of Bluetongue Virus Particles Cryoelectron Microscopy
A. HEWAT, *J TIMOTHY F. BOOTH,: AND POLLY Rout93
*Laboratoire de Biologie Structurale, CEA and CNRS URA 1333, DBMSIDSV, CENG, 85X 38041 Grenoble, France; ?NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, United Kingdom; iLaboratory of Molecular Biophysics, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom; and #Department of Environmental Health Sciences, School of Public Health, The University of Alabama at Birmingham, Universityy Station, Birmingham. Alahama 35294
Received April 15, 1992, and in revised form June 22. 1992
forms tubules and NS2, an ssRNA-binding protein, forms virus inclusion bodies (Lecatsas 1968, Huismans et al., 1987. Urakawa and Roy, 1988, Thomas et al., 1990). BTV is the best-characterized Orbivirus. The complete sequence for BTV serotype-10 tBTV-10) is known (Fukusho et al., 1989) and the genomes of many of the 24 known serotypes have been at least partially sequenced. The biochemistry of BTV infection and assembly has been studied using expression in insect cells of up to five BTV proteins simultaneously using baculovirus vectors (Loudon and Roy, 1991) and structures formed during BTV infection have been identified using immunoelectron microscopy (e.g., Hyatt and Eaton, 1988, Hewat et al., 1992a). Structurally the BTV virus particle is less well characterized. The inner shell has been shown to consist of an almost spherical inner layer composed of 60 copies of VP3 arranged on a T = 1 lattice (Hewat et al., 199213). Attached to the VP3 layer on a T = 13,l lattice are 260 triangular spikes (VP7 trimers) (Prasad et al., (1992); There are pores in the VP3 layer which could allow the passage of metabolites and RNA to and from the core of RNA transcription during infection. Largely on the basis of conventional electron microscopy the BTV outer shell has generally been described as fuzzy or having no well-defined structure (Mertens et al., 1987, Verwoerd et al., 1972). In this paper we describe 3-D reconstructions of BTV-10 virus particles (including data to 4.5 nm) using image analysis of cryoelectron micrographs. This lowresolution structure of the BTV virus particle reveals a well-ordered outer shell. The belief that the outer shell of BTV had no well-defined structure is an example of the problems that can occur in classical negative stain electron microscopy. Using cryoelectron microscopy to observe biological molecules in amorphous ice the problems associated with heavy metal stains, fixatives, dehydration, etc. are avoided.
The structure of the bluetongue virus (BTV) particle, determined by cryoelectron microscopy and image analysis, reveals a well-ordered outer shell which differs markedly from other known Reoviridae. The inner shell is known to have an icosahedral structure with 260 triangular spikes of VP7 trimers arranged on a T = 13,l lattice. The outer shell is seen to consist of 120 globular regions (possibly VP5), which sit neatly on each of the six-membered rings of VP7 trimers. “Sail’‘-shaped spikes located above 180 of the VP7 trimers form 60 triskeliontype motifs which cover all but 20 of the VP7 trimers. These spikes are possibly the hemagglutinating protein VP2 which contains a virus neutralization epitope. Thus, VP2 and VP5 together form a continuous layer around the inner shell except for holes on the 5-fold axis. v 1992 Academic
