VIROLOGY
76,
Polarity
429-432
(1977)
of Stripping
of Tobacco Mosaic Virus by Alkali and Sodium Dodecyl Sulfate
TAKESHI Department
ofBiophysics
and
OHNO Biochemistry, Tokyo Accepted
Polar treatment
stripping occurs
of coat protein from the 5’-end
YOSHIMI
AND
Faculty of Science, 113, Japan August
of Tokyo,
Bunkyo-ku,
23,1976 sulfate
Tritium-labeled TMV-RNA was prepared by periodate oxidation and subsequent reduction with tritium-labeled sodium borohydride (9). By this treatment, both the 5’- and 3’-ribosyl ends of TMVRNA were specifically labeled by tritium (10). The m7G at the 5’-terminal blocked structure was also reduced by sodium borohydride. However, 7-methyl-8-hydro13Hlguanosine was reoxidized in aqueous solution (111, and the C-8 tritium proton of m7G was exchanged with a proton of the solvent (12 1. Analysis by phosphocellulose (P-cellulose) column chromatography (13 1 after alkaline hydrolysis (0.3 N KOH, 37”, 18 hr) of tritium-labeled TMV-RNA showed that about half of the radioactivity was found as m’G’pppGp from the 5’-terminal blocked structure and the other half was found as A’ from the 3’-terminal structure (Fig. 2A) (A’ and G’ represent the nucleoside trialcohol of A and G, respectively). Tritium-labeled TMV-RNA was reconstituted with TMV-protein in 0.1 M phosphate buffer (pH 7.2) at 25” overnight, and reconstituted particles were purified by sucrose density gradient centrifugation and subsequent ultracentrifugation (Fig. 1A). Alkaline degradation of the TMV particles was carried out by dialysis against 0.01 M borate buffer (pH 10.5) at 4” for 24 hr according to the method of Perham (14). After digestion with ribonuclease A to remove the exposed RNA tail, the sample was centrifuged on a sucrose density
429 Q 1977 by Academic Press, Inc. of reproduction in any form reserved.
University
of TMV by both alkaline and sodium dodecyl of the RNA, contrary to previous conclusions.
It has been reported that coat protein is stripped from the 3’-end of the RNA of tobacco mosaic virus (TMV) by sodium dodecyl sulfate (SDS) (I). This conclusion was based on the experiments using two kinds of phosphodiesterases. That is, treatment of partially stripped virus with snake venom phosphodiesterase led to loss of infectivity, but spleen phosphodiesterase did not (I). The polarity of stripping under mild alkaline conditions was also reported to proceed in the same direction as stripping by SDS (2). However, the recent report that TMV-RNA has the blocked structure m7G5’ppp5’Gp as the 5’terminus (3, 4) makes it necessary to reevaluate the experiments using these phosphodiesterases for the determination of the polarity of stripping of TMV. Because spleen phosphodiesterase does not digest the blocked structure at the 5’-terminus (5, 61, and venom phosphodiesterase releases not only the 3’-terminal nucleotide but also the 5’-terminal blocking structure, pm7G (3, 71, we reinvestigated the polarity of stripping of coat protein from TMV by alkali and SDS, using particles reconstituted from TMV-RNA labeled with tritium at both ends. TMV, Japanese common strain OM (81, was purified by polyethylene glycol precipitation and differential centrifugation. TMV-protein and RNA were isolated by acetic acid method and phenol-bentonite extraction, respectively. Copyright All rights
OKADA
SHORT
(A)
COMMUNICATIONS
1 . ;’ : 1
covered from the stable fragment and another half from the ribonuclease-sensitive fraction (supernatant fraction). The stable fragment and the supernatant fraction were collected and hydrolyzed in 0.3 N KOH at 37” for 18 hr and analyzed on Pcellulose chromatography, respectively. From the stable fragment fraction only an adenosine trialcohol originating from the 3’-end of TMV-RNA was recovered as radioactive material (Fig. 2B), while the supernatant fraction contained primarily
(A)
Fraction
Number
