Functional distinctions between the ribonucleic acids from citrus exocortis viroid and plant viruses: Cell-free translation and aminoacylation reactions

VIROLOGY

61, 486-492 (1974)

Functional Citrus

Distinctions

Exocortis

Viroid

Between

the Ribonucleic

and Plant Viruses:

and Aminoacylation T. C. HALL Department

of Horticulture,

Acids from

Cell-free

Translation

Reactions

R. K. WEPPRICH

AND

University

of Wisconsin,

Madison,

Wisconsin 53706

J. W. DAVIES Laboratory

of Biophysics,

University

of Wisconsin,

Madison,

Wisconsin 53706

AND

L. G. WEATHERS Department

of Plant Pathology,

AND

University Accepted

J. S. SEMANCIK

of California,

Riverside,

California

92502

June 27, 1974

A low molecular weight RNA having tRNA-like physical characteristics has previously been shown to be the pathogen causing citrus exocortis disease. The ability of this RNA to serve as a messenger molecule, or as an amino acid acceptor, was compared with that of other small RNA molecules from plant viruses. While the viral RNAs showed both of these activities, the citrus exocortis viroid had neither, contrasting the nature of this pathogen with that of plant viruses. INTRODUCTION

Physical studies of the low molecular weight RNA (125,000) responsible for citrus exocortis disease (Semancik and Weathers, 1972) suggested that the molecule may be tRNA-like (Semancik, et al., 1973; Semancik et al., 1974). The RNA components from several small viruses have been shown to have properties like tRNA, binding specific amino acids in energy and enzyme-dependent reactions (Yot et al, 1970; oberg and Philipson, 1972; Kohl and Hall, 1974). The smallest RNA component (300,000 daltons) from brome mosaic virus (BMV) has been shown to serve as a monocistronic messenger for coat protein synthesis (Shih and Kaesberg, 1973) as well as being capable of accepting tyrosine at its 3’-terminus (Hall et al., 1972). This article describes experiments

conducted to determine if citrus exocortis viroid (CEV) RNA is capable of binding an amino acid in a tRNA-like manner, and to compare its ability to direct protein synthesis in vitro with that of viral RNAs known to be plant pathogens. MATERIALS

AND

METHODS

Sources of RNA. Biologically active CEV RNA was isolated from a mixture of nucleic acids obtained by homogenizing infected Gynura auruntica DC in phenol as reported by Semancik et al. (1974). The three CEV RNA preparations used in these studies were each judged about 95% pure by polyacrylamide gel electrophoresis. Alfalfa mosaic virus (AMV) RNA, top component a, was kindly donated by Dr. Lous van Vloten-Doting, Leiden. Rabbit globin 9s RNA was purchased from Searle