Bluetongue virus (BTV) is the prototype virus of the Orbivirus genus of the family Reoviridae. It is an arthropod-borne virus that infects ruminants both domestic (sheep, goats, cattle water buffalo, etc.) and wild (white-tailed deer, elk, pronghorn, antelope, etc.) in tropical and subtropical regions. The BTV genome consists of 10 dsRNA segments each of which encodes at least one protein, seven structural, and three nonstructural (Huismans, 1979; Martins et al., 1973; Verwoerd et al., 1970, 1972). The outer shell of the viral capsid composed of VP2 and VP5 is removed once the virion has entered the host cell to yield the core particle consisting of two major inner shell proteins (VP3 and VP71 and three minor proteins WPl, VP4, and VP61 plus the dsRNA genome. Synthesis of mRNA occurs within the uncoated core using a particle-associated transcriptase, with both strands of dsRNA remaining within the core. The function of the three nonstructural proteins NSl, NS2, and NS3 in virus replication is unknown. NSl 1To whom correspondence should be addressed at Institut de Biologie Structurale. 41 av. des Martyrs 38027Grenoble Ceden 1, France. 61
Copyright c 1992 by Academic Press, Inc. All rights
III ;iny form
BOOTH, AND ROY
Using the available sequence and biochemical data we make a tentative attribution of the specific features of the reconstructed protein density to VP5 and VP2 which has a hemagglutinating action and contains a virus neutralization epitope. The BTV outer shell forms a relatively impervious layer around the inner shell except for the holes on the 5-fold axes. This differs markedly from the outer shells of the simian strain SAll rotavirus (Prasad et al., 1988, 19901, rhesus rotavirus (Yeager et al., 1990) and the human reovirus (Metcalf et al., 1991) which have a more porous structure. The possible implications of these structural differences for the replication cycle of BTV are discussed. It will be noted that, for simplicity of comparison with the literature on other Reoviridae, we refer to the outer shell (VP5 and VP21 and the inner shell (VP3 and VP7). In the BTV literature the inner and outer shell are often referred to as the inner and outer capsid. The core particle refers to the virus particle with the outer shell removed and is sometimes referred to as the single-shelled particle. The core-like particle refers to the shell formed spontaneously when BTV VP3 and VP7 are expressed simultaneously by baculovirus expression (Loudon and Roy, 1991). MATERIALS Growth
Virus particles and core particles were prepared after the methods of Huismans et al., (1983) and Mertens et al., (1987). Virus particles were purified from plaque-purified BTV-lo-infected BHK 21 cell cultures by centrifugation of cytoplasmic extracts on a discontinuous sucrose gradient (40-66% v/w). Prepamtion
Frozen-hydrated specimens were prepared on holey carbon films as described by Dubochet et al., (1985). A modified Zeiss cryoworking unit was employed which allows manipulation of the specimen grid in gaseous rather than liquid nitrogen. The holey carbon films, supported on 400-mesh copper grids were glowdischarged prior to use. Samples of the virus suspension (4 ~1) were applied to grids, blotted immediately with filter paper for l-2 set, and were rapidly plunged into liquid ethane cooled by nitrogen gas at - 175°C. Specimens were observed at temperatures of - 165 to - 175°C in a Zeiss 1OC electron microscope equipped with a top entry cold stage. The microscope was operated at 100 kV and images were recorded at x 20 000 magnification with a dose of less than 10 e-/A’ on Kodak SO163 electron image film which was developed in full-strength D19 for 12 min at room temperature. The magnification was calibrated in an independent experiment using the 4.0~nm first layer line in computed Fourier transforms of microtubule images (Amos and Klug, 1974, Chretien, 1991). Due to the very low concentration of virus particles it was necessary to locate individual virus particles separately at x 2500 magnification before imaging at high magnification. The total dose in this search mode was less than 0.5 e-/A’.
