Aminoacylation properties of eggplant mosaic virus RNA Separation and association of tRNAs

88.281-285

VIROLOGY

(1978)

Aminoacylation

Properties Separation

and Association

M. PINCK Laboratoire

of Eggplant Mosaic Virus RNA of tRNAs

T. C. HALL’

AND

de Physiologic Vegetale, Institut de Botanique, 28 rue Goethe, 67063-Strasbourg Cedex, France and Department of Horticulture, University of Wisconsin, Madison, WI 63706 Accepted March

30, 1978

After filtration on Ultrogel AcA 34, RNA from eggplant mosaic virus (EMV) retained its ability to bind valine, but no significant aminoacylation by lysine could be detected using synthetases from wheat germ or E. coli. Some association of tRNA with EMV RNA was observed after they were incubated together in a magnesium-containing buffer. However, equal levels of lysine binding to the viral RNA-tRNA complex were observed when the tRNA used for the association was derived from either healthy or EMV-infected Datura leaves. These data appear to preclude the presence of a lysine-accepting tRNA specific to infected tissues being bound to EMV RNA, although the presence of appreciable levels of lysine-accepting tRNA as a contaminant in EMV preparations was confirmed. INTRODUCTION

It was shown previously that in addition to acceptance of valine by the genomic RNA of eggplant mosaic virus (EMV), significant amounts of lysine could also be bound during enzymatic aminoacylation (Pink et al., 1974). Although it appeared that the lysine was accepted by a tRNA specifically bound to the genome, subsequent experiments have suggested that the lysine-accepting ability could be removed from the high molecular weight RNA by appropriate filtration techniques, and that prior dissociation with EDTA or other agents was not mandatory. In this paper we re-evaluate the separation of contaminating tRNAs from EMV RNA, and the phenomenon of association of tRNA from healthy and infected leaves of a host plant with EMV RNA. MATERIALS

AND

METHODS

Virus Isolation. Some 21 days after infection with EMV (type strain), leaves (175 g) of Dab-a stramonium were ground using a Waring blender into a mixture of 10 m&f Tris-HCl, pH 7.6 (250 ml), butanol(20 ml) and chloroform (80 ml). The crude ex’ To whom requests for reprints

should be sent.

tract was centrifuged at 3000 X g and virus precipitated from the supernatant by addition (w/v) of 6% polyethylene glycol and 6% NaCl. After standing at 4’ for 4 hr the pellet was resuspended (overnight) by agitation in 10 mM Tris-HCl, pH 7.6, then centrifuged at 15,000 x g for 10 min. The virus in the supernatant was purified by differential centrifugation (using from one to five cycles) at high speed (40,000 rev/mm in a Spinco 40 rotor, 2 hr) then, after resuspension of the amber pellet, at low speed (4,000 X g, 10 min). Preparation, Dissociation, Association and Gel Filtration of Viral RNA. RNA was extracted using water-saturated phenol containing 0.5% (w/v) SDS. After a second phenolic extraction of the aqueous layer, phenol was removed using three or four ether extractions; usually NaCl was added to give a final concentration of 0.1 M, and the RNA precipitated by addition of 2.5 vol. ethanol. For dissociation of tRNA, the viral RNA was heated at 60” for 10 min in 20 mM EDTA, 10 m&f Na cacodylate (pH 5.7, HCl) and rapidly cooled in ice prior to gel filtration. For association, viral RNA was heated at 60° together with tRNA for 10 min in 10 mM MgCh, 10 mM Tris-HCI (pH 8.0) and

281 0042~6822/78/0882-0281$02.00/0 Copyright 0 1978 by Academic

Press,

Inc.