VIROID

AND VIRAL

RNA DIFFERENCES

487

Co. Ltd., Bucks, England. Brome mosaic magnesium acetate, 0.002 M ATP, 0.0002 M GTP (pH 7.0), 0.003 M 2-mercaptoethavirus (BMV) RNA, broad bean mottle virus (BBMV) RNA, cowpea chlorotic mot- nol, 10m4M of each amino acid, 0.75-1.0 mg tle virus (CCMV) RNA, tobacco mosaic S-30 bacterial extract protein, and 30-32 virus (TMV) RNA, and tRNA were pre- pmole of the appropriate viral RNA. The pared as described previously (Kohl and E. coli and P. aeruginosa systems were Hall, 1974). Ribosomal 16s RNA from incubated at 37”, and the B. stearothermophilus system at 60”. Wheat embryo reEscherichia coli was a gift from Dr. Lilian action mixtures contained 0.005 M Tris Shih. Where individual RNA components and 0.02 M HEPES adjusted to pH 7.6 were used, they were obtained by standard with 5 x 10e3-8 x 1O-3 M KOH, 0.004 M sucrose density gradient techniques (Shih magnesium acetate, 0.095 M potassium and Kaesberg, 1973). Enzyme preparations. Synthetase en- acetate, 0.0025 M of each amino acid, zyme from E. coli, Phaseolus vulgaris L 0.0025 M ATP, 3.75 x lo-’ M GTP, 0.005 0.0005 M dithioand Saccharomyces cerevisiae were pre- M phosphoenolpyruvate, threitol, 0.75-1.0 mg S-23 wheat extract pared as described by Kohl and Hall protein, and 30 pmole viral RNA, incu(1974), and stripped free of endogenous bated at 30”. Samples of 0.01 ml were tRNA by Sephadex DEAE-A25 treatment taken at 0 and 40 min, and spotted onto (Kirkegaard et al., 1972) as reported previWhatman 3MM filter discs, then washed ously (Chen and Hall, 1973). acid (5%’ w/v, SO’), ethaRibosome-enzyme preparations from E. in trichloroacetic bound to the coli (S-30) and Triticum aestivum L (S-23) nol, and ether. Radioactivity discs was counted in 5 ml of scintillation were as described by Davies and Kaesberg fluid containing 6 gm PPO and 0.5 gm (1973), and S-30 preparations from BacilPOPOP per liter of toluene, the efficiency lus stearothermophilus and Pseudomonas aeruginosa were similar to those from E. being about 20%. After removing samples for the disc ascoli (details to be published). say, the remainder of the reaction mixture Aminoacylation and protein synthesis. Aminoacylation reactions were carried out was treated and electrophoresed on 10% at 30” in a final volume of 0.05 ml, and (w/v of monomer) acrylamide gels as decontained 0.0064 M ATP, 0.005 M magne- tailed by Davies and Kaesberg (1973) to obtain the peptidyl products. Gel slices (1 sium acetate, 0.04 M potassium acetate, 0.001 M dithiothreitol, 0.002 M GSH, and mm) were dispensed together with 0.3 ml water into scintillation vials by a Gilson 0.1 M HEPES (N-2-hydroxyethylpiperaModel gel fractionator. After zine-N-2-ethanesulfonic acid; mixture fi- Aliquogel nally adjusted to pH 7.li with NaOH, 30”) standing 12 hr (or heating to 60” for 3 hr), 10 ml of a scintillation fluid containing two in addition to enzyme, RNA, and radioparts toluene, one part Triton X-100, 3.2 active amino acid. Amino acid substrates gm PPO and 0.67 gm POPOP per liter were were [3H]histidine (58 Ci/mmole), [3H]measured. tyrosine (40 Ci/mmole), or a mixture of 20 added and the radioactivity (MW 45,000)) chymotrypsinoradioactive amino acids. Aliquots of 0.01 Ovalbumin gen A (25,000), myoglobin (17,800), cytoml were withdrawn at 10 min intervals, and assayed for amino acid binding as de- chrome C (12,400), and insulin (2,900) were A mixture tailed previously (Tao and Hall, 1971), the obtained from SchwarzlMann. of these proteins was electrophoresed at the 30 min figure being that shown in the text. same time as the reaction products to serve Incorporation of tritium-labeled arginine (22 Ci/mmole), leucine (17 Ci/mmole), and as molecular weight markers. lysine (54 Ci/mmole) into proteins using RESULTS bacterial systems was conducted in 0.1 ml Comparison of amino acid binding by reaction mixtures, each containing 0.045 M CEV and other RNAs. The ability of synHEPES and 0.005 M Tris (adjusted to pH 7.6 with KOH); 0.08 M ammonium chlo- thetase enzymes from heterologous sources ride, 0.04 M potassium acetate, 0.01 M to catalyze the binding of an amino acid to

