VIROLOGY
70,214-216
Biochemical
MARIKO Department
(1976)
Characterization
KURISU, of Biophysics
of Cucumber Green Mottle Mosaic Virus Ribonucleic Acid
TAKESHI
OHNO,
YOSHIMI
OKADA,
AND
YUZO
NOZU
and Biochemistry, Faculty of Science, University of Tokyo, Tokyo, Japan, and Institute for Plant Virus Research, Chiba, Japan Accepted October 21, 1975
CGMMV-RNA has the base composition of 20.6% C, 24.6% A, 31.6% U, and 23.2% Gin molar percent. The 3’-terminal nucleoside is A. The 5’-terminus of CGMMV-RNA is blocked, as appears to be the case for TMV-RNA. CGMMV-RNA, as well as TMV-RNA, is able to be reconstituted effectively with either CGMMV-protein or TMV-protein in vitro.
Chemical and immunological properties of the protein of cucumber green mottle mosaic virus (CGMMV) were reported by Nozu et al. (I), and it was confirmed that CGMMV has an ancestral relationship to tobacco mosaic virus (TMV). Ohashi et al. f2) previously reported that TMV-RNA was able to be reconstituted into an infectious particle. Considerable progress has been made in understanding the assembly mechanism of TMV by means of the TMVRNA-CGMMV-protein hybrid system (310). Thus, it is of interest to examine whether CGMMV-RNA is able to form a hybrid virus particle with TMV-protein and to compare biochemically CGMMVRNA with TMV-RNA. The Japanese watermelon strain of CGMMV (1) and the Japanese common strain OM of TMV (11) were purified as before (2). Viral RNA and protein were prepared by phenol-bentonite extraction (12) and the acetic-acid method (13), respectively. The base composition of CGMMV-RNA was determined as described by Takemura et al. (14). The hydrolysate of RNA was applied on a Dowex-1 column (0.4 x 10 cm) and developed by a concave gradient of two volumes of 0.004 N HCl and one volume of 0.1 M NaCl-0.01 N HCl. The base composition was 20.6% C, 24.6% A, 31.6% U, and Copyright 0 1976 by Academic All rights
of reproduction
Press, Inc. in any form reserved.
23.2% G in molar percent. The CGMMVRNA contained significantly less A and more U than the TMV-RNA (15). According to the results of Knight (15)) cucumber virus 4 (CV 4) RNA also has less A and more U than the TMV-RNA. Earlier works on CGMMV-protein Cl) indicated that CGMMV-protein was closely related to CV-4 protein chemically. These results show that CGMMV and CV 4 are similar in the chemical structure of both RNA and protein, although they are not pathogenically the same. The 5’-terminus of CGMMV-RNA was examined as described by Takanami fl6). CGMMV-RNA (2 nmol), polynucleotide kinase (20 units), and [3zPlATP (200 nmol, 6 x 10y cpm/pmol) were incubated for 60 min at 37” in 0.05 M Tris-HCl buffer (pH 7.4) containing 10 mA4 MgC!l* and 5 mM 2mercaptoethanol (total volume 2.0 ml). A nuclease-free preparation of polynucleotide kinase was kindly prepared by Dr. Takanami. The RNA treated with the enzyme was purified by agarose gel filtration (Bio-Rad, AG-5m) and sucrose gradient centrifugation. The amount of 32Pbound to CGMMV-RNA was always less than 0.1 mol/mol of RNA. The amount of phosphate CGMMV-RNA was incorporated into about the same with and without alkaline phosphatase treatment. The results sug214
SHORT
215
COMMUNICATIONS
gest that CGMMV-RNA has a blocked 5’-terminal end group. During the preparation of this manuscript, a paper has appeared on the blocked 5’-terminal end of TMV-RNA (17) which was identified as m7Gj’ ppp”’ Gp. Therefore, it is most probable that the 5’terminus of CGMMV-RNA is also blocked by the same structure. The 5’-terminus of TMV-RNA has been previously thought to be an unphosphorylated A (18, 19). It is now known to have arisen from hidden breaks in the RNA preparation (17). In the case of CGMMV-RNA whose 5’-ends were labeled only at 10% efficiency as described above, the mixture of terminal bases, U (530/o>,G (25%), A (120/o),and C (lo%), was detected as an unphosphorylated 5’-end. They probably originated from hidden breaks in our RNA preparation. The 3’-terminus of CGMMV-RNA was determined by the tritium-labeling method (20,211. A label was introduced at the 3’-end of peroxidate-oxidized CGMMVRNA by reduction with [3H]borohydride. The alkaline hydrolysate of 3H-labeled RNA was chromatographed with unlabeled nucleoside trialcohols on Avicel SF, thin-layer cellulose, using the mixture oftbutanol:methyZ ethyl ketone: H20: NH,OH (40:30:15:10).The 3’-terminal nucleoside of CGMMV-RNA was identified as A (Fig. 1). As shown in Fig. 1, about half of the labeled material remained at the origin, which did not correspond to any nucleoside trialcohol. Such radioactivity remaining at the origin was previously observed in the hydrolysate of 3H-labeled RNA from cytoplasmic polyhedrosis virus by Furuichi and Miura (221, and later was identified as m7G”’ ppp”’ N2’ mp arising from a blocked 5’-end of RNA (23). If the 5’-end group of RNA has a structure such as m7G3’ppp’ Np, the 5’-end as well as 3’-end is potentially periodate-sensitive. Therefore, these results suggest that the labeled material remaining at the origin in Fig. 1 could have arisen from a blocked 5’-terminal end of CGMMV-RNA. The reconstitution reaction was performed in 0.1 M pyrophosphate buffer (pH 7.2) at 25-30” as described by Fraenkel-
Conrat and Singer (24). The formation of virus particles from CGMMV-RNA and CGMMV-protein or TMV-protein was detected by sucrose gradient centrifugation. As shown in Fig. 2, both proteins were able to be reconstituted in vitro effectively with CGMMV-RNA. The efficiency was 60-70%, which was estimated from the ratio of the amount of RNA incorporated into the sedimentable particles to the total RNA in the reaction mixture. From these results, together with our previous reports n
‘0 hz
E Et
Origin 4
G’ U’ @cB
Dlstance
from
C’ A’ @@
orqln
(cm)
FIG. 1. Chromatogram of the alkaline hydrolysate of 3H-labeled CGMMV-RNA on Avicel SF. The cellulose layer was scrapped at 0.5-cm intervals and the radioactivity was counted. Four nucleoside trialcohols (LJ’, C’, A’, G’) were added as markers.
A
1
c J
LA
01 10
Fract ton
20
number
FIG. 2. Sucrose gradient analysis of reconstituted particles of CGMMV-RNA with CGMMV-protein (A) or TMV-protein (B). One hundred micrograms of RNA was incubated with 3 mg of protein in 0.1 M pyrophosphate buffer (pH 7.2) at 25” for 6 hr. Samples were centrifuged at 24,000 rpm (Hitachi, RPS-25 rotor) for 2.5 hr on a 5-2096 sucrose gradient. The direction of centrifugation was from right to left. Arrows show the position of intact CGMMV.
216
SHORT
COMMUNICATIONS
(2, 3), it is deduced that both TMV- and CGMMV-RNA possess a certain critical sequence near the 5’-terminus for interaction with TMV-protein disks (4, 25) or CGMMV-protein disks (5, 8). It wil be interesting to compare the 5’-terminal sequence of TMV-RNA with that of CGMMV-RNA in order to understand a recognition mechanism between RNA and protein molecules. ACKNOWLEDGMENTS This work was supported in part by a grant from the Scientific Research Fund of the Japanese Ministry of Education, Science, and Culture, the Kurata Foundation, and the Toray Science Foundation. REFERENCES 1. Nom, Y., TOCHIHARA, H., KOMURO, Y., and OKADA, Y., Virology 45, 577-585 (1971). 2. OHMHI, Y., OHNO, T., Nozu, Y., and OKADA,
Y., Proc. Japan Acad. 45, 919-924 (1969). Y., OHASHI, Y., OHNO, T., and Nozu, Y., Virology 42, 243-245 (1970). OKADA, Y., and OHNO, T., Molec. Gen. Genetics 114, 205-213 (1972). OHNO, T., INOUE, H., and OKADA, Y., Proc. Nat. Acad. Sci. USA 69, 3680-3683 (1972). NONOMURA, Y., and OHNO, T., J. Mol. Biol. 90, 523-537 (1974). INOUE, H., KURIYAMA, K., OHNO, T., and OKADA, Y., Arch. Biochem. Biophys. 165, 34-
3. OKADA, 4. 5. 6.
7.
