The Structure of Tobacco Mosaic Virus from Base Analogue Treated Tobacco Leaves

Transcription

1 Japan. J. Microbiol. Vol. 13 (2), , 1969 The Structure of Tobacco Mosaic Virus from Base Analogue Treated Tobacco Leaves Akira NOMURA, Takeshi TANIGUCHI, and Tokuzo HIRAI Plant Pathology Laboratory, Faculty of Agriculture, Nagoya University, Nagoya (Received for publication, February 28, 1969) ABSTRACT Structure of TMV containing the base analogues such as 2-thiouracil, 8-azaguanine, and 5-fluorouracil was studied. TMV containing these analogues had the same anionexchange chromatographic properties as had normal TMV, and 2-thiouracil was incorporated into TMV particles, probably into TMV-RNA. Cation-exchange chromatographic properties of TMV containing these analogues had no significant difference as compared with those of normal TMV. TMV containing 2-thiouracil was more stable to alkali degradation than normal TMV. Alkali-degraded TMV containing 2-thiouracil and normal TMV was characterized by chromatography. These results suggest that not surface structure but internal structure of TMV containing 2-thiouracil was different from that of normal TMV. It has been shown that 2-thiouracil (TU) [3], 8-azaguanine (AG) [11], and 5-fluorouracil (FU) [1, 14] are powerful inhibitors of tobacco mosaic virus (TMV) synthesis. These analogues were incorporated into virus ribonucleic acid (RNA), and TMV containing TU and AG had a lower infectivity than normal TMV [4-7, 10, 12]. It was suggested that TMV-RNA containing TU was unable to initiative virus replication [2]. The serological reactivity of TMV containing FU was not altered from normal TMV [15], although Jeener [9] reported that TU-containing TMV had a serologically altered coat protein. The composition of TMV protein after the incorporation of FU was analyzed by Holoubek [8]. However, the chemical structure of TMV containing the analogues remains obscure. This paper describes the structure of TMV containing the analogues as determined by chromatography and alkali-degradation methods. MATERIALS AND METHODS TMV inoculation. Leaves of Nicotiana tabacum L. var. Bright Yellow were inoculated with 0.1 mg/ml of the ordinary strain of TMV. After washing with water, the leaves were kept under the continuous illumination of 6,000-7,000 lux from fluorescent lamps at 27 C. Twenty four hours (or immediately in the case of 8-azaguanine treatment) after inoculation, the leaves were divided into two halves by cutting the midribs. One half was floated on distilled water and the other half on the solution of 1.4 ~10-4 M TU, 0.1 M AG, or 7.8 ~10-3 M FU, respectively. After 6-8 days, the leaves were harvested, washed, and stored at -20 C until the use. Isolation of virus. The frozen leaves 193

2 194 A. NOMURA, T. TANIGUCHI AND T. HIRAI were thawed and homogenized with distilled water. The homogenate was clarified by one of the following two methods: (1) Virus was isolated by several cycles of low and high speed centrifugation. (2) Three volumes of clarified sap were mixed with one volume of saturated ammonium sulfate and centrifuged at 15,000 rpm for 20 min. The precipitate was resuspended in water and centrifuged. The supernatant was ultracentrifuged at 40,000 rpm for 1 hr. Chromatography on ion-exchange columns. Chromatography on ECTEOLAcellulose columns and on carboxymethylcellulose (CMC) columns was carried out as described by Taniguchi [16, 17]. Sucrose density gradient centrifugation. Test sample was layered onto the density gradient columns consisting of 10-35% (w/w) sucrose solution in distilled water. The tubes were centrifuged at 24,000 rpm for 150 min in SW 25 rotor in the Beckman Spinco model L2 centrifuge. After centrifugation a sample was fractionated into approximately 38 fractions and the optical density at 260 mĐ was measured. Alkali-degradation procedure. One ml of TMV solution (3 mg/ml) obtained from the leaves treated with the analogues was adjusted to ph 10.0 with 0.03 M sodium borate buffer (ph 10.0) [16] and incubated at 0 C. The solution was then analyzed by the following methods. (1) Each solution was adjusted to ph 7.0 by adding 1 ml of 0.1 N HCl and 2 ml of 0.1 M sodium phosphate buffer (ph 7.0). It was centrifuged at 40,000 rpm for 1 hr and the optical density of the supernatant was measured at 260 mĐ. (2) Each solution was adjusted to ph 7.0 and placed on the top of ECTEOLA-cellulose column. (3) Each solution was placed on the top of density gradient centrifugation tube prepared by the method described herein. Radioactivity assay. To 4 ml of each fraction eluted from the column, 1 mg purified bovine serum albumin was added as a carrier. It was incubated at 0 C for about 30 min with 5% trichloroacetic acid (final concentration). The suspension was centrifuged at 1,000 g for 10 min, the pellet obtained was dissolved in 1 N NH4OH and dried in a metal planchette. The radioactivity of the sample was measured by using an automatic thin window gasflow counter. RESULTS AND DISCUSSION Ion Exchange Chromatography of TMV from Base Analogue Treated Tobacco Leaves TMV-inoculated leaves were divided into two halves by cutting the midribs 24 hr after inoculation, and 30 disks (12 mm in diameter) were punched from the interveinal areas. The disks were floated on the solution containing 7 ~10-5 M TU-14C (specific activity: 200 Đc/Đm). Leaf-disks from the other halves were floated on water as a control. Virus was isolated from disks after addition of carrier normal TMV. TU-14Clabeled TMV, containing carrier normal TMV, was fractionated by ECTEOLA-cellulose column and the results show that TMV containing TU is not different in anionic properties from normal TMV (Fig. 1). Chromatographic analysis of TMV obtained from AG- and FU-treated tobacco leaves also indicate the same chromatographic properties as that of normal TMV. TU-14C-labeled TMV, containing normal TMV as a carrier, was fractionated by CMC column. The result indicated that cation exchange chromatographic properties of TMV containing TU had no significant difference from normal TMV (Fig. 2). Jeener [9] reported that the virus containing TU, as well as normal virus, showed a single narrow band in density gradient centrifugation pattern and the both prepa-