FIG. 1. Sedimentation profile of 3H-labeled reconstituted TMV and its degraded intermediates. (A) 3H-labeled reconstituted TMV. (B) Alkaline degraded intermediates. The 3H-labeled reconstituted TMV (6 mg/ml) was dialyzed against 0.01 M borate buffer (pH 10.5) at 4” for 24 hr. After digestion with ribonuclease A (2 pg/ml) in 50 mM Tris-HCl (pH 7.4) at 37” for 30 min, the sample was centrifuged. (Cl Virus partially stripped by SDS treatment. The 3H-labeled TMV (6 mg/ml) was shaken in 0.1% SDS (in 1 mM EDTA, pH 7.5) at 37” for 16 min. The reaction was stopped by the addition of tenfold volume of 0.25 M KC1 (in 1 n&f EDTA, pH 7.5). After low speed centrifugation (10,000 g for 20 min), the supernatant was ultracentrifuged (100,000 g for 2 hr). The pelleted, partially stripped virus was digested with ribonuclease A as described in (B). All the samples were centrifuged at 24,000 rpm on 520% sucrose density gradient in 10 n&f Tris-HCl (pH 7.4) for 2 hr at 4” in a SW-25-1 swinging bucket rotor of a Hitachi ultracentrifuge. Sedimentation is from right to left. Arrows show the position of native TMV. l - - -0 Absorbance; O-O radioactivity.
gradient. The absorbancy profile in Fig. 1B showed two sedimenting fractions, a small alkaline stable fragment and alkaliresistant particles that sedimented to the same position as undegraded virus. About half of the radioactivity was re-
Fraction
Number
FIG. 2. Phosphocellulose column chromatography of alkali hydrolysates of (A) 3H-labeled TMVRNA, (B) alkali-stable fragment (Fig. lB), (Cl supernatant fraction (Fig. lB), (D) intermediate rodlet obtained by SDS treatment (Fig. 10, and (E) supernatant fraction (Fig. 10. Each fraction separated as described in the legend of Fig. 1 was hydrolyzed in 0.3 N KOH at 37” for 18 hr. After neutralization with 5% HClO, and subsequent centrifugation (10,000 g for 10 min), the supernatant added with four nucleoside trialcohols (A’, G’, C’, U’) as internal markers, was chromatographed on P-cellulose column which was equilibrated with 10 m&f HCOONH, (pH 3.85). The column was washed with the same buffer, then eluted with the linear concentration gradient from 10 mM to 0.4 M HCOONH, (pH 3.85). l - - -0 Absorbance; O-O radioactivity.
SHORT
m’G’pppGp and trace amounts of A’ (Fig. 20. The distribution of radioactivity of both terminals in the stable fragment and the supernatant fraction is summerized in Table 1. These results indicate that alkaline stripping of TMV starts not from the 3’-end but from the 5’-end of RNA, contrary to the conclusion previously reported (2). To confirm the direction of the polar stripping of TMV by SDS, a degradation experiment was conducted employing the method of May and Knight (1). The “Hlabeled reconstituted TMV was shaken in 0.1% SDS containing 1 n&f EDTA at 37” for 5 or 16 min. The reaction was stopped by the addition of KCl. From the supernatant, partially stripped virus was collected by ultracentrifugation. The partially stripped virus was digested with ribonuclease A and separated by sucrose density gradient centrifugation (Fig. 1C). The alkali hydrolysate from the rod fragment or from the ribonuclease-sensitive fraction was analyzed on P-cellulose as described above (Fig. 2D and E). Radioactive A’ was recovered primarily from the rod fragment, while the radioactive 5’-terminus (m7G’pppGp) in the rodlet decreased with time of SDS treatment (Table 1). This indicates that the stripping of TMV by SDS also starts from the 5’-end of RNA. From these results we concluded that the direction of stripping of protein from TMV is from the 5’- to the 3’-end of RNA both under alkaline and SDS treatments. TABLE DISTRIBUTION
Dege;ro20n
1
OF TERMINAL
RADIOACTIVITY
Fraction
Radioactivity (cpm) 5’-end
Alkali and RNase SDS (5 min) and RNase SDS (16 min) and RNase
Stable fragment Supernatant Intermediate rodlet” Supernatant Intermediate rodlet Supernatant
r( The intermediate rodlet obtained ment for 5 min sedimented in about tion as native TMV.
