The structure of a 12-segmented dsRNA reovirus: New insights into capsid stabilization and organization

Abstract

Infecting a wide range of hosts, members of Reovirales (formerly Reoviridae) consist of a genome with different numbers of segmented double stranded RNAs (dsRNA) encapsulated by a proteinaceous shell and carry out genome replication and transcription inside the virion. Several cryo-electron microscopy (cryo-EM) structures of reoviruses with 9, 10 or 11 segmented dsRNA genomes have revealed insights into genome arrangement and transcription. However, the structure and genome arrangement of 12-segmented Reovirales members remain poorly understood. Using cryo-EM, we determined the structure of mud crab reovirus (MCRV), a 12-segmented dsRNA virus that is a putative member of Reovirales in the non-turreted Sedoreoviridae family, to near-atomic resolutions with icosahedral symmetry (3.1 Å) and without imposing icosahedral symmetry (3.4 Å). These structures revealed the organization of the major capsid proteins in two layers: an outer T = 13 layer consisting of VP12 trimers and unique VP11 clamps, and an inner T = 1 layer consisting of VP3 dimers. Additionally, ten RNA dependent RNA polymerases (RdRp) were well resolved just below the VP3 layer but were offset from the 5-fold axes and arranged with D5 symmetry, which has not previously been seen in other members of Reovirales. The N-termini of VP3 were shown to adopt four unique conformations; two of which anchor the RdRps, while the other two conformations are likely involved in genome organization and capsid stability. Taken together, these structures provide a new level of understanding for capsid stabilization and genome organization of segmented dsRNA viruses.

Fig 1. Overall structure of mud crab reovirus (MCRV).

(A) A radially colored, shaded surface representation of the MCRV icosahedral reconstruction as viewed along a 5-fold axis is shown. The icosahedral asymmetric unit contains 13 copies of VP12 arranged as 5 trimers: P, Q, R, S and T. Trimer T locates around the icosahedral three-fold axis and thus contributes a monomer to the asymmetric unit. The icosahedral asymmetric unit is shown in (B). (B) The left panel shows an outside view, while the right panel shows the interior view. The inner capsid proteins (VP3A and VP3B), clamp protein (VP11) and outer capsid protein (VP12) are colored in cornflower blue, orange, green and medium purple, respectively. 5-fold, 2-fold and 3-fold axes are indicated by black pentagon, ellipse and triangle respectively. (C) The D₅ reconstruction of MCRV indicates that there are 10 well-resolved densities corresponding to VP1 (RdRp) located near 10 vertices and arranged with D₅ symmetry. Under the pole vertices (arrows indicated), the density of expected VP1 is too weak to be annotated. VP1 is highlighted in dark red. The densities of other proteins are in grey. (D) The density map of one vertex indicates the arrangements of major capsid proteins in the D₅ reconstruction are the same as that of icosahedral reconstruction. The interior view (right) illustrates that the only difference between the icosahedral and D₅ reconstructions is the dark-red colored VP1 near the 5-fold vertex. The left panel is an outside view while the right panel is an interior view. The color scheme is the same with that of Fig 1B and 1C.

Fig 2. Structure of inner capsid protein VP3.

(A) Different conformations of the inner capsid protein VP3A (cornflower blue) and VP3B (orange). Both VP3A and VP3B contain apical (red ellipse), carapace (green triangle) and dimerization domains (purple rectangle). VP3B has an extended N-terminus composed of 2 helices and connecting loops. (B) The five “helix-loop-helix-loop” N-termini of VP3B are indicated using balls and highlighted in different colors. They interact with different VP3 dimers and form a “belt” that stabilizes the inner shell. (C) The N-terminus of VP3A differs from that of VP3B. Three VP3A N-termini, highlighted by purple balls, have similar conformations, while the other two VP3A locations have different conformations (in red and cyan respectively) in the D₅ reconstruction. These two N-termini brace the polymerase (gray density) and may help to hold the polymerase to the inner surface of the capsid. The major parts of VP3A and VP3B are colored in cornflower blue and orange respectively, same as that in Fig 1B. The alignment of all VP3A show in (D) indicates that all VP3As are nearly identical, except for the different conformations of the N-terminus. (E) The alignment of the N-terminus of VP3A and VP3B clearly show four different possible N-terminal configurations for VP3. In (F), the large separations among adjacent VP3 pentamers can be seen. (G) A zoomed-in view of the boxed area in (F) shows the large separation at the VP3 dimerization domains. The black oval indicates the 2-fold axis.

Fig 3. Structure of VP12 and interactions within the VP12 trimers.

