Genome Replication and Packaging of Segmented Double-Stranded RNA Viruses

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Virology 277, 217–225 (2000) doi:10.1006/viro.2000.0645, available online at http://www.idealibrary.com on

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Genome Replication and Packaging of Segmented Double-Stranded RNA Viruses

John T. Patton*,1 and Eugenio Spencer†,2

*Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, 7 Center Drive, MSC 0720, Room 117, Bethesda, Maryland 20892; and †Laboratorio de Virologia, Departamento de Ciencias Biolo´gicas, Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Casilla 40 Correo 33, Santiago, Chile Received July 10, 2000; returned to author for revision September 1, 2000; accepted September 19, 2000

Viruses with segmented double-stranded (ds)RNA ge- Transcription of the Segmented Genome nomes infect a wide range of organisms including ver- by the Multiple Polymerase Units tebrates, invertebrates, plants, and bacteria and belong of the Core to the families Reoviridae, Birnaviridae, and Cystoviridae Structural studies have indicated that all segmented (Fauquet, 1994). The Reoviridae is the largest of the and some nonsegmented dsRNA viruses contain an ico- families and includes many well-studied viruses such as sahedral Tϭ2 core consisting of 120 capsid subunits bluetongue virus (BTV) (Roy, 1996), orthoreovirus (Nibert (core lattice protein: VP3 for BTV, ␭1 for reovirus, VP2 for et al., 1996), and rotavirus (Estes, 1996). Reovirus virions rotavirus), which immediately surrounds the genome are icosahedrons, made up of one or more layers of (Butcher et al., 1997; Caston et al., 1997; Grimes et al., capsid protein that enclose a genome consisting of 10, 1998; Hill et al., 1999; Lawton et al., 1997b; Reinisch et al., 11, or 12 equimolar segments of dsRNA (Hill et al., 1999; 2000). Five dimers of the core lattice protein make up Tyler and Fields, 1996). The segmented dsRNA genomes each of the 12 pentamers of the core (Fig. 1). In the case of the Reoviruses are never detected free in the cyto- of the Reoviruses, a molecule of the viral RdRP is asso- plasm but are transcribed and replicated within viral ciated with the interior base of channels that extend capsids by RNA-dependent RNA polymerase (RdRP). through the center of the pentamers, i.e., the fivefold New insight into the mechanism of RNA replication and axes of the core. Also associated with the fivefold chan- into the relationship between this process and the as- nels are one or more copies of the viral protein respon- sembly and structure of the Reovirus has been revealed sible for capping viral mRNAs, which may extend through by analyses of (i) the structural organization of proteins the core and contact a surrounding capsid shell of the and the dsRNA genome within virus particles by cryo- virion (Luongo et al., 2000; Ramadevi et al., 1998; Sandino electron microscopy and crystallography and (ii) the con- et al., 1994). Crystallographic and cryoelectron micro- tribution of RNA structure and protein function to the scopic analysis of several Reoviruses has indicated that synthesis of dsRNA genome using cell-free replication the dsRNA genome is organized in partially ordered systems. These findings in combination with those ob- concentric layers within the core (Gouet et al., 1999; tained with an in vitro packaging and replication system Prasad et al., 1996; Reinisch et al., 2000). The suggestion has been made that each segment may exist as a tightly for ␾6 (Gottlieb et al., 1990; Mindich, 1999a,b), a Cysto- wound spiral around one of the 12 RdRP-capping com- viridae bacteriophage containing three segments of plexes at the fivefold axes of the core (Gouet et al., 1999). dsRNA, suggest possible pathways by which viral RNAs The fact that the icosahedral nature of the core restricts are coordinately packaged, i.e., assorted, during morpho- the number of RdRP-capping complexes to 12, probably genesis to generate progeny containing equimolar seg- explains why no Reovirus has been isolated which con- ments of dsRNA. tains more than 12 genome segments. Indeed, since most Reoviruses contain less than 12 segments, not all the 12 potential sites in the core may be occupied by dsRNA. 1 To whom correspondence and reprint requests should be ad- Infection activates the RdRP of the core, resulting in dressed. Fax: (301) 496-8312. E-mail: [email protected]. the synthesis of capped mRNAs which are released into 2 Fax: (562) 681-9036. the cytoplasm by extrusion through channels at the five-

