Orthoreovirus and Aquareovirus Core Proteins: Conserved Enzymatic Surfaces, but Not Protein–Protein Interfaces

Virus Research 101 (2004) 15–28

Review Orthoreovirus and Aquareovirus core proteins: conserved enzymatic surfaces, but not protein–protein interfaces

Jonghwa Kim a,b, Yizhi Tao c,d,1, Karin M. Reinisch c,2, Stephen C. Harrison c,d, Max L. Nibert a,∗

a Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA b Department of Biochemistry, University of Wisconsin–Madison, Madison, WI 53706, USA c Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02115, USA d Department of Molecular Medicine, Howard Hughes Medical Institute, Children’s Hospital, Boston, MA 02115, USA

Abstract

Orthoreoviruses and Aquareoviruses constitute two respective genera in the family Reoviridae of double-stranded RNA viruses. Orthore- oviruses infect mammals, birds, and reptiles and have a genome comprising 10 RNA segments. Aquareoviruses infect ﬁsh and have a genome comprising 11 RNA segments. Despite these differences, recent structural and nucleotide sequence evidence indicate that the proteins of Orthoreoviruses and Aquareoviruses share many similarities. The focus of this review is on the structure and function of the Orthoreovirus core proteins ␭1, ␭2, ␭3, and ␴2, for which X-ray crystal structures have been recently reported. The homologous core proteins in Aquare- oviruses are VP3, VP1, VP2, and VP6, respectively. By mapping the locations of conserved residues onto the Orthoreovirus crystal structures, we have found that enzymatic surfaces involved in mRNA synthesis are well conserved between these two groups of viruses, whereas several surfaces involved in protein–protein interactions are not well conserved. Other evidence indicates that the Orthoreovirus ␮2 and Aquareovirus VP5 proteins are homologous, suggesting that VP5 is a core protein as ␮2 is known to be. These ﬁndings provide further evidence that Orthoreoviruses and Aquareoviruses have diverged from a common ancestor and contribute to a growing understanding of the functions of the core proteins in viral mRNA synthesis. © 2003 Elsevier B.V. All rights reserved.

Keywords: Reovirus; Rotavirus; dsRNA; Transcription; RNA capping; RNA transport

1. Introduction Many ORVs are fusogenic, causing the formation of multi- nucleated syncytia within infected cultures (Duncan et al., Orthoreoviruses (ORVs) and Aquareoviruses (AqRVs) 1996; Duncan et al., 1995; Lamirande et al., 1999; Wilcox deﬁne two respective genera in the family Reoviridae. and Compans, 1982), and all known AqRVs are fusogenic ORVs have been isolated from mammals, birds, and rep- as well. Fusion by the fusogenic ORVs is known to be tiles, whereas AqRVs have been isolated from ﬁsh, mol- mediated by a type 1 transmembrane protein translated luscs, and crustaceans (van Regenmortel et al., 2001). The from one of their small genome segments (Shmulevitz and ORV genome comprises 10 double-stranded (ds) RNA seg- Duncan, 2000), and a similar protein is translated from one ments, encoding 11 or 12 unique polypeptides, whereas the of the small AqRV segments (Duncan, personal communi- AqRV genome comprises 11 dsRNA segments, encoding cation). Recent studies involving electron cryomicroscopy 12 unique polypeptides (van Regenmortel et al., 2001). and three-dimensional image reconstruction of AqRV particles (Nason et al., 2000; Shaw et al., 1996) have shown their structures to be very similar to those of ORV parti- ∗ Corresponding author. Tel.: +1-617-645-3680; cles obtained by the same techniques (Dryden et al., 1993; fax: +1-617-738-7664. Metcalf et al., 1991). A sensible proposal for analogous, if E-mail address: [email protected] (M.L. Nibert). 1 not homologous, structural proteins within AqRV and ORV Present address: Department of Biochemistry and Cell Biology, Rice particles has thus been made (Nason et al., 2000). University, Houston, TX 77251, USA. 2 Present address: Department of Cell Biology, Yale University School The deduced amino acid sequences of the major outer of Medicine, New Haven, CT 06510, USA. capsid protein VP7 from genogroups A and B AqRV

et al. (2002) have concluded that maintenance of the current taxonomic division into two genera is justiﬁed, given MR σ3 51- ·CMHCLGVV..GSLQRKLKHLPHHRC· · ·-73 AR σB 51- CSQCCGVLYYGTLPADGNYFPHHKC-75 the differences between viruses from the two groups (e.g., NBR σB 51- CAQCCGVLYYGYRPINLYPFPHHRC-75 host range and genome segment number). ORV BR σB 51- CTRCCGVLHYGEMH.GGNPFIRHIC-74 We have previously reported the X-ray crystal structures SBR VP7 50- CACCFKVLL..NWP.GPPIHITHEC-71 ␭ CSR VP7 50- CALCLKQVC..SYHVTEPCYYPHEC-72 of the mORV core particle and polymerase 3 at respective

AqRV GCR VP7 49- CAFCLTTLA..PHANVKTIQDSHAC-71 resolutions of 3.6 and 2.5 Å (Reinisch et al., 2000; Tao et al., C--C------H-C 2002). Together, these studies have provided high-resolution structures for the nearly complete polypeptide chains of four ␴ ␴ Fig. 1. Conserved CCHC motif in ORV 3 and B proteins and AqRV of the ﬁve mORV core proteins (all but ␮2). Given pub- VP7 proteins. The alignment was generated using the program Pileup in GCG (Genetics Computer Group, Madison, WI) with the blosum30 lished evidence for homologies between these proteins and scoring matrix, a gap creation penalty of 6, and a gap extension penalty of those of AqRV GCR (Attoui et al., 2002; Fang et al., 2000), 3. Periods indicate gaps in the alignment. Fully conserved residues in the we compared the conserved and variable regions of the core alignment are indicated by the consensus at bottom. Filled circles at top proteins relative to the available mORV crystal structures. indicate the CCHC residues that coordinate a zinc ion in mammalian ORV By performing these comparisons for this review, we aimed ␴3 protein (Olland et al., 2001). Amino acid positions at the beginning and end of each sequence are marked. MR, mORV T1L (GenBank to determine whether the regions of these proteins involved X61586); AR, avian ORV 176 (GenBank AF059720); NBR, Nelson Bay in enzymatic activities or protein–protein interactions are ORV (GenBank AF059722); BR, baboon ORV (GenBank AF059723); conserved. Our ﬁndings are described in the following sec- SBR, genogroup A AqRV (striped bass reovirus) (GenBank U83396); tions, starting with the more external proteins in the core CSR, genogroup B AqRV (coho salmon reovirus) (GenBank U90430); and working inward. GCR, genogroup G AqRV (grass carp reovirus) (GenBank AF236688).

