Translation of eggplant mosaic virus RNA in wheat germ extracts and reticulocyte lysates

VIROLOGY

91, 305-311 (1978)

Translation

of Eggplant Mosaic Virus RNA in Wheat Germ Extracts and Reticulocyte Lysates

BBRBNICE

RICARD,

HBLBNE

RENAUDIN

AND

JOSEPH-MARIE

I.N.R.A. and Universiti de Bordeaux II, Laboratoire de Biologie Cellulaire et Molkulaire, Grande Ferrade, 33140 Pont de la Maye (France)

BOVB Domaine de la

Accepted August 18, 1978 Eggplant mosaic virus (EMV) RNA extracted from virions (virion EMV RNA) is a good messenger in wheat germ extracts and reticulocyte lysates but directs surprisingly little synthesis of coat protein. Large amounts of coat protein, however, are synthesized when a low molecular weight RNA purified from virion RNA is used as messenger. Heavy virion RNA directs the synthesis of two proteins of high molecular weight in reticulocyte lysates. The larger of these proteins accounts for almost the entire coding potential of 2 x 10” dalton viral RNA. INTRODUCTION

RNAs isolated from plant viruses with nonsegmented, single-stranded RNA genomes have been studied by several laboratories for their messenger properties in vitro. Tobacco mosaic virus (TMV) RNA does not direct the synthesis of its coat protein in any of the systems in which it has been tested. Instead, a low molecular weight RNA isolated from TMV-infected tissues, is the coat protein messenger (Hunter et al., 1976; Siegel et al., 1976). In the case of turnip yellow mosaic virus (TYMV), a low molecular weight RNA (LRNA) has also been found and shown to support coat protein synthesis (Klein et al., 1976; Pleij et al., 1976; Ricard et al., 1977). Unlike common strain TMV but like the cowpea strain of TMV (Higgins et al., 1976, Bruening et al., 1976), the TYMV L-RNA is encapsidated. If L-RNA is important in the replication cycle of tymoviruses, it should be present in other members of the tymovirus group. Yet Klein et al. (1976) have reported that they were unable to detect L-RNA in virions of eggplant mosaic virus (EMV), a tymovirus serologically unrelated to TYMV (Gibbs et al., 1966). In this paper, we present evidence for the presence in EMV virions of L-RNA, the mRNA for EMV coat protein. In addition virions contain an RNA of molecular

weight 2 x 106, corresponding to the viral genome, and which is the mRNA for high molecular weight protein products. MATERIALS

AND

METHODS

Virus and RNA. The purification

by of EMV propagated on Datura stramonium L. and the extraction of RNA from freshly prepared virions were as described for TYMV (Mouches et al., 1974). RNA was extracted from plants by the technique of Van de Walle (1973) modified by Mamoun (1976) by the addition of 0.2% Macaloid to inhibit ribonucleases. chloroform-butanol

Wheat germ extracts and protein synthesis. Commercial wheat germ (General Mills, Inc., Vallejo, Calif.) was extracted and 50 ~1 of incubation mixtures were prepared following the procedure of Marcu and Dudock (1974). Maximum incorporation using 5 $Zi of [35S]methionine (>500 Ci/ mmol) was obtained with an RNA concentration of 100 pg/rnl after 60 min of incubation at 30” at 1.5 mM mg acetate and 76 mM KCl. Typical incorporations in 5 ~1 of incubation mixtures were: no RNA, 5000 cpm; EMV-RNA at 100 pg/ml, 80,000 cpm.

Reticulocyte Zysates and protein synthesis. Lysates were prepared from rabbits which had been made anemic by acetylphenylhydrazine injection as described by Hunt and Jackson (1974). Exogenous

305 0042~6822/78/0912-0305$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reuroduction in anv form reserved.