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282

PINCK

AND

then stood at ambient temperature (about 25O)for 90 min prior to gel filtration. The polyacrylamide-agarose gel filtration material used for columns was typically LKB Ultrogel AcA 34 (fractionation range 20,000 to 400,000 daltons); in some experiments AcA 22 (separation range 60,000 to lo6 daltons) was used allowing some separation of the viral RNA peak since a shoulder was seen eluting after the main absorbance peak, but flow rates were poor. AcA 54 (separation 6,000 to 70,000 daltons) was used for purification of tRNAs. The columns were run at room temperature (20” to 26”) using 10 nGI4 Na cacodylate buffer (pH 5.7) containing 0.1 iIf NaCl and 3 rniIf NaN3. Preparation of tRNA, Synthetases and Aminoacylation Conditions. tRNA was extracted from the leaves (50 g) of healthy or infected (27 days after inoculation with EMV) Datura stramonium plants by grinding in a mixture of 10 mM MgC12,1% SDS, 10 mM Tris-HCl, pH 7.4 (150 ml) and buffer-saturated phenol containing 5 0.5% 8hydroxyquinoline (165 ml). The aqueous layer was extracted twice with phenol, several times with ether, then precipitated with 2.5 vol. ethanol. After centrifugation the RNA pellet was dissolved in water (44 ml), the solution made to 3 M in Na acetate and stirred overnight at 4’ to precipitate ribosomal and other heavy nucleic acids. The supernatant was precipitated with ethanol, sticky stands of DNA being spooled out with a sterile glass rod at this point. The tRNA was purified by filtration on an AcA 54 column to remove remaining DNA and nucleic acid fragments. Wheat germ tRNA was prepared in essentially the same way, except that the initial steps included washing by floatation on a 5:l CCLcyclohexane mixture and a French Pressure Cell was used to crush (max. pressure 1,500 lb ine2) the wheat germ (8 g in a final volume of 20 ml of 10 mikf Tris-HCl, pH 8, containing 0.5% SDS) into buffer-saturated phenol. Bacterial synthetase (amino acid-tRNA ligase, E.C. 6.1.1) was from E. coli, strain MRE 600 (Yot et al., 1970) and the wheat germ synthetase was prepared essentially as described by Kohl and Hall (1974). Typ-

HALL

ically, 5 ~1 of synthetase containing 9.8 pg protein (Lowry et al., 1951) for the bacterial preparation and 1.5pg protein for the wheat germ preparation were added to start the 50 ~1 incubations. Other components of the reaction were: radioactive amino acid, RNA, ATP (0.64 mM) and Me (5 r&f) in 0.1 M HEPES (pH 7.4 with KOH, giving approximately 50 mM K+/50 ~1incubation). Adjustment of the pH with NaOH or N&OH in place of KOH resulted in decreased aminoacylation activity. Aliquots of 10 ~1 were withdrawn after 1, 10,20, and 30 min incubation at 30” and spotted onto filter paper discs for determination of radioactivity bound to RNA (McLeester and Hall, 1977). RESULTS

& DISCUSSION

Gel Filtration of EMV RNA EMV has been shown to have at least two RNA components, mol wt 2.35 and 1.9 X lo6 (Klein, 1976). Because the relative proportions of the genomic RNAs have not been satisfactorily resolved, aminoacylation values are given relative to weight of RNA rather than to molar quantities. Additionally, it has been reported that the related TYMV has several small RNA components (Pleij et al., 1976) and it is possible that these may also occur in EMV. The profle of EMV RNA after gel filtration is shown in Fig. 1. The large genome RNAs are essentially eluted in the excluded volume (peak A). Contaminating tRNAs eluted in peak B; aminoacylation reactions (wheat synthetase) revealed acceptance values of 0.1 pmol lysine and 0.06 pmol valine per pg RNA. Leucine and tyrosine could also be esterified to this material (0.03 and 0.01 pmol/pg RNA, respectively). Peak C was usually higher when the viral RNA was heated in the presence of EDTA before addition to the column than when similar quantities of untreated RNA were used. Considerable amounts of ATP and amino acid were present in this peak after filtration of viral or tRNA after preparative aminoacylation. Aminoacylation of EMV RNA Prior to gel filtration, RNA extracted from EMV (in this case purified by two