488

HALL

viral RNA is variable (Kohl and Hall, 1974), as in the case of tRNAs. For this reason, synthetase enzymes from diverse sources (plant, bacterial, and yeast) were selected for testing the ability of CEV RNA to be aminoacylated. However, no binding of any amino acid commonly found in proteins to CEV RNA was observed under the conditions described in Table 1. CEV RNA did not exhibit any significant competition for amino acid binding to tRNA, or to RNA from BMV or TMV which binds tyrosine and histidine, respectively. In addition to the experiments shown in Table 1, CEV RNA was melted both at low temperature (with dimethyl sulfoxide) and by heating. These single-stranded forms also showed no ability to bind an amino acid, and attempts to show aminoacylation using both higher and lower levels of CEV RNA (data not shown) failed. Ability of CEV and other RNAs to direct protein synthesis. RNA from several plant viruses has been shown to be an effective amino messenger, markedly stimulating acid incorporation into protein (Klein et al., 1972; Shih and Kaesberg, 1973). The efficiency of Q@ RNA and BMV RNA to function as messengers is dependent on the source of the cell-free system to which they are added (Davies and Kaesberg, 1973). We, therefore, used bacterial and plant cell-free systems to test the ability of CEV

ET AL.

RNA to function as a messenger. The data of Table 2 show that these cell-free systems have differential abilities for amino acid incorporation when directed by various RNAs. For example, incorporation in the B. stearothermophilus and E. coli systems was greatly stimulated by AMV RNA and BBMV RNA, while the highest incorporation due to CCMV RNA was obtained in the T. aestivum system. In contrast to the high stimulation of incorporation due to these viral RNAs, addition of CEV RNA to the cell-free systems resulted in a maximum of only 2.3-fold stimulation, which was similar to that obtained in reactions containing ribosomal RNA as a nonmessenger control. It was important to determine if the small level of incorporation observed in the presence of CEV RNA represented even minimal amounts of a polypeptide product. The three plant viral RNAs tested (AMV RNA, BBMV RNA, and CCMV RNA) each gave detectible products in at least one of the bacterial systems (Fig. l), and incorporation dependent on rabbit globin mRNA (Table 2) was found to give a discrete product peak (not shown). Bacillus stearothermophilus was used as one of the test systems since the 60” reaction temperaure would completely unfold the CEV RNA (Semancik et al., 1974), thereby facilitating ribosomal attachment. How-

TABLE COMPARISON

OF AMINO

Source of synthetase Amino acid substrate

ACID BINDING

Escherichia Mixture

1

BY CEV RNA,

coli

Histidine

tRNA,

BMV

RNA,

Saccharomyces cerevisiae Mixture

Histidine

AND TMV

RNA

Phaseolus vulgaris Mixture

Tyrosine

Radioactivity bound over minus RNA control (cpm/O.Ol ml of 0.05 ml reaction) Source of RNA added to reaction” CEV RNA tRNA rRNA + CEV Viral RNA Viral RNA + CEV RNA

200 68,017 68,415

-93 11,386 11,735

27 93,228 98,305

-71

36,938 32,353

264 90,570 86,237

30 60,281 72,940 42,250 39,711

DThe viral RNA used in the Saccharomyces system was TMV RNA (37 ag), and in the Phaseolus system was BMV RNA (10 pg), The tRNA used in the Escherichia system was Escherichla coli K12 tRNA (15 ag), and in the other systems was wheat germ tRNA (6 pg). Data are shown for CEV RNA at 4 pg per reaction, and the reaction conditions are described under Materials and Methods.

VIROID

AND VIRAL

TABLE AMINO

ACID INCORPORATION

STIMULATED

Bacillus Source of incorporation system stearothermophilu.5 Incorporation” Source of RNA added to reaction ANV RBA (top component a) BBMV RNA (component 4) CCMV RNA (component 4) CEV RNA CEV RNA + DMSO Rabbit globin 9s RNA E. coli ribosomal 16s RNA

2

BY SEVERAL RNAs

Escherichia

wm

stimulation

wm

208,991

105.1

341,310

320,169

160.5

4,381

489

RNA DIFFERENCES

coli stimulation

IN DIFFERENT

PROTEIN-SYNTHESIZING

Pseudomonas aeruginosa

SYSTEMS

Triticum

aestiuum

wm

stimulation

cpm

stimulation

28.3

69,577

103.8

32,277

16.0

370,010

30.6

15,599

24.3

76,680

36.6

3.2

93,863

8.5

3,853

5.8

150,056

70.8

996 1,587

1.8 1.5

7,100 8,872

1.6 1.7

889 388

1.6 2.3

547 14

1.2 1.0

24,006

15.6

57,340

5.6

2,736

5.1

98,576

46.9

1,950

2.0

8,070

4.7

0 Radioactivity (cpm) represents amino acid incorporated into 0.02 ml samples of 0.1 ml reaction mixtures (see Materials and Methods), corrected for zero time and minus RNA controls. Stimulation is the ratio of incorporation in the presence of added RNA: incorporation in the absence of added RNA.

