Journal of General Virology (1992), 73, 3051-3064.

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Printed in Great Britain

Review article Bluetongue virus proteins Polly Roy Laboratory of Molecular Biophysics, University of'Oxford, South Parks Road, Oxford OX1 3QU and NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, U.K. and School of Public Health, University of Alabama at Birmingham, Birmingham, Alabama 35294, U.S.A.

Introduction

The virion

Bluetongue virus (BTV) is the prototype virus of the Orbivirus genus in the Reoviridae family. Orbiviruses that infect and are transmitted by arthropod vectors (e.g. gnats, ticks, mosquitoes, etc.) include viruses that may cause disease in their vertebrate hosts with serious economic consequences in some regions of the world. The BTV group, which consists of at least 24 different serotypes (BTV-1, -2, etc.), infects field and domestic animals (e.g. sheep and cattle), occasionally with high morbidity and mortality but often with almost no apparent clinical symptoms. Other orbiviruses that may cause disease in animals include African horse sickness virus (AHSV; nine serotypes, AHSV-1, -2, etc.) and epizootic haemorrhagic disease virus (EHDV; seven serotypes, EHDV-1, -2, etc.)of deer. Although BTV and AHSV were isolated in 1900, and initial morphological and biochemical characterization were reported as early as 1969 (see review article, Gorman, 1990), much of the current knowledge on the molecular biology, genome structure and encoded products has been obtained only recently. The data are predominantly based on research on BTV. Over the past few years, significant advances have been made in the understanding of the structurefunction relationships of the BTV genes and gene products, and the assembly process for the formation of virus particles. In part this has come from the availability of cDNA clones and the use of novel baculovirus expression vectors. A review article on the BTV genes and genome structure has been published (Roy, 1989). The present article will mainly address recent studies on the structure and function of the virus-encoded proteins and the topography of the structural proteins in the viruses, with particular reference to the assembly process of virion capsids. Some structural similarities of the capsid proteins of BTV, AHSV and EHDV, and an indication of their evolutionary relationships, will be discussed.

Like reoviruses and rotaviruses, BTV is a non-enveloped virus with concentric protein shells. The innermost shell of BTV, otherwise termed the subcore, has icosahedral symmetry and is composed of one major protein (VP3) (Huismans & van Dijk, 1990). This encloses three minor proteins (VP 1, VP4 and VP6) and the 10 segments of the dsRNA genome. Located on this subcore is a second major protein, VP7 (Huismans et al., 1987; Loudon & Roy, 1991; Prasad et al., 1992). Together with the subcore and internal components, the assembly is termed the core. The outer capsid consists of two other major protein species, VP2 and VP5 (Verwoerd et al., 1970, 1972). The 10 discrete dsRNA segments (designated L1 to 3, M4 to 6, $7 to S10) range in Mr from 0"5 x 106 to 2.7 x 106 (Fukusho et al., 1989) resulting in a total M~ of 19 x 106. Each BTV RNA segment, except the smallest (S 10), encodes a single viral polypeptide (Mertens et al., 1984). In addition to the seven structural proteins, there are four other virus-induced proteins, NSI, NS2, NS3 and NS3A, found in the infected cell (Roy & Gorman, 1990). Of these, NS3 and NS3A are the products of the SI0 gene (Mertens et al., 1984; French et al., 1989).

0001-1154©1992 SGM

The five core proteins of BTV In virus-infected cells, parental BTV virions are converted to a core particle (420S) containing VP1, VP3, VP4, VP6 and VP7 and exhibiting an RNA-dependent RNA polymerase activity. This enzymatic activity allows the virus to synthesize mRNA from the virion dsRNA templates (Verwoerd & Huismans, 1972; Huismans et al., 1987). To date, little progress has been made in assigning enzymatic functions to the five proteins of the core and their role in mRNA synthesis still remains largely unknown. However, the availability of cDNA clones for each BTV gene and their subsequent expression in

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P. Roy

Table 1. BTV-IO proteins Genome segment

No. of bp

Encoded protein(s)

No. of amino acids

Predicted Mr

No. of molecules/virion

Location

Function

L1 L2 L3

3954 2926 2772

VP1 VP2 VP3

1302 956 901

149588 111 112 103 344

approx. 6* 180t 601"

Inner core Outer shell Core

M4 M5 M6 $7 $8 $9 S 10

2011 1639 1770 1156 1123 1046 822

VP4 VP5 NSI tubules VP7 NS2 VIBs VP6 NS3 NS3A

654 526 552 349 357 328 229 216

76433 59163 64445 38 548 40999 35750 25 572 24020

approx. 5* 120t

Inner core Outer shell Non-structural Core surface Non-structural Inner core Non-structural

RNA polymerase Type-specific structural protein Structural protein, forms scaffold for VP7 trimers Capping enzyme, guanylyltransferase Structural protein Unknown Group-specific structural protein Binds mRNA Binds ssRNA, dsRNA Glycoproteins, aid virus release

NA~ 780t NA approx. 37* NA NA

* Estimated by Huismans & van Dijk (1990). 1"Calculated by cryoelectron microscopy and image processing techniques (Prasad et al., 1992; Hewat et al., 1992b, c). 3~NA, Not applicable.

baculovirus vectors have allowed some information on their structure-function relationships to be obtained.

The three minor core proteins: VP1, VP4 and VP6 The largest viral protein of BTV, VP1 with an Mr of 150K, is encoded by the largest dsRNA segment (L1) (Urakawa et al., 1989; see Table 1). VP1 is present in a low molar ratio in the virion (estimated at six molecules/ virion) (Huismans & van Dijk, 1990). Based upon its size, location, molar ratio in the core and predicted amino acid sequence, this protein is the prime candidate for the virion RNA polymerase (Roy et al., 1988); the primary sequence of VP1 contains a G D D motif, characteristic of other R N A polymerases. In addition, the predicted sequence has a strong homology not only with various DNA and R N A virus polymerases, but also with other eukaryotic and prokaryotic polymerase subunits (Roy et al., 1988). In extracts from insect cells (Spodoptera frugiperda) infected with a recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the BTV VP1 gene, poly(A) synthesis can be demonstrated when the extract is provided with a poly(A) primer and poly(U) template (Urakawa et al., 1989). Thus it has been proposed that VP1 may at least elongate R N A when supplied with a single-stranded substrate. Similarly to those of reoviruses and rotaviruses, the 5' ends of the m R N A species of BTV are believed to be capped and methylated during transcription, and the viral core proteins are presumed to be responsible for the enzymatic activities for the synthesis and modification of