45 (1974). 8. OHNO, T., ORADA, Y., NONOMURA, Y., and INOUE, H., J. Biochem. 77, 313-319 (1975). 9. OKADA, Y., OHNO, T., and NONOMURA, Y., J. Biochem. 77, 1157-1163 (1975). 10. OKADA, Y., Adv. Biophys. 7, 1-41 (1975). II. Nozu, Y., OHNO, T., and OKADA, Y., J. Biothem. 68, 39-52 (1970). 12. FRAENKEL-CONRAT, H., SINGER, B., and TsucITA, A., Virology 14, 54-58 (1961). 13. FRAENBEL-CONRAT, H., Virology 4,1-4 (1957). 14. TAKEMURA, S., “Methods in Nucleic Acid Research,” pp. 207-214, Kyoritsu Shuppan, Tokyo, 1972. C. A., J. Biol. Chem. 197, 241-249 15. KNIGHT, (1952). 16. TAKANAMI, M., J. Mol. Biol. 23, 135-148 (1967). 17. ZIMMERN, D., Nucleic Acid Res. 2, 1189-1201 (1975). 18. FRAENKEL-CONRAT, H., and FOWLKS, E., Biochemistry 11, 1733-1736 (1972). 19. SUGIYAMA, T., and FRAENKEL-CONRAT, H., Biochemistry 2, 332-334 (1963). 20. LEPPLA, S. H., BJORAKER, B., and BOCK, R. M., “Methods in Enzymology,” 12, 236-240 (1968). 21. STEINSCHNEIDER, A., and FRAENKEL-CONRAT, H., Biochemistry 5, 2729-2734 (1966). 22. FURUICHI, Y., and MIURA, K., J. Mol. Biol. 64, 619-632 (1972). 23. MIURA, K., FURUICHI, Y., SHIMOTOHNO, K., URUSHIBARA, T., and SUGIURA, M., “The 10th FEBS Meeting” (1975). 24. FRAENKEL-CONRAT, H., and SINGER, B., Biochim. Biophys. Acta 33, 359-370 (1959).
70,214-216
Biochemical
MARIKO Department
(1976)
Characterization
KURISU, of Biophysics
of Cucumber Green Mottle Mosaic Virus Ribonucleic Acid
TAKESHI
OHNO,
YOSHIMI
OKADA,
AND
YUZO
NOZU
and Biochemistry, Faculty of Science, University of Tokyo, Tokyo, Japan, and Institute for Plant Virus Research, Chiba, Japan Accepted October 21, 1975
CGMMV-RNA has the base composition of 20.6% C, 24.6% A, 31.6% U, and 23.2% Gin molar percent. The 3’-terminal nucleoside is A. The 5’-terminus of CGMMV-RNA is blocked, as appears to be the case for TMV-RNA. CGMMV-RNA, as well as TMV-RNA, is able to be reconstituted effectively with either CGMMV-protein or TMV-protein in vitro.
Chemical and immunological properties of the protein of cucumber green mottle mosaic virus (CGMMV) were reported by Nozu et al. (I), and it was confirmed that CGMMV has an ancestral relationship to tobacco mosaic virus (TMV). Ohashi et al. f2) previously reported that TMV-RNA was able to be reconstituted into an infectious particle. Considerable progress has been made in understanding the assembly mechanism of TMV by means of the TMVRNA-CGMMV-protein hybrid system (310). Thus, it is of interest to examine whether CGMMV-RNA is able to form a hybrid virus particle with TMV-protein and to compare biochemically CGMMVRNA with TMV-RNA. The Japanese watermelon strain of CGMMV (1) and the Japanese common strain OM of TMV (11) were purified as before (2). Viral RNA and protein were prepared by phenol-bentonite extraction (12) and the acetic-acid method (13), respectively. The base composition of CGMMV-RNA was determined as described by Takemura et al. (14). The hydrolysate of RNA was applied on a Dowex-1 column (0.4 x 10 cm) and developed by a concave gradient of two volumes of 0.004 N HCl and one volume of 0.1 M NaCl-0.01 N HCl. The base composition was 20.6% C, 24.6% A, 31.6% U, and Copyright 0 1976 by Academic All rights
of reproduction
Press, Inc. in any form reserved.