isolates SBR (striped bass reovirus) and CSR (coho salmon 2. Core turret proteins ␭2 and VP1 reovirus), respectively, were published in 1997 and show significant homology (Lupiani et al., 1997a,b). At that time, The mORV core turret protein ␭2 catalyzes the last it was not recognized that these proteins contain a con- three reactions in mRNA 5 cap synthesis: RNA guanylyl- served CCHC motif near their N termini, homologous to the transferase (GTase), 7-N-methyltransferase (MTase), and zinc-binding motif of the ORV ␴3/␴B proteins (Duncan, 2-O-MTase (Reinisch et al., 2000). It also interacts with 1999; Mabrouk and Lemay, 1994; Olland et al., 2001; five of the seven other proteins in virions and may thus play Schiff et al., 1988a)(Fig. 1). The genogroup A AqRV VP7 key roles in particle structure and assembly. Each core turret protein and nonfusogenic mammalian ORV (mORV) ␴3 is a pentameric structure of five ␭2 subunits. The 12 turrets proteins have subsequently been shown to occupy analo- per particle thus contain a total of 60 ␭2 subunits. ␭2 con- gous positions in virions of the respective viruses (Nason tains seven well-demarcated domains mediating different et al., 2000). The deduced amino acid sequence of the ma- activities. In addition to the GTase and two MTase domains jor outer capsid protein VP7 from a genogroup G AqRV, (MTase-1 and MTase-2), three small immunoglobulin-like grass carp reovirus (GCR) (Rangel et al., 1999), has been domains from each subunit are aligned to form an elon- recently reported (Qiu et al., 2001a) (GenBank accession gated “flap” that can fold across the top of the ␭2 pentamer number AF236688) and shown to demonstrate significant (Chandran et al., 1999; Reinisch et al., 2000). These flaps homology to the genogroup A and B VP7 sequences, includ- may be involved in regulating the release of capped tran- ing conservation of the CCHC zinc-binding motif shared scripts (Bartlett et al., 1974; Dryden et al., 1993; Reinisch with mORV ␴3(Qiu et al., 2001a) and the ␴B proteins of et al., 2000; Yeager et al., 1996). The function of the re- fusogenic ORVs (Fig. 1). maining domain, which resides between the GTase and In fact, the nucleotide and deduced amino acid sequences MTase-1 domains in both primary sequence and pentamer of several other AqRV genome segments have been recently structure (Reinisch et al., 2000), is not known. Residues on reported to GenBank (see below for accession numbers) as the bottom and side surfaces of the GTase domain, respec- well as in the literature (Attoui et al., 2002; Fang et al., 2000). tively, contact the top surface of the T = 1 ␭1 core shell and In the case of the AqRV core proteins VP1, VP2, VP3, VP5, the side of the core nodule protein ␴2 in cores (Breun et al., and VP6, respective homologies to mORV core proteins ␭2, 2001; Dryden et al., 1993; Reinisch et al., 2000). Residues ␭3, ␭1, ␮2, and ␴2 have been demonstrated, with sequence in the most carboxyl-(C-)terminal immunoglobulin-like do- identities ranging from 22 to 42% in the pairwise compar- main probably contact the base of the ␴1 receptor-binding isons (Attoui et al., 2002; Fang et al., 2000). The homologous fiber in virions (Chandran et al., 2001; Dryden et al., 1993; sequences include the characteristic SG and GDD motifs in Reinisch et al., 2000). The ␭2 regions that contact the ma- the RNA-dependent RNA polymerases ␭3 and VP2 (Fang jor outer capsid proteins ␮1 and ␴3 in virions remain to be et al., 2000). Based on these findings, Fang et al. (2000) have precisely defined. Evidence for conservation of the GTase proposed that the separation of AqRVs and ORVs into dif- and MTase-2 domains in the ␭C protein of a fusogenic ferent genera may need to be reconsidered; however, Attoui avian ORV has been recently reported (Hsiao et al., 2002). J. Kim et al. / Virus Research 101 (2004) 15–28 17

A morphologically similar, pentameric core turret is found in AqRV SBR particles and is formed by protein VP1 (Nason et al., 2000). The similar features include the flap domains, although in AqRV SBR virions these flaps are oriented more perpendicularly to the particle surface than in mORV virions, opening a larger channel at the five-fold axis (Nason et al., 2000). In addition, this five-fold channel becomes smaller in the AqRV SBR cores, opposite the situation for ␭2 in mORV particles (Dryden et al., 1993; Metcalf et al., 1991; Nason et al., 2000). VP1 has been speculated to have activities in RNA capping comparable to those of ␭2(Attoui et al., 2002; Fang et al., 2000; Nason et al., 2000), and indeed the GTase activity of VP1 has been recently demonstrated (Qiu and Luongo, 2003). mORV T3D ␭2 (144 kDa) and AqRV GCR VP1 (141 kDa) have an overall sequence identity of 28% (Fig. 2B)(Fang et al., 2000). The more highly conserved regions correspond to the enzyme and flap domains (Fig. 2B), as seen in comparisons among the ␭2 sequences of different mORV isolates (Breun et al., 2001). In the ␭2 GTase domain (residues 1–380), the conserved residues include Lys171 and Lys190, which have been shown to be important for the GTase activity (Luongo et al., 2000; Luongo, 2002). In fact, these are the only lysine residues in this domain that are conserved in the alignment, as recently noted by others (Hsiao et al., 2002; Qiu and Luongo, 2003). In the ␭2 MTase-2 domain (residues 805–1022), conserved residues include Asp827 and Gly829, which have been shown to be important for the activity of this domain at binding the methyl donor S-adenosyl-l-methionine (SAM) (Luongo et al., 1998; Hsiao et al., 2002). These two residues are located within the longest consecutive stretch of identity (␭2 residues 826–833) in the entire alignment. Of the six other residues that have been identified to surround the binding site for S-adenosyl-l-homocysteine (SAH, the product of methyl loss from SAM) in this domain (Reinisch et al., 2000), four are conserved in the alignment with GCR VP1 (␭2 residues Asn812, Asp850, Asp871, and Tyr872). No muta- genesis has yet been performed on the ␭2 MTase-1 domain (residues 434–691), but 10 residues have been identified to surround the binding site for SAH in this domain (Reinisch et al., 2000). Of these 10 residues, nine are conserved in the alignment with VP1 (␭2 residues Ser482, Ser518, His521, Tyr552, Asp553, Asp561, Asp577, Asp579, and Asp583). Fig. 2. Averaged sequence identities shared by mORV and AqRV core These findings provide evidence for the conservation of en- proteins. mORV sequences used were: (A) T1L ␭1 (GenBank AF129820), zymatic surfaces in the mORV ␭2 and AqRV VP1 proteins. (B) T3D ␭2 (GenBank J03488), (C) T3D ␭3 (GenBank M31058), (D) To gain more information about conserved aspects of ␭2 T1L ␮2 (GenBank X59945), and (E) T1L ␴2 (GenBank L19774). AqRV and VP1, we mapped the conserved residues in the align- sequences used were (A) GCR VP3 (GenBank AF260513), (B) GCR VP1 ␭ (GenBank AF260511), (C) GCR VP2 (GenBank AF260512), (D) GCR ment onto the T3D 2 crystal structure (Reinisch et al., VP5 (GenBank AF251262), and (E) GCR VP6 (GenBank AF236688).The 2000). Clustering of conserved residues is observed at sev- alignments were generated using the program Gap in GCG with the eral locations in the structure. Particularly striking is the blosum50 matrix, a gap creation penalty of 12 or 14, and a gap extension asymmetric distribution of conserved residues on the top and penalty of 4. After altering the sequence files to reflect gaps from the bottom surfaces of the MTase-1 domain (Fig. 3). The broad alignment, the sequence identity plot was generated using the program PlotSimilarity in GCG with the blosum50 scoring matrix, a gap creation patch of conservation unique to the bottom surface surrounds penalty of 14, a gap extension penalty of 4, and an averaging window of the binding site for SAH (Fig. 3B) and likely delimits the 15. substrate-binding and catalytic surfaces of this domain. 18 J. Kim et al. / Virus Research 101 (2004) 15–28

