J. Mol. Biol. (1992) 228, 687489
CRYSTALLIZATION
Preliminary
NOTES
Crystallographic Study of Bluetongue Virus Capsid Protein, VP7 A. K. Basakly2, D. I. Stuart’
and P. Roy1
‘Laboratory of Molecular Biophysics Rex Richards Building South Parks Road, Oxford OX1 3&U, U.K. ‘NERC
Institute of Virology and Environmental Microbiology Mansfield Road, Oxford, OX1 3SR, U.K.
(Received 27 January
1992; accepted 17 July
1992)
Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6,22 with tz=b=95.2 A, c=181.0 A, cI=fi=90° y=12@0”, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin=3*0 A. Type II crystals belong to space group P2, with a= 69.4 8, b =97-l A, c= 71.4 A, /I= 1094I”, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin= 2.1 A. These results, together with solution studies, show that the molecule is a trimer. Keywords:
virus structure;
X-ray diffraction;
VP7 while the inner layer, termed the subcore, is composed of VP3 (French & Roy, 1990; Loudon & Roy, 1991) and encloses the minor proteins and the dsRNA genome. BTV VP7 accounts for some 36% of the core protein, with 780 copies of the 38 kDa subunit forming a T = 13 surface lattice on the core. It is highly conserved among various BTV serotypes and has amino acid sequence similarity with the VP7 proteins of other orbiviruses (Roy et al., 1991). The protein is therefore a candidate for a diagnostic reagent specific for this group of virus. VP7 has been synthesized in large quantities in Spodoptera frugiperda cells infected with a recombinant baculovirus AcBTV-10.7 containing the VP7 gene of BTV serotype 10 (BTV-10) (Oldfield et al., 1990). We here report the purification of the protein to homothe growth of crystals and their geneity, preliminary X-ray crystallographic analysis. Protein synthesis and purification. BTV-10 VP7 was synthesized as described by Oldfield et al. (1990). The purification of VP7 protein was modified to overcome problems of insolubility. Spodoptera frugiperda cells infected 48 to 72 hours previously with recombinant AcBTV-10.7 were pelleted by centrifugation at 320 g, rinsed in phosphate buffered saline and resuspended in 10 m&r-Tris * HCl, pH 7.5, containing 95 y. NP40 to a concentration of 5 x lo7 cells/ml. Nuclei were then removed from the resulting cell lysate by centrifugation at 570 g for 15 to 20 minutes. Ice-cold
Bluetongue virus (BTVt) belongs to the orbivirus genus of the Reoviridae family. It infects livestock (sheep, cattle, goats) and wild ruminants (deer, pronghorn). The virus is endemic in many parts of the world. In sheep and wild ruminants the disease is acute and mortality is often high, whereas in cattle and goats the disease is mild. To date, twenty four different serotypes of the virus have been identified from different parts of the world. Like other members of the Reoviridae family, BTV has a double-capsid structure (Browne & Ritchie, 1970) with the inner capsid (the core) exhibiting icosahedral symmetry. The BTV genome consists of ten double-stranded RNA segments designated large (L) Ll to L3, medium (M) M4 to M6; and small (S) 57 to SlO, with RNA sizes ranging from 3,954 (Ll) to 822 bp (X10) (Fukusho et al., 1989). The viral proteins are numbered according to the RNA segment that codes for them. The icosahedral core of BTV consists of five types of protein, two major (VP3 and VP7) and three minor (VPl, VP4 and VP6) (Huismans, 1979). This core is surrounded by a loosely bound outer capsid, composed of two proteins, VP2 and VP5. Recent analysis of the BTV core particle has indicated that it is 690 A in diameter (1 A=@1 nm) and may be divided into two concentric layers, the outer layer containing t Abbreviations base-pairs; PEG,
used: RTV, polyethylene
0022-2836/92/22068743
$08.00/O
bluetongue glycol.
virus;
crystallization
bp.
