Specific inhibition of TMV-RNA synthesis by blasticidin S

36, 646-651 (1968)

VIROLOGY

Specific

Inhibition

of TMV-RNA Blasticidin

ATSUSHI Department California

HIRAI,

by

TOKUZO

HIRAI

S’

S. G. WILDMAN,

AND

of Botanical Sciences and Molecular Biology Institute, of Plant Pathology, Paculty 900.24, and Laboratory Nagoya, Japan Accepted August

Synthesis

University of California, of Agriculture, Nagoya

Los Angeles, University,

27, 1968

Immediately after inoculation with TMV, tobacco leaves were treated with blasticidin S, a potent inhibitor of TMV multiplication and normal protein synthesis. After 72 hours, s2P was applied to the infected leaves for 6 hours. The total leaf RNA was extracted, and the amount of newly synthesized 32P-TMV-RNA was determined after resolution from normal leaf RNA by sucrose density gradient centrifugation. The antibiotic strongly inhibited TMV-RNA synthesis whereas synthesis of host RNA was not affected. Evidently, synthesis of new protein, probably a TMV-RNA polymerase, is required before TMV-RNA synthesis can occur. INTRODUCTION

Blasticidin S, a puromycin-like antibiotic, was found to be a potent inhibitor of tobacco mosaic virus (TMV) multiplication provided the antibiotic was applied at the earliest stage of the infectious process (T. Hirai et al., 1966). The antibiotic is known to inhibit protein synthesis by blocking transfer of amino acids from amino acyltRNA’s to polypeptide (Yamaguchi et al., 1965)) and T. Hirai and Shimomura (1965) also found it to be a strong inhibitor of protein synthesis in tobacco leaves. T. Hirai et al., therefore hypothesized that blasticidin S interfered with TMV multiplication by preventing the synthesis of a prot(ein, most likely a TMV-RNA polymerase, necessary for the initiation of TMV-RNA synthesis. However, the extent of TMV multiplication had to be measured by infectivity of the virus, and the possibility that inhibition of TMV coat protein synthesis by the antibiotic was the cause of reduced infectivity 1 Supported by Research Grant AI 00536 from National Institutes of Health, U. S. Public Health Service, and Contract AT (ll-l)-34, Project 8, from the Division of Biology and Medicine, U. S. Atomic Energy Commission. 646

could not be ruled out. The finding (A. H. and S. G. W.) that TMV-RNA can be separated from leaf RNA, and that both “free,” single-stranded, TMV-RNA, as well as TMV-RNA coated with protein can be recovered and identified from TMV-infected leaves has permitted further experiments with blasticidin S which strongly support the view that protein synthesis is required before synthesis of TMV-RNA occurs. MATERIALS

AND

METHODS

Virus and plants. Conditions for the growth of host plants, the use of “third” leaves infected with the Ul strain of TMV, as well as the technique of applying 32P, 3H-uracil, 14C-amino acids and antibiotics to the attached leaves have been previously described (A, Hirai and Wildman, 1967a, b). RNA extraction. A modification of the method of Sanger and Knight (1963) was employed. One gram of the “third” leaves was homogenized with 5 ml of glycine buffer (0.1 Mglycine, 0.1 M NaCI, 0.005 M EDTL4, adjusted to pH 9.5), 0.5 ml of 10 % sodium lauryl sulfate, $0 mg of bentonite, and 10 ml of water-saturated phenol using a Waring Blendor for 1 min. After the homogenate

T>IS’-TlNA

SYNTHESIS

CPM

AND BLASTICIDIN

647

S

CPI X10

a

0. 1000

CPM

,o

50t

‘-h -......_ I10 35 30 25 20 15 10 5 Fraction Number

fraction Number

FIG. 1. Separation of TMV-RNA from tobacco leaf RNA by sucrose density gradient centrifugation. 25 ml, 5-20y0 linear sucrose gradients in 0.05 M NaCl; centrifuged 16 hours at 24,000 rpm, SW 25.1 rotor. Sedimentation, right to left. (a) Mixture of 3H-TMV-RNA and nonradioactive healthy leaf RNA. (b) Mixture of nonradioactive TMV-RNA and 32Phealthy leaf RNA.

