Emerging Themes in Rotavirus Cell Entry, Genome Organization,

8/14/2019 Emerging Themes in Rotavirus Cell Entry, Genome Organization,

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Virus Research 101 (2004) 6781

Emerging themes in rotavirus cell entry, genome organization,transcription and replication

Hariharan Jayarama, M.K. Estesb, B.V. Venkataram Prasada, c,a Program in Structural and Computational Biology and Molecular Biophysics, Houston, TX 77030, USA

b Department of Molecular Virology and Microbiology, Houston, TX 77030, USAc Verna and Marrs McLean Department of Biochemistry and Molecular Biology,

Baylor College of Medicine, Houston, TX 77030, USA

Abstract

Rotaviruses, causative agents of gastroenteritisin younganimalsand humans, are large icosahedral viruses witha complexarchitecdouble-strandedRNA(dsRNA)genomecomposedof11segments,whichcodesfor6structuraland6non-structuralproteins,isenclosethree concentric capsid layers. In addition to facilitating host-specic interactions, the design of the capsid architecture in rotaviotherdsRNAvirusesshould alsobe conducive to the requirementof transcribing theenclosedgenome segmentsrepeatedly andsimultwithin the capsid interior. Several non-structural proteins facilitate the subsequent processes of genome replication and packagingcryomicroscopy studies of intact virions, recombinant virus-like particles, functional complexes, together with recent X-ray crystastudies on rotavirus proteins have provided structural insights into the capsid architecture, genome organization, antibody interaentry, trypsin-enhanced infectivity, endogenous transcription and replication. These studies underscore contrasting features andthemes between rotavirus and other dsRNA viruses. 2003 Published by Elsevier B.V.

Keywords: Rotavirus; dsRNA virus; Cryo-EM; Genome organization; Transcription; Replication

1. Introduction

Rotavirus, a member of the family Reoviridae is a non-enveloped, icosahedral, double-stranded RNA (dsRNA)virus (reviewed inLawton et al., 2000). The rotaviruscapsid, like all virus capsids is structured to protect itsgenome and deliver it successfully into a suitable host cell,in which the genome is replicated and the virus particlemakes copies of itself. Several recent structural and bio-chemical studies have provided crucial insights into howthe different proteins encoded by the virus enable it to(1) transcribe its genome from within the connes of thecapsid, (2) control the translation of the host genes to en-hance translation of the rotaviral genes in the cytoplasmof the infected host cell, (3) enable genome replicationwherein the negative strand is synthesized using the tran-scribed mRNA as a template, and nally (4) packagethe replicated dsRNA genome and assemble the capsid

Corresponding author. Tel.:+ 1-713-798-5686;fax: + 1-713-798-1625.

E-mail address: [email protected] (B.V.V. Prasad).

layers yield the complete virus particles (Estes, 2001).From such studies on rotavirus and other dsRNA virusesincluding other members of the family Reoviridae, common themes are emerging regarding the role of the viralcapsid and non-structural proteins during the virus lifecycle.

The dsRNA viruses, classied into ve major groups,range from the relatively simple viruses with a singledsRNA segment like in the members of the Totiviridae family to more complex viruses in the Reoviridae family, whichcontain 1012 dsRNA segments (Lawton et al., 2000; vanRegenmortel et al., 2000). Other families of dsRNA viruseslike Birnaviridae and Cystoviridae contain two and threedsRNA segments, respectively. Accordingly, there is a con-siderable variation in the complexity of the capsid organization in dsRNA viruses. In the members of Totiviridae anBirnaviridae, a single-layer capsid encloses the genomewhereas viruses in Cystoviridae contain a double-layeredcapsid. In some genera of Reoviridae such asRotaviru s,Orbiviru s, and Reoviru s, viruses exhibit distinct triple-layered capsid architecture. In all the well-characterizeddsRNA viruses, the capsid structure is based on icosahedra

0168-1702/$ see front matter 2003 Published by Elsevier B.V.doi:10.1016/j.virusres.2003.12.007


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68 H. Jayaram et al. / Virus Research 101 (2004) 6781

symmetry. With the exception of cystoviruses (Mindich,1988), which use bacteria as hosts, the dsRNA viruses arenon-enveloped.

The dsRNA genome in rotavirus and other dsRNA virusespresents a unique set of conditions for their survival. Be-cause the host cells do not possess the enzymatic machinery

to convert dsRNA into a translatable mRNA molecule,these viruses have to provide for a mechanism to synthesizemRNA from their genomic dsRNA. Both the transcriptionand the subsequent synthesis of progeny dsRNA have tobe carried out in conned environments not only to avoiddegradation of the genome by cellular nucleases but also toprevent unfavorable antiviral responses in the host cells thatcould be triggered by increased concentrations of dsRNA.All the dsRNA viruses encode their own enzymes necessaryfor transcription and are capable of endogenously transcrib-ing their genomes. Together with this common attribute of endogenous transcription, the capsid architecture in theseviruses also has to account for host-specic interactions.The dsRNA viruses are ubiquitous in nature and infect hoststhat range from bacteria and fungi to species throughoutthe plant and animal kingdoms (van Regenmortel et al.,2000). An interesting question is: how does capsid archi-tecture in these viruses efciently integrate the requirementof host-specic cell entry with the common requirement of endogenous transcription?

Structures of several dsRNA viruses including L-A virusof yeast origin from Tottiviridae family (Caston et al., 1997),infectious bursal disease virus from Birnaviridae (Bottcheret al., 1997), 6 from Cystoviridae (Butcher et al., 1997,2001), and several members of the Reoviridae representing

various genera including rotavirus (Prasad and Estes, 2000;Tihovaet al., 2001), bluetongue virus (BTV) in theOrbivirus(Grimes et al., 1997; Prasad et al., 1992), orthoreovirus(Drydenetal., 1993, 1998), aquareovirus(Nason et al., 2000;Shaw et al., 1996), rice dwarf virus (RDV) in thePhytore-ovirus genera (Zhou et al., 2001), and cypovirus (Hill et al.,1999; Xia et al., 2003), have been analyzed by electron cry-omicroscopy (cryo-EM) techniques. X-ray structures of L-Avirus (Naitow et al., 2002), and transcriptionally competentcores of BTV (Grimes et al., 1998), orthoreovirus (Reinischet al., 2000), and RDV (Naitow et al., 2002), have been de-termined to near 3 resolution. In addition to the structuresof viral capsids, in some cases, individual viral proteins havebeen determined by X-ray crystallography (Butcher et al.,2001; Deo et al., 2002; Dormitzer et al., 2002; Jayaram et al.,2002; Liemann et al., 2002; Mathieu et al., 2001; Tao et al.,2002). Together these structural studies have begun to pro-vide a detailed description of the capsid organization andmolecular insights into themechanisms of various functionalactivities of these dsRNA viruses. This review primarily fo-cuses on the recent structural ndings in rotavirus, and inrelation to other dsRNA viruses, attempts to summarize thisinformation to underscore unifying principles in capsid ar-chitecture, genome organization, endogenous transcriptionand replication.

