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Crystallization of belladonna mottle virus
,I. Mol. Riol. (1981) 146, 629-633
Crystallization
of Belladonna Mottle Virus
Belladonna mottle virus belongs to the turnip yellows mosaic virus group of the small spherical plant viruses. It contains 180 protein subunits, which are arranged in a T = 3 icosahedral surface lattice. The top and bottom viral components crystallize isomorphously in hexagonal space group R3 (a = b = 296 A, c = 729 A).
The unit cell contains three virus particles, while the crystallographic asymmetric unit consists of only one-third of a particle. X-ray diffraction extend to at least 3% A resolution.
Belladonna
virus (BDMV) was first isolated in 1942 from specimens of (Smith, 1943). Three strains of the virus have since been discovered (Peters & Derks, 1974; Moline & Fries, 1974; Lee et al., 1979). The variants differ in virulence, in the relative proportions of the two sedimenting components (Paul, 1969), and in sensitivity to mercury-containing compounds (Koenig, 1969). Nevertheless, the serological relationship among them is very close. Physical data, RNA base composition, and serological behavior show that BDMV belongs to the Andean potato latent virus subgroup of the turnip yellows mosaic virus (TYMV) group of small spherical plant viruses (Jankulowa et al., 1968 : Koenig, 1969; Bercks & Querfurth, 1972). Vesicles produced in chloroplasts infected with BDMV resemble those caused by TYMV (Moline. 1973). RNA extracted from BDMV will associate with nascent TYMV protein capsids as efficiently as TYMV RNA itself (Bouley et al., 1975). Tymoviruses are characterized by relatively large contents of cytidine nucleotides (Gibbs et al.. 1966) ; for BDMV it approaches 33% (Jankulowa et al., 1968). Chemical modification studies of the tymovirus cytidine nucleotides imply that about 30:/o of them are distributed along single-stranded RNA stretches involved in protein interactions (Bouley et al., 1975). Approximately 55% of the TYMV RNA appears to be in the double-helical formation (Haselkorn, 1962: Witz Rr Strazielle, 1973). Other tymoviruses that have been extensively studied include eggplant mosaic virus (cf. Briand et a,Z.. 1977; Bouley et al., 1976) and erysium latent virus (ELV) (Shukla & Schmelzer, 1972; Shukla et aZ., 1980). The tymovirus family contains many members despite weak serological relationships among them (Givard & Koenig, 1974; Koenig, 1976). The molecular weight of the BDMV top (empty protein capsids) and bottom (protein shells containing RNA necessary for infection) components are 3.7 x 1O6 and 5.2 x 106, respectively (Paul, 1969). Thirty-seven per cent of the virus b) weight is RNA (Moline & Fries, 1974); the protein subunit molecular weight is 20,300. indicating 180 subunits per intact particle (T= 3) (Paul et al., 1968; Jankulowa et aZ., 1968). Electron micrographs show the icosahedral virus to be spherical in shape with a diameter between 250 A and 300 A (Paul et al.. 1968). Atropa
