JOURNAL OF VIROLOGY, June 1975, p. 1467-1474

Vol. 15, No. 6

Copyright 0 1975 American Society for Microbiology

Printed in U.SA.

Messenger Activity of RNA Transcribed In Vitro by DNA-RNA Polymerase Associated to Vaccinia Virus Cores G. JAUREGUIBERRY,* F. BEN-HAMIDA, F. CHAPEVILLE,

AND

G. BEAUD

Institut de Biologie Moleculaire du Centre National de la Recherche Scientifique, Universite Paris VII, 75005-Paris, France

Received for publication 27 December 1974

The coding properties of RNA transcribed in vitro by purified vaccinia cores have been investigated using Krebs ascites tumor cells, L cells, and reticulocyte lysates. Six to 10 proteins synthesized in vitro are separated on polyacrylamide gels by electrophoresis in the presence of sodium dodecyl sulfate. Their molecular weights vary from 10,000 to 44,000. The electrophoretic behavior of these proteins is similar to that of early proteins isolated from infected L cells. The tryptic peptide analysis of one of these proteins indicates similarity in amino acid sequences. These results show fidelity of both in vitro transcription and translation. The fact that not all of the early proteins, especially those of molecular weight above 44,000, are synthesized in vitro does not seem due to a competition between 12S mRNA synthesized in excess and RNA of a higher sedimentation coefficient present in a lower amount. The cytoplasmic site of vaccinia virus replication (for a review, see references 13 and 35) and evidence that regulation occurs at the level of translation at both the early (25, 33) and late (9, 19, 28, 31) stages of viral development make this virus a useful tool for studying the control of viral gene expression. To this aim an in vitro system translating exogenous vaccinia RNA would allow better exploration of the mechanisms involved. It has been shown that vaccinia virus contains an RNA polymerase associated with the cores (16, 29) which is able to synthesize in vitro RNA similar to early mRNA's (2, 14, 29). It is known that this RNA stimulates protein synthesis in cell-free systems (1, 5). However, no definite characterization of synthesized proteins was made, and it is the purpose of the present paper to show that vaccinia early mRNA's made in vitro are also faithfully translated. MATERIALS AND METHODS Cell cultures. KB cells were grown on monolayers in Eagle medium (Eurobio), modified I.G.R. (glucose, 4.5 g/liter; fourfold vitamin concentration; tryptose phosphate, 10% [vol/vol; Difco]), and supplemented with 10% heat-denatured (30 min at 56 C) calf serum. L cells (strain CCLI) were grown in suspension (starting from monolayer culture) in minimum Eagle medium supplemented with glucose (3.5 g/liter), tryptose phosphate broth powder (3 g/liter; Difco), and 8% denaturated calf serum. All media contained 100 Ag of penicillin and 500 gg of streptomycin per ml. Vaccinia virus production and purification. KB

cells grown on monolayers were infected by vaccinia virus, Lister strain, at a multiplicity of 3 PFU (180 particles/cell). After 40 h of incubation at 37 C, the virus was purified by sedimentation through two successive sucrose cushions, followed by one sucrose gradient centrifugation (1 absorbancy unit of purified virus at 260 nm [A2,601 corresponds to 1.2 x 1010 particles/ml) (11, 17). Vaccinia virus strain Chambon St-Yves Menard, obtained from the Institut de la Vaccine (Paris), was purified as described above except that three successive sucrose cushions and two successive sucrose gradients were made. Such a viral preparation contains 40 to 50% particles other than virions, as determined by electron microscopy after negative staining. As shown in Results these impurities did not interfere significantly with the in vitro transcription. In vivo labeling. Early vaccinia virus proteins were labeled by '5S-methionine (Radiochemical Centre, Amersham) using a described procedure (4). L cells in suspension were infected at 107 cells/ml by the Lister strain at a multiplicity of 5 PFU/cell in Puck medium A plus 20 mM Mg2+ (2) for 15 min at 37 C. The cells were then diluted 10 times with Eagle medium containing 0.5 AM methionine and 5% dialyzed calf serum. Labeling by [35S Imethionine (25 gCi/ml, 60 Ci/ mM) was done from 15 to 50 min after the adsorption period. The cells were then cooled to 0 C, collected on Whatman GF/C filters, extracted by 0.4 ml of Laemmli sample buffer (18), and finally boiled for 3 min. Isolation and purification of early mRNA's released after in vitro transcription. Vaccinia cores were prepared as described by Kates and Beeson (14). Purified vaccinia virus was treated with 0.2% Triton X-100 and 50 mM 2-mercaptoethanol at 37 C for 25 min. The pellet of the cores obtained by centrifugation at 30,000 x g for 20 min was used for in vitro 467