FIG. 1. Organization of proteins and the dsRNA genome in rotavirus particles. (Upper left) Three-dimensional structure of the mature triple-layered rotavirion, in which portions of the outer (VP4 and VP7) and intermediate shells have been removed. (Upper center) Representation of the Tϭ2 core formed by 120 copies of VP2. The white boundary indicates the interaction between two molecules of VP2 along the twofold axis. Note that of the 10 molecules of VP2 making up the pentamer, 5 are positioned close to the fivefold axis (red) while the other 5 originate further away from the axis. (Upper right) Cutaway of the intermediate and core layers of protein, displaying protrusions of the RNA polymerase, VP1, and capping enzyme, VP3, at the fivefold axes into the interior of the core, where they are surrounded by the dsRNA genome arranged as a dodecahedron. (Lower left) Side view illustrating the three-dimensional organization of proteins through the fivefold axis of a double-layered particle. The VP1–VP3 complex extends as “flower-petals” from the VP2 pentamer. VP2 has RNA binding activity and may form a platform on which the RdRP carries out RNA synthesis. (Lower right) Proposed pathway for exiting of a nascent transcript through channels at the fivefold axis of the core and intermediate shell of transcriptionally active particles. Figure was kindly provided by B. V. V. Prasad (Baylor College of Medicine) and details are given in Prasad and Estes (2000), Prasad et al. (1988, 1996), and Lawton et al. (1997a,b). fold axes of the core and, in some instances, an over- cles in vitro has shown that they have the capacity to laying Tϭ13 layer of capsid protein (Bartlett et al., 1974; synthesize high levels of mRNA for several hours, thus Hill et al., 1999; Lawton et al., 1997a; Prasad et al., 1996) indicating that the RdRP efficiently reengages the 5Ј-end (Fig. 1). During transcription, channels penetrating the of the dsRNA template after completing transcription at core and the overlaying protein layer allow entry of the the 3Ј-end (Banerjee and Shatkin, 1970; Cohen, 1977; nucleoside-triphosphate substrates for the RdRP (Prasad Spencer and Arias, 1981). The efficiency of the process and Estes, 2000). In essence, the core may be viewed as suggests that the proteins of the core hold the 5Ј- and a collection of 12 polymerase units, each representing 3Ј-ends of each dsRNA segment in close proximity to one of the pentamers of the core, which operate inde- one another such that the segments functionally operate pendently, but simultaneously, to transcribe the genome as circular templates during transcription (Yasaki et al., segments. The structural organization of the transcrip- 1986). Based on the affinity of the RdRP-capping enzyme tionally-active particle dictates that only a single type of complex for the core lattice protein (Grimes et al., 1998; mRNA can pass through any one of the fivefold channels Reinisch et al., 2000; Zeng et al., 1996), the RNA poly- of the core. The independent function of each of the merase is probably structurally anchored to the core polymerase units of the core is reflected by the obser- even as mRNA synthesis takes place (Fig. 1). As a con- vation that the mRNAs products are not made in equimo- sequence, the dsRNA template must be free to move lar levels, in contrast to the genome segments which are about within the core, such that it can slide through the produced in equimolar levels during RNA replication RdRP-capping enzyme complex. Indeed, recent studies (Antczak and Joklik, 1992; Patton, 1990). with BTV have indicated that the dsRNA genome exists The analysis of transcriptionally active Reovirus parti- as a liquid crystal in the core, thereby providing the MINIREVIEW 219 fluidity necessary for the template to efficiently move the de novo synthesis of full-length dsRNA from viral during transcription (Gouet et al., 1999). The actual move- mRNA made either in vitro by transcriptionally active ment of the dsRNA template during transcription is likely subviral particles or by T7 transcription of viral cDNAs the product of the translocase activity of the RdRP (Yin et (Chen et al., 1994; Gottlieb et al., 1990). In the rotavirus al., 1995). The exact role of the core lattice protein in system, the source of replicase activity is cores which transcription is not known, although for some Reovi- have been purified from virions and disrupted (“opened”) ruses, the RdRP requires this protein for activity (Patton by incubation in hypotonic buffer. By adding viral tran- et al., 1997). Given that the