Fig. 3. Structural mapping of residues in the MTase-1 domain of mORV T3D ␭2 protein that are variable or conserved in the alignment of ␭2 with AqRV GCR VP1 protein. The crystal structures of the MTase-1 domain (residues 434–691) from five ␭2 subunits are shown in space-filling format in their relative positions within a ␭2 core spike in mORV cores (Reinisch et al., 2000). The images in this and all subsequent figures were rendered with RasMac 2.7.1 and prepared for publication with Photoshop 5.5 and Illustrator 8.0 (Adobe Systems, Mountain View, CA). Residues are color coded as follow: sites of nonconservative changes, pale yellow; sites of conserved residues, dark green; and sites of conservative substitutions, lighter green. A blosum50 matrix was used for defining conservative and nonconservative substitutions in the alignment. The alignment included no gaps between the two sequences in this region. The position of bound SAH (dark orange) (Reinisch et al., 2000) is shown for one subunit. (A) Stereo views from the top relative to the ␭1 core shell. (B) Stereo views from the bottom relative to the ␭1 core shell, reflected across a horizontal axis from the views in (A).

In the case of the GTase domain, only scattered con- residues on the bottom of the GTase domain that approach servation is seen on the bottom surface that interacts with within 4.0 Å of ␭1 in the core crystal structure (Breun et al., ␭1(Fig. 4I), suggesting that this surface is not highly con- 2001; Reinisch et al., 2000), only two are conserved in the served in the two groups of viruses. In fact, of the 32 alignment with VP1 (data not shown). Similarly, of the six

Fig. 4. Structural mapping of residues in the whole mORV T3D ␭2 protein that are variable or conserved in the alignment of ␭2 with the AqRV GCR VP1 protein. The crystal structures of the five ␭2 subunits in one core spike (Reinisch et al., 2000) are shown. Nonconserved residues are color coded according to domain: GTase domain (residues 1–380), pale pink; extended region (residues 381–433), pale gray; MTase-1 domain (residues 433–691), pale yellow; spacer domain of unknown function (residues 692–805), pale green; MTase-2 domain (residues 806–1022), pale blue; and flaps (residues 1023–1289), violet. Nonconserved residues in the three flap domains are shown in progressively darker violet as they approach the C-terminus and five-fold axis. Other residues are color coded as follow: sites of conserved residues, dark green; sites of conservative substitutions, lighter green; and sites of insertions in ␭2 relative to VP1, purple. A blosum50 matrix was used for defining conservative and nonconservative substitutions. Sites of insertions in VP1 relative to ␭2 are not indicated. The two positions of bound SAH (dark orange) (Reinisch et al., 2000) are shown for one subunit, as evident in (A, E and F). Each bottom view is that reflected across a vertical axis from the respective top view. All views are in space-filling format except that in (A). (A) Side view of the ␭2 pentamer spike, with the subunit nearest the viewer shown in thick backbone and the other subunits shown as ribbons indicating secondary structure. The top of the pentamer was rotated toward the viewer by 90◦ to generate all of the other top views in the figure. (B and C) Top (B) and bottom (C) views of the C-terminal flap domains. A conserved surface patch is indicated in one subunit (arrowhead). (D–F) Top (D) and bottom (E) views of the MTase domains. Conserved surface patches representing the MTase-1 and MTase-2 domains are indicated in one subunit (arrowhead and arrow, respectively). A cut-away view from the bottom of the MTase domains (F) better reveals the enzymatic cleft of the MTase-2 domain. Conserved residues Asp827 and Gly829 (see text) are shown in red. Conserved residues 830–833 in the subunit with the arrowhead are removed from the cut-away view to permit better visualization of Asp827 and Gly829. (G and H) Top (G) and bottom (H) views of the extended region and spacer domain. An elongated patch of conservation is indicated in one subunit (arrowhead). (I–K) Top (J) and bottom (I) views of the GTase domain. A conserved surface patch representing the enzymatic cleft is indicated in one subunit (arrow), and is better visualized in a cut-away view from the top of the GTase domain (K). Conserved lysines 171 and 190 (see text) are shown in red. J. Kim et al. / Virus Research 101 (2004) 15–28 19

residues on the side of the GTase domain that approach cally important residues Lys171 and Lys190 (Luongo et al., within 4.0 Å of ␴2 in the core crystal structure (Breun et al., 2000; Luongo, 2002). These ﬁndings provide additional ev- 2001; Reinisch et al., 2000), only two are conserved in idence for conservation of substrate-binding and catalytic the alignment with VP1 (data not shown). On the top sur- surfaces within the GTase enzymatic cleft. Other conserved face of the ␭2 GTase domain, in contrast, a contiguous surface residues in this region of ␭2 include Gln58, Ala168, patch of conserved residues is evident (Fig. 4J) and is made Tyr172, Tyr195, Ala196, His223, Thr228, His232, Glu258, clearer in a cut-away view (Fig. 4K) allowing better vi- Arg278, and Tyr283. Of these, Tyr172, Tyr195, and Glu258 sualization of conserved residues surrounding the catalyti- are notable because they interact with Lys190 and may be 20 J. Kim et al. / Virus Research 101 (2004) 15–28