306

RICARD, RENAUDIN,

mRNA was assayed in 50 ~1 of incubation mixtures according to Darnbrough et al. (1973) except that the potassium ion concentration was brought to 160 mM with potassium acetate and beef liver tRNA was added to 60 pg/ml. Spermine at 30 pg/ml and about 5 PCi of [35S]methionine (>500 Ci/mmol) were also present. Acrylamide-sodium dodecyl sulfate (SDS) gel electrophoresis. Five microliters of lysate assays (about 200,000 cpm) or 50 $ of wheat germ assays (50,000-800,000 cpm) were analyzed on 15% acrylamide SDS slab gels of the type described by Studier (1973) using the gel system of Laemnili (1970). After electrophoresis, the gels were dried down onto Whatman 3MM paper. Radioactive proteins were located by autoradiography or fluorography (Bonner and Laskey, 1974) on Kodak Kodirex film. Limited proteolysis of presumptive and authentic EMV coat protein. Coat protein was prepared from EMV virions by dialysis at 4’ for 12 hr against 1 M CaCh, pH 6.7, followed by extensive dialysis against deionized water. Intact particles were then removed by centrifugation at 105,OOOgfor 90 min. Limited proteolysis of coat protein was carried out essentially as described by Cleveland et al. (1977). Coat protein in 125 mi$f Tris-HCl, pH 6.8, 0.5% SDS and 10% glycerol was heated at 100” for 2 min, then incubated with a-chymotrypsin (Sigma) for 30 min at 37”. Digestion was stopped by bringing the reaction mixtures to 10% 2mercaptoethanol and 2% SDS, then boiling for 2 min. The digestion products were separated on 15% acrylamide SDS gels. Proteins co-migrating with EMV coat protein on 15% acrylamide SDS gels were located by autoradiography after staining, destaining and drying of the gel slab. Protein bands were cut from the gel, swollen in a solution containing 125 m&f Tris-HCl, pH 6.8,0.1% SDS, and 1 m&f EDTA, then digested without prior elution by placing the swollen gel slice in the sample wells of a second 15% acrylamide SDS gel and then overlaying each slice with cu-chymotrypsin. Digestion proceeded directly in the stacking gel during the subsequent electrophoresis.

AND BOW? RESULTS

Virion EMV-RNA directed surprisingly little synthesis of coat protein in wheat germ extracts in contrast to virion TYMVRNA (Benicourt and Haenni, 1976; Ricard et al., 1977). Instead, the majority of the products were larger than coat protein and also very heterogeneous. This could mean that coat protein was synthesized as part of a larger precursor molecule; or, since a small amount of coat protein seemed to be synthesized, this might indicate the presence in virion RNA of very small amounts of coat protein mRNA. Yet coat protein was expected to be the major viral protein synthesized in EMVinfected Datura leaves. This was borne out by studies of coat protein synthesis programmed with RNA extracted from Daturu leaves 3, 5, 6, 7, and 8 days after infection with EMV. RNA extracted from Datura leaves late after infection directed the synthesis of numerous protein products. Among them were large amounts of protein co-migrating with coat protein (Fig. 1, tracks 5 and 7: viral-specific protein, VSP 20). A protein present in Datura inoculated with water (Fig. 1, tracks 3 and 6: host protein, HP 19) migrated slightly faster than VSP 20. HP 19 was, of course, also present in infected Datura and can be seen in tracks 1, 2, and 4. In tracks 5 and 7, HP 19 does not appear to be present; however, VSP 20 was sypthesized in such large amounts that it could be argued that HP 19 was present but is masked by VSP 20. Since conditions for protein synthesis in wheat germ extracts were optimized for the translation of EMV RNA, it seemed more likely that viral RNA was preferentially used as messenger. This idea was reinforced by the fact that the other proteins synthesized with RNA from healthy Datura do not have their counterpart in tracks 5 and 7. Furthermore, although RNA was present at 100 pg/ml in all cases, incorporation was 3-fold greater when RNA was extracted 7 or 8 days after infection, corresponding to the presence of viral RNA in larger proportions. Certain preparations of RNA extracted from healthy Datura were even less active than others (compare track

