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381 J. gen. ViroL (I98O), 46, 381-389 Printed in Great Britain Translation Products of Genome and Satellite R N A s of Tomato Black Ring Virus By C H R I S T I A N E FRITSCH, M.A. MAYO 1 AND A. F. M U R A N T 1 lnstitut de Biologie Mol(culaire et Cellulaire, 15 rue Descartes, 670o0 Strasbourg, France and 1Scottish Horticultural Research Institute, Invergowrie, Dundee, Scotland (Accepted 6 September 1979) SUMMARY In wheat germ extracts and reticulocyte lysates the genome R N A molecules of tomato black ring virus (TBRV), R N A - I (tool. wt. 2.8 × lO6) and RNA-2 (mol. wt. 1.6 × IO6), were translated into products of maximum mol. wt. 2.2 × lO5 and 1.6 × lO5, respectively. These products represent about 8o % and IOO% of the coding capacity of the two R N A species. The 1.6× Io 5 mol. wt. polypeptide reacted with antiserum to TBRV particles but the translation products of R N A - I did not; this is additional evidence that RNA-2 contains the coat protein cistron. Satellite R N A molecules (RNA-3, mol. wt. 4.8 × lO 5) associated with strain S of TBRV, like those associated with strain G, were translated in wheat germ extracts into a polypeptide o f m o l , wt. 4"8 × lO4; this did not react with TBRV antiserum. Protease digestion released peptides from the translation product of strain S RNA-3 which were different from those released from the translation product of strain G RNA-3, suggesting that the two kinds of satellite R N A molecules differ in base sequence. INTRODUCTION The genome of tomato black ring virus (TBRV), like that of other nepoviruses, comprises two R N A molecules (Murant et al. 1973 ; Randles et al. I977) ofmol, wt. 2.8 x lO ~ ( R N A - I ; A. F. Murant, M. Taylor & G. H. Duncan, unpublished data) and 1-6× lO6 (RNA-2; Murant & Taylor, 1978). In addition, some isolates contain a satellite R N A (RNA-3) of mol. wt. 4.8 x io s (Murant et al. 1973; Murant & Taylor, 1978). We showed previously (Fritsch et al. 1978) that satellite R N A is translated in vitro and in vivo into a polypeptide of mol. wt. 4"8 x lO4 and that unfractionated virus R N A directed the formation in vitro of numerous additional polypeptides among which the most prominent had a mol. wt. o f 1-6 × lO5 and the largest had a mol. wt. of 2.2 x 1o5. In this paper we show that the two large polypeptides are translated from RNA-2 and R N A - I respectively, and that satellite R N A molecules from two strains of TBRV are chemically different. METHODS Virus isolates. Three isolates of TBRV contained satellite R N A : TBRV-G, an isolate of the German (potato bouquet) serotype of TBRV (Harrison, I958); TBRV-E, an isolate obtained from Norfolk, England which was serologically close to T B R V - G ; and TBRV-S, the stock culture from Arctium lappa (Harrison, 1958) of the Scottish (beet ringspot) o022-1317/80/0000-3787 $02.00 © I980 S G M Downloaded from www.microbiologyresearch.org by 14 IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00 vIR 46 382 c. FRITSCH, M. A. M A Y O AND A. F. M U R A N T serotype of TBRV. An isolate lacking satellite RNA, TBRV-St2, was derived from TBRVS using the procedure described by Murant et al. (I973)- Tobacco rattle virus (TRV), P R N strain (Cadman & Harrison, 1959), was used in one experiment. Purification. TBRV was extracted from systemically infected leaves of Nicotiana clevelandii by the method used previously (Fritsch et al. I978). TRV was purified from systemically infected leaves of N. clevelandii by differential centrifugation of borax-clarified leaf extracts (Harrison & Woods, I966). R N A extraction. Virus suspensions (about I mg/ml) were shaken with phenol as described by Fritsch et al. (I978) and RNA was recovered from the aqueous phase by ethanol precipitation. Separation of R N A species. Nucleoprotein components of