3 STRUCTURE OF BASE ANALOGUE TREATED TMV 195 structure of TMV was not altered by the analogue incorporation. The replacement of a few uridylic acid residues in TMV- RNA appeared to modify the array of antigenic determinants which exist on many of the protein subunits [9], however. If this is in case, the surface structure of TMV-containing analogues may also be altered, but this alteration could not be detected with the present chromatographic procedures. Fig. 1. Chromatography fo TU-14C labeled TMV on a ECTEOLA-cellulose column (1 ~12 cm). Open circles show O.D. 260 mĐ of normal TMV which was added as a carrier. Solvent for adsorption : 0.01 M ph 7.0 sodium phosphate buffer. Elution: Salt concentration of the eluent was gradually raised. Fraction volume: 4m1. Resistance to Alkali Degradation of TMV from Analogue-Treated Tobacco Leaves TMV from TU-treated tobacco leaves (TU-TMV) and normal TMV was treated with alkali (ph 10.0) at 0 C for 1 hr. Schramm reported that TMV was degraded at ph 9.2 [13], and we also found that TMV degraded moderately at ph Each solution was adjusted to ph 7.0 and centrifuged at 40,000 rpm for 1 hr. Optical density of the supernatant was measured at 260 mĐ. Results indicated TU-TMV was more stable to alkali than was normal TMV (Table 1). Duncan's multiple range test was used to determine the statistical significance of differences between treat- Table 1. Resistance to alkali degradation of TMV from TU-treated tobacco leaves and of normal TMV Fig. 2. Chromatography of 2-thiouracil-2-14C labeled TMV on a carboxymethylcellulose column (1 ~12 cm). Open and closed circles show radioactivity and optical density at 260 mĐ, respectively, as shown in Fig. 1. Solvent for adsorption: ph 3.0 citrate-hci buffer. Elution: ph of the eluent was gradually raised. Fraction volume: 4ml. rations had the same RNA content. The particles under electronmicroscope were identical in appearance and dimensions. The present results show that the surface TMV from TU-treated tobacco leaves and normal TMV was treated with alkali (ph 10.0) at 0 C for 1 hr. Each solution was adjusted to ph 7.0 as described in the text and centrifuged at 40,000 rpm for 1 hr. Optical density of the supernatant was measured. Each value shows the mean of 9 independent values. Value of (a) differs significantly from value of (b) at P=0.05.