mottle
data from the crystals
be&donna
tm2-2836/81/080629-05
$02.00/O
029
630
K. I,. HElTSS.
J. K. MOHANA
RAO
AND
P. ARGOS
The strain
of BDMV used in the present study was originally isolated from in Iowa (Molinr Rr Fries. 1974). BDMV was purified from a tobacco hybrid, Xicotiana gllrtirrosa-clevelandii by the procedure of ($habrial et nl. (1967), with the following modifications. Homogenized tissue was left overnight at pH 4.6 before low-speed centrifugation. The clarified homogenate was adjusted to pH 5.5 with lo:/, NaOH and the virus was precipitated with 8yi1 polyethylene glycol6000 plus @l M-NaCl. The viral components were separated by cesium chloride density-gradient centrifugation (40% (w/w) CsCl in 0.05 Mpotassium phosphate buffer, pH 5.5). Five bands were observed after a 15-hour spin at 38,000 revs/min in a Beckman model 50Ti fixed-angle rotor. The top and bottom bands corresponded with those previously observed for BDMV. The number of bands observed is the same as that found for TYMV. where each band corresponds to a protein capsid containing varying amounts of the total RNA necessary for infection (Higgins et al., 1978). Although it is not unusual for spherical plant, viruses to crystallize, few crystals are suitable for high-resolution X-ray structure analyses. Salient exceptions include tomato bushy stunt virus (Harrison et al., 1978), southern bean mosaic virus (SBMV) (Abad-Zapatero et al., 1980) and satellite tobacco necrosis virus (Unge et al., 1980). Both the BDMV top and bottom components were crystallized by the vapor-diffusion technique (McPherson, 1976). The environmental conditions are listed in Table 1. The longest dimension of crystal growth sometimes approached 3.0 mm. Crystallization experiments for other tymoviruses have been reported by and by Colman et al. (1980) for ELV. The TYMV Klug et al. (1966) for TYMV crystals diffract to 20 a resolution, while the ELV diffraction power extends to 3.7 A. Reflections are observed to at least 3.0 I! resolution from the BDMV crystals. Physalis
heteraphylla
TABLE 1 Vapor
Virus BDMV bottom BDMV top
diffusion
Virus (3oncm in well (mg/ml)
PEG 6000 concn in well PC,. w/v,)
I2 25
3.5 34
coditiotts
PH 4.5 43
for
crystallization
Potassium phosphate buffer In well In reservoir (M) (M) 0.05 0.075
OG5 0.10
Reservoir
PEG6000 conrn (Oo. w/w) 10 10
Time to achieve crystals 33 weeks 2-i days
Both the top and bottom components crystallize isomorphously in hexagonal space group R3 with unit cell dimensions a = b = 296 A and c = 729 8. The principal zone precession photographs clearly showed the ( - h + k + 1 = 31,) systematic absences of the hexagonal space group. If three particles are assumed to be within the bottom component unit cell, the l’,,, value (Matthews, 1968) becomes 3.5 a”/dalton, which is in excellent agreement with those found for other spherical plant viruses (e.g. 3.6 A3/dalton for SBMV: Akimoto et al., 1975). The crystalline viral particles are stacked according to pseudo-cubic close-packing as also found in type II SBMV crystals, which belong to
632
Ii.
1,. HElTSS.
,I. K. MOHAXA
KAO
rZN1) P. AHGOS
hexagonal space group R32 (Akimoto rt nl., 1975). Obviously the BDMV icosahedral 3-fold axes are similarly aligned. Determination of the remaining icosahedral axial orientations must await rotation-function studies subsequent to high-resolution data collection. The particle positions given in fractional cell coordinates in the hexagonal unit cell can be chosen as (0. 0. 0), (l/3.2/3,2/3) and (2/J. l/3, l/3). The unit cell in the rhombohedral setting is characterized by u = 297 AA and ‘I = 59.7”. The rhombohedral unit-cell dimension would correspond to the maximal particle diameter and is in good agreement with the 250 to 300 A range obsefved from BDMV electron micrographs and 300 A determined for TYMP (Finch & Klug. 1967: Mellema &, Amos, 1972). Precession photographs (CL= 2”) of the top and bottom crystal forms (Fig. 1) clearly illustrate their isomorphous nature. The interactive forces necessary to pack the particles in crystalline arrays must therefore depend only on the capsid protein subunits: any effect from the RNA is apparently shielded. However. intensity differences are definitely in evidence to at least 22 A resolution on the top and bottom precession photographs : presumably they derive from the scattering of the RNA core in the bottom component material. High-resolution top and bottom structure determinations, presently underway, nil1 hopefully show differences in the protein subunit conformation resulting from protein-RNA interactions. Drs Andrew Jackson and Richard Lister supplied the BDMV virus and were most helpful in achieving its purification. Appreciation is expressed to Marilyn Anthony for help in the manuscript preparation. P.A. wishes to acknowledge financial support from the National Science Foundation (no. PCMX-20287 and no. PCM804.575) as well as the American Cancer Society (Faculty Research Award no. FRA173). Purdue University Department of Biological Sciences West Lafayette, Ind. 47907 U.S.A.