these species. For reoviruses and rotaviruses, the R N A capping enzyme, guanylyl transferase, has been assigned to specific proteins (22 and VP3 respectively; Cleveland et aL, 1986; Mao & Joklik, 1991; Pizarro et aL, 1991; M. Liu et al., 1992). Data obtained by Mertens & Burroughs (1990) have indicated that a GTP-core complex catalyses the capping of BTV m R N A and suggest that the guanylyl transferase activity of BTV is probably associated with the minor core protein, VP4. Direct evidence for GTP binding by VP4 has been obtained by using a recombinant baculovirus that expresses VP4 (Le Blois et aL, 1992). The data support the view that VP4 is probably the guanylyl transferase of the virus. The third minor protein, VP6, is encoded by small R N A segment 9 ($9). VP6 has an Mr of 35K (Roy et aL, 1990a; Table 1). It is a highly basic protein, abundant in arginine, lysine and histidine residues (Fukusho et aL, 1989); the composition suggests that it may bind to nucleic acids. VP6 exhibits two domains (N- and C-terminal) separated by a glycine-rich region. Since VP6 is a minor component of the virion, it can be conjectured that, like VPI and VP4, it is a component of the mRNA polymerase complex. Baculovirus-expressed and BTV-derived VP6 have been shown to have a strong binding affinity for both ss- and dsRNA species even in the presence of SDS, indicating that its binding capability is independent of tertiary structure (Roy et al., 1990 a). Sequence analyses of VP6 have revealed a motif common to helicases. Such an activity may be involved in unwinding the dsRNA genome prior to the synthesis of cRNA species. VP6 is probably closely associated with the viral genome in virions and may aid the encapsidation of RNA.

R e v i e w : Bluetongue virus proteins

The major core proteins: VP3 and VP7 Proteins VP3 and VP7 are the two major proteins of the core, VP7 being the more abundant. The VP7 protein is located on the surface of core particles, VP3 forming an inner shell (the main structure of the subcore). VP7 is encoded by small RNA segment 7 ($7) and has an Mr of 38K (Yu et al., 1988; Oldfield et al., 1990; Table 1). VP7 protein is extremely hydrophobic, the hydrophobic regions resembling those associated with the lipid-free protein coats of fd-type bacteriophages (Smilowitz et al., 1972; Fukusho et al., 1989). This hydrophobicity is particularly evident in AHSV-4 (Roy et al., 1991 ; Chuma et al., 1992). A striking feature of the BTV VP7 protein is that it contains only one lysine residue (Yu et al., 1988; Kowalik & Li, 1991). This lysine (K 255) is conserved among all BTV serotypes sequenced and is shared by the VP7 of the closely related EHDV-1 (Kowalik & Li, 1991 ; Iwata et al., 1992a). For AHSV-4 this lysine residue is replaced by an arginine, suggesting that the positively charged residue at position 255 may have a particular structural role. Studies involving site-specific mutagenesis will be necessary to determine whether this positively charged residue indeed has any such role. The high level expression of VP7 by recombinant baculovirus and its presence in the soluble fraction of infected insect cells has allowed the purification of the protein to virtual homogeneity (>98~o). In contrast to the BTV VP7 protein, that of AHSV-4 synthesized in insect cells by recombinant baculoviruses self-assembles to form disc-shaped crystalline structures of various sizes. The expressed and purified VP7 protein of BTV-10 has been crystallized and two forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6322, with a --- b = 9.52 nm, c = 18-10 nm, ct = fl--90 °, 2: = 120"0°, and contain a single subunit in the crystallographic asymmetric unit. They diffract to d m i n = 0"3 nm. Type II crystals belong to space group P21 with a = 6.49 nm, b = 9.71 nm, c = 7.14 nm,/3 = 109.0°, and contain a trimer in the crystallographic asymmetric unit. They diffract to d m i n = 0.21 nm (Basak et al., 1992). These results, together with solution studies, conclusively demonstrate that VP7 forms trimers, and the threedimensional (3D) structure has almost been completely characterized. The third largest RNA segment (L3) encodes the VP3 protein (Mr 100K, Table 1). Like VP7, VP3 is also hydrophobic in nature. The protein plays an important role in the structural integrity of the virus core. Both VP7 and VP3 are highly conserved, not only among the various BTV serotypes that have been analysed, but also in AHSV-4 and EHDV-1. Baculovirus-expressed VP3 and VP7 proteins have been demonstrated to contain

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group-specific antigenic determinants (Inumaru et al., 1987; Oldfield et al., 1990). Both proteins are recognized by all anti-BTV antisera, and also react weakly with antiAHSV and anti-EHDV antisera (Oldfield et al., 1990; Chuma et al., 1992).

The outer structural proteins: VP2 and VP5 The icosahedral core of BTV is surrounded by an outer coat composed of VP2 and VP5, which in contrast to that of reoviruses, exhibits no well defined structure in negatively stained preparations. VP2 and VP5 are encoded by RNA segments L2 and M5, respectively (Verwoerd et al., 1972; Mertens et al., 1984), and exhibit the least conservation between virus serotypes (for a review see Roy et al., 1990b). VP2 is the most variable, is the main serotype-specific antigen (Huismans & Erasmus, 1981; Kahlon et al., 1983; Roy et al., 1990¢) and is the viral haemagglutinin (Cowley & Gorman, 1987; French et al., 1990; Loudon et al., 1991). Antisera raised in rabbits following immunization with baculovirus-expressed BTV-10 VP2 protein efficiently neutralize the infectivity of BTV-10 (Inumaru & Roy, 1987). In addition, the antisera neutralize, to lesser extents, certain other BTV serotypes (BTV-20, -11, -17 and -4), and antisera raised against the VP2 proteins of other BTV serotypes (BTV- 1, -2, - 11, - 13 and - 17) also neutralize the respective homologous virus and crossneutralize to lesser extents a number of other BTV serotypes (unpublished observations). These data indicate that some BTV serotypes are more closely related than others, as shown by a dendrogram (Fig. 1a). This is also reflected by VP2 sequence comparisons (39~ to 73~o identity between BTV-1, -2, -10, -11, -13 and- 17; Roy et al., 1990b). In contrast to VP2, very little is known about the function of VP5. The protein is more variable than all the core proteins, but not as variable as the VP2 protein. The primary structures of the VP5 proteins of a number of BTV serotypes (BTV- 1, -2, - 10, - 13, - 11 and - 17) are quite similar (see Fig. 1¢), sharing up to 94~o identical amino acids (Hirasawa & Roy, 1990; Oldfield et al., 1991 ; Yang & Li, 1992). Although the protein is located in the outer capsid, it does not appear to have any distinct neutralizing activity. Antisera raised against baculovirusexpressed VP5 protein do not demonstrate any neutralizing activity in vitro (Marshall & Roy, 1990). However, in contrast to these results, vaccination studies in sheep using VP2 and VP2-VP5 combinations have provided a different view (Roy et al., 1990c). When VP2 is used alone in doses of >50 ~tg/sheep, protection against virulent BTV-10 challenge is obtained. A dose of approx. 50 ~tg of VP2 protects some but not all sheep. However,