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JAUREGUIBERRY ET AL.

transcription. Conditions for this transcription were as follows: 50 mM Tris-hydrochloride, pH 7.5; 2 mM dithiothreitol; 10 mM MgAc2; 1 mM CTP; 1.5 mM GTP; 5 mM ATP; 4 mM phosphoenolpyruvate; pyruvate kinase, 40 Ag/ml; 30 mM NH4Cl; macaloid (Baroid Division, National Lead Co., Houston, Tex.), 200 ug/ml; [3H]UTP (C.E.A., France), 20 Ci/mol, 1 mM; and cores corresponding to 10 A2,0 units of purified virus per ml of reaction mixture. After incubation at 37 C for 60 min, the mixture was centrifuged at 30,000 x g for 30 min. The supernatant containing the RNAs released from the cores was treated with sodium dodecyl sulfate (SDS) to 0.5% and then was shaken for 5 min with an equal volume of water-saturated phenol. Phases were separated by centrifugation at 17,000 x g for 10 min. The aqueous phase was then filtered in the cold through a column of Sephadex G-50 equilibrated with 40 mM Trishydrochloride, pH 7.5, and 0.1% SDS. The fractions containing the greater part of the RNA (corresponding to the exclusion volume) were pooled and precipitated by adding 2 volumes of ethanol and 0.1 volume of potassium acetate (10%, pH 5). The solution was kept at -30 C overnight and centrifuged at 10,000 x g for 15 min. The pellet was washed with a mixture of sterile water (1 volume) and 0.1% potassium acetateethanol (2 volume) and then twice with ethanol; it was dissolved in sterile water and stored in small samples in liquid nitrogen. For analysis a sample of this radioactive mRNA was layered (together with 10 A2,,0 units of cytoplasmic RNA isolated from ascites cells, added as marker) onto a 5 to 20% (wt/vol) linear sucrose gradient prepared in 10 mM Tris-hydrochloride (pH 7.5), 0.1 M NaCl, and 2 mM EDTA. After centrifugation at 24,000 rpm at 4 C for 16 h in a Spinco SW25.1 rotor, the gradient was cut into different fractions; A2.0 and trichloroacetic acidinsoluble radioactivity were measured. Preparation of cell-free extract. Extract was prepared from Krebs II mouse ascites tumor cells and L cells according to Mathews and Korner (23). L cells were grown in suspension to a density of 10" cells/ml and then collected by 10-min centrifugation at 1,200 rpm and washed four times at 4 C with Tris-saline (35 mM Tris-hydrochloride, pH 7.5, and 140 mM NaCl). Rabbit reticulocyte lysates were prepared as described by Housman et al. (10). All the cell-free extracts were stored in small aliquots in liquid nitrogen. Amino acid incorporation directed by vaccinia mRNA's in the cell-free systems. For ascites and L cells the concentrations of components in the reaction mixtures were as follows: preincubated cell-free extracts, 15 to 20 A... units/ml; Tris-hydrochloride, 30 mM (pH 7.5); dithiothreitol, 1 mM; ATP, 1 mM; GTP, 0.1 mM; creatine phosphate, 5 mM; creatine kinase, 0.16 mg/ml; KCl, 50 mM; and MgAc,, 3.5 mM (for L cells: 80 mM KCl and 5 mM MgAc,); 19 unlabeled amino acids, 40MM (each); 38S-methionine, 500 MACi/ml; vaccinia mRNA's isolated and purified after in vitro transcription, 180 Ci/mmol and 100 ug/ ml. The reaction mixture was usually incubated for 70 min at 37 C. Conditions for the reticulocyte cell-free system were as follows: reticulocyte lysate, 0.5 ml/ml;