431
COMMUNICATIONS
3’-end
9 1384 112
1766 147 646
365 35
33 577
645
36
by SDS treatthe same posi-
During the preparation of this manuscript, a paper has appeared (15) on the direction of the polar stripping of TMV under alkaline conditions. Our conclusion is in agreement with their results, which were done on native TMV. The agreement between the two laboratories indicated that the polar nature of our reconstituted TMV was identical with the native TMV. Recently, we have shown that the 5’terminal blocking structure of TMV-RNA is essential for infectivity (IO). The RNA tail with the 5’-terminal blocked structure protruding from the partially stripped virus is insensitive to spleen phosphodiesterase, while the venom phosphodiesterase releases pm7G from the 5’-terminus and leads to the loss of infectivity. This is the reason May and Knight concluded a 3’ -+ 5’ direction for the polar stripping of TMV. Several experiments (16-19) published previously depended on the assumption that the stripping of TMV by alkali or SDS occurs from the 3’-end of RNA. The result, presented here may make it necessary to reconsider the conclusions of some of these studies. After submitting this manuscript, a paper appeared (20) on the polarity of stripping of native TMV in SDS. Both laboratories agree that stripping is 5’ -+ 3’, although the methodology employed is different in the two studies. ACKNOWLEDGMENTS We thank Drs. Y. Nozu and H. Inoue, Institute for Plant Virus Research, for kindly supplying TMV, and Miss M. Takahashi for her technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and a grant from the Toray Science Foundation. REFERENCES 1. MAY, D. S., and KNIGHT, C. A., Virology 25, 502-507 (1965). 2. ONDA, H., TANIGUCHI, T., and MATSUI, C., Virology 42, 551-553 (1970). 3. ZIMMERN, D., Nucleic Acid Res. 2, 1189-1201 (1975). 4. KEITH, J., and FRAENKEL-CONRAT, H., FEBS Lett. 57, 31-33 (1975). 5. WEI, C. M., and Moss, B., Proc. Nat. Acad. Sci. USA 72, 318-322 (1975). 6. REDDY, T., Ro-CHOI, T-S., HENNING, D., and
432
SHORT
COMMUNICATIONS
Busch, M., J. Biol. Chem. 249, 6486-6494 (1974). 7. FURUICHI, Y., and MIURA, K., Nature (London) 253, 374-375 (1975). 8. Nozu, Y., and OKADA, Y., J. Mol. Biol. 35,643646 (1968). 9. RAJ BHANDARY, U. L., J. Biol. Chem. 243, 556564 (1968). 10. OHNO, T., OKADA, Y., SHIMOTOHNO, K., MIURA, K., SHINSHI, H., MIWA, M., and SUGIMURA, T., FEBS Lett. (in press). Il. POCHON, F., PASCAL, Y., PITHA, P., and MICHELSON, A. M., Biochim. Biophys. Acta 213, 273281 (1970). 12. TOMASZ, M., Biochim. Biophys. Acta 199, 18-28 (1970).
13. LEWANDOWSKI, L. J., CONTENT, J., and LEPPLA, S. H., J. Virol. 8, 701-707 (1971). 14. PERHAM, R. N., J. Mol. Biol. 45,439-441 (1969). 15. PERHAM, R. N., and WILSON, T. M. A., FEBS Lett. 62, 11-15 (1976). 16. MANDELES, S. J. Biol. Chem. 243, 3671-3684 17.
(1968). KAM), C.
I.,
and
18. KADO, C. I., and KNIGHT, 15-23
(1968).
19. HASHIMOTO, 20.
C. A. Proc. Nat. (1966). C. A., J. Mol. Biol. 36,
KNIGHT,
Acad. Sci. 55, 1276-1283
J., and OKAMOTO, K., Virology 67, 107-113 (1975). WIISON, T. M. A., PERHAM, R. N., FINCH, J. T., and BUTLER, P. J. G., FEBS Lett. 64, 285-289 (1976).
