Comparison of three different cell-free systems for turnip yellow mosaic virus RNA translation

BIOCHIMIE, 1983, 66, 127-133.

Comparison of three different cell-free systems for turnip yellow mosaic virus RNA translation. Wlodzimierz Z A G O R S K I ** Marie-Dominique M O R C H * and A n n e - L i s e H A E N N I o.

(Requ le 15-7-1982, accept£ aprks rdvision le 8-12-1982).

* Laboratoire de Biochimie du Ddveloppemem, Institut lacques Monod, C.N.R.S., Universitd Paris VII, 2, place lussieu, 75254 Paris Cddex 05. ** lnstytut Biochemii i Biofizyki, Polska Akademia Nauk, Ulica Rokowiecka 36, PL - 02532 Warszawa, Polska.

R~sum~.

Summary.

Les deux prot~ines, de poids moldculaires 150.000 et 195.000, sp~cifiques de la traduction du R N A du virus de la mosdique jaune du navet ( ~ Y M V : turnip yellow mosaic virus) dan~ le lysat de r~ticulocytes, viennent d'Otre d~cel~es dans deux autres syst~mes acellulaires programm~s avec le R N A du T Y M V : l'extrait de .germe de bl~ et le systkme acellulaire d'ascites d'Ehrlich. L'extrait de germe de blO contient des prot{,ases qui affectent les cha~nes polypeptidiques nais~antes de T Y M V . La maturation post-traductionnelle sp£cifique de la prot£ine de poids moldculaire 195.000, dont on sait qu'elle se produit dans le lysat de rdticulocytes, a ~t~ ~tudi~e dans le systkme d'ascites. Un lragment N-terminal et un [ragrnent C-terminal, dont les poids moldculaires sont respectivement 120.000 et 78.000, ont pu Otre d#ceI#s ; ils co-migrent avec les produits de clivage post-traductionnels observes dans le lysat de rdticulocytes. De m~me un fragment marqu~ en C-terminal, d'un poids mol~culaire de 78.000 a pu dtre observJ dans le systbme de germe de bid. Les differences entre ces trois systbmes in vitro, en ce qui concerne la traduction du R N A de T Y M V , ~ont discut£es.

The two proteins o] molecular weights 150,000 and 195,000 specific o] turnip yellow mosaic virus ( T Y M V ) R N A translation in reticulocyte lysates have now also been detected in two other cell-flee systems programmed with T Y M V R N A : the wheat germ extract and the Ehrlich ascites cell-free system. The wheat germ extract contains proteases that affect the nascent T Y M V polypeptide chains. The specific post-translational maturation o[ the protein o[ molecular weight 195.000 known to occur in the reticulocyte lysate has been investigated in the ascites system. A n N-terminal ]ragment and a C-terminal fragment o] molecular weights 120,000 and 78,000, respectively, could be detected co-migrating with the post-translational cleavage products observed in the reticulocyte lysate. Similarly, a C-terminally labelled 78,000 molecular weight fragment could be observed in the wheat germ system. The differences between the three in vitro systems with respect to T Y M V R N A translation are discussed.

Mots-cl~s : traduction in vitro / syst~mes acellulaires de r6ticulocytes de germes de bl~ et d'ascites / RNA du virus de la mosaique jaune du navet.

Key-words : in vitro translation / retieulocyte, wheat germ and ascites cell-free systems / turnip yellow mosaic virus RNA.

<>To whom all correspondence shouM be addressed. Abbreviations: TYMV, turnip yellow mosaic virus; TLCK, N~-tosyl-L-lysylchloromethane; HEPES, N-2-

hydroxyethylpiperazine-N-2-ethane sulJonate; DTT, dithiothreitol ; $105, supernatant from a 105 000 × g centrifugation ; K, kilodaltons.