2. Rotavirus capsid structure and function

Rotavirus is the major causative agent of infantile diarhea accounting for nearly one million deaths annually the world (Cohen, 2001). This fact has led to the virus be-ing extensively studied to understand its pathogenicity a

discover ways to treat and manage rotavirus infections. Trotavirus is a relatively large,1000 in diameter, icosa-hedral virus. Its capsid encloses 11 segments of dsRN(Fig. 1A), each segment codes for one protein with the exception of segment 11, which codes for 2 proteins (reviewin (Estes, 2001). Of these 12 proteins, 6 are structural (VPsand 6 are non-structural (NSPs). The rotavirus capsid composed of three concentric protein layers that enclothe genome (Fig. 1B and C; reviewed inPrasad and Estes,2000). The complete virions are called triple-layered parcles (TLPs,Fig. 1B), particles that lack the outer layer arecalled the double-layered particles (DLPs,Fig. 1E) and arenon-infectious, and particles that lack the outer two layeare called the single-layered particles (SLPs,Fig. 1D).

2.1. The outer capsid layer and cell entry

The viruses in Reoviridae infect a wide variety of hosRotaviruses infect thecells of theintestinal epithelium, whisome viruses of the genusOrthoreovirus spread to the cen-tral nervous system and viruses among the Orbiviruses fect both insect and mammalian hosts. The outermost layin Reoviridae are implicated in cell attachment, membrapenetration and cell entry. In keeping with the wide horange, the outer capsid layers among the Reoviridae sho

a remarkable range of diversity in terms of composition proteins, their organization, their associated activities, athe mechanisms of cell entry.

Cryo-EM reconstructions of rotavirus (Prasad et al.,1988; Prasad and Estes, 2000; Tihova et al., 2001; Yeaget al., 1990), orthoreovirus (Dryden et al., 1993, 1998),BTV (Grimes et al., 1997; Hewat et al., 1992; Schoehet al., 1997), rice dwarf virus (Lu et al., 1998) and cy-povirus (Hill et al., 1999; Xia et al., 2003), have providedstructural insights into the organization of this outermocapsid layer. Accordingly, members of the Reoviridae cabe classied into two types, those that have a compleT = 13 outer layer or the non-turreted viruses and thothat have an incompleteT = 13 layer where the normalT = 13 arrangement is interrupted by the presence of turret-like structure at the ve-fold axes of symmetry.

In rotavirus, the outermost layer is made up of 780 copof VP7 (38kDa) a glycoprotein, and 120 copies of thspike protein VP4 (88kDa) (Prasad et al., 1990) (Fig. 1B).Cryo-EM studies indicate that the 780 copies of VP7 are ranged as 260 trimers that are located at the local and strthree-fold axes of aT = 13 (left-handed) icosahedral lattice.A distinctive feature of the rotavirus structure is the preence of 132 aqueous channels,140 deep, spanning theouter two capsid layers, at all the ve- and six-coordinat


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H. Jayaram et al. / Virus Research 101 (2004) 6781 69

Fig. 1. Architectural features of rotavirus. (A) PAGE gel showing, 11 dsRNA segments comprising the rotavirus genome. The gene segments aron the left and the proteins they encode are indicated on the right. (B) Cryo-EM reconstruction of the rotavirus triple-layered particle. The spVP4 is colored in orange and the outermost VP7 layer in yellow. (C) A cutaway view of the rotavirus TLP showing the inner VP6 (blue) and layers and the transcriptional enzymes (shown in red) anchored to the VP2 layer at the ve-fold axes. (D) Schematic depiction of genome oin rotavirus. The genome segments are represented as inverted conical spirals surrounding the transcription enzymes (shown as red balls) insilayer in green. (E and F) Model from Cryo-EM reconstruction of transcribing DLPs. The endogenous transcription results in the simultaneouthe transcribed mRNA from channels located at the ve-fold vertex of the icosahedral DLP.

positions of theT = 13 lattice. Inserted into the channels(type II channels) that surround the 12 icosahedral ve-foldvertices are the 60 bilobed spikes, each 120 long. Themolecular identity of these spikes as dimers of VP4 wasshown by cryo-EM reconstructions of rotavirus complexedwith anti-VP4 antibodies (Prasad et al., 1990; Tihova et al.,2001).Subsequent cryo-EM studies discovered that VP4 hasa large globular domain that is buried inside the inner layer,making the total length of the spike 200 (Shawet al., 1993;Yeager et al., 1994).

2.1.1. VP4Although earlier studies implicated VP7 in the cell entry

process (Fukuhara et al., 1988; Sabara et al., 1985), sub-sequent studies have increasingly indicated that VP4 is themajor player in this process. VP4 is implicated not only incell attachment and cell penetration but also in hemagglu-tination, neutralization and virulence (Estes, 2001). VP4 issusceptible to proteolysis. Proteolytic cleavage of VP4 en-hances viral infectivity by several fold (Arias et al., 1996;Estes et al., 1981) and facilitates virus entry into cells(Kaljot et al., 1988). During proteolysis, VP4 (88kDa) iscleaved into VP8(28kDa, aa 1247) and VP5(60kDa,aa 248776) and the cleavage products remain associated inthe virion (Fiore et al., 1991). Trypsinized viruses enter cells

more readily and rapidly than those particles that are notrypsinized (Kaljot et al., 1988; Keljo et al., 1988). In vitroexperiments have shown that proteolytically activated particles, aswell as recombinant VP5, possess lipophilic activity(Dowling et al., 2000; Nandi et al., 1992; Ruiz et al., 1994).It is interesting to note here that VP4 contains a putativefusion domain similar to that seen in the enveloped virusesuch as alphaviruses and inuenza viruses. Proteolysis thuconstitutes a key process in the efcient internalization orotaviruses into cells. This is particularly relevant considering that rotavirus replication takes place in enterocytes inthe small intestine, an environment rich in proteases.

The molecular mechanisms of increased infectivity byproteolysis and its role in the membrane penetration are nowell understood. Recent studies comparing the biochemicaand structural properties of rotavirus grown in the presencand absence of trypsin have provided some exciting andnovel insights into this process, and indicated that trypsincleavage stabilizes the spike assembly and confers icosahedral ordering (Crawford et al., 2001). These results are con-sistent with biochemical studies on recombinant VP4, whichshow that proteolysis of monomeric VP4 yields dimericVP5 (Dormitzer et al., 2001). Based on these studies it ap-pears that that trypsin cleavage has an intracellular role inensuring the correct conformation and proper assembly o


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the spikes. Further structural and biochemical studies arerequired to provide a better understanding of how trypsinaffects intracellular spike assembly.