was centrifuged at 10,000 g for 10 min, the aqueous phase was removed and was constantly mixed for 10 min in a hypodermic syringe with 10 ml of water-saturated phenol. The mixture was centrifuged at 10,000 g for 10 min, and the aqueous phase was once more extracted by phenol. RNA in the third aqueous phase was precipitated with 2 volumes of ethanol. After standing for 4 hours at - 15”, the RNA was collected by centrifugation, resuspended in 0.4ml of 0.05 M NaCl, and twice dialyzed against 2 liters of 0.05 M NaCl. Sucrose density grandient centrifugation. Linear gradients of 5-20 ‘3%sucrose in 0.05 M NaC1 were formed with a perfusion pump at a rate of 0.5 ml/min and stabilized overnight at 5”. RNA in 0.4 ml of 0.05 M NaCl was layered on top of a 25-ml gradient, which was centrifuged at 24,000 rpm in the Spinco SW 25.1 rotor for 16 hours. Forty to 42 fractions were collected while monitoring the optical density at 260 rnp by the method used by Chen and Wildman (1967). Radioact’ivit’y in the fractions was determined by the procedure of Byfield and Scherbaum (1966).

EXPERIMENTAL

RESULTS

Resolution of TMV-RNA from Ribosomal RNA by Sucrose Density Gradients Two kinds of experiments showed that TMV-RNA can be resolved from tobacco leaf RNA by sucrose density gradients. In the first kind, nonradioactive TMV-RNA was mixed with 32P-healthy tobacco leaf RNA and the mixture was subjected t,o sucrose density gradient centrifugation with the results shown in Fig. lb. In the optical density profile, four clearly resolved peaks were apparent, but only t,hree peaks were radioactive, showing that the fastest-sedimenting component was T&IV-RNA. In the second kind of experiment, a small amount’ of 3H-TMV-RNA was mixed with unlabeled healthy tobacco leaf RNA. aft,er cent’rifug:ltion, the result,s shown in Fig. la were ohtained. Only three peaks appeared in the optical density profile, but the radioactivity separat.ed as a fast,er-sedimenting conlponent, showing TMV-RNA to consist’ of a single peak clearly resolved from ribosomnl RN.1.

648

HIRAI,

WILDMAN,

AND HIRAI

Recovery of “Free” TMV-RNA Extraction Procedure

CPM

by the RNA

To check whether the RNA extraction method would be capable of recovering “free”, single-stranded, TMV-RNA if it were present in the infected leaves, the following experiment was performed. Nonradioactive, TMV-infected leaves were subjected to the first step of blending for 10 sec. Then 32P-TMV-RNA (ea. lo5 cpm) was added to the blended material and blending was resumed for 60 set more. Total RNA was extracted and subjected to sucrose density gradient centrifugation. As shown by the data in Fig. 3, about 70 % of the added 321’-TMV-RNA was recovered in its native state, a result which provided confidence that extensive degradation of RNA did not occur during the extraction procedure. 40 35 30 25 20 15 10 5 Fraction Number

FIG. 2. Identification of newly synthesized, 82P-TMV-RNA in the presence of leaf RNA from infected leaves. One- and two-hour incorporation periods from separate gradients. TMV-RNA occurs between fractions 30 and 35.

Identijkation of Newly Synthesized TMVRNA in Total RNA Extracts of Tobacco Leaves To judge how small an amount of newly synthesized TMV-RNA could be detected by the sucrose density gradient method, 32P was supplied to “third” leaves 60 hours after TMV inoculation. One set of leaves was harvested 1 hour after 32P application while another set was harvested 2 hours after 32P treatment. Total leaf RNA was extracted from both sets of leaves and resolved by sucrose density gradient centrifugation with the results shown in Pig. 2. By 60 hours, enough TMV-RNA could be extracted from the infected leaves to produce a shoulder in the optical density profile between fractions 30 and 35. With a further l-hour synthesis of virus in the presence of 32P, the radioactivity measurements indicate the beginning of a 32P-TMV-RNA peak, which in 2 hours of synthesis is clearly resolved as 32P-TMV-RNA.