KATHLEEN L. HEUSS *J. K. MOHANA RAO PATRICK ARaost
Received 7 October 1980
REFERENCES Abad-Zapatero,
C”.. Abdel-Meguid,
S. S., Johnson, J. E., Leslie, .4. G. W., Rayment. I.. Rossmann, M. G., Suck, D. & Tsukihara, T. (1980). AV:atur~ (hdon). 286, 33-39. .4kimoto, T., Wagner, M. A., Johnson, J. E. & Rossmann, M. G. (1975). J. Vltrastmct. RPS. 53, 306-318. Bercks, R. & Querfurth. G. (1972). Phytopathol. Z. 75, 215-222. Bouley, J. P., Briand. J. I’.. Jonard, G., Witz, J. & Hirth, L. (1975). l’irology, 63, 312-319. Bouley. .J. I’.. Briand, J. P., (ienevaux, M.. Pinrk. M. Rr Witz, J. (1976). li’rology, 69, 775, 781. Briand, .J. P., Bouley, J. P. & Witz, J. (1977). LFrology, 76, 664469. Colman, I’. M.. Tulloch, 1’. A., Shukla, D. D. 8.1Gough, K. H. (1980). J. Mol. Hiol. 142, 263268. Finch. J. T. & Klug, ;1. (1967). J. Mol. Rio/. 24, 289-302. Ghabrial, S. A.. Shepherd, R. J. B (brogan. R. G. (1967). li’rology, 33, 17-25. t To whom reprint
requests should be atidrrxsed
LETTERS
TO THE
EDITOR
fi33
A. qJ., Hecht-Poinar, E., Woods, R. D. & McKee, R. K. (1966). J. Oen. Microbial. 44. 177-193. (iivard, L. & Koenig, R. (1974). Virology, 58, 119-125. Harrison, S. C’., Olson, A. J., Schutt, C. E., Winkler. F. K. &, Bricogne, 0. (1978). Satwe
(:ibhs,
(Imdm)
, 276, 368-373.
Haselkorn. R. (1962). J. Mol. Riol. 4, 357-367. Higgins. T. J. V., Whitfield, P. R. & Matthews, R. E. F. (1978). Virology, 84, 153-161. .lankulowa. M.. Huth, W., Wittmann, H. G. & Paul, H. L. (1968). Phytopathol. 8. 63. 177 185.
Klug. A., Longley, Koenig, R. (1969). Koenig, R. (1976). Lee. R. F., Niblett,
W. & Leberman, R. (1966). J. Mol. Hiol. 15. 315-343. l&o&y, 88, 14&144. Virology, 72, 1-5. C. L., Hubbard, J. D. 8: Johnson, L. B. (1979). Phytopthd.
2.69,9f&
989.
Mrl’htsrson, A. Jr (1976). In Methods of Biochemid Analysis ((:lick, D.. ed.). vol. 23, pp. 249-345, Wiley, New York. hlatthews, B. W. (1968). J. Mol. Biol. 33, 491497. Mellema. ,J. E. &, Amos, L. A. (1972). J. Mol. Riol. 72, 819-822. Moline, H. E. (1973). l’irolqy, 56, 123-133. Moline, H. E. Nr Fries, R. E. (1974). Phytopthology, 64. M--48. Paul, H. I,. (1969). Phytopthol. 2. 65, 257-262. I’aul. H. L.. Bode, O., Jankulowa, M. & Brandes, J. (1968). Phytoputhol. 2. 61, 342-361. I’et,ers. D. & Derks, A. F. L. M. (1974). Neth. J. Plant Pathol. 99, 124-132. Shukla, D. D. & Hchmelzer, K. (1972). Acta Phytopthol. Acad. Sci. Hung. 7, 157-167. Shukla, D. D., Koenig, R., Cough, K. H., Huth, W. & Lesemann, D. E. (1980). I’hytopathology,
70, 382-384.
Smith. K. W. (1943). Parasitology, 35, 159-161. ITnge, T., Liljas, L., Strandberg, B., Vaara, I., Kannan, K. K., Fridborg, K., Nordman. (‘. E. RTLentz, I’. .J. Jr (1980). Sature (London), 285, 373-377. Witz. ,J. & Strazielle, C’. (1973). In Subunits in Biological Systems (Fasman, (:. 1). & Timashrff. S. h.. rds). part B, pp. 207-252, Marcel Dekker Inc., New York.