Several recent studies have shown that rotavirus cell entryis a multi-step process involving sialic acid (SA)-containingreceptors in the initial cell attachment step and integrins such

as v 3, 4 1, 2 1 during thesubsequent post-attachmentsteps (Coulson et al., 1997; Guerrero et al., 2000; Hewishet al., 2000; Zarate et al., 2000). In this process, the VP8domain is involved in the interactions with SA, whereasVP5 is implicated in interactions with integrins. Involve-mentof SA during rotavirus infections isnot anessential stepfor all rotavirus strains. In the majority of rotavirus strains,including human rotaviruses, cell entry is SA independent(Ciarlet et al., 2001). In these viruses, the majority of neu-tralizing monoclonal antibodies that recognize VP4 selectmutations in VP5, suggesting that cell entry is mediatedmainly by the VP5(Kirkwood et al., 1996; Kobayashi et al.,1990; Padilla-Noriega et al., 1995). Studies with polarizedepithelial cell lines show that with SA-dependent viruses,the viral entry is restricted to the apical membrane, whereasSA-independent viruses enter either apically or basolater-ally thus further documenting variations in the entry mech-anisms between SA-independent and SA-dependent viruses(Ciarlet et al., 2001).

Recently, X-ray structure of a portion (aa 64242) of the sialic acid binding domain of VP8was determinedand this structure strongly suggests that the distal globularheads of the spikes are made of VP8(Dormitzer et al.,2002). Interestingly, the sialic acid binding domain exhibitsa -sandwich fold similar to that seen in galectins, a family

of sugar binding proteins, despite a lack of sequence similar-ity. Analysis of the crystal structure with bound sialic acidrevealed that although the galectin binding site is blockedin VP8, the protein binds sialic acid in a shallow grooveon its surface using residues that are conserved in sialicacid-dependent rotavirus strains. A comparison of this struc-ture with that of the apo-form of VP8, determined by NMR,suggests that the ligand binds by inducing a slight conforma-tional change in the residues involved in binding. The VP8structure further represents one of the rst observed casesof a protein in rotavirus and the dsRNA viruses (other thanthe polymerase) taking on a fold seen among host proteinsand unknown thus far among viral proteins whose struc-tures have been determined. Based on this structural result,it is proposed that VP4 arose from the insertion of a hostcarbohydrate-binding domain into a viral membrane inter-action protein (Dormitzer et al., 2002).

The proposed sequential interactions with various lig-ands in the multi-step rotavirus entry processes may in-volve a series of conformational changes. For instance, inSA-dependent strains of rotavirus, binding of sialic acid mayinduce conformational changes in VP8to enable VP4 tobind to the integrins more efciently. In the SA-independentstrains VP4 may already be in a conformation that is suit-able for directly interacting with the upstream receptors. In

the multi-step entry process, the conformational adaptabiland exibility of VP4 may play an important role. Studon rotaviruses grown in the absence and presence of tryptogether with the observation that VP4 can undergo larpH-inducedconformational changes are consistentwith suca notion (Crawford et al., 2001; Pesavento et al., 2001). A

detailed picture will emerge once more intermediates in trotavirus entry process are characterized structurally.

2.1.2. VP7 The precise role of VP7 during early interactions of t

virus with the cell is not clear, but its has been postulated tVP7 may modulate the function of VP4 during the attacment and entry process (Beisner et al., 1998; Mendez et al.,1996), and may interact with cell surface molecules after tinteraction is initiated by VP4 (Mendez et al., 1999). One of the rst events upon cell entry is the loss of this outer layto expose the transcriptionally active DLP to the cytoplasBiochemical studies indicate that the in vivo decapsidatican be mimicked by treating TLPs with calcium chelatolike EDTA (Cohen et al., 1979). VP7 binds calcium andthe sensitivity of virions to low calcium concentrationsstrain-dependent (Cohen et al., 1979; Gajardo et al., 1997;Ruiz et al., 1996). Several studies also have suggestedcalcium-driven conformational changes in VP7 (Dormitzerand Greenberg, 1992). Studies on baculovirus-expressedrecombinant VP7 have shown a requirement for calciuin the formation of VP7 trimers, which crystallize inhexagonal plates mimicking the arrangement of VP7 othe capsid (Dormitzer et al., 2000). Thus, while appropriatelevels of calcium help maintain the structural integrity

the VP7 layer, low calcium concentrations, similar to thoin the cytoplasm, trigger the disassociation of VP7 trimleading to uncoating of the VP7 layer. Uncoating of thouter layer resulting in transcriptionally competent DLPsa necessary event in the replication cycle of rotavirus. Soantibodies directed against VP7 neutralize the virus by ihibiting the decapsidation of the TLP (Ludert et al., 2002).The effect of these neutralizing antibodies is not overcomby lipofecting virusantibody complexes into cells. In cotrast, some antibodies directed against VP4 neutralize binhibiting the binding of virus to the cell, an effect thcan be overcome by lipofecting these complexes into celIt is possible that neutralizing anti-VP7 antibodies preveVP7 from undergoing necessary conformational changthat facilitate uncoating of the VP7 layer. Thus, VP7 is tkey mediator of the calcium driven uncoating in rotavirand the function of the outer layer composed of VP4 aVP7 is to ensure that the transcriptionally active DLPs adelivered to the host cytoplasm.

2.2. The intermediate layer and endogenous genometranscription

The DLP resultingfrom the removal of the outer layer proteins, VP4 and VP7, is the transcriptionally competent fo


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of rotavirus capable of transcribing the enclosed dsRNA seg-ments within its connes (Fig. 1E). The DLP is composedof the remaining four structural proteins encoded by the ro-tavirus genome of which VP6 is the major constituent. Inthe DLP, VP6 forms the outer most capsid layer with 260trimers of VP6 assembled on aT = 13 icosahedral lattice.

In the context of the rotavirus TLP, the VP6 layer, hence-forth referred to as the intermediate layer, is sandwichedbetween the outerT = 13 layer formed by VP7 and VP4,and the innerT = 1 capsid layer formed by VP2. In the ro-tavirus structure, the VP6 trimers lie below the VP7 trimersso that the aqueous channels in the twoT = 13 layers arein register. In the overall organization of the rotavirus, VP6appears to integrate the two principal functions of the virus,cell entry and endogenous transcription, through its interac-tions with the outer layer proteins VP7 and VP4, and theinner layer protein VP2.