ever, no detectable product due to CEV RNA addition was obtained in any of the cell free systems. Heating the CEV RNA to 90” immediately prior to its addition to the reaction, even in the presence of 70% dimethylsulfoxide, also failed to reveal any products (data not shown, but identical to CEV RNA products in Fig. 1) confirming that any incorporation stimulated by CEV RNA was fortuitous. The small amount of incorporation observed in the presence of E. coli 16 S ribosomal RNA (Table 2) similarly gave no detectable products on electrophoresis. Modification of BMV RNA can influence the nature of the polypeptide product synthesized in vitro (Shih et al., 1974), and the possibility of interaction of CEV RNA with viral RNA was considered. It is also known that some double-stranded RNAs are strongly inhibitory to protein synthesis (Ehrenfeld and Hunt, 1971), hence we thought that CEV RNA might interfere with protein synthesis directed by other messengers. Therefore, as a final test for a translational role of CEV RNA, a compari-

son was made of CEV RNA and BMV RNA separately and together as messengers in the wheat germ system (Fig. 2). Once again, CEV RNA appeared inert, having neither messenger activity nor any influence on translation of BMV RNA. DISCUSSION

The small size of the CEV RNA molecule imposes the restraint that the maximum size of a polypeptide for which it could serve as a messenger template is about 10,000 daltons. If such a pathogen-specific polypeptide functioned as a subunit of replicase, then a metabolic function resulting in the disease symptoms would be established. The association of tRNA-like properties with several plant viruses (Kohl and Hall, 1974) suggests that a physiological function may exist, perhaps facilitating viral RNA transcription or translation. While a combination of tRNA and mRNA functions is theoretically attractive as a basis for the infectivity of small molecules such as the RNA of CEV and of potato spindle tuber viroid (Diener, 1971), the

490

HALL B. steorothermophilus

System:

Mol. wt. x 1o-3:

45

25

148

ET AL. E. coli

Ps. aeruginosa

2.9

AMV RN&-a6

KY 4

2

CEV RNA

1~

1’

o-

0’

0.1 ’ 0%

FIG. 1. SDS-polyacrylamide gel analysis of products from bacterial cell-free systems. Each diagram represents the radioactivity profile (vertical scale = cpm x lOma) of l-mm slices cut from gels (electrophoresed from left to right) containing reaction products treated as described in Materials and Methods. The width of each panel is equivalent to 100 slices. Molecular weight markers were run for the Bacillus sterarothermophih and Pseudomonas aeruginosa systems and their positions are indicated as MW (x 10m3)at the top of the panels.

VIROID T. aestivum

System :

BMV RNA4

6 6

AND VIRAL

491

RNA DIFFERENCES

present data lend no support to this suggestion, despite the several sets of biological conditions used. A possibility exists that the exocortis pathogenic RNA might function more effectively in aminoacylation and translation systems derived from hosts of the exocortis disease. Nevertheless, the systems utilized in these studies have been shown to be competent with the addition of foreign RNA species. Potato spindle tuber viroid RNA also shows no messenger function in vitro (Davies et al., 1974). The present results show a clear distinction between the activity of RNAs from plant viruses and the exocortis viroid, further emphasizing the unusual nature of this pathogenic RNA. ACKNOWLEDGMENTS

14

12

We thank Professor Paul Kaesberg for his interest and use of some of his laboratory facilities, and gratefully acknowledge the gift of wheat seed from Prof. H. L. Shands. This work was supported by NIH Grants AI-11572, AI-1466, and CA-15613, and by Grants GB-38652 and GB-39605 from NSF.