23.2% G in molar percent. The CGMMVRNA contained significantly less A and more U than the TMV-RNA (15). According to the results of Knight (15)) cucumber virus 4 (CV 4) RNA also has less A and more U than the TMV-RNA. Earlier works on CGMMV-protein Cl) indicated that CGMMV-protein was closely related to CV-4 protein chemically. These results show that CGMMV and CV 4 are similar in the chemical structure of both RNA and protein, although they are not pathogenically the same. The 5’-terminus of CGMMV-RNA was examined as described by Takanami fl6). CGMMV-RNA (2 nmol), polynucleotide kinase (20 units), and [3zPlATP (200 nmol, 6 x 10y cpm/pmol) were incubated for 60 min at 37” in 0.05 M Tris-HCl buffer (pH 7.4) containing 10 mA4 MgC!l* and 5 mM 2mercaptoethanol (total volume 2.0 ml). A nuclease-free preparation of polynucleotide kinase was kindly prepared by Dr. Takanami. The RNA treated with the enzyme was purified by agarose gel filtration (Bio-Rad, AG-5m) and sucrose gradient centrifugation. The amount of 32Pbound to CGMMV-RNA was always less than 0.1 mol/mol of RNA. The amount of phosphate CGMMV-RNA was incorporated into about the same with and without alkaline phosphatase treatment. The results sug214
SHORT
215
COMMUNICATIONS
gest that CGMMV-RNA has a blocked 5’-terminal end group. During the preparation of this manuscript, a paper has appeared on the blocked 5’-terminal end of TMV-RNA (17) which was identified as m7Gj’ ppp”’ Gp. Therefore, it is most probable that the 5’terminus of CGMMV-RNA is also blocked by the same structure. The 5’-terminus of TMV-RNA has been previously thought to be an unphosphorylated A (18, 19). It is now known to have arisen from hidden breaks in the RNA preparation (17). In the case of CGMMV-RNA whose 5’-ends were labeled only at 10% efficiency as described above, the mixture of terminal bases, U (530/o>,G (25%), A (120/o),and C (lo%), was detected as an unphosphorylated 5’-end. They probably originated from hidden breaks in our RNA preparation. The 3’-terminus of CGMMV-RNA was determined by the tritium-labeling method (20,211. A label was introduced at the 3’-end of peroxidate-oxidized CGMMVRNA by reduction with [3H]borohydride. The alkaline hydrolysate of 3H-labeled RNA was chromatographed with unlabeled nucleoside trialcohols on Avicel SF, thin-layer cellulose, using the mixture oftbutanol:methyZ ethyl ketone: H20: NH,OH (40:30:15:10).The 3’-terminal nucleoside of CGMMV-RNA was identified as A (Fig. 1). As shown in Fig. 1, about half of the labeled material remained at the origin, which did not correspond to any nucleoside trialcohol. Such radioactivity remaining at the origin was previously observed in the hydrolysate of 3H-labeled RNA from cytoplasmic polyhedrosis virus by Furuichi and Miura (221, and later was identified as m7G”’ ppp”’ N2’ mp arising from a blocked 5’-end of RNA (23). If the 5’-end group of RNA has a structure such as m7G3’ppp’ Np, the 5’-end as well as 3’-end is potentially periodate-sensitive. Therefore, these results suggest that the labeled material remaining at the origin in Fig. 1 could have arisen from a blocked 5’-terminal end of CGMMV-RNA. The reconstitution reaction was performed in 0.1 M pyrophosphate buffer (pH 7.2) at 25-30” as described by Fraenkel-
Conrat and Singer (24). The formation of virus particles from CGMMV-RNA and CGMMV-protein or TMV-protein was detected by sucrose gradient centrifugation. As shown in Fig. 2, both proteins were able to be reconstituted in vitro effectively with CGMMV-RNA. The efficiency was 60-70%, which was estimated from the ratio of the amount of RNA incorporated into the sedimentable particles to the total RNA in the reaction mixture. From these results, together with our previous reports n
‘0 hz
E Et
Origin 4
G’ U’ @cB
Dlstance
from
C’ A’ @@
orqln
(cm)
FIG. 1. Chromatogram of the alkaline hydrolysate of 3H-labeled CGMMV-RNA on Avicel SF. The cellulose layer was scrapped at 0.5-cm intervals and the radioactivity was counted. Four nucleoside trialcohols (LJ’, C’, A’, G’) were added as markers.