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Introduction. The complete genetic information of turnip yellow mosaic virus (TYMV) is contained within a long infectious single-stranded ( + ) R N A (molecular weight 2 × 106) [1]. Besides this genomic R N A , a major subgenomic R N A species of molecular weight 240,000 is found in the viral particles [1, 2] ; this latter species corresponds to the 3' end of the genomic R N A and codes for the coat protein of the virus in various cell-free translation systems [1-3]. Conversely the full-length genomic R N A has so far been shown to code essentially for two overlapping high molecular weight proteins, 150,000 ( 1 5 0 K ) and 195~000 (195 K), only in the reticulocyte lysate [4, 5]. In this system the larger of the two proteins undergoes post-translational cleavage [6]. The N-terminal and C-terminal fragments resulting from this cleavage have been identified as polypeptides of molecular weights 120,000 (120 K) and 78,000 (78 K), respectively. Except for the coat protein, it has as yet not been possible to correlate these in vitro translat i o n products with the proteins observed in vivo in TYMV-infected leaves or plant protoplasts [7]. It was consequently important to verify whether t h e synthesis of high molecular weight proteins as well as the post-translational cleavage observed in the reticulocyte system only reflects particularities of this in vitro system, or whether it is a virus-specific phenomenon. T o this end, we examined the expression of T Y M V R N A in other in vitro systems than the reticulocyte lysate : the wheat germ extract and the Ehrlich ascites system. We demonstrate here that both systems can be improved in such a way that the 150 K and the 195 K proteins can be detected, and that in these systems the 195 K protein can undergo a proteolytic cleavage similar to the one observed in the reticulocyte lysate. This strongly suggests that these aspects of T Y M V R N A expression in vitro correspond to a virus-coded event. Nevertheless, differences were observed between the three systems in the efficiency of translation and cleavage, and the wheat germ extract was shown to contain proteases that specifically affect the T Y M V R N A translation products.

Materials and Methods. Materials.

z.-[e~S]methionine ( > 1 000 Ci/mmol) and L-[3~S] cysteine ( > 1 000 Ci/mmol) were purchased from New England Nuclear (U.K.). Amino acids, dithiothreitol BIOCHIMIE, 1983, 65, n ° 2.

(DTT), spermidine tetrachloride, GTP trisodium salt, creatine kinase, edeine and puromycin were from Sigma Inc. (U.S.A.). N-a-tosyl-t,-lysyl-chloromethane (TIZ~K), ATP dipotassium salt and creatine phosphate dipotassium salt were from Calbiochem-Behring Corp. (West Germany). Acrylamide and bisacrylamide were from Eastman-Kodak and al~ other chemicals from Merck. Fuji medical X-ray films were used for autoradiography. The scintillation fluid for radioactivity measurements was Econofluor from New England Nuclear. Human placental RNase inhibitor was a generous gift from P. Blackburn (Rockefeller University, New York). TYMV-infeeted Chinese cabbage leaves were kindly provided by S. AstierManifacier and P. Cornuet (C.N.R.A., Versailles), and beef liver and wheat germ tRNA by M. Fournier (Institut de Biologic Cellulaire, Bordeaux) and C. B6nicourt, respectively. Crude reticuloeyte initiation factors were a kind gift from H. Trachsel (Biozentrum, Basel) to A. Person (I.J.M., Paris). Wheat germ was a gift of the Grands Moulins de Paris. Sterile conditions were used whenever possible. Virus purification and RNA extraction.

TYMV was isolated from Chinese cabbage leaves, as outlined previously [~] and its RNA extracted from the viral particles, according to Porter et al. [8]. Reticulocyte lysate : preparation and incubation.

The mRNA-dependent reticulocyte lysate was prepared essentially as described by Pelham and Jackson [9] and modified as previously indicated [4]. The standard translation mixtures (20 ~1) contained 10 ~1 of reticulocyte lysate, 1 mM MgCI~, 100 mM KCI, 20 ~M each amino acid except methionine, 10 p.Ci of [35S] methionine, 1 ~g of beef liver tRNA and 1 p~g of viral RNA. Incorporation was about 40 pmoles of [anSl methionine per I~g of TYMV RNA after 90 rain at 30°C. Where stated, N-terminal labelling was pevfx>rmedusing the pulse-chase method [10], and C-terminal labelling as originally described [61. Wheat germ extract : preparation and incubation.