Crystallization
of Belladonna Mottle Virus
Belladonna mottle virus belongs to the turnip yellows mosaic virus group of the small spherical plant viruses. It contains 180 protein subunits, which are arranged in a T = 3 icosahedral surface lattice. The top and bottom viral components crystallize isomorphously in hexagonal space group R3 (a = b = 296 A, c = 729 A).
The unit cell contains three virus particles, while the crystallographic asymmetric unit consists of only one-third of a particle. X-ray diffraction extend to at least 3% A resolution.
Belladonna
virus (BDMV) was first isolated in 1942 from specimens of (Smith, 1943). Three strains of the virus have since been discovered (Peters & Derks, 1974; Moline & Fries, 1974; Lee et al., 1979). The variants differ in virulence, in the relative proportions of the two sedimenting components (Paul, 1969), and in sensitivity to mercury-containing compounds (Koenig, 1969). Nevertheless, the serological relationship among them is very close. Physical data, RNA base composition, and serological behavior show that BDMV belongs to the Andean potato latent virus subgroup of the turnip yellows mosaic virus (TYMV) group of small spherical plant viruses (Jankulowa et al., 1968 : Koenig, 1969; Bercks & Querfurth, 1972). Vesicles produced in chloroplasts infected with BDMV resemble those caused by TYMV (Moline. 1973). RNA extracted from BDMV will associate with nascent TYMV protein capsids as efficiently as TYMV RNA itself (Bouley et al., 1975). Tymoviruses are characterized by relatively large contents of cytidine nucleotides (Gibbs et al.. 1966) ; for BDMV it approaches 33% (Jankulowa et al., 1968). Chemical modification studies of the tymovirus cytidine nucleotides imply that about 30:/o of them are distributed along single-stranded RNA stretches involved in protein interactions (Bouley et al., 1975). Approximately 55% of the TYMV RNA appears to be in the double-helical formation (Haselkorn, 1962: Witz Rr Strazielle, 1973). Other tymoviruses that have been extensively studied include eggplant mosaic virus (cf. Briand et a,Z.. 1977; Bouley et al., 1976) and erysium latent virus (ELV) (Shukla & Schmelzer, 1972; Shukla et aZ., 1980). The tymovirus family contains many members despite weak serological relationships among them (Givard & Koenig, 1974; Koenig, 1976). The molecular weight of the BDMV top (empty protein capsids) and bottom (protein shells containing RNA necessary for infection) components are 3.7 x 1O6 and 5.2 x 106, respectively (Paul, 1969). Thirty-seven per cent of the virus b) weight is RNA (Moline & Fries, 1974); the protein subunit molecular weight is 20,300. indicating 180 subunits per intact particle (T= 3) (Paul et al., 1968; Jankulowa et aZ., 1968). Electron micrographs show the icosahedral virus to be spherical in shape with a diameter between 250 A and 300 A (Paul et al.. 1968). Atropa