EGGPLANT

MOSAIC

VIRUS

307

mRNA

i

,VSP :20K -HP19

1

2

+3

+5

3 -6

4

5

+6

l 7

6

7

-a

+a

20

FIG. 1. Translation in wheat germ extracts of RNA extracted from EMV-infected Datura leaves. Tracks 1, 2,4,5, and 7 are analyses of the radioactive proteins synthesized in wheat germ extracts incubated with 100 ag/ ml of RNA extracted from Datura leaves 3, 5,6, 7 and 8 days after inoculation (+) with EMV. Tracks 3 and 6 are analyses of the proteins directed by RNA extracted from Datum leaves 6 and 8 days after mock inoculation (-). The position and molecular weights of markers (66K, bovine serum albumin (BSA); 46K, ovalbumin; 20K, EMV coat protein) and of VSP 20 and HP 19 are indicated.

3 to 6). This might reflect the fact that cellular mRNAs were especially sensitive to RNAses. VSP 20 can be perceived slightly above HP 19 among the proteins synthesized with RNA extracted as early as 5 days after infection (fig. 1, track 2). This is also the earliest date at which coat protein could be detected by its reaction with specific antiserum in the cell sap of infected plants. VSP 20 is present in larger amounts than HP 19 when the RNA used is extracted 6 days after infection (Fig. 1, track 4) and completely obscures HP 19 when mRNA is extracted 7 or more days after infection (Fig. 1, tracks 5 and 7). To directly characterize VSP 20 as EMV coat protein, peptide mapping was carried out by partial protease digestion directly within the gel slice during a second electrophoresis as described by Cleveland et al., 1977. Figure 2B shows that a-chymotrypsin partial digestion of unlabeled authentic EMV coat protein and labeled VSP 20 gives qualitatively identical

peptide profiles. We conclude that VSP 20 is EMV coat protein. Except for the presence of coat protein, the electrophoretic profiles of the proteins directed by EMV virion RNA and RNA extracted from Daturu plants after 7 and 8 days of infection were comparable. These profiles were very different from those of RNA extracted from healthy Daturu plants (tracks 3 and 6) and Duturu plants up to 6 days after infection (tracks 1, 2, and 4). Viral-specific RNA from EMV-infected Duturu appears to differ from virion RNA only in its ability to program efficient coat protein synthesis. The simplest way to explain this is to hypothesize larger amounts of coat protein mRNA in the former. L-RNA is present in EMV virions. In the case of TYMV, L-RNA present in virions is the mRNA for coat protein (Klein et al., 1976; Pleij et al., 1976; Ricard et al., 1977). TYMV L-RNA can be detected directly by formamide polyacrylamide gel electrophoresis (PAGE) of as little as 5 pg

308

RICARD,

0 .Ol .03 w

.3

1

RENAUDIN,

3

AND

BOVE

10

abchymotrypsin

-2OK

1

2

FIG. 2. Limited proteolysis of authentic (A) and presumptive (B) coat protein. (A) Ten micrograms of coat protein was incubated with 0, 0.01, 0.03, 0.3, 1, 3, and 10 pg of cY-chymotrypsin as described in Materials and Methods. The digestion products were analyzed by SDS-PAGE followed by staining with Coomassie blue. Note that the fastest migrating bands represent the enzyme. (B) The protein co-migrating with coat protein in Fig. 1, track 7, was cut out, placed in sample well (1) of a second gel, and overlaid with 1 ag of cu-chymotrypsin. Ten micrograms of unlabeled coat protein was incubated with 0.1 pg of o-chymotrypsin for 30 min at 37” and placed in sample well (2). Electrophoresis was carried out and the gel was stained then submitted to fluorography. Track 1 is the fluorograph of the labeled protein and track 2 is stained.