4 196 A. NOMURA, T. TANIGUCHI AND T. HIRAI ment means with each experiment. This suggests that the bonding force, which combines the protein subunits with each other or with the nucleic acid, of TU-TMV was different from normal TMV. TMV obtained from AG-treated tobacco leaves was treated with alkali as stated before. The result was variable, probably due to the fact that the rate of incorporation of the analogue into TMV-RNA varied with each incorporation experiment. Characterization of Alkali-Degraded-TU- TMV Chromatography on ECTEOLA-cellulose column of alkaline treated normal TMV (ph 10.0 at 0 C for 1 hr) showed that the component which corresponds to whole virus decreased and new components (designated as P1 and P3 in Fig. 3 right) appeared. The ratios of absorbancies at 260: 280 mĐ of the components P1, P2 and P3 were , 1.2 and 1.5 respectively and Fig. 3 Chromatography of normal TMV (N-TMV) and TMV from TU-treated tobacco leaves (TU-TMV) on ECTEOLA-cellulose columns. Both TMV preparations were treated with alkali (ph 10.0) at 0 C for 1 hr. Column procedures were identical to those described in Fig. 1. Fig. 4. Pattern of density-gradient columns after centrifugation for 150 min at 24,000 rpm in the SW-25 rotor of the Spinco model L2 centrifuge. N-TMV and TU-TMV were layered onto the gradient column prior to centrifugation. Both solutions were treated with alkali (ph 10.0) at 0 C for 30 min before centrifugation.

5 STRUCTURE OF BASE ANALOGUE TREATED TMV 197 the ratio of normal TMV not treated with alkali was 1.2. In the case of TU-TMV, the middle component (P2 in Fig. 3) decreased less than did the corresponding component of normal TMV. TU-TMV as well as the normal virus showed a single narrow band in sucrose density gradient centrifugation. By alkaline treatment of normal TMV at 0 C for 30 min before centrifugation, a portion of the component which corresponds to whole virus decreased and new components (designated as P1, P3, and P4 in Fig. 4 right) appeared. In the case of TU-TMV, the component P2 less decreased than did the corresponding component in normal TMV. These facts, together with the results shown in. Table 1, show that TU-TMV is more stable than the normal TMV. These results suggest that not surface structures but the internal structures of TU-TMV are different from the normal TMV. REFERENCES [1] Davern, C. I., and Bonner, James The influence of 5-fluorouracil on tobaccomosaic virus production in tobacco-leaf discs. Biochim. Biophys. Acta 29: [2] Francki, R. I. B Infectivity of ribonucleic acid from tobacco mosaic virus containing 2-thiouracil. Virology 10: [3] Francki, R. I. B The inhibition of plant virus multiplication in two host species by 2-thiouracil. Virology 17: 1-8. [4] Francki, R. I. B Infectivity of tobacco mosaic virus from tobacco leaves treated with 2-thiouracil. Virology 17: [5] Francki, R. I. B., and Matthews, R. E. F Effect of 2-thiouracil on the infectivity of tobacco mosaic virus. Biochim. Biophys. Acta 34: [6] Gordon, M. P., and Staehelin, M The incorporation of 5-fluorouracil into the nucleic acid of tobacco mosaic virus. J. Amer. Chem. Soc. 80: [7] Gordon, M. P., and Staehelin, M Studies on the incorporation of 5-fluorouracil into virus nucleic acid. Biochim. Biophys. Acta 36: [8] Holoubek, V The composition of tobacco mosaic virus protein after the incorporation of 5-fluorouracil into the virus. J. Mol. Biol. 6: [9] Jeener, R Effect on the antigenic determinants of tobacco mosaic virus of a small number of base replacements in the ribonucleic acid. Virology 26: [10] Jeener, R., and Rossels, J Incorporation of 2-thiouracil-35S in the ribose nucleic acid of tobacco mosaic virus. Biochim. Biophys. Acta 11: 438. [11] Lindner, R. C., Cheo, P. C., Kirkpatric, H. C., and Govindu, H. C Some effects of 8-azaguanine on tobacco mosaic virus replication. Phytopathology 50: [12] Matthews, R. E. F Incorporation of 8-azaguanine into nucleic acid of tobacco mosaic virus. Nature 171: [13] Schramm, G., Schumacher, G., and Zillig, W Uber die Struktur des Tabakmosaikvirus III. Mitt,: Der Zerfall in lakalischer Losung. Z. Naturforsch. 10: [14] Staehelin, M., and Gordon, M. P Effects of halogenated pyrimidines on the growth of tobacco mosaic virus. Biochim. Biophys. Acta 38: [15] Sutic, D Effect of 5-fluorouracil on antigenic properties of tobacco mosaic virus. Nature 203: [16] Taniguchi, T The accumulation in plants of chromatographically separated components of tobacco mosaic virus. Virology 22: [17] Taniguchi, T Chromatography of a mixture of tobacco mosaic virus and ribonuclease. Virology 30: