Orbivirus Structure and Assembly

VIROLOGY 216, 1–11 (1996) ARTICLE NO. 0028

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POLLY ROY

Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford and NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford, OX1 3SR; and University of Alabama at Birmingham, Birmingham, Alabama 35294

Received August 14, 1995; accepted October 13, 1995

Orbiviruses (Reoviridae family) are complex nonenveloped RNA viruses with seven structural proteins and a RNA genome consisting of 10 variously sized double-stranded RNA segments. Significant advances in orbivirus research have been made in recent years through the use of gene manipulation techniques coupled with the baculovirus expression system. Several orbivirus proteins have yielded to crystallization and X-ray crystallographic structure determination and, when combined with the three-dimensional image reconstruction of virion particles and cores obtained by cryoelectron microscopy, consider- able insight has been gained into the intricate organization and topography of the individual viral components. Formal identification of the sites of interaction has been obtained through protein–protein interaction studies on the components of the virion particle, including those that are involved in capsid assembly. Finally, a beginning of the understanding of the sequence of assembly events has also been obtained. ᭧ 1996 Academic Press, Inc.

Orbiviruses are transmitted to vertebrate hosts by cer- The 10 dsRNA segments (designated L1–3, M4 –6, tain arthropods (ticks, gnats, or mosquitoes, depending and S7–10) range in mass from 0.5 1 106 to 2.7 1 106 on the species) and replicate in both hosts. Other than Da, with a total mass of 13 1 106 Da (Fukusho et al., bluetongue virus (BTV), a limited amount of information 1989; Verwoerd et al., 1970). Each BTV RNA segment, is available on certain other orbiviruses such as epizootic except the smallest (S10), encodes a single viral polypep- hemorrhagic disease virus (EHDV) of deer and African tide (Table 1; Mertens et al., 1984; French et al., 1989). horsesickness virus (AHSV). Like BTV, they are transmit- In addition to the seven structural proteins, there are four ted by particular Culicoides (gnat) species. Broadhaven virus-specified nonstructural proteins (NS1, NS2, NS3, virus, a tick-transmitted species, has also been studied. and NS3A) that are found in virus-infected cells (see This article mainly reviews findings with BTV, although review by Roy et al., 1990a). Of these, NS1 is synthesized relevant studies on EHDV and AHSV are also included. abundantly and is assembled into polymeric morphologi- The seven structural proteins of BTV (VP1– VP7) are orga- cal structures that are characteristic features of orbivirus- nized into two shells. There is an outer shell containing infected cells (Lecatsas, 1968; Huismans, 1979; Huis- two major proteins, VP2 and VP5, and an inner core con- mans and Els, 1979; see review by Eaton et al., 1990). taining the five other proteins as well as the dsRNA genome The NS1 protein forms tubules which are present pre- (Verwoerd et al., 1972, 1979; Els, 1973). In virus-infected dominately in peri- or juxtanuclear locations and are cells, parental BT virions are converted into core particles attached to the intermediate filaments of the cell cy- (420 S) which contain five proteins, VP1, VP3, VP4, VP6, toskeleton (Eaton and Hyatt, 1989). These tubules are and VP7, with VP3 and VP7 the major components (Ver- thought to be involved in the virus transportation process woerd and Huismans, 1972; Huismans et al., 1987a). The in infected cells. The NS2 protein is the only virus-spe- three minor proteins VP1, VP4, and VP6 are closely associ- cific phosphoprotein (Huismans et al., 1987b; Thomas ated with the 10 dsRNA segments. Negatively stained core et al., 1990). It is also expressed abundantly. NS2 is particles exhibit an icosahedral symmetry with distinct cap- associated with viral mRNA species and is the major someric units. By contrast, the outer shell exhibits an ob- component of the granular viral inclusion bodies (VIBs) scure morphology when negatively stained particles are that occur in virus-infected cells (Thomas et al., 1990; examined by electron microscopy (Murphy et al., 1971; Els Eaton et al., 1990). VIBs are frequently observed in prox- and Verwoerd, 1969). In this respect, the morphology of BT imity to the cell nucleus and are believed to be involved virions is quite distinct from that of rotaviruses or reoviruses, in the early stages of virion assembly and morphogene- two other members of the family which also exhibit a dis- sis. The NS3 and NS3A proteins are colinear products tinct icosahedral morphology. of the S10 gene among all orbiviruses (Mertens et al.,

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TABLE 1

Bluetongue Virus Coding Assignments

Genome Predicted segment No. of bp Proteins No. of aa size Location Function

L1 3954 VP1 1302 149,588 Inner core RNA polymerase L2 2926 VP1 956 111,112 Outer shell Type specific, structural protein L3 2772 VP3 901 103,344 Core Structural protein, forms scaffold for VP7 trimers M4 2011 VP4 654 76,433 Inner core Capping enzyme–guanylyltransferase M5 1639 VP5 526 59,163 Outer shell Structural protein M6 1770 NS1 552 64,445 Nonstructural Tubules, an unknown function S7 1156 VP7 349 38,548 Core surface Group-specific structural protein S8 1123 NS2 357 40,999 Nonstructural IBs, binds ssRNA S9 2046 VP6 328 35,750 Inner core Binds ssRNA, dsRNA S10 822 NS3 229 25,572 Nonstructural Glycoproteins, aid virus release NS3A 216 24,020

1984; French et al., 1989; van Staden and Huismans, core particles (i.e., avoiding heavy metal stains, fixative, 1991). They originate from different in-frame initiation co- and dehydration; Prasad et al., 1992; Hewat et al., 1992a). dons (French et al., 1989; Wu et al., 1992). Both NS3 and Using image reconstruction methods the micrographs of NS3A are glycosylated and involved in virus release from cores at 30 A˚ resolution revealed that the particles were infected cells (Wu et al., 1992; Hyatt et al., 1993). 690 A˚ in diameter, exhibited an icosahedral symmetry, and contained surface knobs organized with a triangula- Assembly of BTV core-like structures by baculovirus tion number of 13 (Prasad et al., 1992). The surface con- expression systems sisted of clusters of VP7 trimers providing 260 prominent knob-like protrusions (780 VP7 molecules) organized into The flexibility of baculovirus expression vectors and pentameric and hexameric units with channels in be- the capacity of the baculovirus genome to accommodate tween (see Fig. 2). Channels into the CLP interior were large amounts of foreign DNA has allowed us to exploit visualized, a total of 132 per particle, involving all the this system for the simultaneous expression of multiple threefold axes and with dimensions approximately 70 A˚ BTV genes in a single insect cell. Our initial effort was deep and 80 A˚ wide at the surface (Fig. 2B). Some of to assemble the two major core proteins, VP3 and VP7. the channels penetrate through the inner layer and are For this purpose, a dual baculovirus expression vector probably the pathways for metabolites to reach the sites consisting of duplicated polyhedron promoters (PH) of of viral mRNA transcription and for the export of nascent Autographa californica nuclear polyhedrosis virus, with mRNA molecules out of the cores. The underlying smooth downstream transcription terminator sequences were scaffold for the VP7 trimers consists of the second major utilized to express the coding sequences of the L3 (VP3) core protein, VP3, the organization of which was not fully and S7 (VP7) genes of BTV (French and Roy, 1990). Re- revealed at the initial 30 A˚ resolution map. VP7 and VP3 combinant baculoviruses synthesizing both proteins enclose the inner core which comprises the three minor were isolated and produced core-like particles (CLPs) proteins (VP1, VP4, and VP6) and the genomic dsRNA. distributed throughout the infected insect cells. Gradient- How these are organized with respect to each other, or purified CLPs were similar in size and appearance to to VP3 and VP7, is not known. cores prepared from BTV (see Fig. 1). Only VP3 and VP7 The images of baculovirus-synthesized CLPs at rela- were identified as the protein components of the ex- tively low resolution (65 A˚ and 37 A˚ ) revealed a similar pressed particles and the molar ratios of these two pro- icosahedral configuration (T Å 13 for the surface layer) teins were similar to those of VP3 and VP7 derived from and an identical diameter (690 A˚ ) to that of BTV cores infectious BTV. The CLPs appeared to lack nucleic acids (see Fig. 2C). Some of the synthetic CLPs lacked the full when analyzed by phenol–chloroform extraction and al- complement of VP7 and, as a consequence, the shapes cohol precipitation. of the VP7 trimers were more clear (Hewat et al., 1992a). The trimers have tripod-like shapes and each consists Three-dimensional structures of BTV cores and CLPs of an upper (outermost) and lower (innermost) domain. To determine whether CLPs mimic the morphology of The shapes and structures of VP7 trimers have recently BTV-derived core particles, cryoelectron microscopy was been confirmed by X-ray crystallographic data (see later). first used to examine unstained, unfixed virus-derived It appears that the VP3 molecules are roughly disk-

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shaped and that dimers of VP3 form the building blocks for the icosahedral structure. There are a total of 120 VP3 molecules per virion organized with a triangulation number of T Å 1. The disk-shaped configuration of VP3 is seen clearly, even at low resolution (see Fig. 2C). This is due to the fact that CLPs appear to be empty, so that the contrast between the inner shell and the interior is greater than that in virus-derived cores. VP3 subcores have been purified from CLPs by dialysis against a low- salt buffer that removes the VP7 trimers. Subcores may be separated from CLPs by sucrose gradient centrifugation of dialyzed materials. The cryo-EM data obtained for purified subcores appear similar to those deduced for VP3 within CLPs (Hewat et al., 1992a).

Incorporation of the three minor proteins within CLPs

To determine whether the three minor proteins can be incorporated into CLPs, VP1 and/or VP4 and/or VP6 have been coexpressed using baculovirus vectors together with VP3 and VP7. VP1, the putative viral polymerase, was readily incorporated within CLPs. When the VP7 trimers were removed, the derived subcores consisted only of VP3 and VP1, demonstrating that VP1 interacts with VP3 (Loudon and Roy, 1991). Similar results were obtained with VP4 and VP6 and for combinations of all three minor proteins (Le Blois et al., 1991). However, unlike VP1 or VP4, VP6 was only poorly incorporated into CLPs. Since VP6 is a highly basic protein and readily associates with RNA (single or double-stranded), it is possible that VP6 chaperones the incorporation of RNA into particles (or vice versa) and is only poorly incorporated in the absence of RNA (Roy et al., 1990b; Hayama and Li, 1994). To determine whether CLPs without the minor protein components retain the ability to interact with viral RNA species, various assay systems have been developed using purified CLPs and single-stranded RNA species synthesized in vitro. The data indicate that the RNA binding affinity of CLPs involves VP3 and probably not the VP7 (Loudon and Roy, 1992). How RNA interacts with VP3 is not known.

Assembly of BTV virus-like particles (VLPs) using baculovirus vectors

Baculovirus multigene vectors have been developed to cosynthesize up to five BTV proteins in the same cell (Belyaev and Roy, 1993; Belyaev et al., 1995). In addition to the PH promoter, copies of the p10 promoter of AcNPV

FIG. 1. Electron micrographs of negatively stained (A) authentic BTV cores and (B) baculovirus-expressed core-like particles. (C) Cryoelec- tron micrograph of spherical subcores showing the slight thickening of the wall in certain orientations (French and Roy, 1990; Hewat et al., 1992a).

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that these proteins are not necessary for the assembly of double-capsid particles (or CLPs). VLPs express high levels of hemagglutination activity, similar to that of BTV virions. Further, antibodies raised to the expressed particles gave high titers of neutralizing activity against the homologous BTV serotype. Virus challenge studies in sheep immunized with two doses of VLPs (10 mg or more per dose) have confirmed the immu- nogenicity of VLPs and have shown that the sheep were protected against virulent challenge even 15 months postvaccination (Roy et al., 1992, 1994). Since VP2 is the main serotype-specific antigen (Huismans and Erasmus, 1981; Kahlon et al., 1983; Roy et al., 1990c) and the viral hemagglutinin (Cowley and Gorman, 1987; French et al., 1990; Loudon et al., 1991), it is probable that VP2 in VLPs is assembled in the correct conformation and is biologically equivalent to the virion VP2. It is not known whether VP2 is attached to the core through VP5 or is attached directly to VP7. Using baculovirus and in vitro transcription–translation systems data have been obtained which indicate that both VP2 and VP5 may sepa- rately bind to CLPs (Liu et al., 1992).

The three-dimensional structure of BTV virions and VLPs The 3-D structure of BTV has been determined using image analysis of cryoelectron micrographs of virion particles (Hewat et al., 1992b). The reconstruction revealed a morphology that contrasts sharply with that deduced by conventional negative-staining methods. The outer capsid exhibits an icosahedral configuration (860 A˚ in FIG. 3. Electron micrographs of (a) BTV virions and (b) baculovirus- diameter) and a well-ordered morphology (see Fig. 4A). expressed double-capsid virus-like particles (French et al., 1990). The two proteins of the outer capsid have distinctive shapes, one is globular and almost spherical, the other have been utilized to facilitate the high-level coexpres- is sail-shaped. The globular proteins, 120 in number, sit sion of several proteins in each infected cell. For opti- neatly on each of the six-membered rings of the VP7 mum synthesis of VLPs a quadruple gene expression trimers of the core. The sail-shaped spikes are situated vector has been used to synthesize the BTV VP2, VP3, above 180 of the 260 VP7 trimers and form 60 triskelion- VP5, and VP7 proteins (see Fig. 3A). The expressed pro- type motifs which cover all but 20 of the VP7 trimers. It teins assembled into virtually homogenous double-cap- is likely that these spikes are the VP2 hemagglutinating sid particles (see Fig. 3B). Coinfections with single or and neutralization antigens. The two proteins appear to dual gene expression vectors gave VLPs that contained form a continuous layer around the core, except for holes different amounts of the outer capsid proteins, depending on the fivefold axis. on the experiment (French et al., 1990). The formation of Three-dimensional reconstruction of VLPs at 55 A˚ res- complete VLPs in the absence of the NS proteins implies olution has also been undertaken (Hewat et al., 1994).

FIG. 2. Surface representation of the cryoelectron micrographs of the BTV core and baculovirus-expressed core-like particles viewed along the icosahedral threefold axis. (A) BTV core showing the knob-like protusions of VP7 trimers in the outer layer and (B) the density in the outer layer showing the large holes or channels at all the five- and six-coordinated positions formed by the arrangement of the VP7 trimers. (C) An incomplete CLP showing the positions (dots) of five VP7 spikes in CLPs (Prasad et al., 1992; Hewat et al., 1992a). FIG. 4. (A) Surface representation of a cryoelectron micrograph of an icosahedral virus particle viewed along a twofold axis, showing the topography of the two outercapsid proteins, one globular-shaped, VP5 (in yellow), and the other sail-shaped, VP2 (in blue). VP7 trimers, underneath, are indicated in white. (B) Reconstruction of a VLP obtained from a cryoelectron micrograph showing essentially similar structures of the outer surface (Hewat et al., 1992b, 1994). FIG. 5. The X-ray crystallographic structure of VP7 (A) trimer and (B) monomer (Grimes et al., 1995).

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At this low resolution the structure of the VLP (860 A˚ in BTV VP5 sequences have shown that while some regions diameter) is comparable to that of authentic virions and exhibit variability, others (e.g., residues 200 to 270), and exhibits essentially the same basic features and full com- both termini, are highly conserved (Oldfield et al., 1991). plement of the four proteins (see Fig. 4B). Further studies to identify the interactive sites involved in particle assembly within these conserved regions are Assembly of heterologous VLPs using different BTV in progress using site-specific mutagenesis (sequence serotypes deletion, substitution, etc).