Fig. 5. Structural mapping of residues in mORV T1L ␴2 protein that are variable or conserved in the alignment of ␴2 with AqRV GCR VP7 protein. The crystal structures of five-fold-proximal (A–C) and three-fold-proximal (D–F) copies of ␴2 in mORV cores (Reinisch et al., 2000) are shown in the same orientation. A two-fold copy of ␴2 is not shown since its structure was not fully resolved in the core crystal structure (Reinisch et al., 2000). Residues are color coded as follow: sites of nonconservative changes, pale pink; sites of conserved residues, dark green; sites of conservative substitutions, lighter green; and sites of insertions in ␴2 relative to VP7, purple. A blosum50 matrix was used for defining conservative and nonconservative substitutions. Sites of insertions in VP7 relative to ␴2 are not indicated. Regions that interact with ␭1 and ␭2 are indicated in B–F. (A and D) View from the side of each ␴2 subunit (in ribbons format to indicate secondary structures) relative to the ␭1 shell in cores. (B and E) View from the top of each ␴2 subunit (in space-filling format) relative to the ␭1 shell in cores. The top of the subunit in (A) and (D) was rotated toward the viewer by 90◦ to generate these top views. (C and F) View from the bottom of each ␴2 subunit (in space-filling format) relative to the ␭1 shell in cores, reflected across a vertical axis from the views in (B) and (E).

important for its proper orientation and pKa for catalysis. tamer formation may thus be a prerequisite for at least one His223 and His232 have also been recently shown to be of the MTase activities of these proteins (Reinisch et al., essential for the GTase activity of ␭2(Qiu and Luongo, 2000), consistent with the lack of MTase activity by puri- 2003). fied ␭2 monomers (Luongo et al., 1998; Mao and Joklik, In the case of the MTase domains, only scattered con- 1991). servation is found on the top surfaces (Fig. 4D), but broad In the case of the flap domains of ␭2(Chandran et al., contiguous patches of conserved residues are seen on the 1999; Chandran et al., 2001; Luongo et al., 1997; Reinisch bottom surfaces surrounding the sites at which SAH binds et al., 2000), which are dispensable for mRNA capping to each domain (Fig. 4E). The conserved patch surrounding (Luongo et al., 1997), little localized conservation in the the SAH binding site in the ␭2 MTase-1 domain (yellow alignment with VP1 is seen (Fig. 4B and C). Nonetheless, background) is evident to its full extent in the bottom a small conserved patch on the top surface of the flaps view (Fig. 4E), whereas the conserved patch surrounding (Fig. 4B) includes ␭2 residues Pro1131, Gln1133, Leu1134, the SAH binding site in the ␭2 MTase-2 domain (blue Asp1135, Phe1136, Thr1137, Asp1142, Ile1220, Pro1223, background) is made clearer in a cut-away view (Fig. 5F) and Asn1225. The significance of this patch is unknown, but allowing better visualization of the enzymatic cleft in- might reflect a binding activity for viral entry. In addition, cluding conserved residues Asp827 and Gly829 described around the top opening of the five-fold channel, ␭2 residues above. These findings provide additional evidence for con- Gly1244 and Asn1245 are conserved in the alignment. Given servation of substrate-binding and catalytic surfaces in both their locations, these residues may contribute to the ␴1 an- MTase domains of mORV ␭2 and AqRV VP1. Besides choring activity of ␭2(Chandran et al., 2001); however, no the residues identified in ␭2byReinisch et al. (2000) and ␴1 homolog (or structural analog) has been found in AqRVs Luongo et al. (1998), other conserved residues on the en- (Nason et al., 2000). zymatic surface of the MTase-1 domain include Asp480, The remaining domain of ␭2 is the only one formed from Arg481, Lys485, Asp486, Val489, Pro527, Val554, Tyr575, discontinuous regions of sequence: residues 381–433 and Val581, Lys615, Asn617, Phe618, Glu650, and Phe652; 692–805. It exhibits only scattered conservation on its bot- and other conserved residues on the enzymatic surface tom surface (Fig. 4H), but a patch of conserved residues of the MTase-2 domain include Leu828, Thr830, Pro832, on its top surface (Fig. 4G) includes ␭2 residues Leu397, Glu833, Ile836, Leu839, Arg852, Thr887, Gly893, Ala894, Pro398, Gln399, Tyr400, Arg764, Arg765, Arg768, Leu769, Ala895, Asn927, Phe951, Arg955, and Glu958. In addi- Arg770, and Pro773. The function of this domain is not tion, from the MTase-1 domain of an adjacent subunit in known, but several of the conserved residues (397–400 and the ␭2 pentamer, several other conserved residues project 765) project into the bottom portion of the enzymatic cleft into the enzymatic cleft that is predominantly formed by of the MTase-2 domain (data not shown) and may contribute the MTase-2 domain, including Pro567 and Gly569. Pen- to its activities. J. Kim et al. / Virus Research 101 (2004) 15–28 21