of TYMV virion RNA (Renaudin, 1978). LRNA could not be detected in EMV virion RNA even when the gel was heavily overloaded. Hence, if L-RNA is present in EMV virions, it must be present in extremely small amounts. We therefore sought to enrich for the hypothetical L-RNA. We heated EMV virion RNA for 5 min at 70” in sterile, distilled water to disrupt aggregates, then sedimented the preparation through 12 ml of 5-20s linear sucrose gradients made up in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0, at 40,000 rpm for 5 hr in a Beckman SW41 rotor. Eight l-ml fractions were collected from the top of the gradients and the RNA recovered by ethanol precipitation, Five such gradients containing a total of 3 mg of EMV virion RNA were thus processed in order to obtain 0.1-0.2 AZWunits of material from fraction

2, the fraction in which TYMV L-RNA is routinely found. The RNA in each fraction of the sucrose gradients was analyzed by PAGE. As expected, heavy virion RNA was found in fractions 7 and 8. RNA of intermediate molecular weight was present in fractions 4, 5 and 6. Three discrete RNAs of low molecular weight appeared in fractions 2 and 3. They had mobilities similar to that of TYMV L-RNA, enabling us to estimate their molecular weights at about 0.2-0.3 x 106. Transfer RNA was present in fraction 1. The RNAs recovered from the above sucrose gradients were analyzed for their messenger properties in wheat germ extracts. Analysis of the [35S]-methionine-labeled protein products by SDS-PAGE gave the results shown in the autoradiogram of Fig. 3. RNA from fractions 2 and 3 directed the

EGGPLANT

MOSAIC

synthesis of a polypeptide co-migrating with EMV coat protein. This polypeptide has been characterized by partial protease digestion. Figure 4 shows that its peptide profile (track 2) is identical to that of authentic EMV coat protein (track 4) and of VSP 20 synthesized with RNA from EMVinfected Datura (track 3) and different from that of TYMV coat protein (track 1). We conclude that the protein directed by EMV L-RNA is EMV coat protein. Virion EMV-RNA directs the synthesis of two high molecular weight proteins. Coat protein was also synthesized when LRNA was used as messenger in reticulocyte lysates (results not shown). However, with virion RNA as messenger, different results were obtained in reticulocyte lysates and in wheat germ extracts. Figure 5 shows that only 2 proteins of high molecular weight (Rl and R2) were synthesized when virion EMV-RNA was translated in reticulocyte lysates. The highest molecular weight protein could also be detected in wheat germ extracts but usually in very small amounts, most of the translation products being smaller in size.

VIRUS

309

mRNA DISCUSSION

Characterization of protein by partial small protease digestion. Relatively amounts of label, even when high specific activity [J”S]methionine was used, were incorporated into the presumptive EMV coat protein, making it difficult to obtain meaningful labeled immunoprecipitates or tryptic fingerprints. A very sensitive microtechnique was necessary for characterization. Cleveland et al. (1977) have described peptide mapping by limited proteolysis in the presence of SDS and analysis by gel electrophoresis. The technique was particularly useful because digestion of the protein within the gel slice occurred during electrophoresis, eliminating the need for elution and possible loss or modification of the protein. Several proteases were tested and cu-chymotrypsin was chosen because it hydrolyzed EMV coat protein into first two, then the same two plus a third partial digestion product when the protease to coat protein ratio was increased lOO-fold from 0.001 to 0.1 (Fig. 2A). According to Cleveland et al. (1977), the same partial digestion products

0

,66K

2

3

4

5

676

0

FIG. 3. Translation of EMV-RNA fractionated on sucrose gradients. Virion EMV-RNA (100 pg/ml) (0) as well as 106 gg/ml of RNA size-fractionated on sucrose gradients as described in the text into low molecular weight RNA (fractions 2 and 3) and larger molecular weight RNA (fractions 4-8) were used to direct protein synthesis in wheat germ extracts. Radioactive products were analyzed by SDS-PAGE followed by autoradiography. The position and molecular weights of marker proteins are indicated.