The wheat germ extract was prepared essentially as described previously [11] and stored in liquid nitrogen. Translation assays were performed in 25 ~1. They contained 8 p.l of wheat germ extract, 20 mM HEPES-KOH pH 7.5, 5 mM Tris-acetate pH 7.5, 1.5 mM magnesium acetate, 125 mM potassium acetate, 600 ~M spermidine, 0.5 mM DTT, 0.5 mM ATP, 0.375 mM GTP, 20 mM creatine phosphate, 1.25 ~g of creatine kinase, 50 ng of placental RNase inhibitor, 250 p.M each of unlabelled amino acids except methionine or cysteine which were only 8 ~M, 10 p.Ci of [35Sl methionine or [a~S] cysteine, 3 ~g of wheat germ tRNA and 4 Ixg of TYMV RNA. Incubations were performed at 25°C for 2.5 h. Incorporation was normally 10 pmol of [a~S] methionine or 4 pmol of [e~S] cysteine per Ixg of TYMV RNA after 2.5 h under optimized conditions.

Turnip yellow mosaic virus R N A Incubations in the Ehrlich ascites cell-free system. The ascites cell-free system rendered mRNA-dependent by treatment with micrococcal nuclease, according to Pelham and Jackson [9], was prepared and generously provided by A. Person. The incubations (20 ~1) contained 5 Ixl of nuclease-treated ascites lysate, 2 mM magnesium acetate, 160 mM, potassium acetate, 1 mM DTT, 1 mM ATP, 0.5 mM GTP, 40 ~M each of the unlabelled amino acids except methionine, 20 ~tCi of [zsS] methionine, 2.4 fxg of beef liver tRNA and 1 ~g of TYMV RNA. Crude initiation factors from the reticulocyte lysate [12] were added at a final concentration of 0.3 mg/ml. Incubatkon's were performed at 30°C for 2 h. Where indicated TLCK •(final concentration, 4 raM) was added after 60 min incubation. Incorporation was about 5 pmoles of [35S] methionine per ~g of TYMV RNA after 2 h of incubation. In C-terminal labelling experiments, unlabelled methionine was omitted at the beginning of the incubation, edeine (20 ~M) was added after 10 min, and 20 ~Ci of [zsS] methionine after 25 min ; incubation was pursued for another 90 min.

translation in vitro.

129

of the wheat germ extract that allows the synthesis of full-length b r o m e mosaic virus a n d tobacco mosaic virus R N A - c o d e d proteins [11] was adopted here. This system was s u p p l e m e n t e d with

A

ab

B

¸

ab

195K 150K 120K

,4nalysis of the translation products. The radioactivity incorporated into the hot trichloroacetic acid precipitable material was estimated on 1 ~tl aliquots, as previously described [3]. At the times indicated, 5 +xl aliquots were removed from the incubations and analyzed by polyacrylamide-sodium dodecylsulfate slab gel electroph.oresis, as reported elsewhere [10].

J~

, jl

•

+! •

H~;/: + ~ ~i~ ,/

.. ~

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Preparation of wheat germ and reticulocyte supernatants. Aliquots (200 txl) of wheat germ or reticulocyte lysate were centrifuged in a Beekrnarm Airfuge at 105 000 × g (22 psi) for 13 rain. The upper 2/3 of the supernatant were removed and are referred to as wheat germ S105 and reticulocyte S105, respectively.

Results. Full-length translation of T Y M V in a wheat germ extract.