TBRV-SI2 were separated by centrifugation to equilibrium in caesium chloride followed by sedimentation in Io to 40% sucrose gradients (Harrison & Barker, I978). RNA-I and RNA-2 were extracted from B and M nucleoprotein components respectively. Translation in wheat germ extracts. Commercial wheat germ (General Mills Inc., Vallejo, Calif.) was extracted as described by Marcu & Dudock 0974). RNA (5 to t o # g ) was added to I o o # l reaction mixture containing 3o#1 wheat germ extract, I5 to 6o#Ci ~SS-methionine (3oo to 700 Ci/mmol, Radiochemical Centre, Amersham), 20 mM-HEPES (pH 7"8), 98 mM-potassium acetate, a'75 raM-magnesium acetate, o'65 mM-spermidine, 2"5 mM-ATP, 0"375 mM-GTP, 5 mM-phosphoenol pyruvate, 1.8 mM-dithiothreitol and each essential amino acid at o.o25 mM. The reaction mixtures were kept at 30 °C for 2 h. Translation in reticulocyte lysates. Rabbit reticulocyte lysates were prepared as described by Mohier et al. (I975). RNA (5 to IO #g) was added to 50 #1 reaction mixture containing 20#1 lysate, 75 mM-KC1, 0.8 mM-magnesium acetate, i mM-ATP, o.2 mM-GTP, I5]ZMhaemin, I5 mM-creatine phosphate, 2 units creatine phosphokinase, o-I mM-unlabelled amino acids and 0"5/zCi 35S-methionine. Incubation was for I h at 27 °C. In some experiments lysates were treated with micrococcal nuclease (Pelham & Jackson, I976) before adding radioactive amino acids and mRNA. Analysis of polypeptides in polyacrylamide-SDS gels. Reaction mixtures were centrifuged at IO5OOOg for I h and the supernatant fluids were treated with RNase (Mayo et al. I976). Solutions were then made 2 % SDS, 2 % 2-mercaptoethanol and Io % glycerol, and heated in boiling water for 90 s. Samples were stored at - 2o °C or analysed immediately on discontinuous slab gels of 7"5% acrylamide using buffers described by Laemmli (I97O). After electrophoresis the gels were stained, dried and autoradiographed using Kodirex film (Kodak Ltd.). The proteins used to estimate mol. wt. were RNA polymerase from Escherichia coli (Boehringer; mol. wt. I65ooo, 155oo0, 950oo and 395oo), bovine serum albumin (67ooo), pyruvate kinase (57ooo), ovalbumin (45000)and carbonic anhydrase (290o0). Calibration lines of log (mol. wt.) against mobility were slightly curved; lines fitted by eye thus gave estimates with an accuracy of about _+5 %. Serological precipitation. After translation of RNA in wheat germ extracts the reaction mixtures were centrifuged at IO5 ooo g for I h. Trichloroacetic acid was added to the supernatant fluid to give a final concentration of IO% (w/v) and the mixture was kept at 4 °C for ~ h. The precipitate was washed with ethanol and dissolved in o.I to o-a ml oq5 Msodium chloride + o.o~ M-sodium phosphate, pH 7"o (PBS) containing o.z % SDS. Samples containing similar amounts of radioactivity were incubated at room temperature with different amounts of TBRV-S antiserum (titre ~/2o48 in double diffusion tests in agarose) for a h and normal rabbit serum was then added to bring each sample to the same serum concentration. Rabbit antibodies were then precipitated by adding an equal volume o f goat anti-rabbit 3' globulin serum (a gift from Dr M. van Regenmortel) and incubating the mixture overnight at 4 °C. The precipitate was sedimented, washed twice with z'% Triton Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00 Translation of T B R V R N A 383 2 CP 3 .-...