687 0
1992 Academic
Press Limited
688
A. K. Basak et al.
saturated ammonium sulphate in 100 mM-Tris * HCl, pH 7.5, was added to the cytoplasmic cell extracts to a final saturation of 20%. The precipitated protein was pelleted by centrifugation and resuspended in 10 mm-Tris . HCl, pH 7.5 with 1 o/oethylene glycol and then dialysed versus the same buffer at 4°C overnight. Inclusion of ethylene glycol solubilized the protein so that over 95% remained in solution. After dialysis insoluble material was removed by centrifugation at 9000 g for 10 minutes. The supernatant was loaded on a Q-Sepharose column and the bound protein eluted using a gradient of 0 to 95 M-NaCl in the same buffer. Fractions containing VP7 were identified by SDS polyacrylamide gel electrophoresis. The protein was estimated to be over 95% pure by analysis of stained gels. VP7 recovered from the Crystallization. Q-Sepharose column was dialysed against 10 m&r-Tris* HCl, pH 7.5, and concentrated using a Centricon concentrator (with a 10 kDa protein exclusion membrane) until a concentration of 15 mg/ml was reached. Crystals were obtained by the hanging-drop or sitting-drop vapour diffusion method, in the presence of 0.5% fi-octyl glucopyranoside (McPherson, 1982). The sitting-drop experiments used microbridges (Harlos, 1992). The precipitant solution in the reservoir was 10 to 12% (w/v) polyethylene glycol (PEG) 6000 in 10 mllr-Tris. HCI, pH 75, and the protein was mixed with the well buffer in a ratio 1 : 1 and left at 15°C to equilibrate with the reservoir solution. Type I VP7 crystals grew as hexagonal rods. The crystals (@5mm x 92 mm x 0.2 mm) largest appeared within a period of 3 to 4 weeks. Subsequently we have, on occasion, obtained rectangular plate-like crystals with maximum dimensions 95 mm x 63 mm x 0.3 mm. These crystals (type II) grow from somewhat lower PEG concentration and grow much more slowly. In addition, very thin plates of up to diamond-shaped, 64 mm x 94 mm were obtained by the hangingdrop method from protein in 68 to 1.2 M-NaCl solution with the same NaCl concentration in the reservoir. These crystals were unsuitable for X-ray analysis. X-ray analysis of VP7 crystals. The hexagonal type 1 crystals were used initially. The first studies crystal of were performed on a small 0.2 mm x 0.2 mm x 92 mm using 0.96 A monochromatic radiation at station 9.6 of the SERC synchrotron facility at Daresbury, U.K. Subsequently, bigger crystals were used to collect diffraction data area in Oxford using a Siemens (Xentronics) detector with X-rays supplied by a Rigaku rotating anode generator operating at 60 kV and 70 mA. The data (collected as 025” frames) were processed using the XENGEN package of computer programs (Howard et al., 1987: Genex Corp., Gaitthersburg, MD, U.S.A.). The diffraction limit was found to be approximately d,,,=3.O h for data collected at Daresbury. Analyses of the various data indicate that the crystals exhibit hexagonal symmetry with unit cell parameters of: a=b=95.2 h, c= 181.0 h.
a=/?=90',
y= 120.0” and a unit of cell volume of 1.4= IO6 h3. Reflections OOil 3n are absent, and the data merge satisfactorily in point group 622 indicating that the space group is P6,22. Assuming 12 molecules in the unit cell, and one subunit per asymmetric unit, V, = 3.11 h3/Da. This result gives a solvent content of 6Oo/o. i.e. within the normal range of protein crystals (Matthews, 1977). A single type I1 crystal has been analysed using the Xentronics Area Detector. This indicates that the space group is P2,, with a=69.4 h, b=97.1 A. c= 71.4 h and /?= 109”. The crystal diffracted to dmin= 2.1 h. A self rotation function analysis (Rossmann & Blow, 1962) indicated the presence of a noncrystallographic 3-fold axis. Assuming three subunits per crystallographic asymmetric unit l’, = 1.99 h3/Da, corresponding to a solventJ content of 40%. Both crystal forms can accommodate a trimeric molecule and dynamic light scat’tering results indicate that this is also the stat,e of oligomerization in solution (Basak & Stuart, data not, shown). This suggests that the surface of the BTV core is formed by the accretion of preformed VP7 trimers onto the subcore to form a well defined T= 13 lattice. A search for heavy-atom derivatives is in progress in order t,o determine the three dimensional structure of BTV-10 VP7. A.K.B. was supported by the NERC. D.I.S. is a member of the Oxford Centre for Molecular Sciences.
