Cystovirus Maturation at Atomic Resolution

Abstract

Cystoviruses are dsRNA viruses that infect bacteria, and include bacteriophages Φ8 and Φ6. In this issue, two reaserch teams report crystal structures of capsid protein P1 pentamers found in procapsid cores of Φ8 (El Omari et al, 2013) and Φ6 (Němeček et al., 2013). The two structures show striking resemblance in the absence of sequence similarity and offer new mechanistic and evolutionary insights.

In this issue of Structure El Omari et al and Nemecek et al report the crystal structures of the major capsid protein P1 of the Pseudomonas bacteriophages Φ8 (El Omari et al., 2013) and Φ6 (Němeček et al., 2013) and make major contributions that further our understanding of Cystovirus assembly and maturation. These high-resolution structures of the P1 subunit are particularly important because of the dramatic morphological changes occurring in Cystoviruses during genome packaging and associated maturation. Cystoviruses are the only dsRNA viruses known to infect bacteria and the only dsRNA viruses displaying large conformational rearrangements during morphogenesis. The P1 structures bring decades of biochemical, genetic and intermediate resolution electron cryo-microscopy (cryoEM) studies into the realm of specific chemical mechanisms that can now be further tested with structure-based mutations of P1 and other proteins in the capsid.

Structural characterization of dsRNA viruses with both X-ray crystallography and cryoEM has revealed common themes in their overall architecture and mechanisms of replication. These multi-layered virions feature similar inner capsids built from 120 identical gene products organized in a T=1 icosahedral shell containing 60 non symmetric dimers (Grimes et al., 1998; Reinisch et al., 2000). Each dimer is composed of subunit A and Bharboring the same three-dimensional fold but with marked tertiary structure differences resulting from the radically different interactions with neighboring subunits. The genome of these viruses is made of multiple dsRNA segments replicated and transcribed within the inner capsid, where the RNA-dependent RNA polymerase resides. RNA is then secreted for protein synthesis or packaging into progeny virions. Additional layers may be added onto this inner capsid such as the T=13 icosahedral protein shell observed in Φ6, rotavirus and aqua-reovirusor a host-derived lipidic membrane forming the most external layer of bacteriophagesΦ6 and Φ8.

A striking outcome from the crystal structures reported in this issue of Structure is the unambiguous relationship between the major capsid proteins of phages Φ6 and Φ8. They are both large, predominantly α-helical, subunits comprising nearly 800 amino-acid residues and organized in approximately equally sized N- and C-terminal domains (Fig 1 A & B). Although their amino-acid sequences share only 6% similarity, the two P1 structures can be superimposed with a Z-score of 11.8 and a root mean square deviation (rmsd) of 4.5 Å over 445 residues. The super imposition can be further improved if the N- and C-terminal domains of the two structures are treated individually yielding Z-scores of 12.2 and 13.2 with rmsd of 4.4 and 4.2 Å over 253 and 316 residues, respectively (Fig 1 C & D). This structural similarity points to a common origin for the coat subunits of these two Cystoviruses and supports their classification within the same viral family. The observation that both subunits crystallized as comparable pentamers with similar buried surface areas at the interface between each monomer (1635 Ų and 1765 Ų per monomer for Φ6 and Φ8 P1, respectively) further reinforces the similarity between these two proteins.

Figure 1.

Conservation of the P1 coat subunit fold among Cystoviruses. Crystal structures of Φ8 P1 (A) and Φ6 P1 (B) colored from the N-terminus (blue) to the C-terminus (red). Superimposition of the P1 N-terminal domains (C) and C-terminal domains (D) of bacteriophages Φ8 (blue) and Φ6 (orange).

El Omari et al propose that all the subunits forming internal T=1 (120 subunits) dsRNA capsids belong to a single lineage regardless of host. This is based on a comparison of the Φ8 P1 structure to other dsRNA virus capsid proteins using “Homologous Structure Finder”, a software developed by co-authors of this paper (Ravantti et al., 2013). Performing the same analysis with the Φ6 P1 structure would undoubtedly have resulted in the same conclusion due to the striking structural similarity existing between the coat subunits of these two bacteriophages. However, that is not the conclusion reached by Němeček et al as conventional comparison methods, such as the DALI server (Holm and Rosenström, 2010), finds no convincing match linking the Φ6 (or the Φ8) subunit structures to other dsRNA core subunits. However, both studies point out that Φ6 and Φ8 P1 subunits exhibit a compact trapezoid shape that may be required to accommodate the large conformational changes undergone during maturation, while coat subunits observed in eukarytic dsRNA viruses are highly elongated.