-~ iliii!:ii! ; ili!!i!i ii!!i:ii~!i i: ii !Jii~ (a) (b) (c) (d) (e) (f) Fig. L Autoradiograms showing electrophoretic separations in 7"5% acrylamide gels of35Slabelled polypeptides produced in wheat germ extracts; (a to c) are from the same gel and (d to f ) are adjacent tracks. Reaction mixtures contained: (a) R N A from TBRV-S; (b) R N A from TBRV-E; (c) R N A from TBRV-G ; (d) R N A from TBRV-S; (e) R N A - 1 from purified B component o f TBRV-SIz; ( f ) R N A - 2 from purified M component o f TBRV-SI 2. Arrows i, 2 and 3 indicate the largest products from R N A - I , RNA-2 and RNA-3, respectively. The bars indicate the tops of the gels and the position o f TBRV-S coat protein (CP). Xqoo in PBS, dissolved in 50/~1 r M-NaOH and neutralized with HCI. Radioactivity was determined using Bray's scintillator. Partial proteolysis of satellite RNA translation products. Regions of dried acrylamide gels corresponding to the 4800o tool. wt. band in autoradiograms were cut out and the protein was eluted with o-I ~o SDS, I mM-EDTA, o.i25 M-tris-HC1, pH 4"8, for 24 h (Benicourt et al. I978). Samples of ~o to ~5/tl containing about equal amounts of radioactivity were digested for 30 min at 3o °C with 0-2 to 80/~g/ml protease V8 from Staphylococcus aureus (Miles) or 0.o8 to 80/~g/ml subtilisin (Sigma; Cleveland et al. I977). The reactions were stopped by boiling the samples and the polypeptide composition of each was analysed by electrophoresis in I6 ~ acrylamide gels. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00 14-2 384 C. F R I T S C H , M. A. M A Y O (a) (b) (c) AND (d) (e) A. F. M U R A N T (f) Fig. 2. Autoradiograms showing electrophoretic separations in a 7"5 % acrylamide gel of asS-labelled polypeptides produced in: (a to c) untreated reticulocyte lysates; (d, e) reticulocyte lysates treated before use with micrococcal nuclease; ( f ) w h e a t germ extracts. (a) No added RNA; (b, d ) R N A - I from purified B component of TBRV-S I 2; (c, e) RNA-2 from purified M component of TBRV-S t 2; ( f ) unseparated TBRV-S R N A as in Fig, ~(a). Arrows and bars are as for Fig. I. RESULTS Translation in wheat germ extracts TBRV R N A at about Ioo/~g/ml (the optimum concentration) stimulated incorporation of 35S-methionine by wheat germ extracts about 5o- to I oo-fold. Analysis by polyacrylamide gel electrophoresis of the translation products of R N A from TBRV-S, TBRV-E and T B R V G is shown in Fig. I(a to c). With all three strains one of the prominent bands (band 3, arrowed) is the translation product of satellite R N A described previously (Fritsch et al. I978); the remainder result from translation of R N A - I and RNA-2. The largest has a tool. wt. of about 2-2 × I0 5 (band 0 , and a polypeptide of mol. wt. 1.6 x i0 5 (band 2) is prominent in the translation products of TBRV-S and T B R V - G and present in those of TBRV-E. Both band I and band 2 polypeptides of TBRV-S migrated slightly more slowly than those of TBRV-E and TBRV-G. To determine which R N A species gave rise to the different polypeptides in Fig. t (a), R N A was extracted from preparations of the B and M components of TBRV-SI2 to give purified R N A - I and RNA-2. Comparison of the translation products of mixed R N A from TBRV-S (Fig. I d) with those of R N A - I (Fig. I e) and RNA-2 (Fig. I f ) shows that bands I and 2 are translated from R N A - I and RNA-2 respectively. In addition the R N A - I translation products contained a prominent i-9 x ~0s tool. wt. polypeptide. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00 Translation o f T B R V R N A 385 Table I. Precipitation of translation products by antiserum to tomato black ring virus (TBR V) Expt. 2 /zl TBRV antiserum added RNA translated* , Ct/min in •~ supernatant fluid pellet % ct/min in pellet TBRV-S RNA-x TBRV-S RNA-2 TBRV-S satellite IO xo IO 24567 25 586 15260 232 165o 170 o'9 6' I I"t TBRV-S RNA-2 o oq 1 io 43763 4I 6oi 40889 39725 645 55z 1968 3162 I"5 I'3 4"6 7'4 TRV-PRN RNA-I o o.x I 10 48 627 469IO 49903 48807 460 763 690 562 0"9 1.6 i "4 I.I • RNA was translated in wheat germ extracts. Washed protein from translation mixtures was treated as described in Methods. The patterns of bands produced by the translation of R N A from TBRV-S and TBRV-G (Fig. I a, c, respectively) showed many minor differences in the position of