Some 24 serotypes of BTV are recognized (BTV-1, BTV- Assembly of chimeric particles between different 2, etc.). The outercapsid proteins (VP2 and VP5) exhibit orbiviruses the least conservation between BTV serotypes, in con- Sequence comparisons of the four major capsid pro- trast to all the other virus-coded proteins (see review, teins, VP2, VP3, VP5, and VP7, of BTV-10 with those of Roy et al., 1990a). VP2 is the most variable (VP2 sequence EHDV-1 and AHSV-4 have revealed that the three viruses identity comparisons between BTV-1, -2, -10, -11, -13, have generally comparable primary sequences for their and -17 range from 39 to 73%). VP2 is the main serotype- capsid components (see reviews by Roy, 1992; Roy et specific antigen. Antisera raised to the baculovirus-ex- al., 1994). Of the four major capsid proteins, the inner- pressed VP2 of particular BTV serotypes neutralize the most, VP3, is the most conserved and the outermost, homologous virus and, depending on the antigen, cross- VP2, the most variable. The two major core proteins of neutralize to some extent certain other BTV serotypes BTV-10 and EHDV-1 share a high percentage of identical (Inumaru and Roy, 1987; Urakawa et al., 1993). These residues (79% for VP3 and 64% for VP7). Compared to data indicate that some BTV serotypes are more closely AHSV-4, the VP3 and VP7 proteins of BTV and EHDV related to each other but distantly related to other sero- are less similar (44–46%). It is likely that the conserved types (such as BTV-1, -2, and -13). In contrast to VP2, the sequences represent structurally important regions. primary sequences of the VP5 proteins of a number of Intertypic CLP and VLP formation has been investi- BTV serotypes (e.g., BTV-10, -11, and -17) are more simi- gated using combinations of the cloned genes and bacu- lar, sharing up to 94% identical amino acids (Hirasawa lovirus expression vectors. Preliminary studies have and Roy, 1990; Oldfield et al., 1991; Yang and Li, 1992). demonstrated that both the VP3 and the VP7 proteins of The structural compatibilities of various VP2 and VP5 BTV-10 and EHDV-1 are exchangeable, although the species in VLP formation have been investigated. Heter- outer capsid proteins are not. The latter is not unex- ologous VLPs have been sought by co-infection of insect pected, as the VP2 proteins of the three orbiviruses share cells with appropriate recombinant baculoviruses (Lou- only 19–24% identical amino acids, although VP5 is more don et al., 1991). Assembled particles were purified and conserved, scoring up to 64% identity between BTV and analyzed by electron microscopy and SDS–PAGE to con- EHDV and 43–45% between BTV or EHDV and AHSV. firm their authenticity. The presence of VP2 and VP5 on Interestingly, all three minor proteins of BTV can be VLPs was demonstrated by hemagglutination and West- encapsidated into chimeric CLPs that are formed by the ern immunoblotting, respectively. Despite the high level VP3 of EHDV and the VP7 of BTV. Thus it appears that of sequence variation among the different serotypes, the the functional regions of EHDV and BTV VP3 proteins VP2 and VP5 proteins of six different BTV serotypes involved in interacting with each of the minor proteins formed VLPs with the VP3 and VP7 of another source are conserved (Le Blois et al., 1991). The ability of the (Loudon et al., 1991). Combinations of VP2 and VP5 which VP3 of BTV (or EHDV) to interact with the VP7 of AHSV resulted in the assembly of particles (e.g., the VP5 of and the formation of CLPs appears to be restricted. Al- BTV-10 and the VP2 of BTV-11 or -17) are given in Table though occasionally a few particles have been identified, 2. Some combinations (e.g., the VP2 of BTV-2 and the they appear to be highly labile to any physical treatment VP5 of BTV-10) did not form VLPs. The data indicate that (unpublished observations). VP2 may require the VP5 of the same or a closely related serotype (e.g., BTV-10 or BTV-17) but may not form VLPs Intra- and intermolecular interacting domains within efficiently with the VP5 proteins of a more distant sero- VP7 and VP3 molecules type. Alignments of the predicted amino acid sequences of The ability of VP7 trimers to interact with VP3 subcores the VP2 of various BTV serotypes have identified con- and form CLPs has been used to identify the regions of served and variable regions within the proteins (see re- VP7 necessary for the formation of particles. Several site- view by Roy et al., 1990a). VP2 possesses at least five specific mutants and deletion or extension mutants of highly variable regions, although the carboxy terminus VP7 have been evaluated. The role of the carboxy termi- and certain other regions (e.g., residues 360 to 367) are nus of VP7 was investigated in CLP formation by deletion well conserved (Roy, 1989). Similarly, comparisons of and in-frame addition of sequences (Le Blois and Roy,

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TABLE 2

VP2 Origin

VP5 origin BTV-1 BTV-2 BTV-10 BTV-11 BTV-13 BTV-17

BTV-2 //0000 BTV-10 00//0/ BTV-13 ND ND 00/0

Note. S. frugiperda cells were infected with combinations of the following recombinant baculoviruses: a dual recombinant expressing VP3 and VP7; a single recombinant expressing VP2 of serotypes 1, 2, 10, 13, or 17; and an additional single recombinant expressing the VP5 of serotypes 2, 10, or 13. The cells were lysed 3 days postinfection and the resultant particles were isolated on discontinuous sucrose gradients. Purified particles were examined under the electron microscope. Presence (/) and absence (0) of double-shelled virus-like particles is indicated. ND, experiment not done.