Inhibition

of Protein Synthesis by Blasticidin

S

Although blasticidin S had been reported to inhibit protein synthesis of a fungus (Huang et al., 1964), of bacterial and mammalian cells (Yamaguchi et aZ., 1965), and of TMV-infected tobacco leaves (T. Hirai and Shimomura, 1965), experiments were performed to ensure that the antibiotic inhibits protein synthesis under the conditions used in these experiments. In the first experiment, plants were separated into two groups immediately after inoculation with TMV. Blasticidin S (0.5 Hg/ml) together with a 14C-labeled amino acid mixture (10 yCi/ml) in a volume of 0.1 ml per plant was applied to “third” leaves of one group of plants and only the 14Clabeled amino acids to the other group. Twelve hours later, the leaves were harvested and homogenized by razor blade chopping; organelle fractions were separated. Hot TCA-insoluble radioactivity contained in the chloroplast-nuclear fraction (1000 8, crude mitochondrial fraction 10 min), (10,000 8, 15 min), cytoplasmic ribosome fraction (100,000 g, 120 min) and soluble protein fraction (100,000 g supernatant) was determined as previously described (Spencer and Wildman, 1964). The results of the analysis are shown in Table 1. Only a slight inhibition by blasticidin S of amino acid in-

TM\‘-RNA

SYNTHESIS

A?r’D BLASTTCIDW

f-b&

S

before a s-hour treat,ment# with the W-labeled amino acid mixture, a significant, inhibition of protein synthesis was detected.

corporation int’o protein was observed. However, when the experiment was repeated in the same way except that a 3-hour pretreatment with the antibiot,ic occurred

Infected Leaves

I

C.P.M. Just aft’er inoculation of leaves with TJLV, . ...... . plants were separated into two groups. For one group, 0.1 ml of blasticidin S (0.5 pg/ml) was supplied to the “third” leaves and Ivat er was supplied to the other group. Seventytwo hours after inoculation, 32P (50 &i:/ml, 0.3 ml) was supplied for 6 hours; then the RX.4 was extracted. The RX*1 w-as resolved by sucrose density gradient centrifugation (Fig. a), and analysis of t,he dist,ribut,ion of radioactivity showed t.hat the prrsencc of the antibiotic in the early stage of TMV infect’ion inhibited only TAIV-RSA synthesis, lvhile the host RNA synthesis was rather st’imulated compared to the control without the ant,ibioGc. The experiment w:w repeated with closely similar results.

1000

500

40

30

20

FRlCTlON

10

TOP

DISCUSSION

NUMBER

The dat’a in I;ig. 4 show specific inhibition of TI\IV-RXA synt’hesis by blast,icidin S whereas synthesis of host Rn’A was not, affected. Since even a short pretreatment of leaves with the ant’ibiotic caused extensive inhibition of protein sy&hesis, it is most

FIG. 3. Recovery of 32P-T112V-RNh added to partial homogenates of TM\‘-infected leaves. Total leaf RNA extracted with phenol and resolved by sucrose density gradient centrifugation. Counts per minute/O.1 ml of fraction. Recovery of radioactivity in gradient about 7091. TABLE

1

INHIBITION OF in Viino PROTEIN SYNTHETIC OF TMT~~-INFECPEDTOBACCO LEAVES BY BLASTICIDIN S

Control, acids

1%hour

treatment

with

1%Hour treatment

with

amino acids Percent inhibition

by blasticidin

l4C amino

blasticidin

S + W S

Control, 3-hour pretreatment with water followed by 5 hours with 1% amino acids 3-ITour pretreatment with blasticidin S followed by 5 hours with 1°C amino acids Percent inhibition by blasticidin S ’ \.alues

are expressed

Fraction-

-

Condition

as counts

per minute

__._

-_~.~.~__

1000 g pellet

10,000 g pellet

1W~ g pellet

53,477

8,880

8,530

35 ) 497

45 ) GM

8,545

8,465

X-3) 945

11.6

3.8

0.8

39,654

4,510

4,384

18,910

20,846

2,G57

3,562

11,581

47.4 per fraction.