lattice protein has affinity not scripts that contain deletions and site specific mutations only for the RdRP, but for RNA as well (Bisaillon and to the open cores, it has been possible to define features Lemay, 1997; Labbe et al., 1994), it may be that in the of Reovirus mRNAs that promote efficient minus-strand pentameric polymerase units, the lattice protein forms a synthesis (Wentz et al., 1996b). These studies have es- platform on which the dsRNA duplex is melted and tran- tablished that the 3Ј-consensus sequence plays an es- scribed by the RNA polymerase (Prasad et al., 1996). sential role in the synthesis of dsRNA (Patton et al., 1996). Precisely, two significant observations have been Ј Features of the Viral mRNA Template made: (i) viral transcripts lacking the 3 -consensus se- Promoting dsRNA Synthesis quence no longer serve as templates for synthesis of dsRNA, and (ii) the addition of the 3Ј-consensus se- The genome segments of Reoviruses are usually quence to the 3Ј-end of a nonviral RNA confers to the monocistronic and contain 5Ј- and 3Ј-untranslated re- chimeric RNA the ability to be replicated by the viral gions (UTRs) that vary considerably in length. The extent RdRP, albeit, at levels that are lower than that of full- of nucleotide homology between all the genome seg- length viral transcripts. Exhaustive mutagenesis has re- ments of any particular Reovirus is limited to only short vealed that the last one or two nucleotides of the 3Ј- stretches (Ͻ10 nucleotides) at their 5Ј- and 3Ј-termini consensus sequence are critical for RNA replication, (Estes, 1996; Kudo et al., 1991; Nibert et al., 1996; Roy, although other residues of the sequence also contribute 1996), termed the 5Ј- and 3Ј-consensus sequences. Be- to the efficiency of the process (D. Chen and J. T. Patton, cause Reovirus transcripts do not undergo polyadenyla- unpublished results). Based on the commonality of the tion, the viral mRNAs made during replication generally 3Ј-consensus sequence and by analogy with the repli- terminate with the same short consensus sequences. In cation mechanisms of other RNA viruses, the 3Ј-consen- contrast to these sequences, comparison of homologous sus sequence probably represents a cis-acting signal segments of different strains of a Reovirus have revealed recognized by the RdRP (3Ј-essential replication signal that extensive regions of the 5Ј- and 3Ј-termini, including or 3ЈEsRS) that is used in the formation of a ternary the entire 5Ј- and 3Ј-UTRs and in some cases portions of initiation complex for minus-strand synthesis (Chen and the open reading frame (ORF), are highly conserved. The Patton, 2000; Frilander et al, 1995; Miller et al, 1986; Wu fact that these highly conserved termini differ between et al., 1994). the genome segments probably reflects the existence Deletion mutagenesis has indicated that, besides the within them of unique packaging signals that allow the 3Ј-consensus sequence, other sequences are present segments to be distinguished from one another during within Reovirus transcripts that enhance the synthesis of replication. The lack of an effective cDNA-based reverse dsRNA in cell-free replication systems. Most importantly, genetics system for any member of the Reovirus family analysis of viral mRNAs containing deletions of all or part has precluded attempts to directly demonstrate that the of their 5Ј-UTR have demonstrated that this region con- terminal sequences indeed contain such packaging sig- tains sequences that promote the synthesis of dsRNA nals. However, variants have been identified for WTV, (Patton et al., 1996; Wentz et al., 1996a). Similarly, deletion orthoreovirus and rotavirus, whose genome includes a mutagenesis has shown that sequences within the 3Ј- segment which contains a deletion of part or nearly all of UTR but upstream of the 3ЈEsRS also contribute to effi- the ORF (Anzola et al., 1987; Desselberger, 1996; Tanigui- cient replication. From these results, which suggest that chi et al., 1996; Zou and Brown, 1992). Because the both ends of viral mRNAs are involved in minus-strand aberrant segment can undergo packaging and replica- synthesis, and computer modeling of the secondary tion, despite the deletion, indicates that the essential structure of the viral transcripts, the 5Ј- and 3Ј-ends of signals involved in these processes are located within Reovirus mRNAs have been proposed to interact in cis the terminal sequences of the segments (Gottlieb et al., via basepairing of complementary terminal sequences to 1990). form panhandle structures that promote the synthesis of In the infected cell, the mRNAs serve as templates for dsRNA (Chen and Patton, 1998). both the synthesis of protein and the synthesis of minus- Besides the 5Ј- and 3Ј-UTRs, replacement of the ORF strand RNA to form dsRNA. For rotavirus, as well as the of rotavirus mRNAs with foreign sequences of equivalent phage ␾6, efficient cell-free replication systems have size has been shown to cause a decrease in the ability been developed which contain RdRP activity that support of the RNA to undergo replication (Patton et al., 1999). A 220 MINIREVIEW