76,
Polarity
429-432
(1977)
of Stripping
of Tobacco Mosaic Virus by Alkali and Sodium Dodecyl Sulfate
TAKESHI Department
ofBiophysics
and
OHNO Biochemistry, Tokyo Accepted
Polar treatment
stripping occurs
of coat protein from the 5’-end
YOSHIMI
AND
Faculty of Science, 113, Japan August
of Tokyo,
Bunkyo-ku,
23,1976 sulfate
Tritium-labeled TMV-RNA was prepared by periodate oxidation and subsequent reduction with tritium-labeled sodium borohydride (9). By this treatment, both the 5’- and 3’-ribosyl ends of TMVRNA were specifically labeled by tritium (10). The m7G at the 5’-terminal blocked structure was also reduced by sodium borohydride. However, 7-methyl-8-hydro13Hlguanosine was reoxidized in aqueous solution (111, and the C-8 tritium proton of m7G was exchanged with a proton of the solvent (12 1. Analysis by phosphocellulose (P-cellulose) column chromatography (13 1 after alkaline hydrolysis (0.3 N KOH, 37”, 18 hr) of tritium-labeled TMV-RNA showed that about half of the radioactivity was found as m’G’pppGp from the 5’-terminal blocked structure and the other half was found as A’ from the 3’-terminal structure (Fig. 2A) (A’ and G’ represent the nucleoside trialcohol of A and G, respectively). Tritium-labeled TMV-RNA was reconstituted with TMV-protein in 0.1 M phosphate buffer (pH 7.2) at 25” overnight, and reconstituted particles were purified by sucrose density gradient centrifugation and subsequent ultracentrifugation (Fig. 1A). Alkaline degradation of the TMV particles was carried out by dialysis against 0.01 M borate buffer (pH 10.5) at 4” for 24 hr according to the method of Perham (14). After digestion with ribonuclease A to remove the exposed RNA tail, the sample was centrifuged on a sucrose density
429 Q 1977 by Academic Press, Inc. of reproduction in any form reserved.
University
of TMV by both alkaline and sodium dodecyl of the RNA, contrary to previous conclusions.
It has been reported that coat protein is stripped from the 3’-end of the RNA of tobacco mosaic virus (TMV) by sodium dodecyl sulfate (SDS) (I). This conclusion was based on the experiments using two kinds of phosphodiesterases. That is, treatment of partially stripped virus with snake venom phosphodiesterase led to loss of infectivity, but spleen phosphodiesterase did not (I). The polarity of stripping under mild alkaline conditions was also reported to proceed in the same direction as stripping by SDS (2). However, the recent report that TMV-RNA has the blocked structure m7G5’ppp5’Gp as the 5’terminus (3, 4) makes it necessary to reevaluate the experiments using these phosphodiesterases for the determination of the polarity of stripping of TMV. Because spleen phosphodiesterase does not digest the blocked structure at the 5’-terminus (5, 61, and venom phosphodiesterase releases not only the 3’-terminal nucleotide but also the 5’-terminal blocking structure, pm7G (3, 71, we reinvestigated the polarity of stripping of coat protein from TMV by alkali and SDS, using particles reconstituted from TMV-RNA labeled with tritium at both ends. TMV, Japanese common strain OM (81, was purified by polyethylene glycol precipitation and differential centrifugation. TMV-protein and RNA were isolated by acetic acid method and phenol-bentonite extraction, respectively. Copyright All rights
OKADA
SHORT
(A)
COMMUNICATIONS
1 . ;’ : 1
covered from the stable fragment and another half from the ribonuclease-sensitive fraction (supernatant fraction). The stable fragment and the supernatant fraction were collected and hydrolyzed in 0.3 N KOH at 37” for 18 hr and analyzed on Pcellulose chromatography, respectively. From the stable fragment fraction only an adenosine trialcohol originating from the 3’-end of TMV-RNA was recovered as radioactive material (Fig. 2B), while the supernatant fraction contained primarily
(A)
Fraction
Number