1993). In one example, an 11-amino-acid, immunogenic To investigate whether the amino terminus of VP7 pro- epitope representing rabies virus glycoprotein was tein which is generally hydrophilic (Yu et al., 1987) can added to the carboxy terminus of the BTV-10 VP7 gene tolerate additional sequences, an extension mutant with and the chimeric protein was expressed by a recombi- an additional 11-amino-acid sequence was prepared. nant baculovirus. When this virus was used to synthesize The chimeric protein assembled into CLPs in the pres- CLPs in the presence of VP3, no morphological struc- ence of VP3 protein, although the particles were less tures were obtained, suggesting that the presence of stable than normal CLPs when analyzed by CsCl gradient this sequence at the carboxy terminus of VP7 precluded centrifugation. The result suggested that the modified particle formation. Further, when a truncated VP7 lacking VP7 may attach to subcores composed of VP3 but that residues 336 to 349 of the carboxy terminus was ex- the protein was removed under high-salt conditions, such pressed in the presence of VP3 in insect cells, no CLPs as exposure to CsCl. When authentic VP7 was present were recovered following sucrose gradient centrifugation it appeared to stabilize the association of the modified of cell extracts. From these data it was concluded that form. This phenomenon was further investigated by gen- the natural carboxy terminus of VP7 is critical for CLP erating a second N-terminal extended recombinant VP7 formation. A hydropathic plot of VP7 indicates the exis- protein. In this construct, the chimeric protein contained tence of hydrophobic domains involving the last 50 amino 48 amino acid residues of the hepatitis B virus preS2 acid residues of VP7. Such domains may be involved region (Belyaev and Roy, 1992). With this longer exten- in intra- or intermolecular interactions important for the sion, VP7 did not form CLPs in insect cells in the pres- function of the protein in particle formation. ence of VP3 alone. However, CLPs were formed when The 349-amino-acid VP7 protein of BTV-10 contains the cells were co-infected with this recombinant and a two cysteine residues (Cys-15 and Cys-65), both of which recombinant that expressed the unmodified VP7 and VP3 are conserved among various orbivirus VP7 proteins. The proteins of BTV (Belyaev and Roy, 1992). The protein possibility that these two conserved residues are im- profiles indicated that the two forms of VP7 were present portant for the structural integrity of VP7 proteins was in CLPs. It is possible that VP7 is added sequentially to investigated by mutating each cysteine to a serine. The the VP3 subcore, first to positions that stabilize the struc- ability of the mutant proteins to interact with VP3 protein ture and second to positions that complete the surface and to form CLPs was investigated. Particles were iso- arrangement of VP7. By this model a chimeric VP7 that lated with each mutant and formed CLPs with ratios of by itself is unable to produce CLPs with VP3 alone may VP3 and VP7 essentially similar to those of normal CLPs. do so when supplemented with unmodified VP7 protein. These data suggested that one or both cysteines are not The BTV 110-kDa inner capsid protein, VP3, has a low essential for the formation of cores. content of charged amino acids and a high content of The VP7 sequence of all BTV serotypes so far analyzed hydrophobic amino acids (Purdy et al., 1984). VP3 se- contains only one lysine residue (amino acid residue quences are highly conserved among different BTV sero- 255), suggesting that it may have an important role. In types (see review by Roy et al., 1990a; Hwang et al., order to investigate this, a mutant VP7 gene was made 1994). To identify regions within the VP3 protein that may in which the lysine was substituted by leucine. Although be essential for core formation, alignment of the VP3 the protein was expressed to a high level, it did not form protein sequence with the corresponding protein se- CLPs. The results suggested that the VP7 lacking this quences of two closely related viruses (EHDV-1 and lysine either does not fold correctly or does not polymer- AHSV-4) (Iwata et al., 1992) revealed a number of con- ize to form trimers, or that some interactions between served regions. To investigate whether specific amino VP7 and VP3 require this residue. acids within the conserved regions are important for the