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(a)

- -

t

--

BTV-I 1

--

BTV-17

(b)

BTV-2

(c)

(d) BTV-1

-I

BTV-1

BTV-2 BTV-17

BTV-10

BTV-I 1 BTV-I(A) BTV-17

BTV-17 - -

BTV- 10

BTV-2

BTV-10

BTV-13

- -

--EHDV-1

EHDV-1 AHSV-4

AHSV-4

-

-

i

BTV-I(SA)

BTV-13

BTV-13

EHDV-I

EHDV-I

AHSV-4

AHSV-4

Fig. 1. Dendrogram showing relationships between VP2 (a), VP3 (b), VP5 (c) and VP7 (d) of different BTV serotypes, EHDV-1 and AHSV-4 generated using the CLUSTAL V program (Higgins & Sharp, 1989) and the default parameters. The score of the most distant pairs in (a) is 0.95 (e.g. AHSV-4 and BTV-1), in (b) 0.41 (e.g. AHSV-4 and BTV-10 or EHDV-1), in (c) 0-55 to 0.56 (e.g. AHSV-4 and BTV-1 or EHDV-I), and in (d) 0.55 to 0-56 (e.g. AHSV-4 and BTV-1 or EHDV-1).

Table 2. Amino acid sequence identity and similarity between four major structural proteins of three orbiviruses Core

Outer capsid

VP3

EHDV-1 AHSV-4

VP7

VP5

Sequence comparison

BTV-10

EHDV-1

BTV-10

EHDV-I

BTV-10

Identity (~) Similarity (~) Identity (~) Similarity (~)

79 90 58 76

57 75

37 81 44 66

-

62 77 45 63

when used in combination with approx. 20 gg of VP5, 50 lag quantities of VP2 protect all vaccinated sheep and elicit a higher neutralizing antibody response. This study indicates the involvement of VP5 in protection and in virus neutralization. Perhaps VP5 enhances the immune response indirectly by interaction with VP2, and by affecting the conformation of VP2 and, consequently, its serological properties.

Evolutionary relationships among three orbiviruses as evidenced by the sequences of the major capsid proteins AHSV, BTV and EHDV induce seasonal diseases in both domestic and wild ruminants, and in certain regions of the world two or three of these viruses are sympatric, hence there is opportunity for them to infect the same vectors and host species. This raises the question of whether the viruses have evolved from a common ancestor. To obtain such information, the sequences of the four major capsid proteins (namely VP2, VP3, VP5

46 67

VP2 EHDV-1

BTV-10

EHDV-1

42 64

23 49 17 44

20 44

and VP7) of EHDV-1 and AHSV-4 have been deduced from cDNA analyses, and compared with those of BTV-10 (see Table 2 and Fig. 1). The predicted amino acid sequence comparisons revealed that of the four capsid proteins, the innermost protein, VP3, is the most conserved, and the outermost protein, VP2, is the most variable. Some 57 to 58 ~oof the aligned BTV-10 and EHDV-1 VP3 amino acids are identical with those of AHSV-4 VP3. This compares to an identity of 79~ between the BTV and EHDV VP3 sequences. For the VP7 proteins 64~o of the aligned amino acids of BTV-10 and EHDV-1 are identical, whereas 44~ to 46 ~ of amino acid residues are identical with the aligned AHSV-4 sequence (Iwata et al., 1991, 1992 a, b; Roy et al., 199 l). As expected, the VP2 proteins of the three viruses are only 17 to 23~ identical. However, various other comparative analyses of the proteins indicate that the VP2 species of the three orbiviruses are similar. Unlike VP2, the other outer capsid protein VP5 is more conserved among the three viruses. On alignment, the AHSV-4 VP5 is 42 to 45~ identical in amino acid sequence to those of BTV-10 and

Review: Bluetongue virus proteins

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Fig. 2. (a) Cryoelectronmicroscopeimageof BTV NS1 tubulesat a defocusof 3 ~tm Note the step in diameter of a tubule. Short,end-on tubules are also visible. (b) Model showing the proposed unwinding mechanism of the dimer ribbon (Hewat et al., 1992a).

EHDV-I. The identity between BTV and E H D V VP5 is 6 2 ~ (Iwata et al., 1991, 1992b) Clearly, these viruses share strong phylogenetic relationships, and the differences in VP2 structure possibly reflect the immune pressure of the host receptor.

The non-structural proteins: NS1 to 3 At least four different non-structural proteins have been identified in BTV-infected cells. The two major nonstructural proteins, NS1 and NS2, are synthesized abundantly, whereas the two minor, closely related, proteins NS3 and NS3A are barely detectable. The sequences of all four NS proteins are highly conserved (96~) among BTV serotypes (see review by Roy et al., 1990b). The synthesis of NS1 and NS2 in BTV-infected cells coincides with that of two virus-specific structures, tubules and granular viral inclusion bodies (VIBs) which characterize the cytoplasm of cells infected with orbiviruses (Lecatsas, 1968; Cromack et al., 1971). VIBs have both granular and fibrillar characteristics and are found throughout the cell, but predominantly in proximity to the nucleus (Eaton et al., 1990). Virus tubules are present in large numbers predominantly in peri- or juxtanuclear locations. These morphological structures are believed to be attached to the intermediate filament