FIG. 2. Putative replication and packaging signals formed by the interaction of the 5Ј- and 3Ј-termini of rotavirus mRNAs. The mfold program was used to predict the secondary structure of the 1611-nucleotide gene 5 mRNA, and the portion of the computed structure made up of the conserved 5Ј and 3Ј-terminal sequences is shown (Zuker, 1989, 1994). The few nucleotide differences that exist in the terminal sequences for this mRNA among the various strains of group A rotaviruses (indicated with arrows) generally have no effect on the predicted structure because they are located in loops or they do not alter the extent of basepairing within the stem, due to compensatory mutations (lower case letters in italics). The structure displays the three common elements observed upon folding any of the rotavirus mRNAs (i) 5Ј–3Ј complementary sequences that generate a panhandle, (ii) a stem–loop, and (iii) a non-base-paired 3Ј-tail which includes the 3Ј-consensus sequence (*). Although the panhandle is not required for in vitro synthesis of dsRNA, its presence significantly enhances the efficiency of the process. The 3Ј-consensus sequence includes the 3Ј-essential replication signal, a motif that is required for dsRNA synthesis. For the gene 5 mRNA of SA11 rotavirus, an additional A residue is present within the 3Ј-consensus sequence (open arrowhead). The stem–loop structure differs for each rotavirus mRNA, suggesting that it represents a packaging signal. systematic analysis of the importance of the ORF by mentary deoxyoligonucleotides to the 3Ј-consensus se- overlapping deletion mutagenesis has revealed that only quence blocks dsRNA synthesis while annealing of oli- certain regions contained within it promote the synthesis gonucleotides to other regions of the mRNA have little or of dsRNA (J. T. Patton and M. Jones, unpublished results). no effect. From these studies, it is apparent that the Those regions of the ORF that do promote dsRNA syn- secondary structure of the viral mRNA plays a key role in thesis contain sequences that support basepairing be- dsRNA synthesis. In part, the structure is likely to con- tween the ends of the mRNA, and thereby, have an tribute to efficient RNA replication by assuring that the impact on the formation and stability of the panhandle 3ЈEsRS of the 3Ј-consensus sequence is presented in a structures. sterically accessible manner to the RdRP, allowing the While interactions between the ends of the viral mR- formation of an initiation complex (Chen and Patton, NAs cause the formation of panhandles, the nature of the 2000). basepairing is not such that it gives rise to perfect 5Ј–3Ј A key observation made from the predicted secondary duplexes (Fig. 2). Indeed, this feature of the panhandle is structures of rotavirus mRNAs is that one or more highly critical in the synthesis of dsRNA. Specifically, computer conserved stable stem–loop structures are always lo- modeling of the secondary structure of the rotavirus cated near the 5Ј–3Ј terminal end of the panhandles (Fig. mRNAs has indicated that the 3Ј-consensus sequence 2 and Chen and Patton, 1998). One remarkable variation extends as an unbasepaired tail from the end of the of the stem–loops is that, in some cases, they are formed panhandle. Two types of experiments have shown that by the 5Ј-terminal sequence and in other cases they are the single-strandedness of the 3Ј-consensus sequence formed by the 3Ј-terminal sequence. Since the stem is critical for function of the 3ЈEsRS (Chen and Patton, loops differ for each of the mRNAs, and since such 1998): (i) mutations introduced into the 5Ј-end of the structures have been shown to function as specific pack- mRNA that increase the extent of complementarity be- aging signals for several other RNA viruses (Berkowitz et tween the 3Ј-consensus sequence and the 5Ј-terminus al., 1997; Pappalardo et al., 1998; Qu and Morris, 1997), inhibit dsRNA synthesis and (ii) annealing of comple- including that of ␾6 (Pirttimaa and Bamford, 2000), we