3. Core nodule proteins ␴2 and VP6 4. Core shell proteins ␭1 and VP3

No enzymatic activities have been ascribed to the mORV The mORV ␭1 protein is present in 120 copies per viral core nodule protein ␴2. Instead, ␴2 is thought to play a particle and forms the T = 1 core shell through associations role in stabilizing the ␭1 shell (Kim et al., 2002; Reinisch of 60 asymmetric ␭1 homodimers (Reinisch et al., 2000). et al., 2000; Xu et al., 1993) and in binding the base of ␮1 The ␭1 shell is thin and plate-like in morphology. On its outer for outer capsid assembly (Liemann et al., 2002; Reinisch surface in viral particles, it binds core spike protein ␭2 and et al., 2000). The significance of a dsRNA-binding activity core nodule protein ␴2(Dryden et al., 1993; Reinisch et al., of ␴2(Dermody et al., 1991; Schiff et al., 1988b) remains 2000). On its inner surface in particles, it is thought to bind unknown, but a comparable activity of the avian ORV ho- ␭3 and ␮2 to form the viral transcriptase complexes (Dryden molog, ␴A, has been shown to play a role in combatting et al., 1998). ␭1 has been proposed to play a role in tran- cellular antiviral responses (Gonzalez-Lopez et al., 2003). scription (Morgan and Kingsbury, 1980; Powell et al., 1984), Monomeric copies of ␴2 bind atop the T = 1 ␭1 shell in and its capacity to bind RNA (Bisaillon and Lemay, 1997b; three distinct positions: surrounding the five-fold axes, sur- Lemay and Danis, 1994; Schiff et al., 1988a) may be impor- rounding the three-fold axes, and across the two-fold axes, tant in this regard. More recently, ␭1 has been shown to influ- for a total of 150 copies per core (Reinisch et al., 2000). ence the strain-dependent nucleoside triphosphate phospho- The apparent homolog of ␴2 in AqRV, VP6 (Nason et al., hydrolase (NTPase) activities of mORV cores (Noble and 2000), occupies similar positions and thus seems likely to Nibert, 1997a), and recombinant ␭1 has been shown to medi- play similar roles in assembly and structure. One difference, ate NTPase, RNA helicase, and RNA capping 5 triphosphate however, is that no VP6 nodules are found at the icosahe- phosphohydrolase activities (Bisaillon et al., 1997; Bisaillon dral two-fold positions of the VP3 shell: VP6 nodules are and Lemay, 1997a), suggesting multiple roles for ␭1 in viral found only surrounding the five-fold and three-fold axes, for RNA synthesis. A CCHH zinc-finger motif near residue 200 a total of 120 copies per core (Nason et al., 2000). (Bartlett and Joklik, 1988; Harrison et al., 1999) was shown mORV T1L ␴2 (47 kDa) and AqRV GCR VP6 (44 kDa) to bind zinc in the core crystal structure (Reinisch et al., have the lowest level of overall sequence identity among 2000), but other aspects of its function remain unknown. An the core proteins of these viruses, 22% (Fig. 2E)(Attoui amino-(N-)terminal region of ␭1 (approximately residues et al., 2002). Their status as homologs is nonetheless ev- 1–200) is much more hydrophilic than the rest of the protein ident from local regions of greater sequence conservation (Harrison et al., 1999) and contains sequence motifs remi- (Fig. 2E), the distribution of conserved sequences across niscent of NTPases and helicases (Bartlett and Joklik, 1988; most of the protein lengths (Fig. 2E), and their similar sec- Bisaillon et al., 1997; Harrison et al., 1999; Noble and ondary structure prediction profiles (data not shown). The Nibert, 1997a). In the core crystal structure, this N-terminal two regions of greatest conservation are in the C-terminal region was not visualized for the 60 copies of ␭1 that ap- halves of the proteins, corresponding to ␴2 residues 301–321 proach the icosahedral five-fold axes (Reinisch et al., 2000). and 343–356. When mapped on the T1L ␴2 crystal struc- Most of this region was visualized, however, for the 60 ture (Reinisch et al., 2000), the conserved residues are more copies of ␭1 that approach the icosahedral three-fold axes, or less evenly scattered across the protein surface (Fig. 5) in which it displays an extended conformation seemingly and through the protein interior (data not shown). In ad- inconsistent with enzymatic activities. Instead, since the dition, of the 93 different residues in the three-fold- and N-terminal arms extend beneath adjacent ␭1 subunits around five-fold-proximal ␴2 subunits that approach within 4.0 Å the three-fold axes, they have been proposed to play a role in of ␭1 in the core crystal structure (Reinisch et al., 2000) and assembling and/or stabilizing the core shell (Reinisch et al., are presumably important for ␭1 binding, only 20 are con- 2000), but recent studies indicate that these arms are dispens- served in the alignment with VP6. (The two-fold-proximal able for particle assembly and isolation (Kim et al., 2002). ␴2 subunits were not visualized appropriately in the crystal A core shell of similar diameter and morphology is found structure to permit this analysis (Reinisch et al., 2000).) Sim- in AqRV SBR particles and is proposed to be formed by ilarly, of the 11 residues on the side of the five-fold-proximal protein VP3 (Nason et al., 2000). Based on its homology to ␴2 subunits that approach within 4.0 Å of the ␭2 GTase do- mORV ␭1, VP3 is speculated to bind dsRNA and exhibit main in the core crystal structure (Reinisch et al., 2000), NTPase and RNA helicase activities (Attoui et al., 2002; only one is conserved in the alignment with VP6 (data not Fang et al., 2000). shown). Thus, ␴2 and VP6 show little evidence for con- mORV T1L ␭1 (142 kDa) and AqRV GCR VP3 (132 kDa) served enzymatic surfaces or sites of protein–protein inter- have an overall sequence identity of 31% (Fang et al., action. The fact that ␴2 and VP6 share a larger number of 2000)(Fig. 2A). A robust alignment of ␭1 and VP3, re- conserved residues on their top than on their bottom sur- quiring only a few small gaps, was obtained for residues faces (Fig. 5) suggests that the interactions of the respec- 105–1275 of ␭1. The first 104 residues of ␭1, however, tive core nodules with outer capsid protein ␮1 or VP5 may are represented by only 37 residues in VP3 so that the be more conserved than those with core shell protein ␭1or alignment includes several large gaps in that region, the VP3. exact placements of which are sensitive to the gap penalty 22 J. Kim et al. / Virus Research 101 (2004) 15–28