components of the cytoskeleton of the cell (Eaton et al., 1988) and are presumed to be involved in some way in the virus replication or transportation process. Tubules are composed entirely of one type of protein, a 64K protein, NS1, the gene product of middle-size R N A segment 6 (M6) (Urakawa & Roy, 1988; Table 1). This protein is expressed more highly than any other BTV protein (Huismans & Els, 1979). NS1 is rich in cysteine residues, and all 16 are conserved in the NS1 proteins of the different BTV serotypes analysed (for a review see Roy et al., 1990b). This suggests that NS1 has a highly ordered disulphide-bonded structure. In addition, NSI possesses several very strong hydrophobic regions which may play a role in the formation of tubules. When the NS1 gene is expressed in insect cells by a recombinant baculovirus, tubules similar to those found in BTVinfected mammalian cells are formed, confirming that the tubules are multimeric forms of the NS1 protein only (Fig. 2; Urakawa & Roy, 1988). How NS1 forms tubules and the roles of tubules in the course of infection are unknown. Some virions and inner core particles have been observed in association with tubules in the cytoplasm of BTV-infected cells (Eaton et aL, 1988). Detailed structural analyses of tubules have been undertaken using cryoelectron microscopy (Hewat et aL, 1992a). The analyses reveal that the tubules are on

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P. R o y

average 52.3 nm in diameter and up to 1000 nm long with a helical configuration. The structure of the helical surface lattice has been determined using computer image processing to a resolution of 4 nm. The NS1 protein, which is about 5-5 nm in diameter, forms a dimer-like structure. The tubular structure consists of a helically coiled ribbon of these NS1 dimers, with about 21 or 22 dimers per turn. The surface lattice displays P2 symmetry and forms a one-start helix with a pitch of 9-1 nm. The ribbon of dimers may be seen unwinding from the ends of tubules under certain conditions. A possible model for the unwinding of the dimer ribbon is shown in Fig. 2. Biochemical and physical analyses of mutant forms of NS1 are in progress to provide further information on the detailed structure of tubules. None of the BTV structural proteins are 15hosphorylated. The only virus-specific phosphoprotein that can be identified in BTV-infected cells is NS2 (Devaney et al., 1988; Huismans et al., 1979). This protein, which is encoded by RNA segment 8 ($8), is very rich in charged amino acids (Fukusho et at., 1989). In BTV-10-infected mammalian cells (like NS1) NS2 is synthesized to a high level, indicative of its important role in the virus life cycle. To determine the specific location of NS2 protein in BTV-infected cells, electron microscopy studies have been performed. Immunoelectron microscopy in conjunction with gold particles has proved to be the most sensitive and specific method for these studies. Using an anti-NS2 monoclonal antibody (MAb), or polyvalent monospecific antibodies raised against a baculovirusexpressed NS2 protein, it has been demonstrated that NS2 is present mostly in VIBs and that it is not associated with virions (Hyatt & Eaton 1988; Thomas et al., 1990). Purification experiments by FPLC have indicated that the protein exists predominantly in a multimeric form (unpublished data). Expression of NS2 by baculovirus vectors also results in the formation of VIB-like structures (Thomas et al., 1990). The morphology of the expressed VIBs is virtually identical to that of BTV-derived VIBs. In vitro studies with baculovirus-expressed NS2 have shown that NS2 is phosphorylated at specific serine residues (amino acids 185 and 308 in particular, but also residues 182, 289, 291 and 292). To what extent these sites are phosphorylated in the population of NS2 molecules is unknown. The cellular enzyme(s) involved in phosphorylation is not known. In an attempt to determine whether phosphorylation is important in RNA binding, studies have shown that dephosphorylated NS2 protein binds ssRNA efficiently. Thus phosphorylation does not appear to be important in RNA binding (Thomas et al., 1990). Mutagenesis studies are needed to confirm these data.

In one study, deletion of up to 130 amino acids from the C terminus of NS2 did not abolish the RNA-binding activity. In contrast, the removal of even 40 amino acids from the N terminus totally prevents ssRNA binding (Bremer et al., 1992). These data indicate that the N- but not the C-terminal domain of NS2 is needed for proteinRNA interactions. Unlike the NS1 and NS2 proteins, the two smallest non-structural proteins, NS3 and NS3A, are synthesized in low abundance in BTV-infected BHK cells. Both NS3 and NS3A proteins are encoded by BTV S10 dsRNA, as established by in vitro translation (Mertens et ak, 1984) and through the use of expression vectors. Insufficient NS3 and NS3A synthesized by BTV in mammalian cells has prevented detailed analyses of their structure, although some information on their structural and immunological properties have been obtained from cDNA analyses and the use of expression vectors. Peptide maps and immunological analyses have indicated that NS3 and NS3A are related (French et al., 1989). The S10 gene has two conserved in-frame methionine codons, suggesting that NS3 and NS3A may derive from alternative translation initiation sites (Lee & Roy, 1987). Deletion of the first AUG abolishes synthesis of NS3 but not that of NS3A (Wu et al., 1992). Predicted protein sequence analyses of the encoded products of BTV-10 S10 dsRNA have revealed at least two conserved hydrophobic domains (amino acid residues 118 to 147 and 156 to 182, respectively), which may serve as transmembrane domains. In addition, two potential glycosylation sites (amino acid residues 63 to 65 and 150 to 152) are also present. In immunoelectron microscopic studies of recombinant vaccinia virus expressing NS3, Hyatt et al. (1991) have recently reported that NS3 and NS3A are associated with intracellular, smooth-surfaced vesicles and the plasma membrane. This suggests that the proteins may be involved in the final stages of BTV morphogenesis, i.e. the release of BTV from infected cells. NS3 may share some functional similarities with the rotavirus glycoprotein NS28, which mediates the binding of rotavirus particles to the rough endoplasmic reticulum and the acquisition of a transient envelope (Meyer et al., 1989; Au et al., 1989). Recent studies involving the transient expression of NS3 and NS3A proteins in mammalian cells have provided data which indicate that both NS3 and NS3A exist as N-linked glycoproteins containing high-mannose sugars (Wu et al., 1992). Both modified proteins are further converted to an endonuclease H-resistant, high Mr, nested-set of complex carbohydrate chain(s) products, characteristic of polylactosaminoglycans. It is known that N-linked carbohydrate chains can be attached to secretory and integral membrane proteins at the luminal side of the endoplasmic reticulum, and then