EMV I

I

1

f I I

1

RNA-tRNA

283

INTERACTION TABLE

I

1

1.2 -

EMV RNA sample (a/ reaction)

Radioactivity bound” (cpm) Valine

FRACTIONS

FIG. 1. Gel filtration of EMV RNA. RNA, extracted from EMV purified by two cycles of differential centrifugation, was heated with EDTA (see “Materials and Methods”) and 1 mg applied to a 136 X 0.8 cm column of Ultrogel AcA 34. Peak A was genome RNA; peak B was tRNA, peak C contained low molecular weight material, and in separate experiments free ATP or radioactive amino acids eluted at this position.

cycles of differential centrifugation) showed appreciable levels of valine and lysine binding in aminoacylation reactions; however, after gel filtration much lower levels of valine fixation and very small values for lysine fEation were obtained (Table 1). It was noted that a decrease in the amount of tRNA contaminants relative to the genome RNA was obtained by repeated cycles of differential centrifugation, but even after five such cycles there was still approximately 1 pg of tRNA per 66 pg viral RNA. Thus, the contamination of EMV RNA with tRNA appears to derive from both the surface of the virion, and from some tRNA encapsidated within the virion, presumably in light particles (Bouley et al., 1976). In this respect EMV differs from TYMV, as no contaminating tRNA is found in extracted RNA after as little as one differential centrifugation cycle during the preparation of the latter virus. It was noticed from the data of Table 1 that the quantity of lysine bound was not proportional to the amount of gel-filtered genome RNA added to the reactions. Therefore, a series of aminoacylations containing varying levels of viral RNA and enzyme were compared. As a control, brome mosaic virus (BMV) RNA which accepts tyrosine but no other amino acid (Hall et al, 1972) was added in some reac-

1

AMINOACYLATION OF EMV RNA BY VALINE AND LYSINE

Before gel fdtration 2.2 5.5 11.0 After gel filtration 2.2 4.5 6.7

Lysine

Amount of amino acid bound (pmol/ pg RNA) Valine

Lysine

7 783 21 913 43 489

721 2 036 3 518

1.53 1.73 1.71

0.13 0.15 0.13

1 793 3 815 6 117

85 156 133

0.35 0.37 0.40

0.02 0.01 0.01

u Aminoacylation reactions containing 7 &i [3H]valine (specific activity 26 Ci/mmol) or 5 PCi [3H]lysine (specific activity 28 Ci/mmol) were run as described in “Materials and Methods”; the cpm shown (after 30 mm incubation) are for 20 al samples of the reaction containing EMV RNA not subjected to gel filtration, and for 10 4 samples of reactions containing EMV RNA after filtration on an AcA 34 column. Values for control incubations omitting viral RNA (878 or 375 cpm for lysine and 664 or 306 cpm for valine) have been subtracted from the above figures.

tions. From this experiment (Table 2) it became clear that the levels of apparent lysylation of the viral RNA were dependent on the amount of enzyme present, rather than on the amount of RNA. This is reflected by the increased radioactivity bound in the presence of 10 ~1 enzyme compared with 5 4. Note that for tRNA the specific activity (pmol lysine/pg RNA) is constant for different levels of added tRNA, while it decreases with higher levels of added viral RNA. When BMV RNA was added, approximately the same degree of apparent lysine fmation occurred as when EMV RNA was added. Thus, the observed lysylation was probably due to small levels of tRNA present in the wheat germ synthetase preparation; we conjecture that the viral RNA may have served to protect this tRNA. A similar series of aminoacylations was made using synthetase from E. coli. In these experiments (data not shown), we found that EMV RNA was entirely freed of lysine-accepting capacity by column filtration, without any need for prior treatment

284

PINCK

AND

TABLE 2 EFFECT OF ENZYME AND RNA CONCENTRATION ON APPARENT LYSYLATION OF VIRAL RNA Reaction

components

RNA added (I%)