/ m4777$7626 01-12-96 08:00:23 viras AP-Virology 8 POLLY ROY function(s) of VP3, the formation of CLPs and the inclu- 198–208, interacts with both trimeric partners at the top sion of the minor proteins into CLPs have been investi- of the molecule. Below this loop, aa 133–141 form an- gated (Tanaka and Roy, 1994). Domains of BTV-17 VP3 other loop (the A–B loop), clipping onto the inner face (aa 31 –40, 345–350, 371–385, 499–508, 526–538, and of the b-sandwich of an adjacent subunit. The upper 828–837) that are largely conserved between the three domain is structurally very similar to the jelly-roll struc- orbiviruses were chosen for this study. Six different inter- ture of influenza virus hemagglutinin (Wilson et al., 1981). nal deletion mutants were synthesized and recombinant The lower domain of VP7, which contains both the N baculoviruses were constructed. When these viruses terminus (aa 1–120) and the C terminus (aa 250–349) of were tested for assembly into CLPs in the presence of the molecule, is composed entirely of a-helices (nine in VP7 protein, only one mutant (aa 499 to 508) did not form total) and long extended loops. The lysine at position 255 CLPs. Shorter and single amino acid mutations encom- is located in an exposed hinge region of the protein and passing in this region indicated that the M at residue between the upper and the lower domains, reflecting 500 and the R at residue 502 were essential for CLP the critical function of this residue on the folding of the formation. molecule necessary for trimerization and assembly of VP7 into CLPs, as discussed above. The N-terminal por- Three-dimensional X-ray crystallographic structure of tion of VP7 forms five helices preceding the upper do- VP7 trimer at the atomic level main insert and the remaining four helices are formed by the C-terminal region (see Fig. 5). The helices are The 780 molecules of VP7 are the main components packed tightly together. The N-terminal section forms the of the virion and are located in 260 trimeric units as base of the molecule on top of which rests the C-terminal a middle layer bridging the outer capsid and the inner portion. The interactions between the three VP7 lower subcore. These trimers are the basis from which the domains in the trimer are complex. However, most in- complex surface lattice of the viral core is built up. We volve helices numbers 5 and 6 and the associated loops have recently obtained the crystal structure of VP7 at 2.8 (see Fig. 5). A˚ , which has revealed that the three molecules of VP7 Helix 5, which goes through the inside of the trimer interact extensively to form the trimers with relatively flat and interacts with helices numbers 1, 2, 6, and 8, consists bases and that these can be oriented unambiguously of aa 103–118. Helix 6 borders the internal cavity and is within the electron micrographic reconstructions (at 30 the main chain of the molecule. The residues in this helix A˚ resolution) of the cores (Grimes et al., 1995). The three- are not highly conserved and appear not to be involved fold axis of the trimer (85 A˚ long) is perpendicular to the in close interactions with other residues. core surface and the broader base of the trimer (65 A˚ ) The sideways interactions between the VP7 trimers contacts the internal network of the VP3 of the subcore. must be structurally varied as these form a complex T Each subunit consists of two distinct domains, the ‘‘up- Å 13 lattice in the surface of the core. Although on the per’’ and the ‘‘lower,’’ and are twisted such that the top basis of the VP7 structure alone definite assignments domain of one monomer rests upon the lower domain of cannot be made, it seems that the C-terminal residues a threefold related subunit in a clockwise direction (see from aa 333 onward are crucial. Mutagenesis studies Fig. 5). The interactions between subunits are extensive referred to above have demonstrated that deletion of the and there are contacts between the top domains related end residues, or extensions of 5–10 residues on the C by threefold symmetry and also with the symmetry-re- terminus, abolished CLP formation. Crystallographic data lated bottom domain. There are both hydrophobic inter- have revealed that the VP7 helix 9 is formed from the C- actions and specific hydrogen bond interactions which terminal residues (aa 341–347) and is on the outside of bind the two domains strongly. Despite the extensive the trimer, probably interacting with residues from an- interactions there are cavities at the center of the trimer other subunit. Preceding this helix, a hydrophobic loop (along the threefold axis) surrounded by predominantly (sequence NPMPGPL) is particularly flexible, suggesting uncharged residues. that this part of the structure may be able to adopt a The smaller upper domain of VP7 forms the outer sur- number of conformations to provide stabilizing side-to- face of the core and contains the central one-third of the side contacts. polypeptide chain of the molecule (aa residues 121–249). The 260 trimers of VP7 adhere to a scaffold that is In the trimer, the three upper-domain subunits, each of made up of only 120 VP3 molecules, indicating a symme- which is folded into an anti-parallel b-sandwich, are inti- try mismatch in the interactions between the bases of mately associated via two extended loops from each sub- the 260 trimers of VP7 and the 120 molecules of VP3. unit, both with each other and with all possible threefold Nevertheless, such interactions must stabilize the BTV related subunits. The topologies of the two groups of b- subcores during assembly since subcores of VP3 in the sheets are A؅BIDG and CHEF, corresponding to a jelly- absence of VP7 are very unstable. The flat bases of VP7 roll structure. One loop (the D–E loop), consisting of aa trimers are mainly responsible for the interactions with