RNA

E l o n g a t i o n of polypeptide chains is reportedly less efficient in the wheat germ t r a n s l a t i o n system t h a n in the reticulocyte lysate : irt the former system, prepared by c o n v e n t i o n a l methods, unfinished polypeptides p r e d o m i n a t e [14, 15]. This feature of the wheat germ system was particularly deleterious for the synthesis of the long 195 K protein, the final p r o d u c t of c o n t i n u o u s translation of T Y M V R N A in the reticulocyte lysate. Indeed, former experiments of T Y M V R N A translation in the wheat germ system had revealed the viral coat protein (directed by the s u b g e n o m i c R N A species) as being the m a j o r p r o d u c t synthesized, together with several other polypeptides with m o l e c u l a r weights n o t higher t h a n 165:000 [1-3]. A modified procedure for the p r e p a r a t i o n BIOCHIM1E, 1983, 65, n ° 2.

FIG. 1. - - T Y M V RNA translation products in the wheat germ extract (Panel A) and assay [or proteases (Panel B). Panel .4. The TYMV RNA-coded polypeptides were labelled with ['~S] methionine throughout the incubation in the reticulocyte lysate as a control (lane a) and in the wheat germ system under optimal conditions (lane b). The positions ef the 195K, 150K and 20K (coat) proteins, the major translation products of TYMV RNA in the reticulocyte lysate, are indicated on the left. The four wheat germ-specific polypeptides referred to as W1, W2, W3 and W4 are identified on the right of the panel. Panel B. TYMV RNA was translated in the reticulocyte lysate using N-terminal labelling conditions. After 30 min of incubation, puromycin (200 ~M) was added and 10 ~I of the translation mixture were mixed with 5 ~1 of wheat germ S105 (lane a) or water as a control (lane b). Incubation was pursued for 90 min and the samples analyzed. The position of the 120K protein previously characterized during TYMV RNA translation in the reticulocyte lysate [10] is indicated to the right of the panel.

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600 [zM spermidine [16] and with purified placental RNase inhibitor [17]. Under these conditions, the activity of the wheat germ system, as measured by total incorporation of radioactivity into proteins (10 pmoles of [3~S] methionine/lxg of TYMV RNA), was comparable to if not hioher than that reported by other authors [18-20]. Moreover, the 150 K and 195 K proteins specific of TYMV RNA translation in the reticulocyte lysate (fig. 1 A, lane a) could also be identified in the wheat germ system programmed with TYMV RNA (fig. 1 A, lane b). In the latter system however, these translation products and especially the 195 K protein were still only poorly synthesized, at least when compared to three other predominant proteins : W1, W2 and W3 (molecular weights about 102,000, 44,000 and 39,000) that appeared not to be produced to any significant extent in the TYMV RNA-directed reticulocyte lysate. At least three possibilities can be considered to explain the appearance of these predominant short polypeptides in the wheat germ extract. 1) The elongation of polypeptide chains is arrested at discrete sites along the RNA because of a defect in the wheat germ translational machinery, such as putative signal on the RNA that would be overcome in the reticulocyte lysate but not in the wheat germ extract. 2) The RNA of TYMV is degraded in the wheat germ system and its fragmentation products dictate the synthesis of the three new polypeptides. 3) The continuous translation products of TYMV RNA are cleaved by (an) endogenous wheat germ protease(s). The first possibility was examined by supplementing the wheat germ system with reticulocyte S105, with wheat germ or beef liver tRNAs, or by heat-denaturing the viral RNA prior to translation (results not shown). None of these variants decreased the intensity of bands Wl, W2 and W3. Moreover the translation patterns obtained with tobacco mosaic virus or brome mosaic virus RNAs in the wheat germ system were not significantly different from those observed in the reticulocyte lysate (not shown), thus confirming that the plant extract did not lack in any detectable manner a factor essential for full-length translation of viral RNAs. To test the second possibility, TYMV RNA was pre-incubated with wheat germ S105 and then translated in the reticulocyte lysate. This led to a decrease in the efficiency of translation either BIOCHIMIE, 1983, 65, n° 2.

