesis, we also tested the ability of eIF-2 to reverse the protein synthesis inhibition induced by the vaccinia cores. However, only a partial reversal of this inhibition was obtained with purified eIF-2 under the conditions of protein synthesis inhibition in this work. [The highly purified reticulocyte initiation factors used in these
Biochemistry: Ben-Hamida and Beaud experiments were provided by T. Staehelin and B. Ei.( e Institute for Immunology).] Another explanation of the vaccinia core-mediated translational inhibition might be related to the specific phosphorylation of 40S ribosomal proteins which occur in the vaccinia virus-infected HeLa cells (25). Fractionation of the vaccinia core proteins and solubilization of the inhibitor should lead to a better understanding of the mechanism of action of the core-mediated shutoff of host cell protein synthesis. We thank Prof. F. Chapeville for encouragement and helpful advice during this work. We are grateful to Alfred Dru for his excellent technical assistance in preparing and purifying vaccinia virus cores. We thank Dr. Petrissant for his generous gift of tRNAPhe. The wheat germ extract and TYMV RNA were donated by Drs. A. L. Haenni and Cl. Benicourt. Thanks also are due to Drs. Schapira and C. Vaquero for supplying the rabbit reticulocytes. We are grateful to Dr. A. L. Haenni for reading of this manuscript. The work was supported in part by a grant from the Centre National de la Recherche Scientifique (ATP 2114) and from the C.E.A. (Saclay, France). 1. Bablanian, R. (1975) Prog. Med. Virol. 19,40-83. 2. Moss, B. (1974) in Comprehensive Virology, eds. FraenkelConrat, H. & Wagner, R. R. Vol. 3, pp. 405-474. 3. Shatkin, A. J. (1963) Nature 199,357-358. 4. Moss, B. (1968) J. Virol. 2, 1028-1037. 5. Rosemond-Hornbeak, H. & Moss, B. (1975) J. Virol. 16, 3442. 6. Esteban, M. & Metz, D. H. (1973) J. Gen. Virol. 19,201-216.
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7. Joklik, W. K. (1964) J. Mol. Biol. 8, 263-276. 8. Dales, S. (1963) J. Cell. Biol. 18,51-72. 9. Jaureguiberry, G., Ben-Hamida, F., Chapeville, F. & Beaud, G. (1975) J. Virol. 15, 1467-1475. 10. Easterbrook, K. B. (1966) J. Ultrastruct. Res. 14, 484-496. 11. Holloway, P. W. (1973) Anal. Biochem. 53,304-308. 12. Housman, D., Jacobs-Lorena, M., RajBhandary, U. L. & Lodish, H. F. (1970) Nature 227,913-918. 13. Davies, J. W. & Kaesberg, P. (1973) J. Virol. 12, 1434-1441. 14. Ben-Hamida, F., Chapeville, F. & Beaud, G. (1975) "In vitro transcription and translation of viral genomes," INSERM Colloq. 47,355-361. 15. Clemens, M. J., Pain, V. M., Henshaw, E. C. & London, I. M. (1976) Biochem. Biophys. Res. Commun. 72, 768-775. 16. Munyon, W., Paoletti, E., Ospina, J. & Grace, J. T., Jr. (1968) J.
Virol. 2, 167-172. 17. Gold, P. & Dales, S. (1968) Proc. Natl. Acad. Sci. USA 60, 845-852. 18. Paoletti, E., Rosemond-Hornbeak, H. & Moss, B. (1974) J. Biol. Chem. 249, 3273-3280. 19. Paoletti, E. & Moss, B. (1974) J. Biol. Chem. 249,3281-3286. 20. Ehrenfeld, E. & Hunt, T. (1971) Proc. Natl. Acad. Sci. USA 68, 1075-1078. 21. Grill, L. K., Sun, J. D. & Kandel, J. (1976) Biochem. Biophys. Res. Commun. 73, 149-156. 22. Person, A. & Beaud, G. (1978) J. Virol., in press. 23. Paoletti, E. & Moss, B. (1972) J. Virol. 10, 417-424. 24. Kleiman, J. H. & Moss, B. (1974) J. Biol. Chem. 250, 24202437. 25. Kaerlein, M. & Horak, I. (1976) Nature 259, 150-151.