Both groups propose that the crystalline pentamer of P1 is relevant to the in vivo assembly pathway. The Φ8 P1 pentamers are readily visualized in solution with negative staining EM and both groups report straight forwardly fitting the pentamers into the Φ6 procapsid cryoEM reconstructions. Integrating these results with data from the literature, they put forward an assembly mechanism relying on the accumulation of P1 pentamers in solution (forming the A subunits) along with monomeric species (forming the B subunits). Association of A subunit pentamers would be promoted by recruitment of monomeric B subunits with concomitant stabilization of the assembling procapsid by addition of the P2 RNA polymerase, the P7 accessory protein and the P4 genome packaging ATPase. The Cystovirus procapsid has a dodecahedral morphology, with deeply recessed vertices (Nemecek et al., 2012) (Fig. 2 A & B), described in terms of an atomic model in the two manuscripts by fitting the reported P1 subunit structures into Φ6 cryoEM reconstructions. The near-atomic resolution reconstruction of the Φ6 procapsid determined by Nemecek et al allowed them to provide a detailed description of the tertiary structure differences between P1 subunits A and B and of their quaternary interactions.

Figure 2.

Large scale conformational changes occurring during Cystoviruses maturation. Surface representation of the Φ6 procapsid (EMDB 2341) (A) and cross section (B) showing its characteristic dodecahedral shape with recessed vertices. Surface representation of the detergent treated Φ6 expanded capsid (EMDB 1206) (C) and cross section (D) revealing the magnitude of the reorganization associated with genome packaging and maturation. The maps are radially colored as indicated at the bottom of the figures (indicated values correspond to diameter). The orientation of icosahedral axes are indicated in panel B.

Bacteriophages Φ6 and Φ8 contain a dsRNA genome composed of three segments, termed s, m and l (according to their respective lengths), which are sequentially packaged in a conserved order. Specific binding of the first genome segment to the P1 subunit procapsid shell promotes its translocation by the P4 ATPase and initiates the cascade of conformational changes underlying maturation and recruitment of the other two genome segments. The availability of cryoEM reconstructions for two Φ6 expansion intermediates as well as the mature Φ6 and Φ8 capsids enabled the authors of both studies to describe the conformational trajectory of the P1 subunit shell during maturation. It is characterized by modifications of the tertiary structure of subunits A and B, large subunit rotations and changes of quaternary contacts to yield a spherical capsid with a considerably larger diameter (Huiskonen et al., 2006) (Fig. 2 C & D). These results also provide a framework to rationalize mutagenesis data and to propose a structural model for the sequential exposure of genome binding sites on the procapsid surface ensuring the successive recruitment of the three RNA segments. Finally, El Omari et al performed an asymmetric cryoEM reconstruction of the expanded Φ8 capsid into which they fitted the crystal structure of Φ8 P4 to analyze the contacts between the pentameric vertices of the P1 shell and the P4 ATPase hexamers and deduce a mechanism for genome packaging in Cystoviruses.

In summary, the articles reported by El Omari et al and Nemecek et al provide major advances in the field of virus assembly and maturation and place Cystoviruses among the best characterized, large-scale protein reorganizations in biology. Cystovirus capsid maturation is an interesting blend between the dsDNA tailed bacteriophages, which exhibit large morphological changes during genome packaging (Gertsman et al., 2009; Wikoff et al., 2000), and the dsRNA viruses infecting eukaryotic organisms, characterized by the presence of the T=1 inner capsid that appears to be morphologically static during maturation. On the other hand, efforts to assess common ancestry of all the dsRNA virus cores on the basis of the subunit fold are frustrating at best. The dominant and variable helices in the core tertiary structures of these proteins bring to mind trying to align “cans of worms”. A broader perspective is the striking and readily recognizable quaternary structure that is common to all these viruses. While significant variations occur in the details of subunit interactions (Dunn et al., 2013), our overall impression is that the quaternary structures of all these T=1 inner cores are homologous.