2.2.1. Pseudo-atomic model of the VP6 layer Although an X-ray structure of the DLP is yet to be de-

termined, docking of the X-ray structure of VP6 into thecryo-EM density of the DLP has resulted in a pseudo-atomicmodel of theT = 13 VP6 layer (Mathieu et al., 2001). VP6has two domains, the distal domain with eight-strandedanti-parallel -sandwich fold makes contact with the VP7layer, and the lower domain consisting of a cluster of

-helices makes contact with the inner VP2 layer (Fig. 2).VP6 trimers interact laterally to form theT = 13 layer andthere appear to be at least two types of contacts between thetrimers. The contacts, across the quasi two-fold axes, closerto the icosahedral three-fold axes, are similar, whereas as

the trimers approach the icosahedral ve-fold axis, the con-tacts are varied. In contrast to VP7 of BTV, a structural

Fig. 2. Structure of VP6 trimer. Structure of VP6 trimers showing the upper domain with its seven-strand jelly role topology and the predhelical lower domain (Mathieu et al., 2001). The structure of the upper domain is analogous to the domain in BTV VP7 (Grimes et al., 1995, 1998).The docking of the VP6 trimers into electron-density from cryo-EM reconstructions has allowed the construction of a pseudo-atomic model fsurface of the DLP in rotavirus.

equivalent of VP6 in rotavirus, the VP6 trimer exhibitsextensive lateral interactions involving charged residuesThus, modeling has revealed that all the interacting sur-faces (VP6VP4, VP6VP7, and VP6VP2) contain themost conserved residues of VP6. Although the base of theVP6 layer displays an overall negative surface electrostatic

potential, the VP6 interactions with VP2 are predominantlyhydrophobic. In contrast the interactions with VP7 and VPappear to involve several charged residues. In solution, VPforms highly stable trimers. The VP6 trimer has a boundzinc ion that is shown to be necessary for the stability othe trimer. VP6 trimers easily form 2-D crystalline arraysand helical tubes with pseudo-hexagonal packing, but rarelyicosahedral shells that are similar to theT = 13 VP6 layer inthe rotavirus structure. The lateral interactions between VPtrimers do not have all the information to form the closedshell. In contrast, VP2 has the ability to form native-likeicosahedral shells of appropriate size. Therefore, it is likelythat the VP2 layer provides a proper scaffold for the assembly of VP6 trimers into aT = 13 icosahedral organization.

2.2.2. Role of VP6 in endogenous transcriptionEarlier biochemical studies clearly indicated that none of

the components of the DLP alone is capable of transcribinthe dsRNA and that VP6, despite lack of any enzymaticfunctions, is essential for endogenous transcription of thegenome. Based on cryo-EM studies of DLPs,Prasad et al.(1988)were the rst to propose that channels in the VP6layer could be used for mRNA exit. More recent cryo-EMstudies on the actively transcribing DLPs, have shown thaof the three types of the channels in theT = 13 VP6 layer,

the nascent mRNA transcripts exit specically through thetype I channels located at the ve-fold axes (Fig. 1E and F)


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(Lawton et al., 2000). These studies also conrmed thatDLPs maintain their structural integrityduring theprocess of transcription. In the pseudo-atomic model of the VP6 layer,a -hairpin motif of VP6 with a highly conserved sequencethat protrudes into the mRNA exit channel may play afunctional role in the translocation of the nascent transcripts

during endogenous transcription. The net concentration of negative charges on the walls lining the type I channel mayfacilitate the extrusion of the mRNA transcript by increasingits uidity because of the electrostatic repulsion betweenthe interior surface of the channel and the mRNA transcript.

A detailed mutational analysis based on thepseudo-atomicmodel of the VP6 layer has helped to elucidate the deter-minants of VP6 required for assembly on VP2, and howVP6 may affect endogenous transcription (Charpilienneet al., 2002). Thirteen site-specic substitution mutationsof amino acids residues that directly contact the VP2 layer,as identied in the pseudoatomic model, have been stud-ied in terms of their ability to trimerize, to form virus-likeparticles when co-expressed with VP2, and to assembleonto wild-type cores and form in vitro reconstituted DLPsthat could transcribe the enclosed genomes and producerotaviral mRNA. In cases where defects were observed, theamino acids essential for recovery of transcription or as-sembly were identied. All the VP6 mutants formed stabletrimers and self assembled into tubular structures similar towild-type VP6. This is consistent with the observation fromthe X-ray structure of VP6 that the lower-helical domainof VP6 does not contribute to either the trimeric interactionsor the lateral interactions in the assembly of the tubularstructures. Of the 13 VP6 mutants examined, 3 were unable

to assemble with VP2 and 3 others partially assembled.These mutants either did not rescue the transcriptase activ-ity or did so only marginally substantiating further that theproper assembly of VP6 trimers on VP2 is an absolute re-quirement for endogenous transcription. Four other mutantsthat generally preserved the hydrophobic character of thecontact region, assembled and transcribed well, emphasiz-ing the importance of hydrophobic interactions in the properassembly and stability of VP6 on VP2. An interesting resultwas obtained with three of the mutants, which had an extracharge introduced at the VP6VP2 interface. These mutantsassembled well on cores, but surprisingly did not rescuethe transcriptase activity in the reconstituted DLPs. Thisresult prompted the investigators to favor a hypothesis inwhich transcript extrusion during endogenous transcriptionrequires VP6 to undergo subtle conformational changes andthe extra charge in the mutants inhibits such changes.

The idea of conformational changes in VP6 associatedwith endogenous transcription is also suggested by thecryo-EM structural studies on DLP-anti (VP6) MAb com-plexes.Lawton et al. (1999)in their studies showed thatcertain antibodies inhibit transcription as does the presenceof the outermost VP7 layer, which renders the TLP tran-scriptionally inactive. Biochemical analysis of TLP, andDLPantibody complexes in which the antibody inhibited

transcription, revealed that these particles are able to iniate transcription and synthesize capped transcripts of to seven nucleotides in length, thereby indicating that tenzymatic activities of the DLP are not affected by bindiof VP7 or antibody. The short length of transcripts indicathat elongation is not inhibited by any steric narrowing

restriction caused by antibody-binding, or analogously Vbinding, but instead can be attributed to the subtle conformtional changes near the VP6VP2 interface observed constently in the structures of transcriptionally inactive TLP aDLPantibody complexes. A similar cryo-EM study usia different set of antibodies with contrasting effects on trascription however concluded that the inhibitory effect of oof the two antibodies studied may be due to rigidicationthe VP6 trimers upon antibody binding (Thouvenin et al.,2001). Although no conformational changes in VP6 wieither of the antibodies were seen, there were major diffences in the extent of interaction between VP6 and theantibodies. With the antibody that inhibited transcriptiove loops from two of the VP6 subunits in the trimer winvolved in the antibody binding, whereas with the othantibody, which did not inhibit transcription, only one Vmonomer was involved. From theseobservations,Thouveninet al. (2001)proposed that the inhibitory effect of one of thantibodies is caused by preventing conformational changin theVP6layer required for transcription. Although theprposed mechanisms of how a VP6-specic antibody inhibtranscription by these two studies differ, both the studiclearly indicate that the dynamics of the interaction betwethe VP6 and VP2 layers are important for transcription.

The exit of transcripts through the channels at th

ve-fold axes appears to be an emerging common themedsRNA viruses. In orthoreovirus, both conventional EM acryo-EM studies have shown the transcripts exit throuthe turrets at the ve-fold axes. In BTV cores, X-ray crytallographic analysis of core crystals soaked with variosubstrates and products of the transcription reaction hshown that the mRNA exit channel in the BTV core likely to be at the ve-fold vertex (Diprose et al., 2001).In addition to providing conduits for the exit of transcripanother important function of the VP7 layer (equivalent VP6 layer in rotavirus), that became evident from thestudies, is that certain regions surrounding the ve-fold axin this layer function as substrate sinks for the transcriptireaction. Although the dynamic events behind endogenotranscription are still unclear, the structural and biochemical studies on several dsRNA viruses have brought infocus the elegance of capsid organization in these virusand reveal the fact that capsid architecture and genome oganization are coordinated to ensure multiple and repeatrounds of transcription.