mottle
data from the crystals
be&donna
tm2-2836/81/080629-05
$02.00/O
029
630
K. I,. HElTSS.
J. K. MOHANA
RAO
AND
P. ARGOS
The strain
of BDMV used in the present study was originally isolated from in Iowa (Molinr Rr Fries. 1974). BDMV was purified from a tobacco hybrid, Xicotiana gllrtirrosa-clevelandii by the procedure of ($habrial et nl. (1967), with the following modifications. Homogenized tissue was left overnight at pH 4.6 before low-speed centrifugation. The clarified homogenate was adjusted to pH 5.5 with lo:/, NaOH and the virus was precipitated with 8yi1 polyethylene glycol6000 plus @l M-NaCl. The viral components were separated by cesium chloride density-gradient centrifugation (40% (w/w) CsCl in 0.05 Mpotassium phosphate buffer, pH 5.5). Five bands were observed after a 15-hour spin at 38,000 revs/min in a Beckman model 50Ti fixed-angle rotor. The top and bottom bands corresponded with those previously observed for BDMV. The number of bands observed is the same as that found for TYMV. where each band corresponds to a protein capsid containing varying amounts of the total RNA necessary for infection (Higgins et al., 1978). Although it is not unusual for spherical plant, viruses to crystallize, few crystals are suitable for high-resolution X-ray structure analyses. Salient exceptions include tomato bushy stunt virus (Harrison et al., 1978), southern bean mosaic virus (SBMV) (Abad-Zapatero et al., 1980) and satellite tobacco necrosis virus (Unge et al., 1980). Both the BDMV top and bottom components were crystallized by the vapor-diffusion technique (McPherson, 1976). The environmental conditions are listed in Table 1. The longest dimension of crystal growth sometimes approached 3.0 mm. Crystallization experiments for other tymoviruses have been reported by and by Colman et al. (1980) for ELV. The TYMV Klug et al. (1966) for TYMV crystals diffract to 20 a resolution, while the ELV diffraction power extends to 3.7 A. Reflections are observed to at least 3.0 I! resolution from the BDMV crystals. Physalis
heteraphylla
TABLE 1 Vapor
Virus BDMV bottom BDMV top
diffusion
Virus (3oncm in well (mg/ml)
PEG 6000 concn in well PC,. w/v,)
I2 25
3.5 34
coditiotts
PH 4.5 43
for
crystallization
Potassium phosphate buffer In well In reservoir (M) (M) 0.05 0.075
OG5 0.10
Reservoir
PEG6000 conrn (Oo. w/w) 10 10
Time to achieve crystals 33 weeks 2-i days
Both the top and bottom components crystallize isomorphously in hexagonal space group R3 with unit cell dimensions a = b = 296 A and c = 729 8. The principal zone precession photographs clearly showed the ( - h + k + 1 = 31,) systematic absences of the hexagonal space group. If three particles are assumed to be within the bottom component unit cell, the l’,,, value (Matthews, 1968) becomes 3.5 a”/dalton, which is in excellent agreement with those found for other spherical plant viruses (e.g. 3.6 A3/dalton for SBMV: Akimoto et al., 1975). The crystalline viral particles are stacked according to pseudo-cubic close-packing as also found in type II SBMV crystals, which belong to
632
Ii.
1,. HElTSS.
,I. K. MOHAXA
KAO
rZN1) P. AHGOS
hexagonal space group R32 (Akimoto rt nl., 1975). Obviously the BDMV icosahedral 3-fold axes are similarly aligned. Determination of the remaining icosahedral axial orientations must await rotation-function studies subsequent to high-resolution data collection. The particle positions given in fractional cell coordinates in the hexagonal unit cell can be chosen as (0. 0. 0), (l/3.2/3,2/3) and (2/J. l/3, l/3). The unit cell in the rhombohedral setting is characterized by u = 297 AA and ‘I = 59.7”. The rhombohedral unit-cell dimension would correspond to the maximal particle diameter and is in good agreement with the 250 to 300 A range obsefved from BDMV electron micrographs and 300 A determined for TYMP (Finch & Klug. 1967: Mellema &, Amos, 1972). Precession photographs (CL= 2”) of the top and bottom crystal forms (Fig. 1) clearly illustrate their isomorphous nature. The interactive forces necessary to pack the particles in crystalline arrays must therefore depend only on the capsid protein subunits: any effect from the RNA is apparently shielded. However. intensity differences are definitely in evidence to at least 22 A resolution on the top and bottom precession photographs : presumably they derive from the scattering of the RNA core in the bottom component material. High-resolution top and bottom structure determinations, presently underway, nil1 hopefully show differences in the protein subunit conformation resulting from protein-RNA interactions. Drs Andrew Jackson and Richard Lister supplied the BDMV virus and were most helpful in achieving its purification. Appreciation is expressed to Marilyn Anthony for help in the manuscript preparation. P.A. wishes to acknowledge financial support from the National Science Foundation (no. PCMX-20287 and no. PCM804.575) as well as the American Cancer Society (Faculty Research Award no. FRA173). Purdue University Department of Biological Sciences West Lafayette, Ind. 47907 U.S.A.
