Portal de Periódicos da CAPES

Intermediates in the assembly pathway of the double-stranded RNA virus phi 6

1997; Springer Nature; Volume: 16; Issue: 14 Linguagem: Inglês

10.1093/emboj/16.14.4477

1460-2075

Sarah J. Butcher, Terje Dokland, Päivi M. Ojala, Dennis H. Bamford, S.D. Fuller,

Bacteriophages and microbial interactions

Article15 July 1997free access Intermediates in the assembly pathway of the double-stranded RNA virus φ6 S.J. Butcher Corresponding Author S.J. Butcher Structural Biology Programme, European Molecular Biology Laboratory, Postfach 10.2209, 69012 Heidelberg, Germany Search for more papers by this author T. Dokland T. Dokland Present address: Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907 USA Search for more papers by this author P.M. Ojala P.M. Ojala Institute of Biotechnology and Department of Biosciences, Division of Genetics Biocenter, FIN-00014 University of Helsinki, Finland Present address: Haartman Institute, Haartmaninkatu 3, FIN-00014 University of Helsinki, Finland Search for more papers by this author D.H. Bamford D.H. Bamford Institute of Biotechnology and Department of Biosciences, Division of Genetics Biocenter, FIN-00014 University of Helsinki, Finland Search for more papers by this author S.D. Fuller S.D. Fuller Structural Biology Programme, European Molecular Biology Laboratory, Postfach 10.2209, 69012 Heidelberg, Germany Search for more papers by this author S.J. Butcher Corresponding Author S.J. Butcher Structural Biology Programme, European Molecular Biology Laboratory, Postfach 10.2209, 69012 Heidelberg, Germany Search for more papers by this author T. Dokland T. Dokland Present address: Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907 USA Search for more papers by this author P.M. Ojala P.M. Ojala Institute of Biotechnology and Department of Biosciences, Division of Genetics Biocenter, FIN-00014 University of Helsinki, Finland Present address: Haartman Institute, Haartmaninkatu 3, FIN-00014 University of Helsinki, Finland Search for more papers by this author D.H. Bamford D.H. Bamford Institute of Biotechnology and Department of Biosciences, Division of Genetics Biocenter, FIN-00014 University of Helsinki, Finland Search for more papers by this author S.D. Fuller S.D. Fuller Structural Biology Programme, European Molecular Biology Laboratory, Postfach 10.2209, 69012 Heidelberg, Germany Search for more papers by this author Author Information S.J. Butcher 1,2, T. Dokland3, P.M. Ojala4,5, D.H. Bamford4 and S.D. Fuller1 1Structural Biology Programme, European Molecular Biology Laboratory, Postfach 10.2209, 69012 Heidelberg, Germany 2MRC Virology Unit, Institute of Virology, Church Street, Glasgow, G11 5JR UK 3Present address: Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907 USA 4Institute of Biotechnology and Department of Biosciences, Division of Genetics Biocenter, FIN-00014 University of Helsinki, Finland 5Present address: Haartman Institute, Haartmaninkatu 3, FIN-00014 University of Helsinki, Finland *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:4477-4487https://doi.org/10.1093/emboj/16.14.4477 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The double-stranded RNA bacteriophage φ6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E.coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the φ6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures. Introduction Many aspects of the multi-segmented, double-stranded RNA (dsRNA) viruses, such as bacteriophage φ6 and the members of the Reoviridae family, raise fundamental questions about viral nucleic acid packaging and replication. In addition to RNA-dependent RNA polymerase activity, the φ6 polymerase complex must recognize and package one copy of each individual genomic segment to ensure that a full complement of segments is present in the virion. A further intriguing problem is the regulation of first minus strand and then plus strand RNA synthesis within this complex. The development of a unique in vitro reconstitution system in which the complete φ6 RNA replication cycle can be followed (Olkkonen et al., 1990) has enhanced our understanding of these phenomena in φ6 replication and of similar processes in the medically important animal dsRNA viruses [reviewed in Mindich and Bamford (1988) and Bamford and Wickner (1994)]. We have now complemented these functional studies by determining the structures of the key replication intermediates of φ6, including the nucleocapsid (NC), the nucleocapsid core (core) and procapsid (PC), to explore the structural basis of φ6 replication. A summary of the compositions of these particles is shown in Table I. We have determined these structures by a combination of cryo-electron