2.3. The innermost layer

The core of the rotavirus consists of the remaining thrstructural proteins VP1, VP2, and VP3 (Fig. 1C and D). Of


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these three proteins, VP2 is the most abundant and forms theinnermost layer interacting with the VP6 layer on the out-side and the genomic RNA on the inside. The other two pro-teins VP1, an RNA-dependent RNA polymerase (Valenzuelaet al., 1991), and VP3, guanylyl and methyl transferase(Chen et al., 1999; Liu et al., 1992), are present in small

quantities and provide the enzymatic functions required forproducing thecappedmRNA transcripts. Of all thestructuralproteinsof rotavirus, VP2 is the only protein thathas the abil-ity to self-assemble into a native-like icosahedral structure,when expressed in insect cells. This observation stronglyindicates that VP2 possesses the determinants required todirect the proper assembly of other rotavirus proteins.

2.3.1. Unique organization of the inner most capsid layer The VP2 layer consists of 120 molecules organized as 60

dimers on aT = 1 icosahedral lattice. Such an icosahedralorganization with 120 subunits is quite unique and is foundonly in dsRNA viruses. This is the most conserved featurein all structurally characterized members of the Reoviridaeand other dsRNA viruses such as6, a bacterial virus andL-A virus of yeast origin. The atomic level description of such an organization with two subunits in the icosahedralasymmetric units, and hence called aT = 2 icosahedralstructure, is provided by the X-ray crystallographic analy-ses of BTV and orthoreovirus cores (Grimes et al., 1998;Reinisch, 2002; Reinisch et al., 2000). In these structures,one of the two subunits in the icosahedral asymmetric unitpoints toward the icosahedral ve-fold axis and the other isslightly offset from the ve-fold axis. Each subunit has threedomains with the N-terminal residues facing inward toward

the virus genome. Similar subunit structure and organiza-tion is also seen in the high-resolution cryo-EM structuresof RDV and CPV and is likely to be replicated in rotavirusas indicated by the cryo-EM analysis of the recombinantVP2/6 particles with full-length VP2 and a mutant VP2 withN-terminal residues deleted. VP2 exhibits RNA-bindingability through its N-terminal residues. Consistent with thisobservation, several points of contact between the VP2 layerand RNA, presumably through the N-terminal residues of VP2, are observed in the cryo-EM structure of the DLP.Similarly, interactions between VP3 and RNA are seen theX-ray structure of the BTV core. Thus, one of the principalfunctions of the innermost layer in these viruses may beto direct the structural organization of the genome that isconducive for its endogenous transcription.

2.3.2. Transcription enzyme complexWhere are the transcription enzymes located? Using a

comparative cryo-EM analysis of DLP and the recombinantvirus-like particles with and without VP1 and VP3,Prasadet al. (1996)were the rst to show that these minor proteinswere incorporated as a heterodimer anchored to the insidesurface of the VP2 layer at each of the 12 ve-fold ver-tices. A similar structural organization of the transcriptionenzymes anchored to the inner surface of theT = 2 layer

subsequently has been seen in aquareovirus, orthoreovirusBTV, RDV, cypovirus, and6 virus. However, one prin-cipal difference between rotavirus, BTV, or orthoreovirus,and aquareovirus or cypovirus, is that in the latter groupof viruses the capping enzyme is external to the innermoslayer forming a distinct turret-like structure at the ve-fold

axis, although the polymerase is internal. The location of thtranscription enzyme complex at the ve-fold axis is consistent with the nascent transcripts exiting through the channels at the ve-fold axes. In rotavirus, biochemical studieon recombinant VLPs containing VP2 with amino-terminadeletions co-expressed with VP6, VP1 and VP3 indicate tharemoval of 25 N-terminal residues of VP2 completely prevent the incorporation of VP1 and VP3. Thus, in addition tRNA-bindingactivity, theN-terminal residues of VP2are in-volved in anchoring VP1 and VP3. The N-terminal residueof the one of the two subunits in the icosahedral asymmetriunit of the T = 2 that is close to the ve-fold axis may beinvolved in anchoring the transcription enzyme, whereas thN-terminal residues of the other subunit, which is slightlyoffset from the ve-fold axis may involved in the interactions with the underlying genomic RNA. It is possible thathe unique organization of the innermost layer in the dsRNAviruses has evolved to serve the dual purpose of properly positioning the transcription enzyme complex and organizingthe genome to facilitate endogenous transcription.

Although the locations of the transcription enzymes canbe inferred from the structures of the intact virions, the precise molecular structures of these enzymes inside thevirions,with the exception of the capping enzyme in the turretteddsRNA viruses such as orthoreovirus, are not resolved a

they are present in non-icosahedral amounts. Thereforestructural understanding of how these enzymes functionhas to come from the studies on individual componentsRecently, X-ray crystallographic structures of the6 andorthoreovirus polymerases have been determined (Butcheret al., 2001; Tao et al., 2002). These studies have providedsignicant mechanistic insights into how these moleculesfunction both in the transcription and replication processesBoth the structures have a canonical ngerpalmthumbcore, as seen in several other polymeases, surrounded byN- and C-terminal elaborations. These elaborations differsignicantly between the two structures perhaps reect-ing the differences in the transcription strategies in theseviruses. In 6 and birnaviruses, which contain three andtwo dsRNA segments, respectively, the transcription issemi-conservative. In the members of Reoviridae, whichcontain 1012 dsRNA segments, the transcription is conservative. Generally, the polymerase molecules in the memberof Reoviridae are larger than their counterparts in otherdsRNA viruses. The structure of the reovirus polymeraseperhaps a better representative of the rotavirus polymeraseshows a cage-like structure with four channels leading tothe central catalytic core (Tao et al., 2002). Further X-raycrystallographic analyses with appropriate ligands have assigned these channels to specic functions such as substrat


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and template entry, mRNA or dsRNA exit depending uponthe polymerase involvement during transcription or replica-tion (Tao et al., 2002). Interestingly, these structural studiesalso revealed a binding site for the 5cap structure seen inreovirus and all the members of the Reoviridae, suggestinga potential mechanism for tethering of the non-template

strand during transcription which helps localize the tem-plate strand and allows for repeated cycling of the templateduring the continuous endogenous transcription. One pri-mary difference between the rotavirus and orthoreovirus isthe location of the capping enzyme. In contrast to orthore-ovirus, in which the capping enzyme is located on the outersurface of the innermost layer, in rotavirus, biochemicaland structural studies have shown that the capping enzymeis in close proximity to the polymerase (Lawton et al.,1999; Prasad et al., 1996). It remains to be seen how thepolymerase in rotavirus accommodates interaction with thecapping enzyme.