values used in generating the alignment (Figs. 2A and 6B; data not shown). The presence of these large gaps indicates that the structure of the VP3 N-terminal region is difficult to infer from the structure of that region of ␭1 and raises further questions about the proposed roles of this ␭1 region in core assembly, structure, and functions (Bisaillon et al., 1997; Bisaillon and Lemay, 1997a; Harrison et al., 1999; Noble and Nibert, 1997a; Reinisch et al., 2000). Notably, the proposed NTP-binding motifs in ␭1(Bartlett and Joklik, 1988; Bisaillon et al., 1997; Noble and Nibert, 1997a) are not well conserved. Another poorly conserved region in the alignment spans ␭1 residues 750–800 (Fig. 2A) and includes sequences that surround the five-fold axis on the top surface of the ␭1 shell (Reinisch et al., 2000). In contrast, sequences that surround the five-fold axis on the bottom surface of the shell include the longest conserved stretch in the alignment, 562-TVSESTTQTLSP-573 in ␭1 and 499-TISESTTQTISP-510 in VP3 (see below). The CCHH zinc-binding motif in ␭1 (residues 183, 186, 199, and 203 (Reinisch et al., 2000)) is also conserved in VP3 (residues 119, 122, 135, and 140), although VP3 has one additional residue between the two histidine residues, and other residues in the region are not well conserved. The clustering of conserved residues from the alignment in the ␭1 structure (Fig. 6) is less striking than that seen for the capping enzyme domains of ␭2. An exception, however, involves the conserved residues surrounding the icosahedral five-fold axis on the bottom (interior) surface of the ␭1 shell (Fig. 6B). These residues form a star-shaped patch centered at the five-fold axis, with conserved residues concentrated in the center (residues 564–570) and on the periphery of the star. This region of conservation between ␭1 and VP3 might be involved in transport of transcripts through the five-fold channel of the core shell and into the core spike for the completion of mRNA capping. It might also be involved in the first enzymatic activity in RNA capping, the RNA 5 triphosphate phosphohydrolase, which has been attributed to ␭1 in mORVs (Bisaillon et al., 1997; Noble and Nibert, 1997a), or in anchoring the underlying RNA polymerase Fig. 6. Structural mapping of residues in mORV T1L ␭1 protein that protein (see below) to the core shell. Other small patches of are variable or conserved in the alignment of ␭1 with AqRV GCR conservation on the bottom surface of the ␭1 shell (Fig. 6B) VP3 protein. The crystal structures of the 10 ␭1 subunits in one de- are of unknown significance. cameric unit surrounding an icosahedral five-fold axis in cores (Reinisch On the top (exterior) surface of the ␭1 shell, small patches et al., 2000) are shown in space-filling format. The residues in orange, magenta, dark blue, and lighter blue represent the N-terminal arms from of conservation with VP3 are also seen (Fig. 6A). These adjacent ␭1 subunits that extend beneath the subunits in the displayed patches do not seem to relate to interactions with ␭2 and decamer (Reinisch et al., 2000) (shown for three of the five asymmetric ␴2, as they are not concentrated at the binding sites for units only). Nonconserved residues in the 5 two-fold-proximal copies of these other proteins (Fig. 6A). In fact, of the 30 different ␭ 1 in the decamer are shown in white, and those in the three-fold-proximal residues in ␭1 that approach within 4.0 Å of ␭2 in the core copies of ␭1 are shown in pale yellow or orange. Other residues are color coded as follow: sites of conserved residues, dark green or dark blue; crystal structure (Luongo et al., 2002; Reinisch et al., 2000) sites of conservative substitutions, lighter green or lighter blue; and sites of insertions in ␭1 relative to VP3, purple or magenta. A blosum50 matrix was used for defining conservative and nonconservative substitutions. of 2 two-fold-proximal copies of ␴2 (2), 2 three-fold-proximal copies of Sites of insertions in VP3 relative to ␭1 are not indicated. Conserved ␴2 (3), 2 five-fold-proximal copies of ␴2 (5), and the GTase domain from residues Cys183, Cys186, His199, and His203 (see text) are shown in two ␭2 subunits (G) are indicated by outline. (B) View of the bottom red, but mostly obscured by other residues. Examples of corresponding (interior) of the ␭1 shell, reflected across a horizontal axis from the view conserved patches on the top and bottom surfaces of ␭1 are indicated in (A). A star-shaped conserved region surrounding the five-fold axis is (arrows). (A) View of the top (exterior) of the ␭1 shell. Binding positions indicated (arrowhead). J. Kim et al. / Virus Research 101 (2004) 15–28 23 and are presumably important for ␭2 binding, only seven ically important palm and fingers domains of ␭3(Tao et al., are conserved in the alignment with VP3. Similarly, of the 2002). In fact, the two sequences can be aligned without 115 different residues in ␭1 that approach within 4.0 Å of gaps from residues 291 to 851 in ␭3. The homologous se- the three-fold- and five-fold-proximal ␴2 subunits, only 23 quences within the central region include the SG and GDD are conserved in the alignment with VP3. Another notable motifs (motifs V and VI of Koonin, 1992) characteristic feature is that several of the small patches on the top and of many viral RNA polymerases (Fang et al., 2000). These bottom surfaces correspond (Fig. 6, arrows indicate exam- motifs reside within the two longest consecutive stretches of ples) and thus represent regions of conservation that span the identity (␭3 residues 679–700 and 724–736, respectively) in shell in those positions. Such shell-spanning patches might the entire alignment. Little else is specifically known about be involved in movements of small molecules (e.g., nucle- the activities of VP2 or its locations within AqRV particles oside triphosphates) across the shell during RNA synthesis, (Nason et al., 2000). as suggested for bluetongue virus (Diprose et al., 2001). To gain more information about conserved aspects of ␭3 and VP2, we mapped the conserved residues from their alignment onto the mORV T1L ␭3 crystal structure (Tao 5. RNA polymerase proteins ␭3 and VP2 et al., 2002). Clustering of the conserved residues is observed at several locations in the ␭3 structure, but most re- The RNA-dependent RNA polymerase protein ␭3 cat- markably lining almost the entire central cavity and the four alyzes phosphodiester bond formation during both minus- channels that connect it to the outside (Fig. 7). Conservation and plus-strand RNA synthesis by mORVs (Starnes and within the cavity and channels is especially well visualized Joklik, 1993). It may also play a role in recognizing the in cut-away views in which the front half of ␭3 was removed plus-strand transcripts or partial genomic duplexes for from the molecule viewed from different angles (Fig. 7B, packaging into progeny particles. Within mORV particles a D, F and H). Conserved residues completely surround the monomer of ␭3 is thought to form part of the putative tran- conserved GDD motif (␭3 residues 733–735) (Fig. 7B and scriptase complex that is located beneath the ␭1 core shell D, red), but also extend onto the adjacent and opposite walls at or near each five-fold axis and shows a flower-shaped of the cavity. The conservation of such broad regions of sur- morphology in cryomicroscopy-derived image reconstruc- face in the cavity and channels in ␭3 and VP2 suggest roles tions (Dryden et al., 1998). ␭3 probably interacts with ␭1 not only in substrate-binding and catalytic activities imme- for anchoring to the under side of the core shell (Dryden diately related to phosphodiester bond formation but also et al., 1998). The recently determined crystal structure of in the other activities such as movements of template and ␭3 shows it to be a globular, cage-like protein with a large product RNA molecules during synthesis. In addition to the internal solvent cavity in which RNA synthesis occurs (Tao GDD motif, ␭3 residues of particular importance for poly- et al., 2002). Four distinct channels connect the central cav- merase activity (Tao et al., 2002) that are conserved in the ity to the outside environment and represent pathways for alignment with VP2 include Asp590, Ser682, and Gln732 movements of substrates and products during minus- and (specificity for ribo-NTPs versus deoxyribo-NTPs at the plus-strand RNA synthesis (Tao et al., 2002). Palm, finger, priming and incoming positions); Thr558, Ser559, Gly560, and thumb domains analogous to those in other polymerases Ser561, and Val563 (part of a loop that supports the prim- are evident, and portions of these are particularly similar to ing NTP); Arg523, Arg524, Arg526, and Ala588 (proper those in the RNA polymerases of hepatitis C virus and bac- positioning of the triphosphate of the incoming NTP); and teriophage ␾6(Butcher et al., 2001; Lesburg et al., 1999; Ile528 and Pro530 (proper positioning of the templating Tao et al., 2002). nucleoside). Several of these ␭3 residues are in the con- A flower-shaped structure also extends inward from the served motifs described by Koonin (1992): motif I (RxxRxI), VP3 shell at each five-fold axis in AqRV particles and has residues 523–528; motif IV (DxS), residues 590–592; motif been attributed to the viral transcriptase complexes by anal- V (SG), residues 682 and 683; and motif VI (GDD), residues ogy with mORV particles (Nason et al., 2000). Based on its 733–735. size, low stoichiometry, and presence in cores, the AqRV Much less conservation with VP2 is seen on the outer VP2 protein has been proposed to contribute to these struc- surfaces of ␭3 viewed from different angles (Fig. 7A, C, E tures and to represent the functional homolog of the mORV and G), but there are several notable features. In particular, polymerase ␭3(Nason et al., 2000). conservation from the channels spills onto the ␭3 outer sur- AqRV GCR VP2 (142 kDa) is indeed a homolog of face in several locations. Leading toward or away from the mORV T1L ␭3 (142 kDa) (Attoui et al., 2002; Fang et al., channel used for exit of dsRNA product during minus-strand 2000). Their degree of sequence identity, 41%, is the high- synthesis (Tao et al., 2002) are grooves with conserved est among all proteins from the two viruses (Attoui et al., residues aligned along their bases (Fig. 7A). Similar grooves 2002; Fang et al., 2000)(Fig. 2C). A central region of are seen leading toward or away from the channel used for the alignment, spanning residues 450–750 in ␭3, demon- entrance of the plus-strand template during minus-strand strates consistently higher identity scores than the N- and synthesis (Tao et al., 2002). In each case, these grooves C-terminal regions (Fig. 2C) and encompasses the enzymat- may be involved in directing the template and/or product 24 J. Kim et al. / Virus Research 101 (2004) 15–28