microscopy (Adrian et al., 1984; Dubochet et al., 1988) and three-dimensional icosahedral reconstruction (Crowther, 1971; Fuller et al., 1996). The determination of these fragile structures was possible due to the preservation of their icosahedral symmetry in vitrified specimens. Table 1. Properties of φ6 sub-viral particles Particle Origina Composition Functional propertiesb Plus-strand RNA packaging Minus-strand RNA synthesis Plus-strand RNA synthesis Nucleocapsid virion P1, P2, P4, P7, P8, dsRNA no no no Core virion P1, P2, P4, P7, dsRNA no no yes Procapsid recombinant P1, P2, P4, P7 yes yes yes P1, P4 recombinant P1, P4 no no no P1 virion P1 no no no a See Materials and methods for purification details. b Plus-strand packaging is a prerequisite for minus-strand synthesis. dsRNA is a prerequisite for plus-strand synthesis. Bacteriophage φ6 is a spherical enveloped virus of Pseudomonas syringae (for reviews, see Mindich and Bamford, 1988; Bamford and Wickner, 1994). The NC, consisting of the five proteins P1 (85 kDa), P2 (75 kDa), P4 (35 kDa), P7 (17 kDa) and P8 (16 kDa), is enclosed by a lipid envelope containing a further five structural proteins (Mindich and Davidoff-Abelson, 1980). Entry begins by viral attachment, followed by fusion of the viral membrane with the host cell outer membrane. Fusion releases the NC into the periplasmic space (Bamford et al., 1987) from which it penetrates the cytoplasmic membrane via a membrane invagination (Romantschuk et al., 1988). This second translocation requires the presence of the major NC surface protein, P8, which is removed during this process (Romantschuk et al., 1988; Ojala et al., 1990; Olkkonen et al., 1990, 1991). This series of steps releases a transcriptionally active polymerase complex called the core into the cytoplasm (Bamford et al., 1976; Kakitani et al., 1980). Transcriptionally active core can be generated in vitro by removal of the viral envelope and the protein P8 shell (Olkkonen et al., 1991). The dsRNA genome of φ6 consists of three segments: small (S, 2948 bp), medium (M, 4063 bp) and large (L, 6374 bp) (Semancik et al., 1973; Van Etten et al., 1974; McGraw et al., 1986; Gottlieb et al., 1988a; Mindich et al., 1988). The core comprises this segmented dsRNA genome and four protein species (P1, P2, P4 and P7) which are encoded on the L segment (Revel et al., 1986; Mindich et al., 1988). Distinct functions have been assigned to the four proteins. P1 has been identified as the major structural protein of this complex, forming a particle which appears dodecahedral in negatively stained specimens (Ktistakis and Lang, 1987; Olkkonen and Bamford, 1987). Plus strand RNA packaging is dependent on the presence of nucleoside triphosphates (Gottlieb et al., 1991). Protein P4 has been identified as a nucleoside triphosphatase (Gottlieb et al., 1992a). The presence of P7 stabilizes RNA packaging (Juuti and Bamford, 1995). After RNA packaging, the RNA-dependent RNA polymerase, P2, carries out minus strand synthesis and RNA transcription (Koonin et al., 1989; Gottlieb et al., 1990; Bruenn, 1991; Juuti and Bamford, 1995). The in vitro system for φ6 RNA replication relies heavily upon the readily isolatable, functional, naive polymerase complex. The proteins of the polymerase complex can be expressed separately or in various combinations from an L segment cDNA cloned into Escherichia coli (Revel et al., 1986; Gottlieb et al., 1988b). P1 expression leads to the formation of insoluble aggregates; however, soluble dodecahedral particles can be isolated when both P1 and P4 are expressed together (Gottlieb et al., 1988b). Expression of all four proteins gives rise to functional PCs that can sequentially package plus strand φ6 RNA, synthesize the complementary minus strand RNA and then transcribe additional plus strands which are released from the polymerase complex. Isolated cores are only capable of plus strand synthesis, despite having the same protein composition as the PCs. Both RNA-filled PCs and cores can serve as templates for P8 assembly, giving rise to infectious NCs (Gottlieb et al., 1990; Olkkonen et al., 1990, 1991). The availability of the different subviral particles and assembly intermediates makes φ6 a good system for addressing the problems of dsRNA viral assembly and determining their structural basis. One key result of this work is the striking similarity of the bacteriophage φ6 structures to the corresponding ones of mammalian viruses. The NC reconstruction reveals a smooth outer shell with a T = 13 arrangement, similar to that of rotavirus (Prasad et al., 1988; Yeager et al., 1994). The core structure is reminiscent of that of reovirus cores (Metcalf et al., 1991; Dryden et al., 1993). These