3. Structural organization of the genome

Understanding the structural organization of the genomeparticularly in dsRNA viruses assumes a greater impor-tance because of the intimate involvement of the genomein the various enzymatic activities within the connes of the capsid. From all the available biochemical and struc-tural data to date on several members of the Reoviridae,including cypovirus, orthoreovirus, BTV and rotavirus, theemerging consensus is that an independently functioningtranscription enzyme complex, anchored to the inside sur-

face of the innermost capsid layer, transcribes each genomesegment, and that all the genome segments are transcribedsimultaneously (Banerjee and Shatkin, 1970; Bartlett et al.,1974; Gillies et al., 1971; Skehel and Joklik, 1969; Smithand Furuichi, 1982). In vitro studies indicate that the tran-scriptionally competent particles of these viruses are highlyefcient molecular machines capable of repeated cyclesof transcription. During each cycle of transcription, thedsRNA segment, which has to move around the anchoredpolymerase, must be unwound, separated, rejoined, and re-wound for further cycles of transcription. The transcriptionprocess appears to be impressively fast. In orthoreoviruses,it is estimated that transcription proceeds at a rate of 50nucleotides/s. The structural organization of the genome inthese viruses thus must allow for what seems to be a wellorchestrated dynamic process of repeated, simultaneous,high speed transcription of multiple segments.

3.1. Manifestation of genomic RNA inicosahedrally-averaged structures

Although the precise organization of the genome in themembers of Reoviridae remains to be elucidated, recentstructural studies on some of these viruses have provideduseful insights. The rst visualization of the structural or-

ganization of the genome in a dsRNA virus was providby the cryo-EM analysis of rotavirus (Prasad et al., 1996).These studies indicated that the viral dsRNA forms a ddecahedral structure in which the RNA double heliceinteracting closely with the VP2 layer, are packed arouthe transcription complexes located at the icosahedral v

tices. It was suggested that VP2, which is icosahedralordered, with its RNA-binding property is responsible fthe icosahedral ordering of the closely interacting portioof the RNA, and this ordering is diminished at lower radSubsequent X-ray crystallographic studies on BTV (Gouetet al., 1999) and orthoreovirus cores (Reinisch et al., 2000)and cryo-EM studies on RDV, CPV, aquareovirus, have aconsistently shown that a signicant portion of the genomis statistically ordered and manifests as concentric layeof density in the icosahedrally averaged structures of theviruses. These concentric layers of RNA are generalseparated by 2830 . Assuming a local hexagonal pacing of the RNA helices, such separation translates into inter-strand spacing of 3032. The observations in theicosahedrally-averaged structures are consistent with othstudies. A similar inter-strand spacing was deduced froearlier low angle X-ray scattering studies on orthoreovirwhich also suggested that the dsRNA genome is tightpacked as parallel helices in a semicrystalline array (Harveyet al., 1981). Considering the volume available for thgenome inside the capsid layers and the molecular weigof the genome in these viruses, typically the concentratiof the RNA inside the capsid layer is around 400mg/m(Gouet et al., 1999). At such concentrations, dsDNA isknown to exhibit a columnar hexagonal liquid crystalli

packing with an inter-strand spacing of about3032(Livolant et al., 1989), suggesting thereby the packageddsRNA in these viruses behaves like dsDNA. How are tcharges on the condensed dsRNA neutralized? Generalcounter ions like Mg2+ , or organic polyamines like sper-mines or spermidines are involved in nucleic acid charneutralization in viruses. However,Gouet et al. (1999),found that the levels of metal counter ions in BTV coremeasured using a scanning proton microprobe, are too loto be implicated in charge neutralization, and instead hasuggested that the charge neutralization may be througorganic polyamines in this virus.

3.2. Reversible condensation and expansion of therotavirus genome

Recent cryo-EM analysis of rotavirus examined undvarious chemical conditions unraveled a remarkable abilof the rotavirus genome to undergo reversible condensatiand expansion within the capsid interior (Pesavento et al.,2001) (Fig. 3). These studies have provided further insightinto the structural organization of the genome and the nture of interactions between the genome and the internproteins. At high pH in the presence of ammonium ions, genome condenses to a radius of 180 from the origin


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Fig. 3. Reversible condensation and expansion of the rotavirus genome. A cartoon representation of a plausible structural organization of thenormal physiological condition (left) and in the ammonium high pH condition (right). Such a model from several biochemical and structural stuGouetet al., 1999) offers a simple mechanistic explanation of the observed genome condensation in rotavirus (Pesavento et al., 2001). The condensation from aoriginal radius of 220180 is achieved by simply reducing the inter-strand spacing in the spiral from 31 (indicated by pair of arrows in bas observed in the normal physiological conditions, to a spacing of 25 (pair of arrows bottom right) as observed in the ammonium high p

radius of 220 , and when brought back to physiological pHthe genome expands to its original radius. The genome re-mains transcriptionally viable after returning to its originalstate, thereby indicating that this transformation is merelystructural and the dsRNA is not covalently disrupted bythe ammonium high pH treatment. Apart from VP4, whichis irreversibly altered, the rest of the capsid remains intact

under these conditions. These studies illustrate the remark-able stability of the capsid and resilience of the genome;these attributes may be required to carry out the continuoustranscription of multiple segments within the capsid.

Several observations made in this study have direct rel-evance to the roles of VP2, and the transcription enzymecomplexes in the structural organization of the genome. Theobservation that the condensation of the genome is isometricand concentric with respect to the particle center, suggeststhat stronginteractionsbetween thegenomeandthesymmet-rically disposed transcriptional complexes exist, and suchinteractions may play a critical role in the structural organi-zation of the genome. In the condensed state, the reconstruc-tions show strong density linking the RNA core with the in-side surface of the VP2 layer at all the ve-fold vertices, likespokes linking a hub and wheel. Without such symmetricallydisposed interactions, the condensed RNA core once disas-sociated from theVP2layer would nothavebeen constrainedto remain at the particle center. The isometric condensationalso supports the notion that each dsRNA segment may beassociated with a transcription complex and all the segmentsare similarly affected by the ammonium high pH treatment.

The studies byPesavento et al. (2001)also underscorethe importance of VP2 in the structural organization of thegenome. These studies suggest that the VP2, through its

RNA-binding property, plays an important role in maintaining the appropriate spacing between the RNA strands in thnative expanded state. In the native state the genome exhibitseveral points of contact with the VP2 layer. As the genomeproceeds to the condensed state, these contacts are brokenonly to be reestablished when the physiological conditionsare restored. The observed condensation is a synergetic ef

fect of hydroxyl and ammonium ions, as it is not observedat high pH alone, or with increased concentrations of NH+or Mg2+ ions at physiological pH. The two inferences thatcan be made from these observations are that VP2RNA interactions are pH-dependent and that the negative chargeson genomic RNA in the native state may only be partiallyneutralized. The effect of high pH is to disrupt RNAVP2interactions, possibly due to deprotonation of critical VP2residues, and cause conformational changes in the RNA toallow further charge neutralization by ammonium ions tocause condensation. The partial neutralization of the ge-nomic RNA in the native state may be necessary to maintaiappropriate inter-strand spacing to allow the genomic RNAto move around the transcription complex during transcription. In the condensed state because of the reduced volumeand consequent increase in the concentration of RNA, theinter-strand spacing is reduced from 30 to 25 .