Specimens were prepared for conventional electron microscopy using each of the following negative stains: 2% w/v uranyl acetate, pH 4.4, 2% w/v phosphotungstic acid (PTA), pH 7 and 5, or 2% w/v sodium silicotungstate (SST), pH 7. A range of stains is used as a standard measure to check for stain-dependent artifacts. These specimens were also observed in the Zeiss 1OC at room temperature at 100 kV under low dose conditions. Image
Preliminary examination of the micrographs was carried out using a CCD TV camera linked to a dedicated computer which produced the power spectrum of a 512 x 512 pixel image in 2 set (Tietz video and processing systems). Selected micrographs were digitized for further analysis using a CCD TV camera coupled to a PC with an ADC card. A pixel size of 10.5 +rn on the micrograph was employed, which for a magnification of x 20 000, corresponds to a pixel size of 5.2 A at the specimen. Image analysis was performed on a Vaxstation 3200 using the Semper 6 Plus image analysis package and a version of the MRC icosahedral reconstruction programs (Crowther et al., 1970a,b, Crowther, 1971) extensively modified by Fuller (19871 to improve the orientation refinement and analysis of the low-contrast cryoelectron microscope virus images. Images of virus particles from 12 different micrographs, taken at roughly 3 pm defocus, were analyzed. It was necessary to normalize the data by subtracting the mean and normalizing the standard deviation of gray levels as described in Carrascosa and Steven (1978). No correction was made for variation in magnification since previous experience has shown that this is not necessary with the Zeiss 1OC top entry specimen holder. This specimen holder has no tilting facility and returns to the same position in the microscope after extraction and insertion as judged by the remarkably constant objective lens current at focus. The orientation and origin of each particle are determined by the method of common lines (Crowther, 1971). The average cross common lines phases reached a value of 90” (i.e., random phases implying no correlation) at 4.0 nm resolution. Minimization of the phase residual between cross common lines in different images is used to obtain the best agreement between each image and the whole data set. The Fourier transform data from a complete data set is then interpolated at regular intervals on a cylindrical coordinate system using the matrix inversion described by Crowther, 1971, with the axis along one of the 5-fold icosahedral axes. The density map is calculated by Fourier-Bessel inversion with only D5 symmetry and complete icosahedral symmetry is imposed by real space averaging (Fuller, 1987). In the final reconstruction only 12 of the 35 virus particle images analyzed were retained and data out to 4.5 nm were included. Particles from 8 of the 12 micrographs were included. In general the agreement between particles on a given micrograph was no better than the agreement between particles on different micrographs. Of 12 particles selected on one micrograph only 4 were retained. We believe that the major cause of particle rejection, determined by the phase residuals for the self an&or cross common lines tests, was small inhomogeneities in the virus particle population rather than differences in magnification or defocus in the different micrographs. For the final 12-particle reconstruction all reciprocal eigenvalues for the normal equations within a radius of 45-A resolution were less than 1.0 and 99% were less than 0.1. Thus, the reciprocal space was adequately filled to 45 A. The reconstruction with only D5 symmetry showed essentially the same features (although somewhat noisier) as the fully icosahedrally symmetrized density map. Surfaces of reconstructions were visualized using the Explorer program on a silicon graphics computer.
Electron microscope (EM) images of negatively stained BTV virus particles show little surface contrast compared with the well-defined capsomeric structure seen in negatively stained BTV core particles (Figs. la and lb). Cryoelectron microscope images of frozen-hydrated BTV virus particles (Fig. 2) also show a granular surface without the clear capsomeric structure seen on the BTV core particles or on the rotavirus particle for example (Prasad et al., 1988). Occasionally empty virus particles and core particles are found. The absolute hand of the virus particle cannot be determined from the cryo-EM images which are projections of the structure. The BTV-10 core-like particle was determined to be left-handed from the sur-
FIG. 2. Cryoelectron micrographs of BTV virus particles at approximately 3 nm under-focus (al and (b), plus core particle (cl, plus empty virus particle (d), plus empty virus particle and subcore-like particles (arrowheads) (el. The scale bar represents 100 nm.
FIG. 1. Electron micrographs of negatively stained BTV virus particles (SST) (a) and core particles (PTA) (b). An empty virus particle is arrowed in a and an empty core particle is arrowed in b (inset). The scale bar represents 100 nm.