because of a factor present in wheat germ that inhibits elongation in mammalian systems [21] or because of the presence of nucleases [22]. However these wheat germ nucleases did not induce translation in the reticulocyte lysate of new well-defined polypeptides such as the W1, W2 or W3 proteins (results not shown). The third possibility was tested by adding wheat germ S105 to incubation mixtures performed in the reticulocyte lysate after completion of the 195 K protein; puromycin was added simultaneously to prevent further elongation. The results obtained by N-terminal labelling are presented in fig. 1 B. The presence of wheat germ S105 (fig. 1 B, lane a) considerably modified the pattern of the N-terminally labelled products usually observed in the reticulocyte system (fig. 1 B, lane b). Mainly four proteins appeared to the detriment of the 150 K and 120 K proteins. Three of these new bands can be correlated to the W1, W2 and W:~ proteins; the fourth band (W4) also corresponds to a well-defined polypeptide produced in the wheat germ system (fig. 1 A, lane b). Thus, the three major polypeptides (W1, W2, W3) obtained during TYMV RNA translation in the wheat germ system seem to correspond to Nterminal fragments of a series of wheat germinduced cleavages. Based on kinetic data (not shown), it seems that these cleavages occur during elongation, thereby limiting the number of polypeptide chains that can be elongated to yield the 195 K protein. To prevent cleava~e~ proteaseinhibitors such as N-ethyl-maleimide or TI,CK were added to the incubation in the wheat germ extract. However, since these inhibitors markedly reduced overall protein synthesis, no positive influence on the pattern of the TYMV RNA translation products could be detected (results not shown). Translation of T Y M V R N A and product fragmentation in a lysate of Ehrlich ascites cells. The Ehrlich ascites cell-free system was used as a third in vitro system to study TYMV RNA expression. Incubation conditions were optimized to allow the detection of the two TYMV RNAcoded high molecular weight proteins. This entailed an increase in potassium acetate concentration and addition of crude initiation factors from rabbit reticulocytes (see Materials and Methods). However, total incorporation remained fairly low when compared to that obtained with the reticulocyte lysate (5 versus 40 pmoles of ['~S] methionine/~g of TYMV RNA after 2 h incubation) and the synthesis of the 195 K protein was far

Turnip yellow mosaic virus R N A

translation in vitro.

13i

inferior to that of the 150 K protein. Addition of placental nuclease inhibitor improved neither total incorporation nor the ratio between the two high molecular weight proteins.

;

a b c d

e

f

c

Figure 2 A, lanes b-e, presents a kinetic experiment performed in the ascites ceil-free system programmed with T Y M V R N A . The final translation products can be compared to those obtained in the reticulocyte lysate (fig. 2 A, lane a) where the proteins were N-terminally labelled by the pulse-chase method and analyzed after 150 rain of incubation in the presence of puromycin. This led to complete cleavage of the 195 K protein, allowing easy visualization of the 120 K protein. In the ascites system the 150 K and 195 K proteins can be detected although the latter protein is produced in much smaller amounts than the former. In addition a protein accumulates after the completion of the 195 K protein that co-migrates with the N-terminal 120 K fragment observed in the reticulocyte system. When T L C K was added after 60 min of incubation, this 120 K polypeptide did not accumulate (fig. 2 A, lane f). Kinetic experiments using formyl-[35S]Met-tRNA to label the translation products in the ascites system enabled us to identify the 120 K polypeptide as the N-terminal fragment of the 195 K protein (not shown). Search for C-terminal fragment.