structural similarities match the shared biological properties of these segmented dsRNA viruses despite their limited sequence homology (Bruenn, 1991). The PC-related structures produced by E.coli expression show a number of elaborations on the dodecahedral shape described previously. Comparison of the core structure with the PC structures reveals differences in size and shape corresponding to extensive structural rearrangements and an associated expansion on maturation. We also find that PCs are metastable and undergo a partial expansion in vitro, in the absence of RNA. Results The nucleocapsid and its core NCs were isolated from virions by removing the envelope (see Materials and methods). The NC contains proteins P1, P2, P4, P7 and an outer shell of P8 (Table I). A cryo-electron micrograph of NCs reveals smooth and rounded RNA-filled particles with an average diameter of ∼58 nm (Figure 1A, black arrows). This morphology made the identification of the initial orientations problematic, although the cross-correlation method (Baker and Cheng, 1996) was effective once a model was constructed by the common lines approach. Figure 1.Cryo-electron micrographs of the different polymerase complex-related particles. (A) The NCs are indicated by black arrows. The white arrow points to a partially disrupted NC where the angular inner layer, corresponding to the core, is visible. (B) A field of the cores, isolated from the virion. The small arrows indicate the positions of the extensions projecting out from the surface of the cores. In favourable positions, a maximum of six projections can be seen. The large arrow points out a strand of RNA in the background. The white arrow indicates a core which has lost its RNA. (C) A field of recombinant P1, P4 particles in unexpanded (black arrows) and expanded (white arrows) conformations. '5', '2' and 'g' indicate particles in 5-fold, 2-fold and general (non-axial) orientations respectively. (D) The P1 particles isolated from the virion. The scale bar represents 100 nm. Download figure Download PowerPoint The double-shelled nature of the NC is apparent in partially disrupted particles (Figure 1A, white arrow), where an internal, angular layer is seen. Comparison of NCs (Figure 1A) with the isolated cores (Figure 1B) shows that the outer layer corresponds mainly to the major NC surface protein P8, while the inner corresponds to the four proteins and dsRNA of the core. P8 can be released from the NC by treatment with EGTA. This results in cores (Figure 1B) which appear rounded with a number of small densities protruding from the surface (Figure 1B, small black arrows). These densities extend to a radius similar to that of the NC surface (29 nm), suggesting that a proportion of the surface of the NC is occupied by core proteins. The distribution of these densities suggests that they are located on the icosahedral 5-fold axes. The average diameter of the body of the core is ∼50 nm, which matches that of the inner layer of the NC. A 2-fold view of an NC reconstruction (Figure 2A) made from 24 particles to a resolution of 3.2 nm reveals the smooth outer layer arranged on a holey net with T = 13 symmetry (Caspar and Klug, 1962). This arrangement is illustrated schematically on a facet of the NC reconstruction, viewed down a 3-fold axis (Figure 2B). There are three different types of holes on the surface, denoted I, II and III, giving an appearance similar to that of the spikeless rotavirus (Prasad et al., 1988). Difference imaging with the core (see below) reveals the single layer of P8 (Figures 2C, 3, 4A and B) which forms the majority of the outer NC surface. Negative staining previously demonstrated similar 10 nm holes in P8 assemblies derived by NC disruption or P8 self-assembly in the presence of calcium ions (Ktistakis et al., 1988). Individual monomers of the 16 kDa protein P8 within the structural units of the surface cannot be resolved at the present resolution. However, the hexavalent nature of the type II and type III holes yields an estimate of the number of copies of P8. We assume, as in the case of the other T = 13 structures described thus far in the literature, that there are six subunits around each hexavalent hole. However, the difference map showing just P8 (Figure 2C) illustrates that approximately one-third of the density around the type II hole is occupied by core proteins. Hence, we assume that P8 occupies only four out of a possible six positions here. This gives a total of 600 P8 units in the lattice, each of which could be an oligomer of P8. The volume which would be occupied by 600 copies of the P8 polypeptide (assuming a protein density of 1.3 g/cm3) is too small to account for the density shown