References J. Q. & Ritchie. E. A. (1970). Some Browne, morphological features of bluetongue virus. Virology. 40, 903-911. French, T. J. & Roy, I’. (1990). Synthesis of bluetongue virus (BTV) core like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J. J’irol. 64, 1531)-1536. Fukusho. A.. Yu, Y., Yamaguichi. Y. & Roy. 1’. (1989). Completion of the sequence of bluetongue virus serotype 10 by the characterization of structural protein. VP6, and a non-structural protein, NS2. J. C:en. Viral. 1677-1689. Harlos, K. (1992). Micro-bridges for sitting-drop crystallizations. J. Appl. Crystallogr. 25, 536-538. Howard, A. J., Gilliand, G. L., Finzel, B. C., Poulos. T. L.. Ohlendorf. D. H. & Salemme, F. R. (1987). The use of an imaging proportional counter in macromolecular crystallography. J. Appl. Cystallogr. 20, 383-387. Huismans, H. (1979). Protein synthesis in bluetongue virus-infected cells. Virology, 82, 385-396. Loudon, P. T. CG Roy, P. (1991). Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of largest protein VP1 in the core and virus like particles. Virology, 180. 789-802. Matthews, B. W. (1977). In The Proteins (Neurath, H. and Hill, R. L., eds), 3, pp. 403-590, Academic Press. New York. McPherson. A. ,J. (1982). In Preparath and Anulysis oj Protein Crystals, pp. 82~160, John Wiley and Sons. New York.
Crystallization Oldfield, S., Adachi, A., Urakawa, T., Hirasawa, T. & Roy, P. (1990). Purification and characterization of the major group-specific core antigen VP7 of bluetongue virus synthesized by a recombinant baculovirus. J. Gen. Viral. 71, 26492656. Rossmann, M. G. & Blow, D. M. (1962). The detection of Sub-units within the crystallographic asymmetric unit. Acta Crystallogr. 15, 24-31.
Notes Roy, P., Hirasawa, T., Fenandez, M., Blinov. V. M. & Sanchez-Vixcain Rodrique, J. M. (1991). The complete sequence of the group-specific antigen, VP7, of African homesickness disease virus serotype 4 reveals a close relationship to bluetongue virus. J. Gen. Viral. 72, 1237-1241.
Edited by R. Huber
CRYSTALLIZATION
Preliminary
NOTES
Crystallographic Study of Bluetongue Virus Capsid Protein, VP7 A. K. Basakly2, D. I. Stuart’
and P. Roy1
‘Laboratory of Molecular Biophysics Rex Richards Building South Parks Road, Oxford OX1 3&U, U.K. ‘NERC
Institute of Virology and Environmental Microbiology Mansfield Road, Oxford, OX1 3SR, U.K.
(Received 27 January
1992; accepted 17 July
1992)
Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6,22 with tz=b=95.2 A, c=181.0 A, cI=fi=90° y=12@0”, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin=3*0 A. Type II crystals belong to space group P2, with a= 69.4 8, b =97-l A, c= 71.4 A, /I= 1094I”, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin= 2.1 A. These results, together with solution studies, show that the molecule is a trimer. Keywords:
virus structure;
X-ray diffraction;
VP7 while the inner layer, termed the subcore, is composed of VP3 (French & Roy, 1990; Loudon & Roy, 1991) and encloses the minor proteins and the dsRNA genome. BTV VP7 accounts for some 36% of the core protein, with 780 copies of the 38 kDa subunit forming a T = 13 surface lattice on the core. It is highly conserved among various BTV serotypes and has amino acid sequence similarity with the VP7 proteins of other orbiviruses (Roy et al., 1991). The protein is therefore a candidate for a diagnostic reagent specific for this group of virus. VP7 has been synthesized in large quantities in Spodoptera frugiperda cells infected with a recombinant baculovirus AcBTV-10.7 containing the VP7 gene of BTV serotype 10 (BTV-10) (Oldfield et al., 1990). We here report the purification of the protein to homothe growth of crystals and their geneity, preliminary X-ray crystallographic analysis. Protein synthesis and purification. BTV-10 VP7 was synthesized as described by Oldfield et al. (1990). The purification of VP7 protein was modified to overcome problems of insolubility. Spodoptera frugiperda cells infected 48 to 72 hours previously with recombinant AcBTV-10.7 were pelleted by centrifugation at 320 g, rinsed in phosphate buffered saline and resuspended in 10 m&r-Tris * HCl, pH 7.5, containing 95 y. NP40 to a concentration of 5 x lo7 cells/ml. Nuclei were then removed from the resulting cell lysate by centrifugation at 570 g for 15 to 20 minutes. Ice-cold