3.3. A model for structural organization of the genome

Any model for the structural organization of the genomeparticularly in the members of Reoviridae should allow fosimultaneous, independent and repeated transcription of thgenome segments. Studies byPesavento et al. (2001)imposefurther constrains of reversible condensation and expansion


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on such a model. From the elegant X-ray crystallographicanalysis of the BTV cores, which provide a more detailedand discernible electron density distribution of the genomicRNA, particularly after including lower resolution diffrac-tion data and reducing the noise using real space Gaussianltering,Gouet et al. (1999)proposed a plausible model

for the arrangement of dsRNA segments in the BTV core.In this model, each dsRNA segment is spooled around atranscription enzyme complex at the ve-fold vertex. Thismodel allows for up to 12 independent transcription com-plexes, each attached to an individual dsRNA segment forconcurrent transcription. Consistent with this idea, to dateno dsRNA virus with more 12 segments has been observed.In addition to allowing for the observed simultaneous, re-peated, and independent transcription of the genome, thismodel also appears to be consistent with the isometric andconcentric condensation of the genome observed in rotavirusas shown schematically inFig. 3.

4. Genome replication and packaging

One of the least understood processes in the rotavirusreplication cycle is the genome replication and packaging.This also is true with other members of the Reoviridae.Our understanding of the molecular basis of these processesparticularly in the members of Reoviridae has been limitedmainly because of lack of a reverse genetics system. In twoof the dsRNA viruses,6 (Mindich, 1999) and birnavirus(Mundt and Vakharia, 1996), which contain three and twodsRNA segments, respectively, a reverse genetics system has

been successfully established. However, several in vivo andin vitro studies on rotavirus, reovirus, and BTV have pro-vided signicant information on the downstream events tak-ing place following endogenous transcription. Subsequent toendogenous transcription and release of the transcripts, therotavirus replication cycle may be viewed as having threemajor stages: (1) translation and synthesis of the viral pro-teins; (2) replication, genome packaging and DLP assem-bly; (3) budding of the newly formed DLPs into the ERand assembly of the outer layer to form mature TLPs (re-viewed in (Estes et al., 2001). The capped positive-strandedRNA transcripts encode the rotaviral proteins and functionas templates for production of negative strands to make theprogeny dsRNA. The synthesis of the negative strand and thesubsequent duplex formation is facilitated by the viral poly-merase VP1. The genome transcription and the replicationare thus complimentary processes both involving the viralpolymerase. Just as transcription takes place in the connedenvironment of the capsid interior, the genome replicationalso appears to take place inside a protected environment(Patton and Spencer, 2000). In none of the dsRNA viruseshas free dsRNA been found in infected cells.

Genome replication and packaging in rotavirus and theviruses in the Reoviridae, take place within the cytoplasmicinclusions called viroplasms (Petrie et al., 1984). In most of

the dsRNA viruses, each virus particle is thought to contaa full complement of the genome. The only known excetion is chrysovirus, in which the genome consisting of fodsRNA segments are packaged separately into four partic(Wood andBozarth, 1972).In multi-stranded dsRNA virusessuch as rotavirus, BTV, and reovirus, it remains a myste

as to how each particle procures a correct set of dsRNA sments. Although the molecular mechanisms are presentunclear, it is evident that the virus-encoded non-structurproteins play a major role in choreographing the entire pcess of genome replication, packaging, and perhaps segmeassortment in these viruses. Recently, structuresof twoof thnon-structural proteins encoded by rotavirus have been dtermined. These structural studies have begun to shed somlight into the molecular mechanisms of genome replicatiand packaging.

4.1. Structural studies on NSP3

In rotavirus, translation of the viral mRNA transcripis facilitated by NSP3, a basic 63kDa protein that reconizes the consensus 3sequence on the viral transcripts. Asmentioned above, rotaviral transcripts are capped at the5 end by the action of the structural protein VP3 durintranscription but their 3ends are not poly-adenylated. Ro-taviruses rely on the host translation machinery to produthe proteins encoded by the genome. In the host cellonly poly-adenylated and capped messages are efcienttranslated. This is brought about by recognition of the cap by eIF4E and the poly-A tail by PABP (poly-A bining protein) which then interacts with a cellular fact

eIF4G, a multipurpose adaptor protein that is responsibfor delivering capped and poly-adenylated messages the ribosome. Rotaviruses overcome the lack of a poly-tail, which would hamper their efcient translation, by uing a consensus sequence at their 3ends that specicallybinds virus-encoded NSP3. While the N-terminal domaof NSP3 binds this consensus sequence, the C-terminal hinteracts with eIF4G with an afnity greater than PABto give translation of the rotavirus messages a selectivboost following infection. Thus, NSP3 is a protein ecoded by the virus to subvert the host translation machineand selectively enhance the translation of virally encodmRNA. How NSP3 achieves this was demonstrated by tX-ray structure of the two domains of NSP3 bound to tconsensus 3mRNA sequence (Deo et al., 2002) and thestructure of the other NSP3 domain bound to a peptide cresponding to the binding site on eIF4G (Groft and Burley,2002). The asymmetric NSP3 homo-dimer with an unusually large dimeric interface binds the 3consensus sequence(5-GUGACC-3) and completely buries most of it within abasic deep cleft on the surface of the homo-dimer creatia dead end for the 3terminal nucleotides (Fig. 4). Thistight interaction not only promotes translation of the rtavirus mRNA but also prevents degradation of the rotavimessage by cellular nucleases. The NSP3 homo-dim


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Fig. 4. NSP3 structure. Structure of the rotavirus NSP3 N-terminal domainin complex with a 3consensus sequence of rotaviral mRNA (Deo et al.,2002). A single 5-GUGACC-3RNA segment is buried within a basic

deep tunnel formed in the asymmetric dimer. Each subunit of the dimerparticipates in different interactions with the mRNA segment.

is thought to co-fold with the 3consensus end into thisextremely stable complex, which increases the stability of the NSP3 hetero-dimer. These studies further underscore thefunctional signicance of the conserved sequences at the ter-mini of the transcripts in genome translation via their recog-nition by NSP3 (Poncet et al., 1994; Wentz et al., 1996).

4.2. Structural studies on NSP2

Several in vivo and in vitro studies on rotavirus havestrongly implicated two of the non-structural proteins NSP2and NSP5 in genome replication and packaging. In vivostudies have shown that these two proteins along with VP1,the RNA polymerase, are co-localized in the viroplasms

Fig. 5. NSP2 Structure. Structure of the NSP2 monomer (left), showing a deep cleft (arrow) which may be the site for NTP binding (Jayaram et al., 2002).Structure of the functional NSP2 octamer showing a view down the four-fold (middle) and a view down one of the two-fold axis in octamer deep grooves shown (right, arrows) are lined by basic residues and may be the sites for binding viral mRNA during genome replication and

and are the main constituents of the replication intermediates (Aponte et al., 1996; Gallegos and Patton, 1989).Biochemical studies on recombinant NSP2 have shownthat it readily forms octamers and has NTPase (nucleotidetriphosphatase), RNA-binding and nucleic acid helix destabilizing activities (Taraporewala et al., 1999; Taraporewala

and Patton, 2001). Based on these studies, it is hypothesizedthat NSP2 may function as a molecular motor to facilitategenome packaging using the energy derived from NTPhydrolysis (Taraporewala et al., 1999).