J. VIROL.

MgAc2, 3 mM; KCl, 50 mM. The other components were similar to those described above. Incubation was at 25 C for 40 min. Incorporation of [3SS]methionine into proteins was measured as described in ref. 23. For analysis by polyacrylamide gel electrophoresis, the samples were treated with pancreatic RNase (50 Mg/ml), 0.04 M EDTA, 5 mM L-methionine at 37 C for 15 min and then precipitated by the addition of an equal volume of 10% trichloroacetic acid. After 1 h at 4 C, the precipitate was washed five times with cold 5% trichloroacetic acid, twice with ethanol, and subsequently with ethanol-ether (1:1, vol/vol) and ether to remove residual trichloroacetic acid. The samples of in vitro products were finally prepared for analysis by dissolving them in Laemmli sample buffer (18). SDS-polyacrylamide gel electrophoresis of proteins and tryptic peptides analysis. The in vitro and in vivo proteins were analyzed on slab gels (34) in the discontinuous buffer system described by Laemmli (18). Fifteen percent polyacrylamide gels were used unless otherwise indicated. The electrophoresis was carried out at 100 V for 4 h. For immediate visualization proteins stained with remazol dye according to Griffith (7) were used as standards. After electrophoresis (100 V, 4 h), the gel slab was dried as previously described (22) and autoradiographed on X-ray film (Kodirex). For tryptic digest analysis, regions of gel corresponding to bands of similar migration were excised, and the proteins were eluted by shaking for 24 h at 37 C with 10 ml of 0.1% SDS. The polypeptides were precipitated from the SDS solution after addition of 2 mg of ovalbumin with 20% trichloroacetic acid and dissolved in 1 N NaOH. After two more cycles of precipitation and dissolution to remove the SDS, the proteins were washed with acetone and dried (3). The dried proteins were dissolved in 98% formic acid (100 Ml/mg of protein) and then oxidized with 2 volumes of freshly prepared performic acid (8) for 2 h at room temperature. The sample was frozen, and most of the acid evaporated off under vacuum. A 1-ml amount of cold water was added, and the mixture was lyophilized. Proteolytic digestion (27) was initiated with 10 Mg of TPCK trypsin (Worthington) at 37 C for 6 h in 200 Ml of 0.1 M NH,HCO,. An additional 10 Mg of trypsin was added, and 18 h later the digests were lyophilized an additional three times, finally taken up in a small volume of buffer (pH 3.5), and applied to a thin-layer cellulose sheet (20 by 20 cm; DC-Fertigplatten cellulose F; E. Merck AG) for ascending chromatography in N-butanol-pyridine-acetic acidwater (20:20:6:24). After drying, the sheet was autoradiographed.

RESULTS AND DISCUSSION In vitro translation of the RNAs synthesized by vaccinia cores. The synthesis of RNA by cores derived from the Chambon St-Yves Menard strain presented optimal concentrations for ATP and Mg2+ slightly different from those used for the Lister strain (for both strains the yield was 10 Mg of RNA per A 2610 unit of vi-

IN VITRO TRANSCRIPTION OF mRNA ACTIVITY

VOL. 15, 1975

rus); yet hydrodynamic properties of the transcription products were similar. Moreover, both RNAs gave similar results concerning the size of the proteins synthesized in vitro. Nevertheless the RNAs derived from the Lister strain stimulated somewhat less the synthesis of protein above 25,000 mol wt. The tryptic peptides analysis described below was performed with proteins synthesized in the presence of Lister strain RNA, since this strain was used for in vivo labeling experiments. The characteristics of the stimulation of protein synthesis by vaccinia RNA added to a lysate derived from a Krebs ascites tumor cell have already been described (1, 5). We did not succeed in obtaining suitable antibodies for immunization of rabbits using either virions or cell extracts containing early proteins. Therefore the proteins (labeled by [85S ]methionine),

1469

either made in the cell-free system in response to exogenous early mRNA's or the early viral proteins synthesized in vivo, were compared on the basis of their mobilities using polyacrylamide disc gel electrophoresis in the presence of SDS. Labeled viral proteins were prepared by infecting L cells at a multiplicity sufficient to stop essentially all cellular protein synthesis in 15 min and by adding the label from 15 to 50 min after absorption. This procedure allows the labeling of early viral proteins since identical results were obtained after labeling in the presence of 20 Ag of cytosine arabinoside per ml (data not shown). Experiments of incorporation of radioactive amino acids into proteins of L cells infected at different multiplicities and in the presence of 5 ,ug of actinomycin D per ml (33) show a very rapid shut-off of host protein synthesis (data not shown; ref. 24). Figures 1

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FIG. 1. Autoradiogram showing separation by SDS-polyacrylamide slab gel electrophoresis of in vitro and in vivo translation products of vaccinia RNAs. In the upper insert INF. and N.J. designate infected early proteins and noninfected L cells; +RNA and -RNA correspond to in vitro products synthesized in the presence or in the absence of added vaccinia early RNAs prepared in vitro. The molecular weight of similar in vivo and in vitro proteins is indicated on the left insert. Exposure of autoradiogram, 3 days.

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JAUREGUIBERRY ET AL.