Bluetongue virus (BTVt) belongs to the orbivirus genus of the Reoviridae family. It infects livestock (sheep, cattle, goats) and wild ruminants (deer, pronghorn). The virus is endemic in many parts of the world. In sheep and wild ruminants the disease is acute and mortality is often high, whereas in cattle and goats the disease is mild. To date, twenty four different serotypes of the virus have been identified from different parts of the world. Like other members of the Reoviridae family, BTV has a double-capsid structure (Browne & Ritchie, 1970) with the inner capsid (the core) exhibiting icosahedral symmetry. The BTV genome consists of ten double-stranded RNA segments designated large (L) Ll to L3, medium (M) M4 to M6; and small (S) 57 to SlO, with RNA sizes ranging from 3,954 (Ll) to 822 bp (X10) (Fukusho et al., 1989). The viral proteins are numbered according to the RNA segment that codes for them. The icosahedral core of BTV consists of five types of protein, two major (VP3 and VP7) and three minor (VPl, VP4 and VP6) (Huismans, 1979). This core is surrounded by a loosely bound outer capsid, composed of two proteins, VP2 and VP5. Recent analysis of the BTV core particle has indicated that it is 690 A in diameter (1 A=@1 nm) and may be divided into two concentric layers, the outer layer containing t Abbreviations base-pairs; PEG,
used: RTV, polyethylene
0022-2836/92/22068743
$08.00/O
bluetongue glycol.
virus;
crystallization
bp.
687 0
1992 Academic
Press Limited
688
A. K. Basak et al.
saturated ammonium sulphate in 100 mM-Tris * HCl, pH 7.5, was added to the cytoplasmic cell extracts to a final saturation of 20%. The precipitated protein was pelleted by centrifugation and resuspended in 10 mm-Tris . HCl, pH 7.5 with 1 o/oethylene glycol and then dialysed versus the same buffer at 4°C overnight. Inclusion of ethylene glycol solubilized the protein so that over 95% remained in solution. After dialysis insoluble material was removed by centrifugation at 9000 g for 10 minutes. The supernatant was loaded on a Q-Sepharose column and the bound protein eluted using a gradient of 0 to 95 M-NaCl in the same buffer. Fractions containing VP7 were identified by SDS polyacrylamide gel electrophoresis. The protein was estimated to be over 95% pure by analysis of stained gels. VP7 recovered from the Crystallization. Q-Sepharose column was dialysed against 10 m&r-Tris* HCl, pH 7.5, and concentrated using a Centricon concentrator (with a 10 kDa protein exclusion membrane) until a concentration of 15 mg/ml was reached. Crystals were obtained by the hanging-drop or sitting-drop vapour diffusion method, in the presence of 0.5% fi-octyl glucopyranoside (McPherson, 1982). The sitting-drop experiments used microbridges (Harlos, 1992). The precipitant solution in the reservoir was 10 to 12% (w/v) polyethylene glycol (PEG) 6000 in 10 mllr-Tris. HCI, pH 75, and the protein was mixed with the well buffer in a ratio 1 : 1 and left at 15°C to equilibrate with the reservoir solution. Type I VP7 crystals grew as hexagonal rods. The crystals (@5mm x 92 mm x 0.2 mm) largest appeared within a period of 3 to 4 weeks. Subsequently we have, on occasion, obtained rectangular plate-like crystals with maximum dimensions 95 mm x 63 mm x 0.3 mm. These crystals (type II) grow from somewhat lower PEG concentration and grow much more slowly. In addition, very thin plates of up to diamond-shaped, 64 mm x 94 mm were obtained by the hangingdrop method from protein in 68 to 1.2 M-NaCl solution with the same NaCl concentration in the reservoir. These crystals were unsuitable for X-ray analysis. X-ray analysis of VP7 crystals. The hexagonal type 1 crystals were used initially. The first studies crystal of were performed on a small 0.2 mm x 0.2 mm x 92 mm using 0.96 A monochromatic radiation at station 9.6 of the SERC synchrotron facility at Daresbury, U.K. Subsequently, bigger crystals were used to collect diffraction data area in Oxford using a Siemens (Xentronics) detector with X-rays supplied by a Rigaku rotating anode generator operating at 60 kV and 70 mA. The data (collected as 025” frames) were processed using the XENGEN package of computer programs (Howard et al., 1987: Genex Corp., Gaitthersburg, MD, U.S.A.). The diffraction limit was found to be approximately d,,,=3.O h for data collected at Daresbury. Analyses of the various data indicate that the crystals exhibit hexagonal symmetry with unit cell parameters of: a=b=95.2 h, c= 181.0 h.