MINIREVIEW 221 propose that the stem–loops mediate the packaging and empty cores (Labbe et al., 1994; Patton et al., 1997). assortment of the viral mRNAs of the Reoviridae. Analysis of a rotavirus mutant with a temperature-sensitive assembly-defect mapping to the core lattice protein Protein Function in Genome Replication supports the idea that this protein is necessary for RdRP activity, and moreover, that assembly of the lattice pro- The segmented dsRNA genome of the Reoviruses tein into cores is necessary for RdRP activity (Mansell does not exist in a free, naked form in infected cells, but and Patton, 1990; Munoz and Spencer, 1996; Vasquez et rather exists in association with subviral particles. Based al., 1993). The requirement of the core lattice protein in on the study of replication intermediates (RIs) from in- dsRNA synthesis results in the coordination of RNA rep- fected cells, the simplest intermediates that carry out the lication and virion morphogenesis such that dsRNAs are synthesis of dsRNA through an associated replicase not produced unless cores are available in which they activity are core-like in structure (core RIs) (Gallegos and can be packaged. Patton, 1989; Morgan and Zweerink, 1974; Sandino et al., In contrast to the rotavirus RdRP, analysis of the re- 1988; Zweerink et al., 1976). The treatment of purified RIs combinant RdRPs of BTV and ␾6 have indicated that with single-strand-specific nuclease destroys their ability these polymerases can synthesize dsRNAs in vitro in the to synthesize dsRNA, providing evidence that the mRNA absence of the core lattice protein (Devi et al., 1999; template for replication is at least partially exposed on Makeyev and Bamford, 2000). While this may be the the surface of the intermediates with replicase activity case, the fact that free dsRNAs have not been detected (Acs et al, 1971; Patton, 1986/87). Notably, in vitro assays in cells infected with BTV or ␾6 indicates that the activity have indicated that as rotavirus RIs synthesize dsRNA, of these RdRPs are also regulated in vivo such that only they undergo a continuous decrease in size that only core-associated RdRPs can carry out the synthesis of the stops once the synthesis of dsRNA completed (Patton dsRNA genome. The importance, if any, of other struc- and Gallegos, 1990). The post replication size of the tural proteins in RNA replication is not known. smallest RIs of rotavirus approximates that of cores, and The replication of the dsRNA genome and the assem- nuclease treatment of RIs which have not undergone bly of cores occurs in cytoplasmic inclusions that form in replication reduces their size to those of RIs which have Reovirus-infected cells. One or two nonstructural RNA- undergone replication. In summary, such analyses have binding proteins play a role in the formation of the inclu- indicated that (i) packaging precedes replication, (ii) the sions and the same proteins are components of RIs mRNA template moves from outside to inside RIs during isolated from infected cells (Antczak and Joklik, 1992; replication, and (iii) functional packaging signals reside Fabbretti et al., 1999; Mbisa et al., 2000; Thomas et al., on viral mRNAs, and not on dsRNAs. It is believed that 1990). Studies on what appears to be homologous pro- the same molecule of RdRP that catalyzes the synthesis teins in different Reoviruses (NS2 for BTV, ␴NS for or- of a dsRNA from an mRNA, remains associated with that thoreovirus, and NSP2 for rotavirus) has shown that one dsRNA product, eventually serving as the RdRP respon- of the nonstructural proteins of the inclusions self as- sible for its transcription. sembles into large homomultimeric complexes (Gillian et The analysis of core RIs has indicated that both struc- al., 2000; Taraporewala et al., 1999; Uitenweerde et al., tural and nonstructural proteins are involved in the pack- 1995). Characterization of different members of this ho- aging and replication of mRNAs. In vitro assays per- mologous group of proteins has revealed that some have formed with purified recombinant proteins of rotavirus helix destabilizing activity (orthoreovirus and rotavirus) have indicated that its RdRP only catalyzes the synthesis (Gillian et al., 2000; Taraporewala and Patton, unpub- of dsRNA when the core lattice protein is present (Patton lished results), NTPase activity (rotavirus) and undergo et al., 1997; Zeng et al, 1996). This despite the ability of phosphorylation (BTV and rotavirus) (Ojala et al., 1994). the rotavirus RdRP to specifically recognize the 3Ј-end of The collective