face structure observed on freeze-etched, heavy metal shadowed core-like particles (Hewat et al., 1992b). We used the convention of Caspar and Klug (1962). Prasad et al. (1992) found the core particle lattice to be left-handed by a technique involving comparison of tilted images. The hand of the virus particle is determined by the T = 13,l lattice of the core. Freeze-etched, heavy metal shadowed BTV virus particles have a knobby appearance but location of the symmetry axes is too uncertain to determine the hand of the structure. This is perhaps not surprising given the shape of the outer surface (Fig. 3), which is determined by the T = 13,l lattice of the core particle but does not have a T = 13,l lattice, i.e., it does not have all the local 3- and 6-fold axes of the T = 13,l lattice. A surface representation of the icosahedrally symmetrized reconstructed virus particle is shown in
HEWAT, BOOTH, AND ROY
FIG. 3. (a,b) Surface representations of the icosahedrally symmetrized reconstructed virus particle viewed along a 3-fold axes. Icosahedral5-, 3-, and a-fold axes are marked on (b). Also the globular regions and sail-shaped spikes arranged in a triskelion motif are marked on b. The scale bar represents 10 nm. FIG. 5. Surface representation of the outer shell of the reconstructed virus particle viewed along a 3-fold axis. The globular regions (VP5) are colored in yellow and the sail-shaped spikes (VP2) in blue. The scale bar represents 10 nm. FIG. 7. An enlarged view of the BTV outer shell proteins. The local 3-fold axis of the triskelion motif is marked with a triangle. The perfection of this triskelion motif is witness to the validity of the reconstruction since the icosahedral averaging does not average over the three subunits. The globular regions of density (marked with asterisks) make more or less close contact (see arrowheads) with four surrounding monomers of the neighboring triskelion motifs. The scale bar represents 2.5 nm.
Fig. 3 viewed along the 3-fold axes. At first sight the surface representation of the complete virus appears alarmingly disorganized, rather like the negatively stained images! However, the organization of this complex structure can be better appreciated by ob-
serving the surface representation of selected layers of density, that is by setting the density to zero everywhere except between two chosen radii (Fig. 4). The inner shell is visualized in the layer of radius 22 to 32 nm. The outer tips of the spikes on the inner
OF BTV VIRUS PARTICLES
FIG. 4. Surface representations of shells of the icosahedrally symmetrized reconstructed virus particle viewed along a 3-fold axes. The !nner capsid (radius 22 to 32 nm) is shown in a seen from outside the particle and b from inside the particle. Dots mark the position of some of the VP7 spikes in a. icosahedral 5-fold and local 6-fold axes of the 7’ = 13.1 lattice are also marked and the additional density riot seen in the core-like particle reconstruction is narrowed Cb). The outer 4 nm of the virus particle (radius 39 to 43 nm) is shown in r +nd the outer 8.5 nm (radius 34.5 to 43 nm) is shown in d. Local 3-fold axes of triskelion motifs (small 3) and an icosahedral 3-fold axis I large 3) are marked on c. The globular regions (VP5) are marked with asterisks on d. The scale bar represents 10 nm.
shell have been cut short (by 2.5 nm) to avoid confusion from density contributions from the outer shell proteins. The diameter of the complete inner shell is 69 nm. In agreement with previous reconstructions of the core particle (Prasad et al., 19921, the T = 13,l lattice of VP7 spikes is visible on the outer surface of the inner shell (Fig. 4a). The largest pores in the VP3 layer, located around the icosahedral 3-fold axes, are visible most clearly from inside
the capsid (Fig. 4b). One difference with the core-like particle reconstruction (Hewat et al., 1992131 are the lumps of density inside the VP3 layer. These may be due to one or more of the minor core proteins, VPl, VP4, or VP6. However, the presence and identity of this density must be confirmed, for example, by a higher resolution reconstruction of core-like particles plus one or more of the minor core proteins. The surface representation of the outer 4 nm of