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VP3. The interacting base of the trimers is formed largely by the three copies of helix 2 and the segments of the chain (aa 22–27 and 44–52) leading into and out of this helix. Thus, the base is a relatively flat surface with a large number of hydrophobic residues including two me- thionines (aa 30 and 50). The interactions between the VP7 trimers and the VP3 subcores are currently being investigated via mutational analyses of key residues.

CONCLUSION

The BTV structure, which contains multiple protein components in nonequimolar ratios, each of which is encoded by a discrete gene (mRNA species), presents a challenging system to understand the mechanism of virus assembly. Synthesis of subviral particles in eukary- otic cells by heterologous expression systems, such as the baculovirus expression vectors, has revealed a win- dow of opportunity for defining the types of protein–protein interaction involved under natural conditions. This FIG. 6. The cartoon of VP7 trimers on VP3 scaffold. has allowed us to produce and manipulate particulate entities representing BTV and, together with deletion, site-specific, and protein extension mutants, the impor- particles. How the three minor proteins interact with each tance of various proteins relevant to the BTV assembly other and with VP3 protein and how VP3 and VP7 trimers process is being resolved. assemble into CLPs and generate the complex structure From the data obtained to date, it can be hypothesized lattice of the core is also under investigation. Synthesis that the process of virion assembly may start with a nu- of altered forms of subviral structures by molecular ge- cleocapsid complex involving the various mRNA species netics is being complemented by the physical analysis and at least one nonstructural protein, NS2 (which binds of such structures and their components. For example, ssRNA but not dsRNA). This nucleocapsid complex is high resolution (at 22–25 A˚ ) of the three-dimensional subsequently encapsidated by 60 dimers of disk-shaped structure of synthetic particles containing combinations VP3 molecules. Either shortly after, or simultaneously, of proteins is under investigation. In parallel, BTV and 260 trimers of VP7 are added to the icosahedral scaffold other orbivirus proteins synthesized by baculovirus vec- of VP3. The lower domains of VP7 appear to pack down tors are being crystallized for detailed analysis at atomic against six corners of each VP3 molecule, such that 12 level. Recently, a preliminary report of X-ray diffraction trimers of VP7 are directly in association with each dimer studies of core particles of BTV has been reported (Bur- of VP3. However, the 13th trimer of VP7 (organized in the roughs et al., 1995), showing diffraction to be high, be- icoshedron as T Å 13) does not appear to be directly in yond 4.8 A˚ . Therefore, resolution structural data of these contact with a VP3 molecule. Instead, the contacts of crystals can be expected in the near future, which un- one trimer appear to be with subunits of an adjacent doubtedly will reveal a better understanding of the as- trimer (see cartoon in Fig. 6). sembly of the core structure. At some stage during or before core formation, dsRNA The combined information gained from these studies is synthesized, presumably involving the release of NS2 will provide fundamental insights into the macromolecu- and its replacement with either or all of the minor pro- lar interaction of virus proteins that, hitherto, have not teins (VP1, the putative polymerase; VP4, the guanylyl- been possible for orbiviruses and may have implications transferase; and VP6, which binds dsRNA and ssRNA for virus assembly in general. and may be a helicase), each of which interacts specifi- cally with the VP3 subcore. At a later stage of virus repli- REFERENCES cation the VP5 and VP2 proteins are added onto the surface of the core, possibly interacting with VP7 and Belyaev, A. S., and Roy, P. (1992). Presentation of hepatitis B virus preS2 each other. epitope on bluetongue virus core-like particles. Virology 190, 840– Our current studies involve further mapping the molec- 844. Belyaev, A. S., and Roy, P. (1993). Development of baculovirus triple ular determinants for BTV particle formation and analysis and quadruple expression vectors: Co-expression of three or four of the effects of altered forms of each of the seven struc- bluetongue virus proteins and the synthesis of bluetongue virus-like tural proteins on the assembly and morphology of the particles in insect cells. Nucleic Acids Res. 21, 1219–1223.

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