Review: Bluetongue virus proteins

transported to the Golgi stacks, where modification of the complex carbohydrate chains occurs (Kornfeld & Kornfeld, 1985). It has been shown that the O- and N-linked carbohydrate chains attached to the proteins can be modified by the Golgi apparatus enzymes into polylactosaminoglycan proteins, which possess heterogeneous saccharides often having high Mrs (Williams & Lamb, 1988). The side chains of the lactosaminoglycan proteins are susceptible to endo-fl-galactosidase H (Oand N-linked) and endonuclease F (N-linked). Data have been obtained which indicate that NS3 and NS3A containing N-linked high-mannose sugars are transported to the Golgi apparatus and modified into N-linked complex glycoproteins. Apparently these are further converted into lactosaminoglycan proteins (Wu et al., 1992). Almost all proteins containing polylactosaminoglycan are membrane-bound (Kornfeld & Kornfeld, 1985). In addition to these biochemical data, intracellular immunofluorescence studies have shown that the NS3 gene products are transported to the Golgi apparatus in the presence or absence of tunicamycin. Surface immunofluorescence signals of cells in which NS3 and NS3A are expressed suggest that the proteins may be transported onto the cell plasma membrane (in the presence or absence of tunicamycin), suggesting that the carbohydrate chains are not required for the NS3 protein to be transported to the cell surface (Wu et al., 1992). As discussed later, virus-like particles (VLPs) synthesized by recombinant baculoviruses are associated with the cytoskeleton of the insect cell cytoplasm (A. D. Hyatt & P. Roy, unpublished data). However, such an association is perturbed and these particles are secreted by budding through the cellular membrane if the insect cells are co-infected simultaneously with a recombinant baculovirus expressing the S10 gene products along with the two dual recombinant viruses that are capable of VLP formation (A. D. Hyatt & P. Roy, unpublished data). These findings indicate a role for the NS3 proteins in the egress of BTV from the infected cell.

The associations and assembly of the seven structural proteins (VP1 to 7) Electron microscopic analyses of negatively stained preparations of BTV indicate that the outer capsid of the virus has a fuzzy appearance (Verwoerd et al., 1972; Martin & Zweerink, 1972). It lacks any discernible structure, although virus-derived cores exhibit icosahedral symmetry. Although BTV has been the subject of extensive molecular and genetic studies, little is known about the intracellular assembly process and the stoichiometry of the virus components in the morphogenetic pathways of

3057

virions. Until recently, the mechanism by which an architecturally complex virus such as BTV is synthesized, the temporal order of virion construction and the specific interactions of the protein components to produce the icosahedral core particle were not known. Two different approaches have provided some understanding of the 3D structure of the core and the virion, as well as some clues to the temporal order of BTV particle construction. One of these is the application of cryoelectron microscopy and computer image reconstruction methods to determine the 3D structure (at 3 to 4 nm resolution) of the single- and double-shelled BTV particles. This has been complemented by the synthesis and analysis of core-like particles (CLPs) and VLPs using baculovirus expression vectors. The results obtained from these studies are discussed below.

(i) The 3D structure o f B T V core particles The 3D structure of BTV core particles has been determined to a resolution of 3 nm by using cryoelectron microscopy and image-processing techniques. Cryoelectron micrographs of cores derived from BTV confirm that the structures have a diameter of 69 nm and icosahedral symmetry with a triangulation number of 13 (Prasad et al., 1992). The core structure is divided into two concentric layers of protein enclosing the inner core (see Fig. 3). The first layer, which forms the outer surface of the core, is made up of five- or six-membered rings or clusters of VP7 trimers. The clusters are arranged so that there are 132 channels at all 5- and 6-coordinated centres. The VP7 trimers form prominent knob-like protrusions, 260 in number (i.e. a total of 780 VP7 molecules per particle), and are located at all the local and strict threefold axes. These trimers extend outward to a radius of 34-5 nm, from an inner radius of 30 nm. The aqueous channels are about 8 nm deep and 8 nm wide at the surface, and some penetrate the inner layer. The inner layer, with a radius of between 21.5 and 28 nm, makes a smooth bed upon which the knobby VP7 trimers are located. Therefore it is tempting to speculate that these channels are probably the pathways for metabolites to reach the transcriptional sites and for the transportation of nascent mRNA molecules out of cores to the cytoplasm of the host cell to initiate viral protein synthesis. The VP7 structures are the points for the deposition of the two surface proteins of the particle, VP2 and VP5. The knobby morphological units are connected at a lower radius to produce a saddle-like appearance at all the local and strict twofold axes. Under VP7 is a shell of VP3 molecules; these are arranged as 12 pentamers (60 molecules per core) (Hewat et al., 1992b). The two layers enclose the inner core, which contains the remaining three minor proteins, VP1,

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(ii) 3D structure o f virus particles The 3D reconstruction (including data to 4.5 nm) of BTV particles using image analysis of cryoelectron micrographs has revealed a well ordered morphology of the outer capsid different from that of human reovirus and rotavirus (Metcalf et al., 1991 ; Prasad et al., 1988, 1990; Yeager et al., 1990). This structure is unlike the morphology deduced by negative staining methods. The proteins of the outer capsid have distinctive shapes, one globular and almost spherical, the other sail-shaped (Hewat et al., 1992b; see Fig. 4). The globular proteins, 120 in number, sit neatly in the channels formed by each of the six-membered rings of VP7 trimers. The sailshaped spikes, which project 4 nm beyond the globular proteins, are located above 180 of the VP7 trimers and form 60 triskelion-type motifs which cover nearly all the VP7 molecules. It is likely that these spikes are the haemagglutinating protein VP2 which contains the virus-neutralizing epitope, and that the globular proteins are VP5. However, confirmation of the identity of the globular and spike structures must await reconstruction of synthetic VLPs composed only of VP2 or VP5 attached to the CLPs (see later), and the use of VP2specific MAbs, similar to the procedures used to define the structure of rotaviruses (Prasad et al., 1990). The two proteins appear to form a continuous layer around the inner shell except for holes on the fivefold axis (see Fig. 4). This differs markedly from the structure of the outer capsids of the rhesus rotavirus and mammalian reoviruses, which have a more porous structure. It will be interesting to see how these differences in capsid structure between members representing different genera of the Reoviridae family are associated with differences in virus infection course (vector, route of infection, interaction with cell receptors, etc.).

Fig. 3. Representationof the surface of the reconstructedstructure of the BTV core showing the mass density of the three regions: (a) first layer, (b) second layer and (c) subcore (Prasad et at., 1992).