Fig. 7. Structural mapping of residues in mORV T3D ␭3 protein that are variable or conserved in the alignment of ␭3 with AqRV GCR VP2 protein. The crystal structure of ␭3(Tao et al., 2002) is shown in space-filling format in four different orientations, each progressively differing by a 90◦ rightward rotation as the views shift down the page. Nonconserved residues are color coded according to region of the primary sequence: N-terminal region (residues 1–380), pale blue; central region encompassing the palm and fingers domains (residues 381–790), pale violet; C-terminal region (residues 791–1267), pale pink. Other residues are color coded as follow: sites of conserved residues, dark green; sites of conservative substitutions, lighter green; and sites of insertions in ␭3 relative to VP2, purple. A blosum50 matrix was used for defining conservative and nonconservative substitutions. Sites of insertions in VP2 relative to ␭3 are not indicated. Conserved residues Gly733, Asp734, and Asp735 (see text) are shown in red. Bound cap 0 analog is shown in blue, sticks format. (A, C, E and G) Surface views in stereo. Surface grooves with conserved residues at their bases are indicated in (A) and (C) (arrowheads). Conserved residues visible through the putative nucleoside triphosphate entry channel (Tao et al., 2002) are indicated in (E) (arrow-head). A conserved surface patch adjacent to the putative transcript exit channel (Tao et al., 2002) is indicated in (G) (arrowhead). (B, D, F and H) Cut-away views in stereo, generated by removing approximately the front half of the molecule oriented in the same position as in the respective view at left. Two channels connecting the central cavity to the outside are visible in each view (arrows).

RNA molecules during the synthetic process. The channel through which conserved residues within the channel and proposed to be used for entrance of nucleoside triphosphate central cavity are evident to the viewer. The largest contigu- substrates and exit of pyrophosphate by-product during ous patch of conserved surface residues (Fig. 7G) is adjacent RNA synthesis (Tao et al., 2002) appears to be surrounded to the channel proposed to be used for exit of plus- strand by conserved residues (Fig. 7E), but the stereo view re- transcripts (Tao et al., 2002). This patch is comparable in veals that this channel has a conical opening to the outside, surface area to that surrounding the SAH-binding site in the J. Kim et al. / Virus Research 101 (2004) 15–28 25