in Figure 2C. The volume shown in Figure 2C corresponds to a total of 19.2 MDa or 1200 copies of P8. Hence the observed 600 P8 units probably correspond to dimers. Analytical ultracentrifugation measurements and equilibrium labelling experiments gave a value of 16.9 MDa or 1056 P8 monomers (Day and Mindich, 1980), consistent with our interpretation. Figure 2.Surface organization of the NC. (A) A surface representation of the NC reconstruction viewed down an icosahedral 2-fold axis of symmetry. The particle has two distinct layers; the inner layer can be glimpsed through the 132 holes present in the outer layer. Only the front hemisphere of the structure is shown. (B) A close-up of the NC surface viewed down an icosahedral 3-fold axis of symmetry (black triangle). A 2-fold (black oval) and a 5-fold (black pentagon) axis are also marked. A partial T = 13 lattice and the three different types of holes penetrating the surface (I, II and III) are marked. There are 120 hexacoordinated holes: 60 5-fold-adjacent holes (type II) and 60 3–fold-adjacent holes (type III). (C) Surface representation of a difference map calculated by subtraction of the core reconstruction from the NC reconstruction viewed down an icosahedral 2-fold axis of symmetry. Only the front hemisphere is shown. This volume reveals the position of the major NC surface protein P8 (16 kDa). The NC surface comprises rings of P8, except the ring on the 5-fold axis which is composed of core proteins. The cut-off level is ∼1.5 standard deviations above the mean density level, which gives a volume corresponding to ∼1200 copies of P8. Download figure Download PowerPoint Figure 3.Surface representation of the reconstructions of the core (A) viewed down an icosahedral 5-fold axis of symmetry; (B) a cut-open representation showing the inner surface of the structure viewed down a 2-fold axis of symmetry. Download figure Download PowerPoint Figure 4.Comparison of the NC and the core reconstructions. (A) A surface representation of the core (in orange) inserted into a cut-away surface representation of the P8 volume (in white) showing the internal connection of the P8 shell to the core around the 5-fold tower of the core. (B) A surface representation of the core (in orange) inserted into a surface representation of the P8 volume (in white) showing the contribution of the core to the NC. This also highlights the surface exposure of the core within the NC. (C) Central sections through the NC (left-hand side) and the core reconstructions (right-hand side). P8, which forms the majority of the outer layer of the nucleocapsid, is released by chelation of calcium ions. These sections illustrate the resulting major conformational changes; the 5-fold tower is freed from contact with P8, and the connections from the core to P8 on either side of the 2-fold axis are lost, leaving no obvious protuberances at this position. There is some concentric density just above the background level inside both reconstructions which may correspond to loosely ordered RNA and/or protein. High density is red, low density is black. Download figure Download PowerPoint Figure 3 shows a reconstruction of the core isolated from the virion (made from 28 particles to 3.2 nm resolution). This reconstruction is nearly spherical, with a turret projecting from each 5-fold axis. These turrets correspond to the small projecting densities observed in the micrographs (Figure 1B). They are tethered to the body of the core by five slim connections, spaced ∼2 nm apart. The turret is penetrated by an axial channel, as shown in the cutaway view in Figure 3B. The diameter of the channel is ∼2 nm at its narrowest point. The highest T number that can be assigned to the core at this resolution is T = 1. Comparing the NC and core reconstructions reveals that the ring of density around the 5-fold axis on the surface of the NC reconstruction is contributed by the turret of the core (Figures 2A, 3A, 4A and B). The remainder of the core mass (orange in Figure 4A and B) lies underneath the P8 layer (white in Figure 4A and B), and is visible through the holes in this layer. A section through the NC reconstruction shows the double-layered nature of the NC (Figure 4C, left-hand side). The core only has a single layer (Figure 4C, right-hand side). Figure 4 also shows the contact points between the P8 layer and the core. There are contacts between the two layers near the icosahedral and local 2-fold axes, indicated by white arrows in Figure 4C. Obviously there is also a major interaction around the 5-fold axes of symmetry between the turret of the core and the P8 layer where the core forms part of the NC surface (Figure 4A, arrow). The φ6 procapsid PCs were made by