A
1
c J
LA
01 10
Fract ton
20
number
FIG. 2. Sucrose gradient analysis of reconstituted particles of CGMMV-RNA with CGMMV-protein (A) or TMV-protein (B). One hundred micrograms of RNA was incubated with 3 mg of protein in 0.1 M pyrophosphate buffer (pH 7.2) at 25” for 6 hr. Samples were centrifuged at 24,000 rpm (Hitachi, RPS-25 rotor) for 2.5 hr on a 5-2096 sucrose gradient. The direction of centrifugation was from right to left. Arrows show the position of intact CGMMV.
216
SHORT
COMMUNICATIONS
(2, 3), it is deduced that both TMV- and CGMMV-RNA possess a certain critical sequence near the 5’-terminus for interaction with TMV-protein disks (4, 25) or CGMMV-protein disks (5, 8). It wil be interesting to compare the 5’-terminal sequence of TMV-RNA with that of CGMMV-RNA in order to understand a recognition mechanism between RNA and protein molecules. ACKNOWLEDGMENTS This work was supported in part by a grant from the Scientific Research Fund of the Japanese Ministry of Education, Science, and Culture, the Kurata Foundation, and the Toray Science Foundation. REFERENCES 1. Nom, Y., TOCHIHARA, H., KOMURO, Y., and OKADA, Y., Virology 45, 577-585 (1971). 2. OHMHI, Y., OHNO, T., Nozu, Y., and OKADA,
Y., Proc. Japan Acad. 45, 919-924 (1969). Y., OHASHI, Y., OHNO, T., and Nozu, Y., Virology 42, 243-245 (1970). OKADA, Y., and OHNO, T., Molec. Gen. Genetics 114, 205-213 (1972). OHNO, T., INOUE, H., and OKADA, Y., Proc. Nat. Acad. Sci. USA 69, 3680-3683 (1972). NONOMURA, Y., and OHNO, T., J. Mol. Biol. 90, 523-537 (1974). INOUE, H., KURIYAMA, K., OHNO, T., and OKADA, Y., Arch. Biochem. Biophys. 165, 34-
3. OKADA, 4. 5. 6.
7.
45 (1974). 8. OHNO, T., ORADA, Y., NONOMURA, Y., and INOUE, H., J. Biochem. 77, 313-319 (1975). 9. OKADA, Y., OHNO, T., and NONOMURA, Y., J. Biochem. 77, 1157-1163 (1975). 10. OKADA, Y., Adv. Biophys. 7, 1-41 (1975). II. Nozu, Y., OHNO, T., and OKADA, Y., J. Biothem. 68, 39-52 (1970). 12. FRAENKEL-CONRAT, H., SINGER, B., and TsucITA, A., Virology 14, 54-58 (1961). 13. FRAENBEL-CONRAT, H., Virology 4,1-4 (1957). 14. TAKEMURA, S., “Methods in Nucleic Acid Research,” pp. 207-214, Kyoritsu Shuppan, Tokyo, 1972. C. A., J. Biol. Chem. 197, 241-249 15. KNIGHT, (1952). 16. TAKANAMI, M., J. Mol. Biol. 23, 135-148 (1967). 17. ZIMMERN, D., Nucleic Acid Res. 2, 1189-1201 (1975). 18. FRAENKEL-CONRAT, H., and FOWLKS, E., Biochemistry 11, 1733-1736 (1972). 19. SUGIYAMA, T., and FRAENKEL-CONRAT, H., Biochemistry 2, 332-334 (1963). 20. LEPPLA, S. H., BJORAKER, B., and BOCK, R. M., “Methods in Enzymology,” 12, 236-240 (1968). 21. STEINSCHNEIDER, A., and FRAENKEL-CONRAT, H., Biochemistry 5, 2729-2734 (1966). 22. FURUICHI, Y., and MIURA, K., J. Mol. Biol. 64, 619-632 (1972). 23. MIURA, K., FURUICHI, Y., SHIMOTOHNO, K., URUSHIBARA, T., and SUGIURA, M., “The 10th FEBS Meeting” (1975). 24. FRAENKEL-CONRAT, H., and SINGER, B., Biochim. Biophys. Acta 33, 359-370 (1959).