Radioactivity bound” kpm)

Extent of lysylation bnol/ R#A,

5 pl enzyme EMV RNA

BMV RNA

tRNA

(wheat germ)

10 d enzyme EMV RNA

BMV RNA 15 pl enzyme EMV RNA

BMV RNA

None 5.2 10.4 15.6 4.0 8.0 12.0 6.2 12.5

1 2 2 2 1 2 1 17 35

340 004 210 250 804 081 925 848 389

0.05 0.03 0.02 0.05 0.04 0.02 0.98 1.03

None 5.2 10.4 15.6 8.0

1 2 2 2 2

261 555 861 742 338

0.09 0.06 0.04 0.05

None 5.2 10.4 8.0

1 2 3 2

433 711 047 626

0.10 0.06 0.05

a Values shown are for 10 pl samples taken after 30 min incubation from reactions containing 7 PCi [3H]lysine (specific activity 28 Ci/mmol) catalyzed by synthetase from wheat germ (see “Materials and Methods”). For calculation of the extent of lysylation, the amounts of radioactivity bound were corrected for zero time binding and for the controls to which no RNA was added. The amounts of RNA added are for the total 50 d reaction. The EMV RNA used was from virus purified by four cycles of differential centrifugation; it had been heated in the presence of EDTA and subjected to filtration on a column of AcA 34 gel prior to ethanolic precipitation and solution in water for this experiment.

with EDTA or other agent for dissociating hydrogen-bound material. Association

of tRNA with EMV RNA

Since the above experiments appeared to exclude the presence of a lysine-accepting 4s RNA specifically bound to the RNA genome of EMV, we re-investigated the ability of tRNA from healthy or infected leaves of Datura stramonium to associate with column-filtered EMV RNA. The lysine-accepting capacity (tested with the

HALL

wheat germ synthetase) of tRNA from infected leaves was somewhat higher than for that from healthy leaves (2.1 pmol compared with 1.7 pmol lysine/pg tRNA), while valine binding was nearly identical (2.84 and 2.81 pmol vaIine/pg tRNA for the infected and healthy extracts, respectively). RNA from EMV prepared using four cy cles of differential centrifugation was treated with EDTA and separated from all contaminating tRNA by filtration through an AcA 34 column. Using a ratio of 6.25 pg viral RNA to 1 pg tRNA (a molar ratio of approximately 1:15), a mixture of EMV RNA and tRNA from either infected or uninfected plants was heated to 60” for 10 min in 10 mil4 MgCb buffered at pH 8.0 with 10 mM Tris-HCl. After cooling and standing at ambient temperature (approximately 26”) for 90 min the mixture was added to a 73 x 0.8 cm column of AcA 34, and the peak eluting in the position of the viral genome (peak A, Fig. 1) precipitated with ethanol. Aminoacylation of this material showed that, compared with an EMV RNA control, a small ability to bind lysine was apparent (Table 3). Complete association of EMV RNA with added tRNAi, (1 molecule of viral RNA with 1 molecule of tRNA) would have yielded acceptance values of about half those shown in Table 3 for the tRNA control (although the tRNAs were added in a 15-fold molar excess, tRNAl,, would be expected to constitute only about %5 to % of the tRNA species present). The ability of the associated fraction to bind lysine was, however, identical after complexing with tRNA from either healthy or infected leaves. Additional experiments (data not shown) revealed a low level of binding capacity for leucine and tyrosine (other amino acids were not tested) for the associated, filtered RNA. It appears, therefore, that small amounts of tRNA can be complexed with EMV RNA under certain conditions, but that this reaction is not necessarily specific for tRNAi,,, nor for tRNA for infected as compared with healthy leaves. Thus, the data of Tables 1 and 2 show that the low lysine binding activity observed in the presence of EMV RNA (after column filtration) can be ascribed to trace amounts of tRNA in the synthetase prepa-