FIG. 1. Sedimentation profile of 3H-labeled reconstituted TMV and its degraded intermediates. (A) 3H-labeled reconstituted TMV. (B) Alkaline degraded intermediates. The 3H-labeled reconstituted TMV (6 mg/ml) was dialyzed against 0.01 M borate buffer (pH 10.5) at 4” for 24 hr. After digestion with ribonuclease A (2 pg/ml) in 50 mM Tris-HCl (pH 7.4) at 37” for 30 min, the sample was centrifuged. (Cl Virus partially stripped by SDS treatment. The 3H-labeled TMV (6 mg/ml) was shaken in 0.1% SDS (in 1 mM EDTA, pH 7.5) at 37” for 16 min. The reaction was stopped by the addition of tenfold volume of 0.25 M KC1 (in 1 n&f EDTA, pH 7.5). After low speed centrifugation (10,000 g for 20 min), the supernatant was ultracentrifuged (100,000 g for 2 hr). The pelleted, partially stripped virus was digested with ribonuclease A as described in (B). All the samples were centrifuged at 24,000 rpm on 520% sucrose density gradient in 10 n&f Tris-HCl (pH 7.4) for 2 hr at 4” in a SW-25-1 swinging bucket rotor of a Hitachi ultracentrifuge. Sedimentation is from right to left. Arrows show the position of native TMV. l - - -0 Absorbance; O-O radioactivity.
gradient. The absorbancy profile in Fig. 1B showed two sedimenting fractions, a small alkaline stable fragment and alkaliresistant particles that sedimented to the same position as undegraded virus. About half of the radioactivity was re-
Fraction
Number
FIG. 2. Phosphocellulose column chromatography of alkali hydrolysates of (A) 3H-labeled TMVRNA, (B) alkali-stable fragment (Fig. lB), (Cl supernatant fraction (Fig. lB), (D) intermediate rodlet obtained by SDS treatment (Fig. 10, and (E) supernatant fraction (Fig. 10. Each fraction separated as described in the legend of Fig. 1 was hydrolyzed in 0.3 N KOH at 37” for 18 hr. After neutralization with 5% HClO, and subsequent centrifugation (10,000 g for 10 min), the supernatant added with four nucleoside trialcohols (A’, G’, C’, U’) as internal markers, was chromatographed on P-cellulose column which was equilibrated with 10 m&f HCOONH, (pH 3.85). The column was washed with the same buffer, then eluted with the linear concentration gradient from 10 mM to 0.4 M HCOONH, (pH 3.85). l - - -0 Absorbance; O-O radioactivity.
SHORT
m’G’pppGp and trace amounts of A’ (Fig. 20. The distribution of radioactivity of both terminals in the stable fragment and the supernatant fraction is summerized in Table 1. These results indicate that alkaline stripping of TMV starts not from the 3’-end but from the 5’-end of RNA, contrary to the conclusion previously reported (2). To confirm the direction of the polar stripping of TMV by SDS, a degradation experiment was conducted employing the method of May and Knight (1). The “Hlabeled reconstituted TMV was shaken in 0.1% SDS containing 1 n&f EDTA at 37” for 5 or 16 min. The reaction was stopped by the addition of KCl. From the supernatant, partially stripped virus was collected by ultracentrifugation. The partially stripped virus was digested with ribonuclease A and separated by sucrose density gradient centrifugation (Fig. 1C). The alkali hydrolysate from the rod fragment or from the ribonuclease-sensitive fraction was analyzed on P-cellulose as described above (Fig. 2D and E). Radioactive A’ was recovered primarily from the rod fragment, while the radioactive 5’-terminus (m7G’pppGp) in the rodlet decreased with time of SDS treatment (Table 1). This indicates that the stripping of TMV by SDS also starts from the 5’-end of RNA. From these results we concluded that the direction of stripping of protein from TMV is from the 5’- to the 3’-end of RNA both under alkaline and SDS treatments. TABLE DISTRIBUTION
Dege;ro20n
1
OF TERMINAL
RADIOACTIVITY
Fraction
Radioactivity (cpm) 5’-end
Alkali and RNase SDS (5 min) and RNase SDS (16 min) and RNase
Stable fragment Supernatant Intermediate rodlet” Supernatant Intermediate rodlet Supernatant
r( The intermediate rodlet obtained ment for 5 min sedimented in about tion as native TMV.