10

REFERENCES CL RNA

8

J. M., HALL, T. C. (1973). Comparison of tyrosyl transfer ribonucleic acid and brome mosaic 6 virus tyrosyl ribonucleic acid as amino acid donors in protein synthesis. Biochemistry 12, 4570-4574. 4 DAVIES, J. W., KAESBEHG, P. (1973). Translation of virus mRNA: Synthesis of bacteriophage Qp pro2 teins in a cell-free extract from wheat embryo. J. Viral. 12, 1434-1441. DAVIES, J. W., KAESBERG, P., and DIENER, T. 0. (1974). Potato spindle tuber viroid. XII. An investigation of viroid RNA as a messenger for protein synthesis. Unpublished manuscript. CEV 2 DIENER, T. 0. (1971). Potato spindle tuber “virus.” RNA IV. A replicating, low molecular weight RNA. Virology 45, 411-428. EHRENFELD, E., and HUNT, T. (1971). Double-stranded poliovirus RNA inhibits initiation of protein synthesis by reticulocyte lysates. Proc. Nut. Acad. Sci. USA 68, 1075-1078. HALL, T. C., SHIH, D. S., and KAESBERG, P. (1972). Enzyme-mediated binding of tyrosine to bromeFIG. 2. Electrophoretic analysis of the products mosaic-virus ribonucleic acid. Biochem. J. 129, from the wheat embryo system. The radioactivity 969-976. profiles are shown for incorporation using the wheat system in the presence of BMV RNA component 4 KIRKEGAARD, L. H., JOHNSON, T. J. A., and BOCK, R. M. (1972). Ion filtration chromatography: A powerand CEV RNA, both separately and together. Details ful new technique for enzyme purification applied are as for Fig. 1. the vertical scale being cpm (x 10m3) to E. coli alkaline phosphatase. Anal. Biochem. 50, and the width of each panel representing 100 l-mm 122-138 slices. CHEN,

HALL KLEIN, W. H., NOLAN, C., LAZAR, J. M., and CLARK, J. M., Jr. (1972). Translation of satellite tobacco necrosis virus ribonucleic acid. I. Characterization of in vitro procaryotic and eucaryotic translation products. Biochemistry 11, 2009-2014. 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. Unpublished manuscript. (ZBERG, B., and PHILIPSON, L. (1972). Binding of histidine to tobacco mosaic virus RNA. Biochem.

Biophys. Res. Commun. 48, 927-932. SEMANCIK, J. S., MORRIS, T. J., and WEATHERS, L. G. (1973). Structure and conformation of low molecular weight pathogenic RNA from exocortis disease.

Virology 53, 448-456. SEMANCIK, J. S., MORRIS, T. J., WEATHERS, L. G., RORDORF, F. B., and KEARNS, D. R. (1974). Physical properties of a minimal infectious RNA (viroid) associated with exocortis disease. Virology, in press.

ET AL. SEMANCIK, J. S., and WEATHERS, L. G. (1972). Exocortis disease: Evidence for a new species of “infectious” low molecular weight RNA in plants. Nature

New Biol. 237, 242-244. SHIH, D. S., and KAESBERG, P. (1973). Translation of brome mosaic viral ribonucleic acid in a cell-free system derived from wheat embryo. Proc. Nat. Acad. Sci. USA 70, 1799-1803. SHIH, D. S., KAESBERG, P., and HALL, T. C. (1974). Messenger and aminoacylation functions of brome mosaic virus RNA after chemical modification of S’terminus. Nature (London), in press. TAO, K. L., and HALL, T. C. (1971). Factors controlling aminoacyl-transfer-ribonucleic acid synthesis in uitro by a plant system. Biochem. J. 121, 495-501. YOT, P., PINCK, M., HAENNI, A., DURANTON, H. M., and CHAPEVILLE, F. (1970). Valine-specific tRNAlike structure in turnip yellow mosaic virus RNA. Proc. Nat. Acad. Sci. USA 67, 1345-1352.