Backbone model of an aquareovirus virion by cryo-electron microscopy and bioinformatics

Abstract

Grass carp reovirus (GCRV) is a member of the aquareovirus genus in the Reoviridae family and has a capsid with two shells-a transcription-competent core surrounded by a coat. We report a near-atomic-resolution reconstruction of the GCRV virion by cryo-electron microscopy and single-particle reconstruction. A backbone model of the GCRV virion, including seven conformers of the five capsid proteins making up the 1500 molecules in both the core and the coat, was derived using cryo-electron microscopy density-map-constrained homology modeling and refinement. Our structure clearly showed that the amino-terminal segment of core protein VP3B forms an approximately 120-A-long alpha-helix-rich extension bridging across the icosahedral 2-fold-symmetry-related molecular interface. The presence of this unique structure across this interface and the lack of an external cementing molecule at this location in GCRV suggest a stabilizing role of this extended amino-terminal density. Moreover, part of this amino-terminal extension becomes invisible in the reconstruction of transcription-competent core particles, suggesting its involvement in endogenous viral RNA transcription. Our structure of the VP1 turret represents its open state, and comparison with its related structures at the closed state suggests hinge-like domain movements associated with the mRNA-capping machinery. Overall, this first backbone model of an aquareovirus virion provides a wealth of structural information for understanding the structural basis of GCRV assembly and transcription.

Fig. 1

Cryo-EM density map and backbone models of GCRV. (a) Density map of an asymmetric unit (left panel) segmented out from the GCRV virion reconstruction (upper right). The trimers (Q, R, S, T) are labeled using the nomenclature of bluetongue virus and are colored differently. (b) Density map of an asymmetric unit of the core (left). The core map (upper right) was computationally extracted from the GCRV virion map shown in (a). (c) Cryo-EM densities (wire frames) and models (ribbons) of α-helices (left and middle) and a β-sheet (right) selected from VP6A and VP3A. Densities corresponding to some bulky side chains and partially resolved β-strands are resolved. (d) Backbone models of the virion (left) and an asymmetric unit (right). (e) Backbone models of the core (left) and an asymmetric unit (right). Different proteins (VP1, VP3A, VP3B, VP6A, VP6B and VP1) are shown in different colors.

Fig. 1

Cryo-EM density map and backbone models of GCRV. (a) Density map of an asymmetric unit (left panel) segmented out from the GCRV virion reconstruction (upper right). The trimers (Q, R, S, T) are labeled using the nomenclature of bluetongue virus and are colored differently. (b) Density map of an asymmetric unit of the core (left). The core map (upper right) was computationally extracted from the GCRV virion map shown in (a). (c) Cryo-EM densities (wire frames) and models (ribbons) of α-helices (left and middle) and a β-sheet (right) selected from VP6A and VP3A. Densities corresponding to some bulky side chains and partially resolved β-strands are resolved. (d) Backbone models of the virion (left) and an asymmetric unit (right). (e) Backbone models of the core (left) and an asymmetric unit (right). Different proteins (VP1, VP3A, VP3B, VP6A, VP6B and VP1) are shown in different colors.

Fig. 2

Molecular interactions involved in the assembly of the core. (a and b) VP3A and VP3B models (ribbons) superimposed with their cryo-EM density maps (semitransparent gray). Their subdomains are shown in different colors. (c) Three copies of VP3B organized as a group of three around a 3-fold axis, as viewed from inside the virion. The icosahedral symmetry axes are designated by numbers (“2”, “3” and “5”, for 2-, 3- and 5-fold axes, respectively). The area enclosed in the red dashed box is enlarged and rotated 90° and shown in the box on the right. (d) The extended loops under the icosahedral 2-fold axis (indicated by “2”) and their zoomed-in views. In the zoomed-in view on the right, the atomic model is superimposed in the semitransparent density map and the amino acids contributing to the formation of the kinked bridge are indicated. (e) Organization of the core. Left: ribbon model of the core showing the organization of VP3A (green), VP3B (red) and VP6 (cyan); right: zoomed-in view of a region containing VP3A, VP3B, VP6A and VP6B. The backbone models are shown as ribbons and are superimposed with the cryo-EM density maps shown semitransparently.

Fig. 3

VP3B–RNA interactions and conformational changes of VP3B between the virion and the transcription-competent core. (a) Stereo zoomed-in view of the loop region of VP3B facing the dsRNA genome inside the virion. The dashed region in the inset illustrates the region of the VP3B shown. The N-terminal extension is highlighted in blue, while the C-terminal loop (residues 175–186) of VP3B is highlighted in red. (b) Side view of a 12-Å-thick slab cut along the line drawn in the inset icon view of (a). (c) Negative-stain EM image of transcription-competent transcribing cores. The strands attached to the icosahedral vertices are nascent RNA molecules being released from the cores. (d) Negative-stain EM image of the intact non-transcribing virions. (e) A region of the core inside the virion, containing three copies of VP3B and a copy of VP3A, as viewed from outside of the virion. (f) Structure of the transcription-competent core (gray) superimposed with the virion molecules (color) shown in (e). (g) Zoomed-in view of the area marked by the red box in (f) after a rotation of 180°. (h) Top: Side view of the yellow boxed region in (g); bottom: backbone models of the same shell proteins (same view and color scheme). (i) Identical with (g) except that the blue virion VP3B was removed. The blue amino-terminal region is disordered and not visible in the transcription-competent core. (j) Top: Side view of the yellow boxed region in (i); bottom: backbone models of the same shell proteins (same view and color scheme).

Fig. 4

Comparison between GCRV VP1 and MRV λ2. (a) Ribbon diagram of VP1 with functional domains shown in different colors. (b) Superposition of VP1 (red) and MRV λ2 (green), with enlarged views of the flap and the GTase domains. The channel for nascent mRNA release is marked by a dotted blue circle. (c) A slab of the cryo-EM map of pentameric turret (transparent) superimposed with five copies of VP1 models shown as ribbons. The arrow points to a protruding β-hairpin (Leu47–Thr57). The inset shows the core and the position of the slab (pointed by the two arrows). (d) Schematic illustration of VP1 depicting putative conformational changes (arrows) associated with the transition from the open state to the closed state of the turret protein. The right inset shows the VP1 GTase domain density (pink) superimposed with its backbone model (green ribbon).

Fig. 5

Molecular interactions involved in the assembly of the outer shell. (a) A copy of VP1 and adjacent trimer Q and a zoomed-in view of their contact sites. Key residues at the contact sites are indicated. (b) Top view of the trimers R and S. (c) Side view of trimers R and S. (d) Two slabs as indicated in (c). (e) Zoomed-in view of the contact sites between the VP5 molecules in two neighboring trimers.