310

RICARD,

RENAUDIN,

could be obtained by digestion directly within an SDS-gel, provided that staining was shortened to 30 min and destaining to less than 1 hr. We discovered that normal staining (3 hr) and destaining (16 hr) could be used if the protease to coat protein ratio was increased lo-fold. For these reasons, 1 pg of cu-chymotrypsin was used for digestion directly within the gel slice although only 0.1 pg of a-chymotrypsin was needed for digestion of 10 ag of control EMV coat protein into two or three peptides (Fig. 2A). By combining these slight modifications of the Cleveland technique with fluorography of labeled proteins, we were able to characterize EMV coat protein synthesized in

AND

BOVE

RlR2-

vitro. Comparison of EMV RNA and TYMV RNA. The translation of EMV L-RNA gave results similar to those obtained using TYMV L-RNA. Both directed the synthesis of coat protein. However, L-RNA was present in much smaller amounts in EMV

-0

O-

L $

20K

1

2

3

4

FIG. 4. Limited proteolysis of presumptive EMV coat protein. The protein co-migrating with coat protein and whose synthesis was directed by RNA from fraction 2 (Fig. 3) was cut out, placed in sample well 2 of a second gel and overlaid with 1 ag of cy-chymotrypsin. Radioactive TYMV coat protein was also cut from a gel, placed in sample well 1 and overlaid with 1 ag of a-chymotrypsin. Authentic EMV coat protein was incubated with 0.1 pg of a-chymotrypsin as described in Fig. 2 and placed in sample well 4. VSP 20, whose synthesis was directed by RNA extracted from EMVinfected Datura leaves, was cut from a gel, overlaid with 1 pg of a-chymotrypsin and placed in sample well 3. Electrophoresis was carried out. Track 4 was stained and tracks 1-3 were submitted to fluorography.

1

I

Lc)

FIG. 5. Translation of virion EMV-RNA in rabbit reticulocyte lysates. Rabbit reticulocyte lysates prepared as described in Materials and Methods were incubated in the presence (1) and absence (2) of 1 ag/ ml of virion EMV-RNA. In vitro products were analyzed by SDS-PAGE followed by autoradiography. The position and molecular weights of marker proteins (p/I’, E. coli RNA polymerase, 150K, 160K; BSA, 66K; creatine kinase, 40K; EMV coat protein, 20K) are indicated.

virions. This may be due to diminished encapsidation. Or, the difference in quantity of L-RNA found in TYMV and EMV virions may reflect different amounts of LRNA in vivo. It thus seems likely that all tymoviruses have a monocistronic coat protein mRNA but that it may be encapsidated in variable amounts. Full-length viral RNA can act as a monocistronic mRNA in vitro, directing the synthesis of a protein corresponding to most of its coding potential. A second, slightly smaller protein is also an important translation product. In the case of EMV, the two high molecular weight proteins of molecular weight 170,000 and 140,000, are slightly smaller than the corresponding TYMV-specific proteins, of molecular weight 195,000 and 150,090 (Benicourt et al., 1978; Ricard, unpublished results). Ben-

EGGPLANT

MOSAIC

icourt et al. (1978) report that the two TYMV-specific proteins have common amino acid sequences. Pelham (1978) has shown that not only do the two high molecular weight proteins programmed by TMVRNA in rabbit reticulocyte lysates share common amino acid sequences, but they have the same initiation site. The larger protein is probably the result of readthrough of the termination codon of the smaller protein. In the case of TMV, the large polypeptides appear to be synthesized in Go. We are presently comparing in viva and in vitro products to determine whether such is also the case for TYMV and EMV.