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FIG. 2. Autoradiogram of SDS-polyacrylamide slab gel electrophoresis comparing in vitro translation products of vaccinia RNA in reticulocytes and ascites cell extracts. In the upper insert -RNA and +RNA correspond to the in vitro product synthesized in the absence or presence of added vaccinia RNAs made in vitro. The arrows show the position of protein synthesized in the reticulocyte system, which corresponds to the migration of a similar protein in the ascites extract and in vivo early viral proteins. Hg denotes hemoglobin (mol wt, 16,500). Exposure of autoradiogram, 3 days. Gel concentration, 12.5%.

and 2 show the autoradiogram of proteins synthesized in vivo or by Krebs ascites cell lysate and then separated by SDS-polyacrylamide gel electrophoresis. The following conclusions can be drawn. (i) Early proteins labeled in vivo are resolved into at least 20 proteins of apparent mol wt from approximately 10,000 to more than 70,000 (in this study the gel concentration used precluded determination of mol wt above 70,000). Our results concerning the mol wt of proteins between 20,000 to 70,000 are in accordance with those reported by Esteban and Metz (4). However, some differences were observed for the protein of mol wt 10,000 to 20,000; this especially concerns at least two proteins of mol wt 11,500 and 14,500, which were detected by us in all the preparations. It is likely that the stacking effect of the discontinuous gel electrophoresis we have used allowed a better resolution. (ii) Proteins synthesized in vitro in Krebs ascites extracts in the presence of vaccinia RNA

are separated into at least 10 components, the mol wt of which varies from about 11,500 to no more than 38,000. (iii) Most of the proteins made in vitro correspond to the early viral proteins. However, for a few of them no corresponding proteins made in vivo are found (e.g., see the band located between 20,500 and 28,000 in Fig. 1). This discrepancy could result from a post-translational modification not operating in the in vitro system; it is possible that the corresponding proteins are present in vivo in very low amounts. Only about one-half of the total proteins synthesized in vivo are made in vitro, and most of the high-molecular-weight proteins are absent. This is not peculiar to the Krebs ascites tumor cell lysate, since similar results were obtained with lysates from uninfected L cells and from rabbit reticulocytes (Fig. 2; data not shown). The reticulocyte lysate was chosen because reovirus proteins of higher molecular weight can be synthesized in this type of extract

VOL. 15, 1975

IN VITRO TRANSCRIPTION OF mRNA ACTIVITY

and not in extracts from L cells or Krebs ascites cells (6, 20). Translation of 12S and 18S mRNA's. The absence of synthesis in extracts of Krebs ascites cells, L cells, and reticulocytes of the larger proteins could result from competition for a ribosomal site between the 12 to 14S RNAs present in excess and the RNAs sedimenting faster (and presumably of higher molecular weight). To eliminate this possibility the [3H ]UMP-labeled RNA synthesized in vitro was fractionated by sucrose gradient centrifugation into three classes, VA, VB, VC (Fig. 3). After sedimentation on a sucrose gradient, the migration of the VA and VB fractions was still heterogeneous, but the mean values of the sedimentation coefficients were approximately 12S for VA and 18S for VB (Fig. 4); the RNA of the Vc fraction sedimented at the bottom of the tube in these conditions (data not shown). The sedimentation patterns of these RNA-containing

1471

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sucrose gradient centrifugation. VA and VB fractions were separated as shown in Fig. 3. Sucrose gradient centrifugation was performed as described in Materials and Methods.

x

VB 200-

fractions on sucrose gradients prepared in 85% formamide (21) show that the RNA from the VB fraction is not an aggregated form of RNA present in the VA fraction but contains RNA of 100; higher molecular weight; a part of the RNAs of the V, fraction has the same sizes as those of the VA and VB fraction, in addition to RNAs sedi10 menting faster than the 18S marker (Fig. 5). Fractions The coding capacity of the VA and V. RNA Fractions fractions was tested with reticulocyte lysates. FIG. 3. Fractionation by sucrose gradient centrifu- The results show (Fig. 6) that the proteins gation of early RNAs synthesized in vitro. H1-Iabeled synthesized by these two classes of RNAs are vaccinia RNAs were layered on a linear 15 to 30%s .n.a (wt/vol) sucrose gradient prepared in 20 mM Tris- similarsand are no i initi orpofte hydrochloride, pH 7.4, 0.1 M NaCl, and 0.5% SDS hypothesils. However, to completely elminate (Spinco SW25-1 rotor, 16 h at 24,000 rpm, 4 C). The possible competition for a ribosomal site befractions were pooled as described in the figure and tween mRNA's of different sizes, a better separation is required (i.e., by gel electrophoresis). concentrated after ethanol precipitation.

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JAUREGUIBERRY ET AL.

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Messenger activity of RNA transcribed in vitro by DNA-RNA polymerase associated to vaccinia virus cores.

The coding properties of RNA transcribed in vitro by purified vaccinia cores have been investigated using Krebs ascites tumor cells, L cells, and reti...
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