431
COMMUNICATIONS
3’-end
9 1384 112
1766 147 646
365 35
33 577
645
36
by SDS treatthe same posi-
During the preparation of this manuscript, a paper has appeared (15) on the direction of the polar stripping of TMV under alkaline conditions. Our conclusion is in agreement with their results, which were done on native TMV. The agreement between the two laboratories indicated that the polar nature of our reconstituted TMV was identical with the native TMV. Recently, we have shown that the 5’terminal blocking structure of TMV-RNA is essential for infectivity (IO). The RNA tail with the 5’-terminal blocked structure protruding from the partially stripped virus is insensitive to spleen phosphodiesterase, while the venom phosphodiesterase releases pm7G from the 5’-terminus and leads to the loss of infectivity. This is the reason May and Knight concluded a 3’ -+ 5’ direction for the polar stripping of TMV. Several experiments (16-19) published previously depended on the assumption that the stripping of TMV by alkali or SDS occurs from the 3’-end of RNA. The result, presented here may make it necessary to reconsider the conclusions of some of these studies. After submitting this manuscript, a paper appeared (20) on the polarity of stripping of native TMV in SDS. Both laboratories agree that stripping is 5’ -+ 3’, although the methodology employed is different in the two studies. ACKNOWLEDGMENTS We thank Drs. Y. Nozu and H. Inoue, Institute for Plant Virus Research, for kindly supplying TMV, and Miss M. Takahashi for her technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and a grant from the Toray Science Foundation. REFERENCES 1. MAY, D. S., and KNIGHT, C. A., Virology 25, 502-507 (1965). 2. ONDA, H., TANIGUCHI, T., and MATSUI, C., Virology 42, 551-553 (1970). 3. ZIMMERN, D., Nucleic Acid Res. 2, 1189-1201 (1975). 4. KEITH, J., and FRAENKEL-CONRAT, H., FEBS Lett. 57, 31-33 (1975). 5. WEI, C. M., and Moss, B., Proc. Nat. Acad. Sci. USA 72, 318-322 (1975). 6. REDDY, T., Ro-CHOI, T-S., HENNING, D., and
432
SHORT
COMMUNICATIONS
Busch, M., J. Biol. Chem. 249, 6486-6494 (1974). 7. FURUICHI, Y., and MIURA, K., Nature (London) 253, 374-375 (1975). 8. Nozu, Y., and OKADA, Y., J. Mol. Biol. 35,643646 (1968). 9. RAJ BHANDARY, U. L., J. Biol. Chem. 243, 556564 (1968). 10. OHNO, T., OKADA, Y., SHIMOTOHNO, K., MIURA, K., SHINSHI, H., MIWA, M., and SUGIMURA, T., FEBS Lett. (in press). Il. POCHON, F., PASCAL, Y., PITHA, P., and MICHELSON, A. M., Biochim. Biophys. Acta 213, 273281 (1970). 12. TOMASZ, M., Biochim. Biophys. Acta 199, 18-28 (1970).
13. LEWANDOWSKI, L. J., CONTENT, J., and LEPPLA, S. H., J. Virol. 8, 701-707 (1971). 14. PERHAM, R. N., J. Mol. Biol. 45,439-441 (1969). 15. PERHAM, R. N., and WILSON, T. M. A., FEBS Lett. 62, 11-15 (1976). 16. MANDELES, S. J. Biol. Chem. 243, 3671-3684 17.
(1968). KAM), C.
I.,
and
18. KADO, C. I., and KNIGHT, 15-23
(1968).
19. HASHIMOTO, 20.
C. A. Proc. Nat. (1966). C. A., J. Mol. Biol. 36,
KNIGHT,
Acad. Sci. 55, 1276-1283
J., and OKAMOTO, K., Virology 67, 107-113 (1975). WIISON, T. M. A., PERHAM, R. N., FINCH, J. T., and BUTLER, P. J. G., FEBS Lett. 64, 285-289 (1976).