Note added in proof. U. Szybiak, J. P. and C. Fritsch (1978) in Nucleic Acids Res. 5, 1821-1832, find evidence for

Bouley,

the existence of a coat protein messenger RNA associated with the top component of eggplant mosaic virus and two other ty moviruses. REFERENCES B~NICOURT, C., and HAENNI, A. L. (1976). In vitro synthesis of turnip yellow mosaic virus coat protein in a wheat germ cell-free system. J. Viral. 20, 196-202. B~NICOURT, C., P~RI$ J. P., and HAENNI, A. L. (1978). Translation of TYMV RNA into high molecular weight proteins. FEBS Lett. 86, 268-272. BONNER, W. M., and LASKEY, R. A. (1974). A fiim detection method for tritium-lahelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Bio-

them. 46,83-W BRUENING, G., BEACHY, R. N., SCALLA, R., and ZAITLIN, M. (1976). In vitro and in viuo translation of the ribonucleic acids of a cowpea strain of tobacco mosaic virus. Virology 71,498-517. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106. DARNBROUGH, C., LEGON, S., HUNT, T., and JACKSON, R. J. (1973). Initiation of protein synthesis; evidence for messenger RNA-independent binding of methionyl-transfer RNA to the 40 S ribosomal subunit. J.

Mol. Biol. 76, 379-403. GIBBS, A. J., HECHT-POINAR, E., and WOODS, R. D. (1966). Some properties of three related viruses; andean potato latent, Dulcamara mottle, and Ononis yellow mosaic. J. Gen. Microbial. 44, 177-193. HIGGINS, T. J. V., GOODWIN, P. B., and WHITFELD, P.

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R. (1976). Occurrence of short particles in beans infected with the cowpea strain of TMV. II. Evidence that short particles contain the cistron for coat protein. Virology 71,486-497. HUNT, T., and JACKSON, R. J. (1974). The rabbit reticulocyte lysate as a system for studying mRNA. In “Modern Trends in Human Leukemia” (R. Neth et al., eds.), pp. 300-307, J. F. Lehmanns, Verlag, Munich. HUNTER, T. R., HUNT, T., KNOWLAND, J., and ZIMMERN, D. (1976). Messenger RNA for the coat protein of tobacco mosaic virus. Nature 260, 759-764. KLEIN, C., FRITSCH, C., BRIAND, J. P., RICHARDS, K. E., JONARD, G., and HIRTH, L. (1976). Physical and functional heterogeneity in TYMV RNA; evidence for the existence of an independent messenger coding for coat protein. Nucleic Acids Res. 3, 3043-3061. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. MAMOUN, R. Z. (1976). Replication du virus de la mosaique jaune du navet: caracterisation et Bvolution des RNA cellulaires et viraux dans les cellules de Brassica sinensis L. infectees. These de 3eme cycle, Universite de Bordeaux II. MARCU, K., and DUDOCK, B. (1974). Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ. Nucleic Acids Res. 1, 1385-1397. MOUCHES, C., BovB, C., and Bovf, J. M. (1974). Turnip yellow mosaic virus RNA replicase; partial purification of the enzyme from the solubilized enzyme-template complex. Virology 58, 409-423. PELHAM, H. R. B. (1978). Leaky UAG termination codon in tobacco mosaic virus RNA. Nature 272, 469-471. PLEIJ, C. W. A., NEELEMAN, A., VAN VLOTEN-DOTING, L., and BOSCH, L. (1976). Translation of turnip yellow mosaic virus RNA in vitro; a closed and an open coat protein cistron. Proc. Nat. Acad. Sci.

USA 73,4437-4441. RENAUDIN, H. (1978). Replication des tymovirus; contribution a l’etude des proprietes messageres du RNA du Virus de la Mosaique Jaune du Navet et du RNA du Virus de la mosai’que de l’Aubergine. These de 3eme cycle, Universite de Bordeaux II. RICARD, B., BARREAU, C., RENAUDIN, H., MOUCHES, C., and Bovi, J. M. (1977). Messenger properties of TYMV-RNA. Virology 79,231-235. SIEGEL, A., HARI, V., MONTGOMERY, I., and KOLACZ, K. (1976). A messenger RNA for capsid protein isolated from tobacco mosaic virus-infected tissue.

Virology 73.363-371. STUDIER, F. W. (1973). Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248. VAN DE WALLE, C. (1973). Polyadenylic sequences in plant RNA. FEBS Lett. 34,31-34.