N-terminal MTase domain of ␭2(Fig. 3B) and represents ded in one of the most highly conserved regions (Fig. 2D): a pocket on the ␭3 surface. The function of this conserved 413-LPKGSFKSTI-422 in ␮2 and 408-LPKGSYKSTI-417 patch is not known, but one possibility is that it forms part of in VP5. This region may be one of structural and/or func- the first capping enzyme, the RNA 5 triphosphate phospho- tional importance for both proteins. An overlapping region hydrolase, which has been attributed to ␭1(Bisaillon et al., (␮2 residues 410–420) has been previously suggested as an 1997) but remains to be fully characterized. The external A motif for the putative NTP binding and hydrolysis activi- cap (mGpppG)-binding pocket in ␭3(Tao et al., 2002)isnot ties of ␮2(Noble and Nibert, 1997b), and indeed Kim et al. a region of consistently high sequence conservation in VP2 (2004) have recently shown that Lys415 and/or Lys419 is/are according to the alignment, although residues lining the essential for the triphosphate phosphohydrolase activities of base of the pocket are conserved (Fig. 7C). In addition, the ␮2. binding site for the (positively charged) methylguanine base Several recent articles have described a role for mORV ␮2 appears to include an acidic residue in both ␭3 (Asp1035) in formation of the “factories” in which new core particles and VP2 (␭3 residue Glu1032), whereas the binding pocket are assembled in infected cells (Mbisa et al., 2000; Parker for the nonmethylated guanine base includes a conserved et al., 2002; Broering et al., 2002). As a part of that recent arginine (␭3 residue Arg851). work, ␮2 has been shown to associate with and stabilize cellular microtubules, thereby defining the morphology of the viral factories (Parker et al., 2002). ␮2 residue Pro208 6. Structurally minor core proteins ␮2 and VP5 has been identified as an important sequence determinant of this property. Since proline is conserved at that position The mORV ␮2 protein (80 kDa) is present in low copy in the VP5 alignment, we speculate that VP5 may also be number (approximately 20) per viral particle (Coombs, capable of microtubule association. 1998) and appears to be located internal to the ␭1 shell (Dryden et al., 1998). It is an RNA-binding protein (Brentano et al., 1998) and has been indicated by reassortant genetics 7. Evolution and classification of Orthoreoviruses and to play a role in the NTPase activities of mORV core par- Aquareoviruses ticles (Noble and Nibert, 1997b). It has also been similarly indicated to play a role in transcription by cores (Yin et al., The extent of homology shared by ORV and AqRV pro- 1996), but its precise roles in these enzymatic activities teins indicates that they diverged from a common ancestor remain undefined. Kim et al. (2004) have recently reported much more recently than from other members of the Re- that purified mORV ␮2 protein mediates both nucleoside oviridae family (Attoui et al., 2002). A reasonable question and RNA 5’ triphosphate phosphohydrolase activities in is therefore whether AqRVs should be combined with ORVs vitro consistent with ␮2 playing one or more role in viral in the same genus or subfamily. The host range of this ex- mRNA synthesis. panded group would include mammals, birds, reptiles, and AqRV SBR has been reported to lack a protein similar to fish, thus spanning the vertebrate subphylum. mORV ␮2 in its core particles (Nason et al., 2000). However, Despite their homologies, viruses in the two genera ex- the deduced sequence of AqRV GCR VP5 (80 kDa) shows it hibit a number of differences including (i) the presence in to be a ␮2 homolog, with an overall sequence identity of 24% AqRVs of an additional (11th) genome segment encoding (Fig. 2D). This fact was also noted by Attoui et al. (2002). one or two unique nonstructural proteins, (ii) the lack of a ␴1 (Note that the numbers for AqRV proteins VP4 and VP5 homolog in AqRVs, (iii) the absence of the core nodule pro- have been swapped in recent papers, i.e., ␮2 homolog VP5 tein VP6 at the two-fold axes of AqRV particles, and (iv) the was previously named VP4 and ␮1 homolog VP4 was previ- shortened N-terminal region of the AqRV core shell protein ously named VP5.) Considering the ␮2–VP5 homology and VP3 relative to its mORV homolog ␭1. The demonstration the strong evidence for ␮2 in mORV cores (Coombs, 1998; of an additional (fusion) protein encoded by the fusogenic Dryden et al., 1998; Noble and Nibert, 1997b; Yin et al., avian and mammalian ORVs (Duncan, 1999; Shmulevitz and 1996), it seems likely that AqRV cores contain VP5, but in Duncan, 2000) has not caused them to be segregated into low copy numbers that have been hard to demonstrate to a separate genus from the nonfusogenic mORV isolates date. Since the crystal structure of ␮2 has not been reported (Duncan, 1999; van Regenmortel et al., 2001) and so the (Reinisch et al., 2000), it is not yet possible to ascertain the discrepancy in genome segment number between ORVs and three-dimensional distribution of the residues conserved be- AqRVs need not necessarily relegate them to separate gen- tween it and VP5 as done for the other core proteins. In era. Although not discussed in this article, these homologies addition, few residues of importance to the putative enzy- extend to the major outer capsid proteins ␮1 and VP4, the matic activities of ␮2 have been identified for comparison major nonstructural proteins ␮NS and NS1, and the major with the conserved sequences in VP5. Pro414 in ␮2, which nonstructural proteins ␴NS and NS2 as well (Attoui et al., is changed to histidine in an ORV temperature-sensitive 2002; Liemann et al., 2002; Qiu et al., 2001b). With re- mutant mapped to the M1 segment encoding this protein gard to ␮1 and VP4 (formerly called VP5 (Liemann et al., (Coombs, 1996), is conserved in GCR VP5 and embed- 2002; Nason et al., 2000)), it is interesting to note that two 26 J. Kim et al. / Virus Research 101 (2004) 15–28 more recently reported sequences for VP4 (Attoui et al., but in fact the orientation of those domains has been seen 2002) include a myristoylation signal at its N-terminus, to vary among cores from different mORV isolates (Dryden which is missing from the initially reported VP4 sequence et al., 1993; Luongo et al., 1997). We therefore speculate that we used in our previously published ␮1–VP4 alignment that any difference in orientation of the VP1/␭2 flap domains (Liemann et al., 2002); thus, ␮1 and VP4 appear to share a that indeed exists between AqRV and mORV cores does not myristoylated N-terminus in addition to other apparent ho- have a significant effect on core functions in mRNA synthe- mologies. Regardless of the taxonomic issue, the homolo- sis. Another difference between AqRV and mORV cores is gies demonstrated in this and other recent studies indicate the absence of a monomer of the ␴2 homolog VP6 atop each that further comparisons of ORVs and AqRVs will likely icosahaedral two-fold axis of the core shell (Nason et al., yield new insights into the biology of each. 2000). As a result, only 120 copies of VP6 are present in Sequence conservation at protein–protein interfaces is AqRV cores, versus 150 copies of ␴2 in ORV cores. Since likely a strong selective force among cocirculating strains of the functions of ␴2 remain poorly understood, it is diffi- segmented viruses that are competent to exchange genome cult to speculate about functional effects of this difference. segments through reassortment. Once two groups of such Since ␴2 is required for forming stable core-like particles viruses have diverged to the extent that they are no longer with ␭1(Xu et al., 1993; Kim et al., 2002), one idea is that capable of such exchange, however, the pressure to main- ␴2 acts as a clamp for stabilizing the ␭1 shell (Reinisch tain conserved protein interfaces between the groups is et al., 2000). It may be that the two-fold interactions be- gradually lost. Genetic drift within each group can then tween VP3 subunits in AqRV cores are stronger than those yield divergence at the protein interfaces as suggested in between ␭1 subunits in mORV cores, so that VP6 clamps this study for ORVs and AqRVs. In contrast, because the are not needed at those sites. Indeed, given that ␴2 can sit basic activities demanded of the virally encoded enzymes atop each two-fold axis of the ␭1 shell in either of two possi- within the two groups remain the same even after they have ble orientations (Reinisch et al., 2000), it may be that those become genetically isolated, selective pressure to maintain copies of ␴2 contribute little to stabilizing the shell and may conserved enzymatic surfaces continues to operate and ge- themselves be dispensable. The latter idea should be possi- netic drift in those protein regions proceeds more slowly. As ble to test using our system for assembling ORV core-like larger numbers of ORV and AqRV sequences and structures particles from recombinant proteins (Kim et al., 2002). become available for comparison, these fundamental as- How ORV and AqRV cores are assembled in infected pects of evolution between and within these groups should cells remains largely a mystery, particularly with regard to become even more evident. how the 10 (ORV) or 11 (AqRV) different genomic RNA segments are specifically packaged as a part of this process. Similarities among the core proteins emphasized in this re- 8. General conclusions concerning core functions, view suggest that these two virus groups may have solved structure, and assembly the assembly/packaging problem in a similar manner and thus comparative studies with both groups may expedite our Based on the findings in this review and other recent re- understanding of this mechanism. ports (Attoui et al., 2002; Fang et al., 2000; Nason et al., 2000), it appears likely that the core particles of ORVs and Note added in proof AqRVs will be found to mediate mRNA synthesis using the same set of homologous proteins. Conserved enzymatic Zhang et al. (2003) have recently reported evidence for mechanisms and arrangements of enzyme domains within how and where the polymerase ␭3 is anchored beneath the ␭1 the particles to allow for efficient performance of the se- shell in mORV virions. Incorporating descriptions of these quential reactions in mRNA synthesis appear almost certain authors’ findings into the current review was beyond the for the polymerases (␭3 and VP2), GTases (␭2 and VP1), scope of allowable revisions, but the reader is encouraged and MTases (␭2 and VP1) of the two virus groups. Given to view the regions of sequence conservation identified in uncertainties about the relative roles of ␭1 and ␮2inmORV, Figs. 6 and 7 in light of these new observations. the presence of large deletions in the N-terminal region of the ␭1 homolog VP3 in AqRV, and the absence of crystal structure data for ␮2 or its homolog VP5 in AqRV, it Acknowledgements remains possible that some functional variations involving these two protein pairs will be found. However, we consider We are grateful to Teresa Broering, Roy Duncan, and this unlikely given the extent of homology that is present. John Parker for comments on a draft of this manuscript Previous structural analyses of AqRV cores by electron and to Cindy Luongo and Tao Qiu for helpful discussion. cryomicroscopy (Nason et al., 2000) have demonstrated their This work was supported by NIH grants R29 AI-39533 and striking similarities to mORV cores. A difference in the ori- R01 AI-47904 (to M.L.N.) and R01 CA-13202 (to S.C.H.). entation of the flap domains in AqRV turret protein VP1 rel- S.C.H. is an investigator in the Howard Hughes Medical ative to that in mORV ␭2 has been noted (Nason et al., 2000), Institute. J. Kim et al. / Virus Research 101 (2004) 15–28 27

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