KATHLEEN L. HEUSS *J. K. MOHANA RAO PATRICK ARaost
Received 7 October 1980
REFERENCES Abad-Zapatero,
C”.. Abdel-Meguid,
S. S., Johnson, J. E., Leslie, .4. G. W., Rayment. I.. Rossmann, M. G., Suck, D. & Tsukihara, T. (1980). AV:atur~ (hdon). 286, 33-39. .4kimoto, T., Wagner, M. A., Johnson, J. E. & Rossmann, M. G. (1975). J. Vltrastmct. RPS. 53, 306-318. Bercks, R. & Querfurth. G. (1972). Phytopathol. Z. 75, 215-222. Bouley, J. P., Briand. J. I’.. Jonard, G., Witz, J. & Hirth, L. (1975). l’irology, 63, 312-319. Bouley. .J. I’.. Briand, J. P., (ienevaux, M.. Pinrk. M. Rr Witz, J. (1976). li’rology, 69, 775, 781. Briand, .J. P., Bouley, J. P. & Witz, J. (1977). LFrology, 76, 664469. Colman, I’. M.. Tulloch, 1’. A., Shukla, D. D. 8.1Gough, K. H. (1980). J. Mol. Hiol. 142, 263268. Finch. J. T. & Klug, ;1. (1967). J. Mol. Rio/. 24, 289-302. Ghabrial, S. A.. Shepherd, R. J. B (brogan. R. G. (1967). li’rology, 33, 17-25. t To whom reprint
requests should be atidrrxsed
LETTERS
TO THE
EDITOR
fi33
A. qJ., Hecht-Poinar, E., Woods, R. D. & McKee, R. K. (1966). J. Oen. Microbial. 44. 177-193. (iivard, L. & Koenig, R. (1974). Virology, 58, 119-125. Harrison, S. C’., Olson, A. J., Schutt, C. E., Winkler. F. K. &, Bricogne, 0. (1978). Satwe
(:ibhs,
(Imdm)
, 276, 368-373.
Haselkorn. R. (1962). J. Mol. Riol. 4, 357-367. Higgins. T. J. V., Whitfield, P. R. & Matthews, R. E. F. (1978). Virology, 84, 153-161. .lankulowa. M.. Huth, W., Wittmann, H. G. & Paul, H. L. (1968). Phytopathol. 8. 63. 177 185.
Klug. A., Longley, Koenig, R. (1969). Koenig, R. (1976). Lee. R. F., Niblett,
W. & Leberman, R. (1966). J. Mol. Hiol. 15. 315-343. l&o&y, 88, 14&144. Virology, 72, 1-5. C. L., Hubbard, J. D. 8: Johnson, L. B. (1979). Phytopthd.
2.69,9f&
989.
Mrl’htsrson, A. Jr (1976). In Methods of Biochemid Analysis ((:lick, D.. ed.). vol. 23, pp. 249-345, Wiley, New York. hlatthews, B. W. (1968). J. Mol. Biol. 33, 491497. Mellema. ,J. E. &, Amos, L. A. (1972). J. Mol. Riol. 72, 819-822. Moline, H. E. (1973). l’irolqy, 56, 123-133. Moline, H. E. Nr Fries, R. E. (1974). Phytopthology, 64. M--48. Paul, H. I,. (1969). Phytopthol. 2. 65, 257-262. I’aul. H. L.. Bode, O., Jankulowa, M. & Brandes, J. (1968). Phytoputhol. 2. 61, 342-361. I’et,ers. D. & Derks, A. F. L. M. (1974). Neth. J. Plant Pathol. 99, 124-132. Shukla, D. D. & Hchmelzer, K. (1972). Acta Phytopthol. Acad. Sci. Hung. 7, 157-167. Shukla, D. D., Koenig, R., Cough, K. H., Huth, W. & Lesemann, D. E. (1980). I’hytopathology,
70, 382-384.
Smith. K. W. (1943). Parasitology, 35, 159-161. ITnge, T., Liljas, L., Strandberg, B., Vaara, I., Kannan, K. K., Fridborg, K., Nordman. (‘. E. RTLentz, I’. .J. Jr (1980). Sature (London), 285, 373-377. Witz. ,J. & Strazielle, C’. (1973). In Subunits in Biological Systems (Fasman, (:. 1). & Timashrff. S. h.. rds). part B, pp. 207-252, Marcel Dekker Inc., New York.