VP4 and VP6, and the viral d s R N A genome with a radius of less than 21 nm. The arrangement of inner core components is not known.

(iii) Assembly o f CLPs and VLPs in § cells Although BTV has been well characterized at the molecular level in terms of component R N A s and proteins, little is known about the interactions or precise stoichiometry of the seven structural proteins. To develop an understanding of the morphology and the temporal order of the construction of the core and virus particle, and to understand how the three minor proteins are physically associated and involved in these constructions, a system has been established in which CLPs and VLPs can be produced by co-expression of two or more BTV proteins. The baculovirus expression system has been used to co-express VP3 and VP7 to determine whether cores can be synthesized in the absence of the d s R N A species, or structural or non-structural proteins. For this purpose a dual expression plasmid transfer vector has been made to receive the c D N A genes and to introduce them into a

Review: Bluetongue virus proteins

Fig. 4. Representations of the surface of the reconstructed virus particle (obtained by cryoelectronmicrographs)viewedalonga twofold axis. Icosahedral five-,three- and twofoldaxes are marked in (b). The globular regions and sail-shaped spikes arranged in a triskelion motif are also marked in (b). The bar marker represents 10 nm (Hewat et al., 1992b).

baculovirus. Such vectors have two baculovirus promoter and transcription termination sequences (see Emery & Bishop, 1987) with unique restriction enzyme sites downstream of each promoter (to allow the insertion of two foreign genes). Recombinant baculoviruses expressing VP3 and VP7 have been isolated after cotransfection of insect cells with infectious viral D N A

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and the recombinant transfer vector containing R N A segment 3 of BTV-17 and R N A segment 7 of BTV-10 (French & Roy, 1990). Electron micrographs of S. frugiperda cells infected with the recombinant baculovirus show large aggregates of foreign material in the cytoplasm which, under higher magnification, appear to consist of spherical particles (French & Roy, 1990). These particles were isolated by lysing cells with NP40 and purified by sucrose or CsCI gradient centrifugation. When examined under the electron microscope, the products are found to consist of empty CLPs whose size and appearance are similar to authentic cores prepared from BTV (see Fig. 5). The stoichiometry of VP3 to VP7 in the expressed particles is estimated to be similar to that of these components derived from infectious BTV. Although these observations per se do not prove that the structures are identical to authentic cores, the fact that assembly occurs is indicative that they at least mimic the assembly process. From the observed synthesis of empty BTV CLPs by the expressed VP3 and VP7 proteins, it can be concluded that their formation is dependent on neither the presence of the three minor core proteins nor the d s R N A genome. In addition, the four non-structural proteins are not required. 3D reconstructions of BTV-10 CLPs at resolutions of 6.5 and 3-7 nm have been obtained using image analysis of cryoelectron micrographs (Hewat et al., 1992c). The determination of the structure of the CLPs has yielded information not available from analyses of the structure of the BTV-derived core particle (see above). First, since the CLP is empty, the contrast between the inner shell and the interior is greater. Second, it provides information on the order of assembly of the core particle through the analysis of incomplete CLPs, i.e. CLPs that lack a full complement of VP7 (Hewat et al., 1992c). The CLP analyses have revealed an icosahedral structure similar to the virion core structure, with a complete T = 13 lattice of 260 spikes for the complete structure (B. V. V. Prasad & P. Roy, unpublished observation). The spikes, attributed to VP7 trimers, appear as triangular columns 8.0 nm in height with distinct inner and outer domains. The inner shell (subcore) of the CLP is composed of 60 copies of VP3 (see Fig. 6). The subcore-like particle is noticeably thicker around the fivefold axes. Large pores in the subcore-like particle are situated near each of the local sixfold axes, below each six-membered ring of VP7 spikes. To determine whether double capsid particles can be synthesized by the baculovirus expression system, a second dual recombinant baculovirus was constructed to express the two outer coat proteins, VP2 and VP5. Coexpression of VP2, VP3, VP5 and VP7 in insect cells using both dual expression vectors led to the assembly of double-shelled VLPs containing all four major structural

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Fig. 5. Electron micrographs of baculovirus-expressed CLPs and VLPs. The micrographs show the appearance of expressed CLPs composed of VP3 and VP7 (a) and double-shelled VLPs containing VP2, VP3, VP5 and VP7(b). Bar marker represents 50 ~tm.

p r o t e i n s ( F r e n c h et al., 1990), w i t h v a r i o u s sizes c o n t a i n i n g different a m o u n t s o f the o u t e r c a p s i d p r o teins. I n a d d i t i o n , s o m e C L P s were also p r e s e n t in the p r e p a r a t i o n s ; these m a y reflect different stages in p a r t i c l e assembly. H o w e v e r , the c o m p l e t e e m p t y particles were s i m i l a r in d i a m e t e r to v i r i o n s (81 n m ) a n d

Fig. 6. Representations of the surface of truncated density maps of reconstructed CLPs with a cut-off radius of 36-3, 31.9 and 28-2 nm (a to c) viewed down a twofold axis. They show (a) whole CLP, (b) CLP minus the distal domains of the VP7 spikes and (c) the inner shell (Hewat et al., 1992c).