the virus particle (radius 39 to 43 nm) reveals “sail”shaped spikes linked together in threes to form a triskelion-type motif which covers four VP7 trimers (Fig. 4~). These outer shell spikes each sit on top of a VP7 trimer. Thus, all the VP7 is covered by outer spike proteins except the VP7 trimers on the icosahedral3-fold axis. It will be noted that the apparent 3-fold axis at the center of each triskelion is on a local 3-fold of the T = 13) lattice. These local 3-fold axes are not enforced by the reconstruction or icosahedral averaging process (Fig. 7). Each outer spike is shaped like a sail or right-angled triangle with an oval cross section. The overall height of these spikes is approximately 8.5 nm. The surface representation of the outer 8.5 nm of the virus particle (radius of 34.5 to 43 nm) reveals more of the outer spikes plus globular regions which sit on each of the 6-membered rings of VP7 trimers (Fig. 4d). Thus, the locations of proteins of the outer shell are determined by the T = 13,l lattice of the inner shell but they do not have a T = 13,l lattice. Sections through the density map also help to show the relative positions and shapes of the four capsid proteins. Sections through the particle origin perpendicular to 2-, 5, and 3-fold axes are shown in Figs. 6a-6c). Sections perpendicular to an icosahedral 3-fold through the VP7 trimers of the inner shell (d), through the globular regions of the outer shell (e) and through the sail-shaped spikes of the outer shell (f) are shown in Figs. 6d-60. Inside the capsid, that is in the core, the average density is lower than for the protein capsid as expected for dsRNA plus minor proteins. The density variations within the core cannot be considered significant as errors tend to accumulate at the center. DISCUSSION
VP3 and VP7
The present reconstruction of BTV-10 virus particles shows an inner shell which resembles at modest resolution (4.5 nm) the previous reconstructions of the BTV core particle (Prasad et al., 1992) and the BTV core-like particle (Hewat et al., 1992b). The complete core particle has been shown to consist of an almost spherical inner layer composed of 60 copies of VP3 arranged on a T = 1 lattice (Hewat et al., 1992b) with 260 triangular spikes (VP7 trimers) attached to the VP3 layer on a T = 13,l lattice (Prasad et al., 1992). The 60 VP7 spikes missing on the core-like particle are present both in the core particle and the virus particle reconstructions.
Attribution of the Outer Shell Proteins and VP5
The reconstruction shows an outer shell consisting of multiple copies of two regions of protein density which are easily identified by their distinctive shapes, almost spherical and sail-shaped. They can be seen in Fig. 7 with the near spherical regions colored in yellow and the sail-shaped spikes in blue. The outer shell is known to contain two major component proteins VP2 (i&l11 kDa) and VP5 (M, 59 kDa). VP2 is the principal serotype-determining antigen. It has a hemagglutinating activity (Cowley and Gorman, 1987; Loudon et al., 1991) and elicits neutralizing antibodies (Huismans and Erasmus, 1981, Kahlon et al., 1983, Inumura and Roy, 1987). From its sequence VP2 is seen to be hydrophilic in nature and contains many charged residues (Roy, 1989). It is rich in aromatic residues and conserved cysteine residues which may indicate a highly ordered, disulfide-bonded structure. VP5 is rich in nonpolar residues and has several hydrophobic regions (Roy et al., 1990). There is evidence that VP5 plays a role in serum neutralization of BTV but no neutralizing monoclonal antibodies to VP5 have been produced (Mertens et al., 1989). Thus, it is probable that the sail-shaped spikes, which project 4 nm beyond the globular regions, are VP2 and the globular regions are VP5. There is no evidence in the VP2 sequence for a three-domain structure, thus we suppose each spike is a VP2 monomer. This tentative assignment of VP2 and VP5 implies that the virus particle contains 180 copies of VP2 and 120 copies of VP5. A schematic diagram of the proposed locations of the four major capsid proteins is shown in Fig. 8. The reconstructed virus particle shows that both outer shell proteins are exposed to a certain extent to antibodies and enzymatic action and may have sites for cell recognition. The accessibility of VP2 and VP5 is in agreement with the observation that not only VP2 but also VP5 plays a role in eliciting an immune response in animals both in terms of a humoral IgG or IgM response and a cell mediated response (Roy et al., 1990b). The outer shell covers nearly all of the inner shell, but significantly, leaves 20 of the VP7 trimer spikes accessible to possible antibody binding. This is in agreement with immunogold-labeling experiments which show that virions react with VP7 monoclonal antibodies (Hyatt and Eaton, 1988). All anti-BTV antisera react with VP7 which is a group-specific antigen. The accessi-
FIG. 6. Sections of the reconstructed density map through the particle origin, viewed down (al a a-fold, (b) a &fold, and (c) a 3-fold axis. Sections viewed down a 3-fold axis (d) through the VP7 spikes, (e) through the globular regions (VP51, and (D through the sail-shaped spikes (VP2). Asterisks mark the position of the globular regions and the sail-shaped spikes are marked with a small arrow. Icosahedral axes are marked where appropriate. In d and e dots mark VP7 spike positions. It is noted that the VP7 spikes on the 3-fold axis (open arrow) in a appear to be pulled toward the exterior of the virus particle. In f one of the triskelion motifs is marked. The scale bar represents 10 nm.