the bands, whereas those produced by the translation products of R N A from the serologically related strains TBRV-E and TBRV-G (Fig. I b, c, respectively) differed much less. Translation in reticulocyte lysates In untreated lysates there was considerable endogenous protein synthesis (Fig. 2a); nevertheless a polypeptide of mol. wt. 1.6 × IO5 was detected in lysates containing purified RNA-2 from TBRV-SI2 (Fig. 2c), although no new products were detected in lysates containing R N A q (Fig. 2b). However, when the lysates were pre-treated with nuclease (Pelham & Jackson, I976) endogenous protein synthesis was abolished and autoradiographs of controls without added R N A showed no bands. In the treated lysates RNA-2, as before, was translated into a polypeptide of mol. wt. 1.6 × IO5 (Fig. 2e) and R N A - I was translated into many polypeptides (Fig. 2 d), of which the largest had a mol. wt. of 2.2 × 105; there was also a prominent product of mol. wt. I"9 × Io 5 (Fig. 2d). These three polypeptides comigrated with similar products produced in wheat germ extracts containing TBRV-S R N A (Fig. 2f). No attempt was made to suppress the formation of lower mol. wt. bands by adding extra t R N A (Pelham & Jackson, I976). Detection of coat protein sequences in the translation product of RNA-2 Experiments with pseudo-recombinant isolates (Randles et al. I977) indicated that TBRV RNA-2 contains the cistron for the virus coat protein. However, although RNA-2 translation products made in nuclease-treated reticulocyte lysates included a minor polypeptide that migrated to the position of TBRV coat protein (Fig. 2e), no similar product was produced when RNA-2 was added either to untreated lysates (Fig. 2 c) or to wheat germ extracts (Fig. If). We conclude therefore that there was no significant translation into coat protein molecules. In experiments to detect coat protein sequences the products of translation of different R N A species in wheat germ extracts were recovered by acid precipitation, washed and incubated with rabbit antiserum to TBRV-S. Addition to the mixtures of goat antiserum t o rabbit y-globulin precipitated up to about 7 % of the radioactivity in TBRV RNA-2 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00 386 C. FRITSCH, M. A. MAYO AND A. F. MURANT (a) (b) (c) (d) (e) (I3 (g) in) Fig. 3. Autoradiograms showing electrophoretic separations in 16% polyacrylamide gels of polypeptides released by partial proteolysis from: (a, c, e, g) 48ooo mol. wt. polypeptide translation product of satellite RNA from TBRV-S; (b, d, f, h)480oo tool. wt. polypeptide translation product of satellite RNA from TBRV-G. (a, b) Undigested polypeptide; (c, d) polypeptide incubated with 8o #g/ml Staphylococcus aureus V8 protease; (e,f) polypeptide incubated with o'o8/zg/ml subtilisin; (g, h) polypeptide incubated with 0'4 #g/ml subtilisin. Incubation was for 3o rain at 3o °C. Arrows indicate the position of undigested 48 ooo mol. wt. polypeptide, arrow heads indicate the position of the main products of enzyme digestion. translation products, but only about 1% of the radioactivity in the translation products o f T B R V R N A - I , TBRV satellite R N A or tobacco rattle virus R N A - I (Table I). An increasing proportion of the TBRV RNA-2 translation products precipitated as increasing amounts of anti-TBRV serum were added (Table I, expt. 2). Electrophoresis in polyacrylamide-SDS gels showed that the 1.6 x io 5 tool. wt. polypeptide was present in the serological precipitate of TBRV RNA-2 translation products. Comparison o f translation products o f satellite R N A species f r o m different TBR V strains We showed previously (Fritsch et al. I978) that the 48 ooo tool. wt. polypeptides translated from satellite R N A molecules associated with T B R V - G and TBRV-S migrated at slightly different rates in 7"5 ~ acrylamide gels. Further evidence for differences between these polypeptides came from experiments in which the 48ooo mol. wt. bands were cut from acrylamide gels of RNA-3 translation products, treated with proteases and the resulting peptides compared by electrophoresis in I6 % acrylamide gels (Fig. 3)- The number and position of the peptides produced from either translation product by V8 protease (Fig. 3 c, d) or subtilisin (Fig. 3 e to h) were different. Increasing the duration of protease treatment or the concentration of enzyme used did not affect this result; we conclude that the satellite R N A species from the two TBRV strains produce different translation products, and therefore probably have different base sequences. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00 Translation o f T B R V R N A 387 DISCUSSION Translation of TBRV RNA-I in vitro resulted in the synthesis of many polypeptides although fewer were produced in reticulocyte lysates than in wheat germ extracts. Two high mol. wt. polypeptides were produced by both systems, the larger being of mol. wt. 2.2 × lO 5 which represents about 8o % of the coding capacity of the RNA molecule. The other large polypeptide produced in both translation systems was of mol. wt. I-9 x io 5 and must therefore contain much of the sequence of the 2.2 × lO5 mol. wt. polypeptide. It may be produced by some specific breakdown of the larger polypeptide or by partial translation. The major product of translation o f T B R V RNA-2 (mol. wt. 1.6 x lOS; Mutant & Taylor, 1978) in both in vitro systems was a polypeptide of tool. wt. 1.6 × lO5. The RNA molecule is therefore translated into a single polypeptide. RNA-2 determines the serological specificity of TBRV coat protein (Randles et al. 1977) and our tests showed that some of the 1.6 × lO5 mol. wt. polypeptide reacted with antiserum prepared against virus particles. In contrast the translation products of RNA-1 and satellite RNA failed to react with TBRV antiserum. The serological reaction was inefficient, possibly because the coat protein sequence was in a relatively denatured state within the larger polypeptide; however, the results provide direct evidence that RNA-2 is a plus strand and that the mRNA used in vitro could be that used in vivo. As was shown previously (Fritsch et al. I978) the band 2 polypeptide translated from TBRV-S RNA migrated in gels slightly more slowly than that from TBRV-G RNA (Fig. I a, c) and the general patterns of polypeptide bands produced in wheat germ extracts by RNA from TBRV-G and TBRV-S were similar. However in the present work the correspondence of the bands of RNA translation products was closer between TBRV-G and TBRV-E than between TBRV-G and TBRV-S. presumably most of these polypeptides arise from premature termination, translation of degraded RNA or proteolysis of larger translation products, and their sizes depend on the primary or secondary structure of the RNA molecules. It is interesting that the degree of similarity between these translation artifacts should reflect the serological relatedness of the viruses. We showed previously that TBRV satellite RNA (RNA- 3, tool. wt. 4"8 × lO5; Murant & Taylor, I978) is translated in both in vitro systems into a polypeptide of tool. wt. 4"8 × IO4. The results of partial proteolysis of the translation products provide the first chemical evidence that satellite RNA molecules associated with different strains of TBRV are themselves different. It is not possible to measure the extent of the difference using this technique but few if any peptides produced by the proteases were common to the two polypeptides suggesting that they had significantly different amino acid sequences. The 480oo tool. wt. polypeptide translated from the satellite RNA associated with TBRV-S was detected in extracts of infected protoplasts (Fritsch et aL 1978) suggesting that relatively large quantities were synthesized. Moreover, all or nearly all of the coding potential of the satellite R N A is used during translation. Taken together, these results suggest that the protein may have a specific role, perhaps facilitating the replication of the satellite RNA in some way. In most plant viruses so far studied, coat protein is produced in vivo by translation of a sub-genomic monocistronic m R N A (e.g. Lane, 1974; Hunter et al. 1976; Siegel et al. I976). With TBRV, if it can be assumed that translation behaviour in vitro is followed in vivo, it seems that a different strategy is adopted: the genome RNA is translated in its entirety into a large polypeptide from which the coat protein is presumably cleaved by protease action. In this respect TBRV appears to resemble cowpea mosaic virus (CPMV). CPMV M-RNA (equivalent to RNA-2) codes for the smaller coat protein polypeptides of mol. wt. about 25ooo (Gopo & Frist, I977), whereas both in reticulocyte lysates (Pelham Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00 388 C. F R I T S C H , M. A. M A Y O AND A. F. M U R A N T & Stuik, I977) and in wheat germ extracts (Davies et aL I977) it is translated into two large polypeptides of mol.wt, about I IOOOO but not into coat protein. Recent work has shown other similarities between R N A molecules of TBRV and CPMV. R N A molecules from both viruses contain polyadenylate (El Manna & Bruening, I973; Mayo et al. I979a) and also have small proteins covalently attached (Daubert et aL I978; Stanley et aL I978 ; Harrison & Barker, x978; Mayo et aL ~979b)- Perhaps the similarities between nepoviruses and comoviruses in their translation strategies are related to the structural features common to the two groups of viruses. We thank Mme Odile Hemmer and Erica Bell for able and skilled technical assistance and Dr M. van Regenmortel for advice on serological techniques. REFERENCES BENICOURT, C., P~R~, J. & HAENNI, A. 0978). T r a n s l a t i o n o f T Y M V R N A into high molecular weight proteins. FEBS Letters 86, 268-272. CADMAN, C. H. & HARRISON, B. O. 0959). Studies on the properties of soil-borne viruses of the tobacco rattle type occurring in Scotland. Annals of Applied Biology 47, 542-556. CLEVELAND, D. W., FISCHER, S. O., KIRSCHNER, M. W. & LAEMMLI, U. K. 0977). 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MURANT, A. F. & TAYLOR) M. 0978). Estimates o f molecular weights o f nepovirus R N A species by polyacrylamide gel electrophoresis u n d e r d e n a t u r i n g conditions. Journal of General Virology 4I, 53-61. PELHAM, H. R. a. & JACKSON, R. S. (1976). A n efficient m R N A - d e p e n d e n t translation system f r o m reticulocyte lysates. European Journal of Biochemistry 67, 247-256. PELHAM, H. R. B. & STUIK, E. J. (1977). T r a n s l a t i o n o f cowpea mosaic virus R N A in a messenger d e p e n d e n t cell free system f r o m rabbit reticulocytes. Proceedings of the Colloquium on Nucleic Acids and Protein Synthesis in Plants, 1976, pp. 691-695. Paris: C N R S . Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00 Translation o f T B R V R N A 389 RANDLES, J. W., HARRISON, B. D., MURANT, A. F. & MAYO, M. A. (1977). P a c k a g i n g a n d biological activity of the two essential R N A species o f t o m a t o black ring virus. Journal of General Virology36, 187-193. SIEGEL, A., HARd, V., MONTGOMERY, I. & KOLACZ, K. (I976). A messenger R N A for capsid protein isolated from TMV-infected tissue. Virology73, 363-371. STANLEY, J., ROTTIER, P., DAVIES, J. W., ZABEL, P. & VAN KAMMEN,A. 0978). A protein linked to the 5' termini o f both R N A c o m p o n e n t s o f the eowpea mosaic virus g e n o m e . Nucleic Acids Research 5, 45o5-4522- (Received 14 May I979) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:47:00