Recently, the X-ray structure of NSP2 to a resolution of2.6 has been determined (Fig. 5). These studies have pro-vided a rm ground to begin developing a mechanistic understanding of how NSP2, in concert with NSP5 and VP1may facilitate genome replication and packaging. NSP2crystallizes as an octamer using the crystallographic 422symmetry, with one monomer per asymmetric unit. Theintrinsic ability of NSP2 to form octamers in solution is supported by other biophysical studies including cryo-EM studies. In vivo studies also strongly suggest that the oligomeristructure is the functional form of the NSP2 (Kattoura et al.,1994).

NSP2 is a two-domain protein, which can be classi-ed as an / protein based on the observed secondarystructures. A characteristic feature in the NSP2 monomeis a 25 deep cleft between the N- and C-terminal do-mains of the protein. The N-terminal domain, predomi-nantly made of -helices, exhibits a novel fold, whereasthe C-terminal domain, surprisingly, despite any notice-able sequence homology, exhibits a fold that is observedin the Histidine Triad (HIT) proteins, a family of ubiqui

tous cellular proteins that hydrolyze nucleotides (Brenner,2002; Lima et al., 1997; Lima et al., 1996). This struc-tural similarity with the HIT proteins led to the proposathat the cleft might correspond to the active site for NTPhydrolysis.


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The nucleic acid binding activity of the NSP2 is inde-pendent of the NTPase activity unlike in a classical helicasewhere the two activities are coupled. In that respect, NSP2cannot be described as a helicase. In many of the oligomerichelicases, nucleic acid binds inside a large central hole (Yuet al., 1996). The donut-shaped NSP2 octamer also displays

a central hole with a diameter large enough for bindingnucleic acid. However, both the rim and interior surface of the hole are mostly hydrophobic, and thus it is unlikely thatthe nucleic acid binds inside the hole in the NSP2 octamer.Instead, the prominent grooves, lined by positively-chargedresidues at the sides of the octamer are possible locationsfor nucleic acid binding. The proposed NTP binding sitesin the monomeric subunits are located on either side of this groove. Although NTP hydrolysis is not directly linkedeither to the RNA binding or to the helix destabilizing ac-tivity of NSP2, it is possible that binding of nucleotide intothe cleft alters the conformation of the monomer and affectsoctamerRNA interactions. Such allosteric changes mayfacilitate translocation of the bound RNA through the poly-merase during replication resulting in concomitant synthesisand packaging of duplex RNA into assembling particles.

Indeed the octameric structure of NSP2 makes it a tempt-ing platform or a scaffold around which the replicationcomplex is organized. It is possible that the hydrophobicside of the octamer, around the four-fold axis, may bindto the VP1; given that NSP5 is an acidic protein, the basicgrooves of the NSP2 octamer may be the sites for bindingNSP5. Although the role of NSP5 in the overall process of replication remains to be elucidated, it is plausible, that byhaving its binding site on NSP2 overlap with that of its RNA

binding site, the function of NSP5 is to regulate the bindingof nucleic acid by NSP2 during replication and packaging.In other members of Reoviridae, the existing biochem-

ical data suggest that NSP2 may be functionally homol-ogous to NS2 of BTV and sNS of reovirus (Fillmoreet al., 2002; Gillian and Nibert, 1998; Gillian et al., 2000;Taraporewala et al., 2001). Another protein with which ro-tavirus NSP2 may have similarities is P4 of 6, which is anNTPase (Gottlieb et al., 1992). However, one critical differ-ence is that, unlike rotavirus NSP2 and its putative counter-parts in BTV and reovirus, P4 is a structural protein. It is anintegral partof the transcriptionallycompetent6 core struc-ture, located at each of the ve-fold vertex as a hexamer (deHaas et al., 1999). Because of the well-established in vitroreplication and packaging system,6 is the only dsRNAvirus for which there is more denitive understanding of how the genomic dsRNA segments are packaged inside(Mindich, 1999). Assisted by the packaging protein P4, thethree dsRNA segments in6 are packaged sequentially intoa preformed core, which undergoes signicant conforma-tional change upon packaging. It remains to be seen whethersuch a model for packaging RNA into preformed cores is ap-plicable to dsRNA viruses with larger numbers of segments.Based on the existing biochemical and structural data onReoviridae members, alternative packaging models assisted

by non-structural proteins including co-assembly of coproteins and genome segments remain a distinct possibili

5. Concluding remarks

Despite the lack of a reverse genetics system, in receyears signicant progress has been made in our understaning of the structurefunction relationships in rotavirus. Thas been primarily due to the advances in molecular bology of rotaviruses that have resulted in cloned rotavigenes and purication of protein complexes and virus-liparticles. Starting with the rst cryo-EM reconstructionrotavirus in 1988, which provided a low resolution pictuof the virus architecture (Prasad et al., 1988) subsequentcryo-EM studies on virusantibody complexes, recombnant virus particles (Prasad et al., 1990, 1996), and morerecent X-ray crystallographic analyses of several rotaviproteins have enabled a better understanding of the moleular mechanisms underlying cell entry (Dormitzer et al.,2002), antibody neutralization (Prasad et al., 1990; Tihovaet al., 2001), trypsin-enhanced infectivity (Crawford et al.,2001), assembly (Mathieu et al., 2001), genome organiza-tion (Pesavento et al., 2001; Prasad et al., 1996), endogenoustranscription (Lawton et al., 1997, 1999), mRNA translation(Deo et al., 2002), and genome replication and packaging(Jayaram et al., 2002). The outcome of such studies shouldbe potentially useful in the development of vaccines anidentication of suitable targets for rational drug designcounteract rotavirus.

In parallel, cryo-EM and X-ray crystallographic studi

on other dsRNA viruses have underscored how the moular architecture of the virus can integrate disparate hospecicities with the common requirement of transcribithe dsRNA segments within the capsid interior. Despite tlack of extensive sequence identity, the structural and bichemical studies thus far indicate a remarkable convergenin both form and function necessitated by the endogenotranscription. Further biochemical and structural studies arequired to establish whether these viruses share any comon themes in the events downstream of transcription.major achievement in the near future may be the establisment of reverse genetics systems for some of the membof Reoviridae. Recent developments in the use of RNA terference techniques are exciting and likely to provide futher insights into the specic role of each of the viralencoded protein in the pathogenesis and morphogenesis these viruses (Dector et al., 2002).

Acknowledgements

This work is supported by grants from the Robert WelFoundation (BVVP), National Institutes of Health AI 360(BVVP) and DK 31044 (MKE). We thank Drs. J.A. Lawtand J.B. Pesavento for help with the gures.


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