BOOTH. AND ROY
FIG. 8. Schematic drawings of the proposed location of the four major capsid proteins of the BTV virus particle. a, b, and c represent an icosahedral triangular face. (a) The T = 13,l lattice of VP7 trimers represented by small triangles. Icosahedral2-, 3-, and 5-fold axes are marked. (b) The inner core proteins VP3 and VP7. VP3 has a T = 1 lattice with three copies per triangular face. The precise division of the density around each &fold axis into five VP3 is not known at present. (c) Schematic representation of VP7, VP5, and VP2. The globular regions siting on each sixmembered ring of VP7 spikes are supposed to be VP5 and the sailshaped spikes are supposed to be VP2. The VP2 spikes are linked in threes to form triskelion motifs which cover four VP7 spikes. Only the VP7 spike on the icosahedral 3-fold is left uncovered.
bility of some VP7 spikes at the virus surface is also in agreement with suggestions that VP7 plays a role in the attachment of cores and/or virus particles to the cytoskeleton or virus inclusion bodies during viral morphogenesis.
It is interesting to compare the structure of the BTV-10 to that of other members of the Reoviridae, the rotavirus (Prasad et al., 1988, 1990; Yeager et al., 1990) and the human reovirus (Metcalf et al., 1991). Each of these viruses has a double-shelled capsid with a T = 13) lattice for the core particle. While the outer shells of the rotavirus and the human reovirus also have a T = 13,l lattice with holes on the 5-fold and local 6-fold axes, BTV has an outer shell which fits on to the T = 13,l lattice and forms a relatively impervious shell around the core but is not a T = 13,l lattice. The outer shell of the rotavirus has 760 copies of a structural protein which covers all the inner spike proteins, and apparently ensures the structural integrity and protection of the virus. It is improbable that the fine spike hemagglutinating protein of the rotavirus is involved in maintaining the structural integrity. In contrast it is the hemagglutinating protein VP2 of BTV which covers the inner spike proteins (VP7) and thus may also play a role in ensuring the structural integrity and protection of the virus particle along with the second outer shell protein VP5. This difference in the outer shell of BTV and rotavirus explains why negatively stained images of BTV are relatively featureless compared with the high contrast, detailed images of the rotavirus. With BTV virus particles the stain does not penetrate into the interior of the outer shell, whereas with rotavirus the stain can penetrate via the holes in the outer shell. The core particle of BTV and the rotavirus have very similar features while the human reovirus has a remarkably different core particle with trumpetshaped projections around each of the &fold axes and very little evidence of other spikes on the core particle. Both rotavirus and human reovirus have fine spike-like proteins, which project from their outer shell and have a hemagglutinating activity, whereas the BTV hemagglutinating protein is a comparatively short compact spike according to the assignment given here. It will be interesting to see if these remarkable differences in capsid structure between different genus of the Reoviridae are associated with differences in the virus replication cycles. Is the apparent difference in permeability of the outer layer important or is this layer simply required to maintain the structural integrity of the virus particle?