EMV TABLE

RNA-tRNA

3

LYSYLATION OF VIRAL RNA COMPLEXED WITH tRNA FROM Datura RNA added to reaction Wheat enzyme EMV RNA + tRNA.1 EMV RNA + tRNA.H EMV RNA tRNA.1 Bacterial enzyme EMV RNA + tRNA.1 EMV RNA + tRNA.H EMV RNA tRNA.1

w

Radioac- Extent of tivity lysylation bpp;y (P~;F

None 13.2 13.2 10.5 10.5 12.2 24.4 4.0

413 220 307 384 363 520 620 9 739

0.02 0.03 0.04 0.03 0.00 0.00 0.92

None 13.2 13.2 10.5 10.5 12.2 24.4 4.0

510 967 985 1 048 1 095 700 631 18 937

0.01 0.01 0.02 0.02 0.01 0.00 1.77

1 1 1 1

n Values were calculated as described for Table 2 for 10 81 samples of reactions containing 7 pC!i [3H]lysine and catalyzed by either wheat germ or E. coli synthetase (5 d in each case). EMV RNA was viral RNA treated with EDTA and subjected to AcA 34 gel chromatography to remove ah tRNA contaminants. This RNA was also incubated in the presence of Mg2+ with tRNA from either healthy (tRNA.H) or infected (tRNA.1) Daturu leaves (see text for details). These mixtures were aIso subjected to AcA 34 gel filtration, the viral RNA peak precipitated with ethanol, then dissolved in water to give associated fractions EMV RNA + tRNA.H and EMV RNA +

tRNA.I.

rations. The additional finding of similar lysine-accepting activities of tRNA from healthy and infected Datura leaves, together with the identical levels of association of these tRNAs with viral RNA (Table 3) does not permit confirmation of the previous report (Pinck et al., 1974) of a 4s RNA specific to infected tissues being hydrogen-bound to the genome of EMV. It

285

INTERACTION

remains to be determined if the encapsidated tRNAs serve any function related to the infective processes of EMV. ACKNOWLEDGMENTS We thank Prof. H. M. Duranton for laboratory facilities for this work, which was supported by C.N.R.S., N.S.F. (grant BMS 73-008), N.I.H. (grant AI 11572), and the U.W. Graduate School. We are sincerely grateful for the excellent technical assistance of Marie-France Cast&Ii, and for a gift of BMV RNA from Dr. L. Pinck. REFERENCES BOULEY, J. P., BRIAND, J. P., GENEVAUX, M., PINCK, M., and WITZ, J. (1976). The structure of eggplant mosaic virus: Evidence for the presence of low molecular weight RNA in top component. Virology 69, 775-781. HALL, T. C., SHIH, D. S., and KAESBERG, P. (1972). Enzyme-mediated binding of tyrosine to brome mosaic virus ribonucleic acid. Biochem. J. 129, 969-976. KLEIN, C. (1976). Variete structurale du RNA de deux tymovirus. Ph.D. Thesis, Strasbourg, France. KOHL, R. J., and HALL, T. C. (1974). Aminoacylation of RNA from several viruses: Amino acid specificity and differential activity of plant, yeast and bacterial synthetases. J. Gen. Virol. 25, 257-261. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MCLEESTER, R. C., and HALL, T. C. (1977). Simplification of amino acid incorporation and other assays using falter paper techniques. Anal. Biochem. 79, 627-630. PINCK, M., GENEVAUX, M., and DURANTON, H. (1974). Studies on the amino acid acceptor activities of the eggplant mosaic virus RNA and its satellite RNA.

Biochimie 56,423-428. 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. Nut. Acud. Sci.

USA 73,4437-4441. YOT, P., PINCK, M., HAENNI, A. L., DURANTON, H. M., and CHAPEVILLE, F. (1970). Valine-specific tRNAlike structure in turnip yellow mosaic virus RNA. Proc. Nut. Acud. Sci. USA 67, 1345-1352.