41.1

19.9

Soluble protein

.--.---

4.4

38.8

_

650

HIRAI, CPM X10-’

i I

WILDMAN,

CON1

AND HIRAI CPM x10-

R it -CPM

iiI I i i

6

I

1 i,

,i 4 Fraction Number

Fraction Number

FIG. 4. Effect of blasticidin S on s2Pincorporation into TMV-RNA and host RNA in tobacco leaves. TMV-RNA peak is fraction 35. Right: with blasticidin S; left, without blasticidin S.

likely that TMV-RNA synthesis was inhibited because the antibiotic inhibited the synthesis of a protein or proteins necessary for viral RNA synthesis. Apparently, these proteins are different from host RNA polymerases which were already present and remained active in the presence of the antibiotic in the infected leaves. That the antibiotic is not a specific inhibitor of already formed enzymes required for TMV-RNA synthesis seems clear from previous results which showed that maximum inhibition of TMV multiplication was achieved only when the antibiotic was present at the early stage of infection (T. Hirai et al., 1966). If the antibiotic merely inhibited the activity of preformed enzyme, the extent of inhibition would be expected to be greater when the antibiotic was applied during the later stage of infection when the rate of TMV-RNA synthesis was maximal. Thus, it can be concluded that the synthesis of at least one kind of protein after infection is necessary for TMV-RNA biosynthesis.

ACKNOWLEDGMENTS We are indebted to Dr. K. K. Tewari for his advice and criticism, and t,o Dr. T. Misato for a generous supply of blasticidin S. REFERENCES BYFIELD, J., and SCHERBAUM,0. (1966). A rapid radioassay technique for cellular suspensions. Anal.

Biochem.

17, 434-443.

CHEN, J. L., and WILDMAN, S. G. (1967). Functional chloroplast polyribosomes from tobacco leaves. Science 155, 1271-1273. HIRAI, A., and WILDMAN, S. G. (1967a). Similarity in symptoms produced in tobacco plants by actinomycin D and TMV. Virology 31, 721722.

HIRAI, A., and WILDMAN, S. G. (1967b). Intracellular site of assembly of TMV-RNA and protein. Virology 33, 467473. HIRAI, T., and SHIMOMURA,T. (1965). Blasticidin S, an effective antibiotic against plant virus multiplication. Phytopalhology 55, 291-295. HIRAI, T., HIRASHIMA, A., ITOH, T., TAKAHASHI, T., SHIMOMURA, T., and HAYASNI, Y. (1966). Inhibitory effect of blasticidin S on tobacco mosaic virus multiplication. Phytopathology 56, 1236-1240.

TMV-RNA

SYNTHESIS

K. T., MISATO, T., and ASUYAMA, H. (1964). Effect of blasticidin S on protein synthesis of Piricularia oryzae. J. Antibiot. 17, G-70. SINGER, H. L., and KNIGHT, C. A. (1963). Action of actinomycin D on RNA synthesis in healthy and virus-infected tobacco leaves. Biochem. Biophys. Res. Commun. 13, 455-461. SPENCER, D., and WILDMBN, S. G. (1964). The

R~AKG,

SND BLASTICIDIN

S

6.51

incorporation of amino acids into protein bl cell free extracts from tobacco leaves. Biochemistry 3, 954-959. ~~~~~~~~~~~ H., YAMAMOTO, C., and TANAKA, N. (1965). Inhibition of protein synthesis by blasticidin S. I. Studies with cell-free system from bacterial and mammalian cells. J. Biochem. (Tokyo) R,667-677.