It is a pleasure to thank C. Closse and J. R. Lalisan for expert technical assistance, F. Metoz for his assistance with the Explorer surface representation programs, R. H. Wade for encouragement and helpful comments on this manuscript, and S. D. Fuller for supplying the icosahedral reconstruction programs used in this work. This work was funded partly by NIH Grant A126879, MRC Grant G9020032CA, and EMBO and NERC.
REFERENCES Amos, L. A., and Klug, flagellar microtubules,
A. (1974) Arrangement J. Cell Sci. 14, 523-549.
Carrascosa, J. L., and Steven, A. C. (1978) A procedure for evaluation of significant structural differences between related arrays of protein molecules, Micron 9, 199-206. Caspar, D. L. D., and Klug, A. (1962) Physical principles of the construction of regular viruses, Cold Spring Harbor Symp. Quant. Biol. 24, l-24. Chretien, D. (1991). Apports de la cryomicroscopie electronique a l’etude de microtubules assembles in vitro, Thesis, Universite Joseph-Fourier, Grenoble. Cowley, J. A., and Gorman, B. M. (1987) Genetic reassortments for identification of the genome segment coding for the bluetongue virus haemagglutinin, J. Viral. 61, 2304-2306. Crowther, R. A. (1971) Procedures for the three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs, Philos. Trans. R. Sot. London Ser. B 261, 221-230. Crowther, R. A., Amos, L. A., Finch, J. T., De Rosier, D. J., and Klug, A. (1970a) Three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs, Nature 226, 421425. Crowther, R. A., De Rosier, D. J., and Klug, A. (1970b) The reconstruction of three-dimensional structure from projections and its application to electron microscopy, Philos. Trans. R. Sot. London Ser. A 317, 319-340. Dubochet, J., Adrian, M., Lepault, J., and McDowall, A. W. (1985) Cryo-electron microscopy of vitrified biological specimens, Trends Biochem. Sci. 6, 143-146. Fukusho, A., Yu, Y., Yamagouchi, Y., and Roy, P. (1989) Completion of the sequence of bluetongue virus serotype 10 by the characterization of structural protein VP6 and a non-structural protein, NS2. J. Gen. Viral. 70, 1677-1689. Fuller, S. D. (1987) The T = 4 envelope of sindbis virus is organized by interactions with a complementary T = 3 capsid, Cell 48, 923-934. Hewat, E. A., Booth, T. F., Wade, R. H., and Roy, P. (1992a) 3-D reconstruction of bluetongue virus tubules using cryo-electron microscopy, J. Struct. Biol., 108, 3548. Hewat, E. A., Booth, T. F., Loudon, P. T., and Roy, P. (199213) Three dimensional reconstruction of baculovirus expressed bluetongue virus core-like particles by cryo-electron microscopy, Virology, 189, 10-20. Huismans, H. (1979) Protein synthesis in bluetongue virusinfected cells, Viro1og.y 92, 385-396. Huismans, H., and Erasmus, B. J. t1981) Identification of t,he serotype-specific and group specific antigens of bluetongue virus, Onderstepoort J. Vet. Res. 48, 51-58. Huismans, H.. Van Der Wait. N. T.. Cloete. M.. and Erasmus B. J. (1983, The biochemical and immunologicai characterization of bluetongue virus outer capsid polypeptides, In Compans R. W., and Bishop, D. H. L.. (Eds.) Double-stranded RNA viruses pp. 165-181. Elsevier, Amsterdam. Huismans, H., Van Dijk, A. A., and Bauskin, A. R. ( 1987) In vitro phosphorilation and purification of a nonstructural protein of bluetongue virus with affinity for single-stranded RNA, J. VIr-01. 61, 3589-3595. Hyatt, A. D., and Eaton, B. T. (1988) Ultrastructural distribution of the major capsid proteins within bluetongue virus and infected cells, J. Gen. Viral. 69, 805-815. Inumaru, S., and Roy, P. (1987) Production and characterization of the neutralization antigen VP2 of blue tongue virus serotype 10 using baculovirus expression vector. Virology 157, 4722479.