expressing an L segment cDNA (Gottlieb et al., 1988b) in E.coli as described in Materials and methods. The particle produced was not stable, having a tendency to lose some of its components. The minimum requirement for particle assembly is the presence of both P1 and P4 (Gottlieb et al., 1988b), which produces the PC-related P1, P4 particle. Our description of the PC structure is based primarily on the P1, P4 particle, although the overall features are common to all the PC-related particles (P1, P2, P4; P1, P4, P7; P1, P2, P4, P7; data not shown) that we have studied. Figure 1C shows a cryo-electron micrograph of a P1, P4 preparation. The particles have a diameter of ∼46 nm and display three characteristic views (Figure 1C, black arrows): a ring of 10 dense spots surrounded by a fainter ring, a hexagonal outline and a 'star of David'. These views reflect the PC's dodecahedral shape (Mindich and Davidoff-Abelson, 1980; Steely and Lang, 1984; Yang and Lang, 1984) and correspond to a 5-fold view, a 2–fold view and a general (non-axial) view respectively. A dodecahedron shares icosahedral (532) symmetry with an icosahedron; the symmetry axes are the same. However, the dodecahedral object has 12 pentagonal faces instead of the 20 triangular ones of an icosahedron. The particles exhibited a marked preference for the 5–fold view, while 3-fold views were virtually absent. This is perhaps not surprising in view of the flat surface that the pentagonal face of a dodecahedron presents to the environment. Unfortunately, the multitude of 5-fold views does not contribute much independent information to the reconstruction (Crowther, 1971). This problem was overcome by tilting the specimen by 10–15° before taking the low dose image so that a broader range of projections was obtained (Stewart et al., 1991). A reconstruction of the P1, P4 particle viewed down a 2-fold axis is shown in Figure 5A and B. This reconstruction was made from 14 particles to a resolution of 3.5 nm. The dodecahedral framework is immediately apparent, defining the overall shape of the particle. This framework consists of 30 twisted bars connected at the 3-fold axes (Figure 5A and B). The most striking elaboration on this basic scheme is the presence of 'cups' on each of the 5–fold axes, suspended from the bars of the dodecahedral skeleton. In the inside view (Figure 5B) the cups are seen to terminate in a projecting domain. The cups are responsible for generating the ring of 10 dense spots seen in the 5-fold views (Figure 1C). Figure 5.Surface representations of the reconstructions of the polymerase complex-related particles. (A and B) The unexpanded P14 particle; (C and D) the expanded P14 particle; (E and F) the P1 particle. All reconstructions are shown in the 2-fold view. (A, C and E) External views; (B, D and F) cut-away views showing the inner surface. Download figure Download PowerPoint The PC structure is labile. Elevating the sample temperature from 4 to 22°C caused a gradual change in particle appearance. The heat-treated particles are ∼8% larger in diameter and display fewer internal features (Figure 1C, white arrows). Two distinct views of this larger particle dominate: 5-fold views which display two concentric rings and 2-fold views which show a simpler, more hexagonal outline than the original PC (Figure 1C). Thus, gentle heat treatment of PCs causes their expansion and other structural changes. A reconstruction of these expanded particles was made from 12 particles to 3.8 nm resolution and is shown in Figure 5C and D. The expanded particles display the outline of a dodecahedral cage similar to that of the PC (Figure 5A and B), but the bars of the cage have moved outward and widened, giving the particle an inflated appearance (Figure 5C and D). The cups now appear as shallow depressions on the 5-fold axes, and show no protruding domain (Figure 5D). Particles containing P1 alone cannot be isolated from the recombinant system due to instability and non-specific aggregation in vivo (Gottlieb et al., 1988b). However, they can be isolated from the virion in the presence of high salt (see Materials and methods) by treatment of the core with EDTA. Several rounds of dissociation were necessary to yield particles which lacked P4. A micrograph of these particles is shown in Figure 1D. The P1 particles look very similar to both the expanded PCs (Figure 1C) and empty cores (Figure 1B, white arrow). A reconstruction was made from 18 P1 particles to 3.4 nm resolution (Figure 5E and F). Difference imaging verified that the P1 structure is remarkably similar to that of the P1, P4 expanded particle (Figure 5C and D), suggesting that the P1, P4 particle reconstruction is dominated by P1. Day and