(A) The model of a VP12 trimer is shown. Three monomers are in medium purple, goldenrod and gray respectively. (B) A single VP12 subunit is annotated. The strand nomenclature is indicated using the upper-case letters A to I. The exterior loops of strands A’A” and BC give rise to a roughly 8Å wide hydrophobic concavity indicated by a red asterisk. (C) A zoomed-in view of the orange boxed area in (A) is shown. Gly164 in one VP12 interacts with the Ser223 and Asn226 of a neighboring VP12 by hydrogen bonds. (D) A zoomed-in view of the green boxed area in (A) illustrates that the PPPG motif in the linker loop interacts with Lys96 of one VP12 and the Arg31 of a neighbor VP12 via hydrogen bonding. There are multiple hydrogen bonds, such as the one between Arg273 and Lys32 of an adjacent VP12.

Fig 4. Locations and structure of VP11 implies that it may function as a clamp protein in MCRV.

(A) The outside view of MCRV capsidis shown. In (B), a zoomed-in view of the boxed regions in (A) can be seen; relative orientation of this view can be inferred based on the indicated location of the 5-fold vertex. Type I, II and III channels are labeled and show the potential aqueous channel positions. The color scheme is the same as that of Fig 1B. VP11 (green) not only inter-digitates the VP12 trimers, the two copies of VP11s in an asymmetric unit also block type II (VP11A) and III (VP11B) channels. (C) VP11 sits in the middle of the six VP12 trimers and interacts with the 6 VP12 trimers in different manners, clamping these trimers together. (D) The atomic model of VP11 is shown in rainbow color. The colors from blue to red indicate the N-terminal to C-terminal. (E) VP11 has a core hydrophobic α-helix (L82-Y98) with five aromatic residues (red). Panel (F) shows the same view in (A) after removing all VP12 trimers. Panel (G) shows a zoomed-in view of the boxed region in (F). VP11 rests on top of VP3 dimers from the inner capsid. (H) VP11B is located on the top of the separation between VP3 dimers from adjacent pentamers, interacting with VP3A from one pentamer and VP3B from the other pentamer. Through these interactions, VP11B clamps the two neighbor pentamers together. The color schemes are the same as that in Fig 1B, except for that in (D).

Fig 5. Structure of VP1 (RdRp).

(A) The model of the RNA dependent RNA polymerase (RdRp), VP1, is shown. Like other Reovirus polymerases, VP1 contains an N-domain (yellow), C-domain (purple) and polymerase domain. The polymerase domain is further divided into the finger subdomain (blue), palm subdomain (dark red) and thumb subdomain (green). The Polymerase domain of the RdRp is shown in (B). The purple arrow indicates the primer grip, the black arrow indicates the priming loop and the orange arrow indicates the unique long loop blocking the gap between the thumb and finger subdomain. (C) The N-domain has a unique protrusion (black circle). (D) The C-domain is shown. The red arrow indicates the C-terminal plug. The overall view of the inner VP3 layer and the RdRp protein VP1 is shown in (E), while (F) shows a zoomed-in view of the green box in E. The VP3A1 N termini (residues 59–69) (red arrow) interacts with the finger subdomain of the RdRp VP1 (blue). (G) A zoomed-in view of the black box area in E is shown. N-terminal residues (40–48, black arrow) of VP3A2, and residues 1220–1226 (green arrow), 67–74 (blue arrow) and 1390–1395 (red arrow) of VP1 form an anti-parallel β-sheet. (H) The zoomed-in view of the cyan-dashed box in F demonstrates the interactions between the apical domain of VP3B and the VP1. Residues 318–346 of VP3B apical domain (orange) form a helix-loop-helix structure along the inner surface and interact with residues 642–657 (blue) of finger subdomain of an RdRp. Residues Lys642 and His646 in the RdRp appear to form a salt bridge with Glu318 of VP3B.

Fig 6. The extra densities in the transcribing MCRV (tMCRV).

(A) The difference map between tMCRV and qMCRV shows the extra density (light blue) at the 5-fold vertex that has RdRp density underneath. The color schemes for models VP12 and VP3A are same as that of Fig 1B. The extra density is displayed at a contour level of about 8.3. (B) The extra density together with the RdRp of tMCRV is shown. Part of two copies of VP3A model is shown to indicate the position of the inner capsid. The extra density appears to be an RNA transcription product, together with the noncontinuous, non-template single strand RNA (also see S11 Fig). The RdRp density is displayed at a contour level of about 2.6. The color scheme for RdRp is same as that in Fig 5. In right panel, red dotted line indicates the transcript product exiting the RdRp and then through the gap among five VP3As at the 5-fold vertex.