Review: Bluetongue virus proteins

could be purified to homogeneity by a one-step process involving sucrose gradient centrifugation of infected cell lysates (see Fig. 5). When purified, the complete VLPs are found to have a size and appearance similar to those of authentic BTV virions and exhibit high levels of haemagglutination activity (French et al., 1990). Antibodies raised to the expressed particles contain high titres of neutralizing activity against the homologous BTV serotype. By protein analyses the purified material obtained from infected cells is found to contain all four structural proteins of the virus (VP2, VP3, VP5 and VP7). A baculovirus expression vector that is capable of expressing four BTV major structural proteins has been prepared that synthesizes VLPs, obviating the need t o use two expression vectors (unpublished data). Reactions of baculovirus-synthesized CLPs with VP2 or VP5 translated in vitro has indicated that each outer capsid protein has the capacity to bind to the preformed CLP. This has been confirmed by in vivo expression of the appropriate genes using baculovirus vectors (H. M. Liu et al., 1992). The results support the data obtained from 3D reconstruction of virus particles. Co-expression experiments involving the dual recombinant viruses (expressing VP2, VP3, VP5 and VP7) and a single recombinant virus expressing either VP 1, VP4 or VP6, have demonstrated that each of the minor proteins can be encapsidated within the derived CLPs. To determine whether the minor proteins interact specifically with VP3 or with VP7, CLPs containing VP1 were dialysed against low salt to remove the VP7 capsomers. The derived subcores consist only of VP3 and VP1, demonstrating that VP3 serves as a framework with which VP1 interacts (Loudon & Roy, 1991). Similar data have been obtained with VP4 or VP6 and combinations of all three minor proteins (Le Blois et al., 1991). By employing dual expression viruses (expressing VP3 and VP7, and VP2 and VP5) and one triple expression vector (expressing VP1, VP4 and VP6), it has been demonstrated that the minor proteins can be encapsidated within the derived VLPs, i.e. forming particles containing all seven proteins (VP1 to VP7; unpublished data). These entities should facilitate determination of the precise role of each protein in the morphogenic process of BTV. In summary, the assembly of all seven structural proteins of BTV into VLPs has been demonstrated in the absence of the viral genome and the four non-structural proteins. The baculovirus expression system has provided a method to understand how and which viral proteins interact and assemble to form virus particles. The current understanding of the topography of the seven structural proteins in virions is illustrated in a schematic diagram (Fig. 7).

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Fig. 7. Schematicdiagramof the BTV particlebasedon data obtained from cryoelectronmicroscopicanalysestogetherwith the baculovirus protein expression system.The diagram shows VP2 and VP5 in the outer capsidsurroundingthe VP7 trimersof the core. PentamericVP3 forms the scaffoldfor VP7 trimers and encasesthe innermostpart of the core,whichcontainsthe threeminorproteinsVPI, VP4and VP6 as well as the 10 dsRNA segments.

Can VLPs be used to vaccinate sheep? Since VLPs elicit higher titres of neutralizing antibodies in guinea-pigs than does VP2 alone (or VP2 and VP5), the efficacy of VLPs as a vaccine has been tested in sheep in the presence of different adjuvants (Roy et al., 1992). The VLPs elicit the highest levels of neutralizing antibodies with Montanide incomplete Seppic adjuvant (ISA-50), and without any adjuvant elicit low levels of neutralizing antibodies. With ISA-50 adjuvant, as little as 10 ~tg of VLPs elicit neutralizing antibodies and protects all the vaccinated sheep. Four control sheep inoculated with saline alone remain seronegative and are not protected against challenge with virulent BTV-10. Similar protection data have been obtained in subsequent trials in sheep with BTV-1 VLPs (performed in collaboration with S. Johnson et al. in Townsville, Australia). It is noteworthy that preliminary studies indicate that VLPs have protective capabilities not only against homologous virus challenge, but also against certain heterologous viruses. For example, BTV-17 VLPs may protect against BTV-4 or BTV-10, BTV-13 against BTV-16, etc. The data clearly demonstrate that

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VLPs are highly immunogenic even at low doses. Sheep given only 10 ~tg of VLPs actually received 1 to 2 ~tg of VP2 (10 to 20~ of the VLP mass). This compares well with the much higher amounts of VP2 alone, or of mixtures of VP2 and VP5 required to elicit a response. There are several possible explanations. First, the conformational presentation of the relevant epitopes on VP2 probably mimics those on the authentic virus. Second, both VP2 and VP5 are present. Third, VP3 and VP7 may provide a necessary scaffold for VP2 and VP5 antigen presentation. Fourth, any of the four BTV proteins might have a direct role in eliciting cellmediated immunity induced by the BTV VLPs. Recent vaccination trials in South Africa have indicated that sheep show neutralizing antibodies up to 14 months postvaccination which are capable of complete protection against virulent virus challenge. In summary, there is no doubt that these particles are highly immunogenic and are potential candidates for bluetongue disease vaccines. It can also be anticipated from the results obtained that this technology has much to offer for the development of vaccines for both animal and human disease. Moreover, there is every reason to believe that it should be possible to make vaccine chimeras representing different BTV serotypes (e.g. involving the expression of several BTV VP2 genes), as well as chimeras containing immunogens representing other selected viral immunogens, or other pathogens (e.g. chimeric proteins involving VP2, and/or VP5, and/or VP3, and/or VP7). This may lead to a novel prospect for future vaccine technology.

Conclusion and future research Virus structures which contain multiple polypeptide species encoded by separate genes (mRNA species) and which involve non-equimolar ratios of proteins, such as those of BTV, present one of the more difficult and challenging objectives to studies of the processes of viral morphogenesis. Synthesis of subviral particles in eukaryotic cells by an expression system opens up a wealth of possibilities for defining mechanisms of protein-protein assembly under natural intracellular conditions. For example, by manipulating the structures of various proteins it will be possible to define experimentally the essential morphogenic interactions of the seven structural and four non-structural proteins. The future direction of BTV research will involve such questions as how VP3 and VP7 proteins assemble into CLPs, how VP2 and VP5 interact with CLPs to form VLPs and how the minor proteins (VP1, VP4 and VP6) interact with the structures. Deletion, site-specific and domain-switched mutant proteins are currently being

generated to identify the motifs relevant to proteinprotein interactions in the BTV assembly process and their effect on the intrinsic functions (ss- and dsRNA binding, GTP binding, RNA polymerase activity, and tubule and inclusion body formation). The results and systems that are established will eventually allow us to determine whether these events can be selectively inhibited in an infected cell. In addition, it can be anticipated that the availability of large quantities of viral components, and of CLPs and VLPs will facilitate an understanding of their 3D structure at the atomic level.