Mindich (1980) suggest that there are 120 copies of P4 per virion, based on gel electrophoresis analyses of radioactive virus, so we expected to be able to identify it. The lack of density attributable to the P4 protein raises the possibility that it may have been lost from the expanded particles. SDS–PAGE analysis showed that P4 was still present in the sample. However, it may not have been associated stoichiometrically with all of the expanded particles used in the P1, P4 reconstruction. Hence the density may have been averaged out during the reconstruction. This could also be a problem if the P4 is in a flexible conformation. Alternatively, the low resolution of the reconstructions may be limiting the differences seen. Sequence alignment and secondary structure prediction The P8 amino acid sequence was aligned to the VP7 amino acid sequence using the Maxhom multiple sequence alignment method (Sander and Rost, 1994) in the EMBL PredictProtein WWW server (Rost, 1996). The pairwise identity between the two sequences was 21%, with an additional 11% homology, generating the N-terminal alignment in Figure 6. When the C-terminal 263–349 residues of VP7 were used alone, the pairwise identity with P8 was only 11%, with an additional 11% homology (C–terminal alignment, Figure 6). The prediction of secondary structure was performed by a system of neural networks (Rost and Sander, 1993, 1994; Rost, 1996) using the sequence alignment between P8 and VP7. A control prediction using the 20 best hits from the PDB database of experimentally determined structures gave a similar result for the prediction of helical secondary structure. The highest pairwise identity from this larger set was only 24%. The low homology of P8 to proteins in the three-dimensional database means that the prediction has to be interpreted with due care. Figure 6.Amino acid sequence comparison between blue tongue virus VP7 (SwissProt accession no. vp7_btv10) and φ6 P8 (SwissProt accession no. vp8_bpph6). The N-terminal alignment is between residues 1–131 of VP7 and residues 5–134 of P8. The C-terminal alignment is between residues 263–349 of VP7 and 54–130 of P8. ** marks a deletion in the P8 sequence of 19 residues to improve the N-terminal alignment. In addition, a gap was inserted in the P8 sequence between residues 99 and 100 of P8, indicated by full stops. Residues in known α-helices of VP7 (Grimes et al., 1995) are marked by αs. Residues which are predicted to have an α-helical secondary structure in P8 (probability ≥0.8) are indicated by α (Rost and Sander, 1993, 1994; Rost, 1996). Homologous residues are highlighted in bold. Download figure Download PowerPoint Discussion Multi-layered segmented dsRNA viruses infect animals, plants and bacteria. Of these, the best studied examples are the Reoviridae [reovirus, rotavirus and blue tongue virus (BTV)] and the enveloped bacteriophage φ6. An in vitro system using purified components which carries out the entire RNA replication cycle has only been developed for φ6 (Olkkonen et al., 1990). One of the reasons for the success of the φ6 system is that assembly of the NC is independent of non-structural viral proteins. This has allowed detailed dissection of the ssRNA binding, packaging, replication and transcription events in the assembly pathway (Gottlieb et al., 1990, 1991, 1992b, 1994; Casini et al., 1994; Mindich et al., 1994; Frilander and Bamford, 1995; Frilander et al., 1995; Qiao et al., 1995; Van Dijk et al., 1995). Previous structural studies of the φ6 NC revealed a dodecahedral cage-like core structure (Steely and Lang, 1984; Yang and Lang, 1984). However, these studies were hampered by the fact that the particles were poorly characterized biochemically. In particular, what was considered to be the NC had actually lost several proteins because of the negative staining technique used (Ktistakis and Lang, 1987; Olkkonen and Bamford, 1987). Here, we have used cryo-electron microscopy, which ensures the best attainable preservation of the native structure, to visualize directly the three-dimensional structure of the virus core and the NC as well as recombinant polymerase complex particles that have never packaged RNA (P1, P4 particle). This has allowed us to address the question of φ6 assembly from a structural point of view. We show that major structural transitions occur during assembly, and also demonstrate surprising structural similarities to other dsRNA viruses. The T = 13 outer shell of the nucleocapsid The NC of φ6 has a smooth, T = 13 surface structure, strikingly similar to that of equivalent substructures of other segmented dsRNA viruses. In BTV, VP7 comprises the outer layer of the core with T = 13 symmetry (Hewat et al., 1992;