properties of these proteins suggest that viral mRNA in the absence of the core lattice protein they function alone or in concert with other proteins to (Patton, 1996). The rotavirus replicase activity is maximal relax RNA secondary structures during the packaging of when the ratio of the polymerase to lattice protein ap- mRNA. proximates that of virion-derived cores (1:10). As suggested earlier for transcription, this finding is consistent Models for Selective Packaging with the idea that pentamers formed by the core lattice of the mRNA Templates proteins serve as platforms on which the polymerase is active, in this case, in the synthesis of dsRNA. The role of The selective packaging mechanism that leads to the the core lattice protein in dsRNA synthesis may be re- presence of equimolar genome segments within Reoviri- lated to its affinity for RNA, since deletion of the domain ons is not understood. Much more is known about this of the protein responsible for RNA binding renders it process for ␾6, as described below. However, it remains unable to support replication by the RdRP, although the uncertain to what extent the packaging pathway for ␾6, mutated protein retains the ability to self-assemble into with its 3 dsRNA segments, serves as an appropriate 222 MINIREVIEW model of the packaging pathway for the Reoviruses, and RNA-pentamer precursor model. The organization of their 10 to 12 dsRNA segments. Nonetheless, for both ␾6 proteins within the Tϭ2 core of Reoviruses indicates that and the Reoviruses, it appears certain that packaging of such structures represent a collection of functionally the mRNA precedes dsRNA synthesis and that dsRNA separate pentameric units, each with RdRP activity and synthesis takes place within Tϭ2 cores. As a conse- each responsible for transcription of one of the genome quence, the cis-acting signals for selective packaging segments. Based on this, it can be proposed that the are present on the mRNA template. Many of the Reovirus RdRP and capping enzyme interact with a molecule of proteins exhibit RNA-binding activity, some specifically mRNA to form a complex. The RdRP-capping enzyme and some nonspecifically (Brentano et al., 1998; Patton, complex and its associated mRNA then serve as a nu- 1995). But none of the Reovirus RNA-binding proteins cleation site to which 10 molecules of the core lattice have the ability to distinguish between the different viral protein bind and assemble into a pentamer. This process RNAs in a manner that would suggest that selective results in the formation of 10 to 12 distinct pentameric packaging is driven simply by the action of one or more units, depending on the type of Reovirus. Specific RNA- of the viral proteins. Instead, it seems more reasonable RNA interactions between the mRNAs of the pentameric ϭ to assume that selective packaging is mediated by RNA- units then drive the assembly of the T 2 core from RNA interactions occurring in trans between the viral pentameric units. This process is perhaps initially cata- mRNA templates. The RNA-binding proteins may func- lyzed and subsequently stabilized by the affinity of the tion in this process to stabilize the RNA–RNA interac- core lattice protein of one pentamer for that of another. tions or to alter the structure of the mRNA templates in a Structural changes in the core lattice protein, as a con- manner that makes the RNA packaging sites sterically sequence of pentamer-pentamer binding, may activate accessible. the RdRP, thereby allowing minus-strand synthesis to Based on existing information, three models can be commence and the dsRNA genome to be formed. proposed by which Reovirus cores acquire their com- Empty core precursor model. Coexpression of recombinant structural proteins of Reovirus and ␾6 has been plete set of viral RNAs. used to generate core particles that contain the viral Precore precursor model. Characterization of RIs from RdRP but lack the dsRNA genome. When incubated with rotavirus-infected cells has indicated the existence of ␾6 empty cores, the 3 mRNAs of this phage [small (s), precore RIs, serving as precursors to core RIs. The medium (m), and large (l)] are packaged in the order s Ͼ precore RI contains viral mRNA and the viral RdRP and m Ͼ l, due to differences in the binding affinities of the capping enzyme, but lacks the core lattice protein. It is mRNAs for the core (Frilander and Bamford, 1995; Qiao proposed that the formation of the precore RI is mediated et al., 1995; Poranen et al., 1999). Indeed, it is believed by RNA-RNA interactions and, when mature, contains