References Au, K.-S., CHAN, W.-K., BURNS, J. W. & ESTES, M. K. (1989). Receptor activity of rotavirus non-structural glycoprotein NS28. Journal of Virology 63, 4553-4562. BASAK, A. K., STUART, D. I. & ROY, P. (1992). Preliminary crystallographic study of bluetongue virus capsid protein VP 7. Journal of Molecular Biology (in press). BREMER, C., THOMAS, C. P. & ROY, P. (1992). Interaction of ssRNA with NS2, the non-structural phosphoprotein of bluetongue virus and formation of inclusion bodies. In Bluetongue, African Horsesickness and Related Orbiviruses. Edited by T. E. Walton & B. I. Osburn. Boca Raton: CRC Press (in press). CHUMA, T., LE BLOIS, H., SANCHEZ-VIZCAiNO,J. M., DIAZ-LAV1ADA, M. & ROY, P. (1992). Expression of the major core antigen VP7 of African horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent. Journalof General Virology73, 925-931. CLEVELAND, n . R., ZARBL, H. & MILLWARD, S. (1986). Reovirus guanylyltransferase is L2 gene product lambda 2. Journal of Virology 60, 307 3ll. COWLEY, J. A. & GORMAN, B. M. (1987). Genetic reassortments for identification of the genome segment coding for the bluetongue virus hemagglutinin. Journal of Virology 61, 2304-2306. CROMACK, A. S., BLUE, J. L. & GRATZEK, B. (1971). A quantitative ultrastructural study of the development of bluetongue virus in Madin-Darby bovine kidney cells. Journal of General Virology 13, 229-244. DEVANEY, M. A., KENDALL, J. & GRUBMAN, M. J. (1988). Characterization of a non-structural phosphoprotein of two orbiviruses. Virus Research I1, 51-164. EATON, B. T , HYATr, A. D. & WHITE, J. R. (1988). Localization of the nonstructural protein NS2 in bluetongue virus-infected cells and its presence in virus particles. Virology 163, 527-537. EATON, B. T., HYATr, A. D. & BROOKES, S. M. (1990). The replication of bluetongue virus. Current Topics in Microbiology and Immunology 162, 89 118. EMERY, V. C. & BISHOP, D. H. L. (1987). The development of multiple expression vectors for high level synthesis of AcNPV polyhedrin protein by a recombinant bacnlovirus. Protein Engineering 1, 359-366. FRENCH, T. J. & ROY, P. (1990). Synthesis of bluetongue virus (BTV) core-like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. Journal of Virology 64, 1530-1536. FRENCH, T. J., INUMARU,S. & ROY, P. (1989). Expression of two related non-structural proteins of BTV-10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization of the gene product in BTV-infected BHK cells. Journal of Virology 63, 3270-3278. FRENCH, T. J., MARSHALL, J. J. A. & ROY, P. (1990). Assembly of double-shelled, virus-like particles of bluetongue virus by the simultaneous expression of four structural proteins. Journal of Virology 64, 5695-5700.

Review." Bluetongue virus proteins

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LE BLOIS, H., MERTENS, P. P. C., FRENCH, T., BURROUGHS, J. N. & ROY, P. (1992). The expressed VP4 protein of bluetongue virus is the guanylyl transferase. Virology 189, 757-761. LECATSAS, G. (1968). Electron microscopic study of the formation of bluetongue virus. Onderstepoort Journal of Veterinary Research 35, 139 149. LEE, J. W. & RoY, P. (1987). Nucleotide sequence of a eDNA clone of RNA segment 10 of bluetongue virus (serotype 10). Journal of General Virology 67, 2833 2837. LIU, H. M , BOOTH, T. F. & RoY, P. (1992). Interactions between bluetongue virus core and eapsid proteins translated in vitro. Journal of General Virology 73, 2577-2584. LIU, M., MAYrION, N. R. & ESTES, M. K. (1992). Rotavirus VP3 expressed in insect cells possesses guanylyltransferase activity. Virology 188, 77-84. LOUDON, P. T. & ROY, P. (1991). Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: inclusion of the largest protein VP1 in the core and virus-like particles. Virology 180, 798-802. LOUDON, P. T., HIRASAWA,T., OLDFIELD, S., MURPHY, M. & RoY, P. (1991). Expression of outer capsid protein VP5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses. Virology 182, 793-801. MAD, Z. & JOKLm, W. K. (1991). Isolation and enzymatic characterization of protein 22, the reovirus guanylyltransferase. Virology 185, 377-388. MARSHALL, J. J. A. & ROY, P. (1990). High level expression of the two outer eapsid proteins of bluetongue virus serotype 10: their relationship with the neutralization of virus infection. VirusResearch 15, 189-196. MARTIN, S. A. & ZWEERINK, H. J. (1972). Isolation and characterization of two types of bluetongue virus particles. Virology 50, 495-506. MERTENS, P. P. C. & BURROUGHS, J. N. (1990). Guanylyltransferase activity associated with protein VP4 of South African bluetongue virus serotype 1 (BTVI(SA)). Abstracts of the Vlllth International Congress of Virology, p. 44, abstract W16-004. MERTENS, P. P. C., BROWN, F. & SANGAR,D. V. (1984). Assignment of the genome segments of bluetongue virus type 1 to the proteins they encode. Virology 135, 207-217. METCALE, P. M., CYRKLAFF, M. & ADRIAN, M. (1991). The threedimensional structure of reovirus obtained by cryo-electron microscopy. EMBO Journal 10, 3129-3136. MEYER, J. C., BERGMANN,C. C. & BELLAMY,A. R. (1989). Interaction of rotavirus cores with the non-structural glycoprotein NS28. Virology 171, 98-107. OLDFIELD, S., ADACHI, A., URAKAWA, T., HIRASAWA, T. & RoY, P. (1990). Purification and characterization of the major groupspecific core antigen VP7 of bluetongue virus synthesized by a recombinant baculovirus. Journal of General Virology" 71, 2649 2656. OLDFIELD, S., HIRASAWA,T. & ROY, P. (1991). Sequence conservation of the outer capsid protein, VP5, of bluetongue virus, a contrasting feature to the outer capsid protein VP2. Journal of General Virology 72, 449-451. PIZARRO, J. L., SANDINO, A. M., PIZARRO, J. M., FERN.~NDEZ, J. & SPENCER, E. (1991). Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3. Journal of General Virology 72, 325 332. PRASAD, B. V. V., WANG, G. J., CLERX, J. P. M. & CHIU, W. (1988). Three-dimensional structure of rotavirus. Journal of Molecular Biology 199, 269-275. PRASAD, B. V. V., BURNS, J. W., MARIETFA, E., ESTES, M. K. & CHIU, W. (1990). Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy. Nature, London 343, 476-479. PRASAD, B. V. V., YAMAGUICHI, S. & ROY, P. (1992). Threedimensional structure of single-shelled BTV. Journalof Virology 66, 2135 2142. RoY, P. (1989). Bluetongue virus genetics and genome structure. Virus Research 13, 179-206.

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Bluetongue virus proteins.

Journal of General Virology (1992), 73, 3051-3064. 3051 Printed in Great Britain Review article Bluetongue virus proteins Polly Roy Laboratory of M...
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