FIG. 2. ~ TYMV RNA translation in the Ehrlich ascites cell-lree system (Panel A) and search for C-terminal ]ragment (Panel B). Panel A : kinetic experiment. The translation products were labelled with [35Sl methionine throughout the incubation and 5 ~,1 aliquots were removed and analyzed after 30 (lane b), 60 (lane c), 120 (lane d) and 180 rain (lane e). In lane f, "I~LCK was added after 60 min and the incubation pursued for 120 min ; to compensate for the very low radioactivity incorporated, this part of the same gel was exposed twice as long. TLCK modifies the migration of the 20K (coat) protein in sodium dodecylsulfate-polyacrylamide gels (unpublished observati,3ns). TYMV RNA translation products have been run as markers (lane a) after 180 min of an incubation performed in the reticulocyte lysate, as described elsewhere [BI]: N-terminal labelling conditions by the pulse-chase method were used, puromycin was added after 30 rain and incubation pursued for 150 rain. Panel B : C-terminal ~abelling in the ascites cell-free system (lane a) and in the reticulocyte lysate (lane b). In the wheat germ system (lane c) [zsS] cysteine was used throughout the incubation as equiva.lent of C-terminal labelling. The position of the 78K C-terminal cleavage product of the 195K protein is indicated ; <~ENDO >> refers to the Nbelling of an endogenous protein of the mammalian systems. BIOCHIM1E, 1983, 65, n ° 2.

The search for the C-terminal fragmentation product in the ascites system was rendered difficult due to the high background of unfinished polypeptides under conditions of total labelling. However, C-terminal labelling experiments revealed a faint band in the ascites system (fig. 2 B, lane a) co-migrating with the 78 K cleavage product observed in the reticulocyte lysate (fig. 2 B, lane b). This fragment could not easily be further characterized because of the very low level of total radioactivity incorporated. A polypeptide co-migrating with the 78 K protein was also detected in the wheat germ system (fig. 2 B , lane c). As shown here, this band appeared very clearly when conditions were optimized for the synthesis of the 195 K protein and when [35S] cysteine was used to preferentially label the C-terminal region of T Y M V R N A coded proteins [6]. Thus, and as stated elsewhere [10], this 78 K protein most likely corresponds to the C-terminal cleavage product of the 195 K protein in the wheat germ system. These results suggest that specific cleavage of the 195 K protein can occur in the wheat germ

132

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system and in the ascites system at the same site as in the reticulocyte lysate although with lower efficiency.

Discussion. Translation of viral mRNA in in vitro systems has often contributed to elucidate the strategy of virus expression, such as competition between RNAs of multipartite genomes [5, 23] and readthrough phenomena [24], and it has permitted synchronous kinetic studies of post-translational cleavages of viral proteins [25-27]. To confirm that such results truly reflect steps in the lifecycle of the virus, the in vitro observations must be correlated with the in vivo data and/or they must derive from various in vitro systems. In the case of TYMV RNA translation, the results presented here show that in three in vitro systems, namely the rabbit reticulocyte lysate, the wheat germ extract and the Ehrlich ascites system, the same TYMV RNA-coded high molecular weight proteins (150 K and 195 K) are synthesized and the same specific maturation of the 195 K protein seems to occur, suggesting that virus-coded mechanisms are involved in both phenomena. However this study has revealed differences between these three systems with regard to the efficiency of translation of TYMV RNA and the resulting polypeptide patterns. Such a divergence between exogenous mRNA translation in various in vitro systems has already been reported in the case of the RNA of southern bean mosaic virus [18], turnip crinkle virus [19] and encephalomyocarditis virus [28]. In most cases however, and as shown here, the improvements of incubation conditions allow the synthesis of identical full-length translation products in the different systems. In our case, addition of nuclease inhibitor was beneficial in the wheat germ system, whereas it had no effect in the two mammalian systems, confirming previous reports that these latter two systems can contain an endogenous nuclease inhibitor [17]. Our results indicate that the wheat germ extract also contains proteases that induce, during TYMV RNA translation, very specific cleavages yielding a well-defined number of stable polypeptides (Wl, W2 and W3). A recent report [29] states the existence of several proteases in the wheat germ extract. Among these a prominent trypsinlike activity, absent from the reticulocyte lysate, was detected in this system. This observation can BIOCHIMIE, 1983, 65, n* 2.