the that the empty core initially displays binding sites for s, full complement of mRNA templates. In this scenario, which leads to its recruitment and packaging. The com- assortment of the viral mRNAs occurs prior to core as- plete packaging of s leads to the disappearance of s sembly. The interaction of the RdRP and capping enzyme packaging sites and the appearance of m packaging with the mRNAs may be required in the formation of the sites. After packaging of m, its packaging sites are lost precore RI, by altering the structure of the mRNAs such and the l packaging sites appear, leading to the forma- that their packaging sites are generated and become tion of cores containing the three viral mRNAs. Packag- sterically accessible. The viral RdRP and capping en- ing of empty ␾6 cores results in their expansion, and in zyme of the precore RI could promote the formation of doing so, stimulates the RdRP of the core to initiate the core RI by serving as nucleation sites for binding of minus-strand synthesis on the mRNA templates (Frilan- ϭ the core lattice protein and for the assembly of the T 2 der et al., 1992, 1995). As a consequence, ␾6 cores core (Vasquez et al., 1993). Given that the synthesis of containing the segmented dsRNA genome are formed. dsRNA by core RIs is extremely RNAse-sensitive, the Importantly, these cores are infectious when reconsti- formation of the core does not appear to engulf the entire tuted appropriately with outer capsid components indi- mRNA template prior to the onset of dsRNA synthesis. cating that the empty core precursor models probably More likely, only regions of the template which are as- reflects the authentic pathway by which its RNAs are sociated with the RdRP-capping enzyme complex are packaged and replicated (Olkkonen et al., 1991). initially located within the core. Following formation of Preparations of empty rotavirus cores, made by coex- the core lattice, the RdRP initiates the synthesis of the pression of appropriate recombinant proteins, will syn- minus-strand RNA, which results in the formation of the thesize dsRNA when incubated with any one of the viral dsRNA genome within the core. During the synthesis of mRNAs in vitro (Zeng et al., 1996). Therefore, unlike the the minus strand, the model predicts that the mRNA ␾6 system, there seems to be no requirement for com- template is drawn into the core via the translocase ac- plete packaging of all the viral mRNAs into rotavirus tivity of RdRP and with the assistance of nonstructural empty cores prior to dsRNA synthesis. Notably, there is packaging proteins. also no evidence that the dsRNA products made by MINIREVIEW 223

rotavirus recombinant cores are packaged into the par- Acknowledgments ticles. Instead, under the conditions used to perform the This work was supported in part by grants to E.S. from the Chilean assay, the recombinant cores are probably unstable and National Commission for Scientific Research (Grants 1980632 and become open, as virion-derived cores do under the same 7980051). conditions. Because of their open nature, the dsRNA products of the recombinant cores are likely released, References which allows the RdRP to be recycled and to reinitiate Acs, G., Klett, H., Schonberg, M., Christman, J., Levin, D. H., and minus-strand synthesis on other mRNA templates in the Silverstein, S. C. (1971). Mechanism of reovirus double-stranded reaction mixture (Chen and Patton, 1999). These results ribonucleic acid synthesis in vivo and in vitro. J. Virol. 8, 684–689. illustrate that while rotavirus empty cores and, for that Antczak, J. B., and Joklik, W. K. (1992). Reovirus genome segment matter, open cores are also important tools for analyzing assortment into progeny genomes studied by the use of monoclonal antibodies directed against reovirus proteins. Virology 187, 760–776. elements of dsRNA synthesis, they apparently are inad- Anzola, J. V., Xu, Z. K., Asamizu, T., and Nuss, D. L. (1987). Segment- equate for studying the packaging process. Thus, it re- specific inverted repeats found adjacent to conserved terminal se- mains unclear whether the mechanism of packaging as quences in wound tumor virus genome and defective interfering determined for ␾6 with its in vitro system is fundamen- RNAs. Proc. Natl. Acad. Sci. USA 84, 8301–8305. Banerjee, A. K., and Shatkin, A. J. (1970). Transcription in vitro by tally the same process by which Reoviruses package reovirus-associated ribonucleic acid-dependent polymerase. J. Virol. their mRNAs into cores. 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