Kahlon, J., Sugiyama, K., and Roy, P. (1983) Molecular basis of bluetongue virus neutralization, J. Virol. 48, 627-632. Lecatsas, G. (1968) Electron microscope study of the formation of bluetongue virus. Onderstepoort J. Vet. Res. 35, 139-150. Loudon, P. T., and Roy, P. (1991) Assembly of five BTV proteins expressed by recombinant baculovirus: Inclusion of the largest protein VP1 in the core-like and virus-like particles, Virology 180, 798-802. Loudon, P. T., Hirasawa. T., Oldfield, S., Murphy, M., and Roy, P. ! 1991) Expression of outercapsid protein VP5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses, Virology 182, 793-801. Martins, S. A., Pett, D. M., and Zweerink, H. J. (1973) Studies on the topography of reovirus and bluetongue virus capsid proteins, J. Viral. 12, 194198. Mertens, P. P. C., Burroughs, J. N., and Anderson, J. (1987) Purification and properties of virus particles, infectious subviral particles and cores of bluetongue virus serotypes 1 and 4, Virology 157, 375-386. Mertens, P. P. C., Pedely, S., Cowley, J., Burroughs, J. N., Corteyn, A. H., Jeggo, M. H., Jennings, D. M., and Gorman, B. M. (1989) Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype, Virology, 170, 561-565. Metcalf, P. M., Cyrklaff, M., and Adrian, M. (1991) The threedimensional structure of reovirus obtained by cryo-electron microscopy, EMBO J. 10, 3129-3136. Prasad, B. V., Yamagouchi, S., and Roy, P. (1992) Threedimensional structure of single shelled core particles of BTV, J. Viral., 66, 2135-2142. Prasad, B. V. V., Wang, G. J., Clerx, J. P. M., andChiu, W. (1988) Three-dimensional structure of rotavirus, J. Mol. Biol. 199, 269-275. Prasad, B. V. V., Burns, J. W., Marietta, E., Estes, M. K., and Chiu, W. (1990) Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy, Nature 343, 476479. Roy, P. (1989) Bluetongue virus genetics and genome structure: Review article, Virus Res. 13, 179-206. Roy, P., Marshal, J. J. A., and French, T. J. (1990) Structure of bluetongue virus genome and its encoded proteins, in Roy, P., and Gorman, B. M. (Eds.) Current Topics in Microbiology and Immunology: Bluetongue Virus, Vol. 162, pp. 43-87, SpringerVerlag, Heidelberg. Roy, P., Urakawa, T.. Van Dijk, A. A., and Erasmus, B. J. (1990br Recombinant virus vaccine for bluetongue disease in sheep, J. Viral. 64, 1998-2003. Thomas. C. P., Booth, T. F., and Roy, P. (1990) Synthesis of bluetongue viral-coded phosphoprotein and formation of inclusion bodies by recombinant baculovirus in insect ceils: It binds the single stranded RNA species, J. Germ. Vwol. 71. 2073-2083. Lrakawa, I’., and Roy, P. (1988) Bluetongue virus tubules made in insect cells by recombinant baculovirus: Expression of the NSl bluetongue virus serotype 10, J. Viral. 62, 39193927. Verwoerd, D. W., Els, H. J., de Villiers, E. M., and Huismans, H. (1972) Structure of the bluetongue virus capsid, J. Virol. 10, 783-794. Verwoerd, D. W., Louw, H., and Oellermann, R. A. (1970) Characterization of bluetongue virus ribonucleic acid, J. Viral. 5, 1-7. Yeager. M., Dryden, K. A.. Olsen, N. H., Greenberg, H. B., and Baker, T. S. (1990) Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconsutrction, J. Cell Biol. 110, 2133-2144.