be correlated with the cleavage of nascent polypeptides occurring in the former system and not in the latter. At the translational level, both the 150 K and the 195 K proteins were synthesized in the three systems, although in varying ratios. These results strongly support the previously hypothesis [30] that the arrest of translation at the end of the 150 K protein gene is not due to particularities of the translation system but that it corresponds to a signal encoded in the TYMV genome itself. This (~ stop ~> signal is recognized and overcome by in vitro systems as different as mammalian and plant cell-free systems. Experiments are in progress to define the mechanism involved in this process. We have searched for the cleavage products of the 195 K protein in the wheat germ extract and the ascites cell-free system. In the former system, the 78 K polypeptide observed in figure 2B, lane c, has been identified by other results such as kinetic experiments [10 and results not shown] as the C-terminal cleavage product of the 195 K protein; the corresponding N-terminal fragment of 120 K could not be identified, because of the wheat germ-induced cleavages that yielded a very high background of N-terminally labelled proteins and that were shown here to act on this 120 K polypeptide. In the ascites cell-free system, an Nterminal fragment of 120 K presumably deriving from the 195 K protein was identified; TLCK prevented the accumulation of this fragmentation product. A C-terminal fragment of 78 K was also detected. Thus in both systems, the 195 K protein seems to undergo a specific although not very efficient proteolytic maturation that involves the same cleavage site as in the reticulocyte lysate. Since the addition of reticulocyte lysate or S105 supernatant did not enhance the cleavage of the 195 K protein in the wheat germ extract (not shown), some particularities of this latter system must prevent efficient cleavage of the 195 K protein. Analogous results have been reported when the RNA of cowpea mosaic virus B component or the encephalomyocarditis virus RNA were translated in the wheat germ system [20, 27] ; as discussed by Shih et al. [27], inefficient maturation of the polyprotein could be caused by the ionic conditions used, specific protease inhibitors or an undefined deficiency inherent to the wheat germ extract. Because of their intrinsic properties and deficiencies (lack of compartmentalization, lack of turnover of the components, thermal instability, etc.), the cell-free translation systems cannot ac-

Turnip yellow mosaic virus R N A translation in vitro.

count for all the successive steps of gene expression of ( + ) RNA viruses. However, when in vivo studies are lacking, a comparison of viral expression in different cell-free systems can help in supporting the validity of in vitro observations. Yet, among the three in vitro systems studied here, the reticulocyte lysate appears to be the most adequate for TYMV RNA translation with regard to total incorporation of amino acids, elongation rate and completion of polypeptide chains. Acknowledgements. We thank F. Chapeville /or his encouragement and interest. This work w ~ per]ormed whilst W. Z. held the position o] Pro]esseur Associ~, Universitd Paris VII ; it was financed partly by the grant A T P Phytopathologie (n ° 3608) form the C N R S and I N R A , and partly by the Ecole Pratique des Hautes Etudes. Thanks are due to A. Person ]or kindly provMing the ascites system and advice concerning the incubation conditions. We are also grate/al to P. Blackburn /or the gift of placental RNase inhibitor. It is a pleasure and an honor to dedicate this article to Professor Edgar Lederer. REFER, ENCES. 1. Pleij, C. W. A., Neeleman, L., Van Vloten-Doting, L. e, Bosch, L. (1976) Proc. Natl. Acad. Sci. U.S.A., 73, 4437-4441. 2. Klein, C., Fritsch, C., Briand, J. P., Richards, K. E., Jonard, G. ~ Hirth, L. (1976) NucL Acids Res., 3, 3043-3061. 3. B6nicourt, C. ,~ Haenni, A. L. (1976) J. Virol., 20, 196-202. 4. B6nicourt, C., P6r6, 1. P. ,~ Haenni, A. L. (1978) FEBS Lett., 86, 268-272. 5. B6nicourt, C. a Haenni, A. L. (1978) Biochem. Biophys. Res. Commun., 84, 831-839. 6. M~rch, M. D. ~ B6nicourt, C. (1980) J. Virol., 34, 85-94. 7. Sugimura, Y., Given, N. K. a Matthews, R. E. F. (1981) Fifth International Congress of Virology, Strasbourg, W25/01.

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