a=/?=90',
y= 120.0” and a unit of cell volume of 1.4= IO6 h3. Reflections OOil 3n are absent, and the data merge satisfactorily in point group 622 indicating that the space group is P6,22. Assuming 12 molecules in the unit cell, and one subunit per asymmetric unit, V, = 3.11 h3/Da. This result gives a solvent content of 6Oo/o. i.e. within the normal range of protein crystals (Matthews, 1977). A single type I1 crystal has been analysed using the Xentronics Area Detector. This indicates that the space group is P2,, with a=69.4 h, b=97.1 A. c= 71.4 h and /?= 109”. The crystal diffracted to dmin= 2.1 h. A self rotation function analysis (Rossmann & Blow, 1962) indicated the presence of a noncrystallographic 3-fold axis. Assuming three subunits per crystallographic asymmetric unit l’, = 1.99 h3/Da, corresponding to a solventJ content of 40%. Both crystal forms can accommodate a trimeric molecule and dynamic light scat’tering results indicate that this is also the stat,e of oligomerization in solution (Basak & Stuart, data not, shown). This suggests that the surface of the BTV core is formed by the accretion of preformed VP7 trimers onto the subcore to form a well defined T= 13 lattice. A search for heavy-atom derivatives is in progress in order t,o determine the three dimensional structure of BTV-10 VP7. A.K.B. was supported by the NERC. D.I.S. is a member of the Oxford Centre for Molecular Sciences.
References J. Q. & Ritchie. E. A. (1970). Some Browne, morphological features of bluetongue virus. Virology. 40, 903-911. French, T. J. & Roy, I’. (1990). Synthesis of bluetongue virus (BTV) core like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J. J’irol. 64, 1531)-1536. Fukusho. A.. Yu, Y., Yamaguichi. Y. & Roy. 1’. (1989). Completion of the sequence of bluetongue virus serotype 10 by the characterization of structural protein. VP6, and a non-structural protein, NS2. J. C:en. Viral. 1677-1689. Harlos, K. (1992). Micro-bridges for sitting-drop crystallizations. J. Appl. Crystallogr. 25, 536-538. Howard, A. J., Gilliand, G. L., Finzel, B. C., Poulos. T. L.. Ohlendorf. D. H. & Salemme, F. R. (1987). The use of an imaging proportional counter in macromolecular crystallography. J. Appl. Cystallogr. 20, 383-387. Huismans, H. (1979). Protein synthesis in bluetongue virus-infected cells. Virology, 82, 385-396. Loudon, P. T. CG Roy, P. (1991). Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of largest protein VP1 in the core and virus like particles. Virology, 180. 789-802. Matthews, B. W. (1977). In The Proteins (Neurath, H. and Hill, R. L., eds), 3, pp. 403-590, Academic Press. New York. McPherson. A. ,J. (1982). In Preparath and Anulysis oj Protein Crystals, pp. 82~160, John Wiley and Sons. New York.
Crystallization Oldfield, S., Adachi, A., Urakawa, T., Hirasawa, T. & Roy, P. (1990). Purification and characterization of the major group-specific core antigen VP7 of bluetongue virus synthesized by a recombinant baculovirus. J. Gen. Viral. 71, 26492656. Rossmann, M. G. & Blow, D. M. (1962). The detection of Sub-units within the crystallographic asymmetric unit. Acta Crystallogr. 15, 24-31.
Notes Roy, P., Hirasawa, T., Fenandez, M., Blinov. V. M. & Sanchez-Vixcain Rodrique, J. M. (1991). The complete sequence of the group-specific antigen, VP7, of African homesickness disease virus serotype 4 reveals a close relationship to bluetongue virus. J. Gen. Viral. 72, 1237-1241.
Edited by R. Huber
