Subnanometer-resolution structures of the grass carp reovirus core and virion

Abstract

Grass carp reovirus (GCRV) is a member of the Aquareovirus genus of the family Reoviridae, a large family of double-stranded RNA (dsRNA) viruses infecting plants, insects, fishes and mammals. We report the first subnanometer-resolution three-dimensional structures of both GCRV core and virion by cryoelectron microscopy. These structures have allowed the delineation of interactions among the over 1000 molecules in this enormous macromolecular machine and a detailed comparison with other dsRNA viruses at the secondary-structure level. The GCRV core structure shows that the inner proteins have strong structural similarities with those of orthoreoviruses even at the level of secondary-structure elements, indicating that the structures involved in viral dsRNA interaction and transcription are highly conserved. In contrast, the level of similarity in structures decreases in the proteins situated in the outer layers of the virion. The proteins involved in host recognition and attachment exhibit the least similarities to other members of Reoviridae. Furthermore, in GCRV, the RNA-translocating turrets are in an open state and lack a counterpart for the sigma1 protein situated on top of the close turrets observed in mammalian orthoreovirus. Interestingly, the distribution and the organization of GCRV core proteins resemble those of the cytoplasmic polyhedrosis virus, a cypovirus and the structurally simplest member of the Reoviridae family. Our results suggest that GCRV occupies a unique structure niche between the simpler cypoviruses and the considerably more complex mammalian orthoreovirus, thus providing an important model for understanding the structural and functional conservation and diversity of this enormous family of dsRNA viruses.

Fig. 1. CryoEM images of GCRV particles

(A) GCRV cores. Arrowhead indicates a turret. (B) GCRV virion. Arrow points to an electron-lucent boundary between inner and outer layers.

Fig. 2. Structural representation of GCRV core and virion

(A) Radially coloured shaded surface representation of GCRV core viewed along twofold axis. The VP6 nodules are in green and the VP1 turrets are in purple. (B) A cartoon illustrating the protein organization in an asymmetric unit (in darker colours) and their symmetry-related proteins (in lighter colours). (C) A central slice from (A). A transcriptase complex under a fivefold vertex is indicated by a red arrow. The green arrow indicates an immunoglobulin-like flap domain. (D) Radially coloured shaded surface representation of GCRV virion. Four kinds of quasi-equivalent trimers are marked by 1, 2, 3 and 4. Three types of conduits -- P1, P2 and P3 -- are also indicated. (E) A central slices of GCRV virion from (D). A transcriptase complex under a fivefold vertex is indicated by a red arrow. The green arrow indicates an immunoglobulin-like flap domain. (F) A cartoon illustrating organization of capsid proteins within three adjacent asymmetric units. Triangles represent VP5-VP7 complex on the virion.

Fig. 3. VP3A and VP1

(A) VP3A segmented from density map of core capsid. (B) Simulated density map of λ1A from crystal structure of MRV core (PDB code: 1EJ6), which was gaussian-filtered to 9-Å resolution (left) and superposed with its crystal structure (cyan) in ribbon (right) to delineate secondary structural elements. (C) Fitting of λ1A crystal structure of into VP3A density map of GCRV. Helices are in red, an N-terminal helix that cannot be assigned to any density of VP3A is in green, and the rest is in blue (cyan). (D) and (E) are zoom-in views of apical domain and carapace domain, respectively. (F) Fitting of MRV λ2 crystal structure (ribbon, PDB code: 1EJ6) into VP1 density map of GCRV with different domains shown in different colours as labelled.

Fig. 4. Trimers of VP5-VP7 heterodimers

(A) Top view (left) and side view (right) of the superposition of GCRV VP5-VP7 trimer density map with MRV µ1σ3 atomic model (PDB code: 1JMU) shown in ribbon. The three σ3 molecules are located on top and are shown in red, purple, and yellow. The three molecules of µ1 are at the bottom and are shown in blue, green, and cyan. (B) Left: the µ1N (red spheres) and µ1C (gray ribbon) atomic models are fitted into the density map of the VP5-VP7 trimer (semi-transparent purple surface). Right: the crystal structures of one µ1 (purple ribbon) and one σ3 (cyan ribbon) are fitted in our density map.

Fig. 5. Molecular interactions in GCRV virion

(A) Zoom-in view of the core with segmented parts shown in colour. (B) Interactions of VP3 and clamping protein VP6. The two right panels reveal the contact points (highlighted using a colour roughly opposite to the surface colour of the molecule of interest itself) between VP3 and VP6 by computationally rotating VP6A and VP6B away as shown. (C) Cartoon illustrating the interactions between the clamping protein VP6 (yellow) and VP5-VP7 trimers (pink, labelled 1–4) located on the outer capsid. (D) From left to right are segmented trimer 1, 2, 3 and 4 as defined in (C). Colour codes: VP1 in green, VP6 in yellow, VP5-VP7 trimer in pink. Fivefold and threefold vertices are indicated by pentagons and triangles, respectively.