Asymmetric reconstruction of mammalian reovirus reveals interactions among RNA, transcriptional factor µ2 and capsid proteins

Figures

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  Fig. 1. Transcription enzyme complex (TEC) and dsRNA organization revealed by asymmetric reconstruction of mammalian reovirus (MRV) infectious subvirion particle (ISVP). a Surface representation of the asymmetric reconstruction of MRV ISVP. The Y-axis lies along a pseudo-3-fold symmetry axis. b Cut-open view of the capsid, exposing the RNA genome and TECs. The twelve vertices are numbered from 1 to 12 (not all shown). NTPases and RNA-dependent RNA polymerases (RdRps) colored in blue and purple, respectively, and RNA duplexes colored in gray. The red circle indicates the unoccupied 5-fold vertex. The inset (black circle) is the zoom-in view of a TEC and its surrounding RNA duplexes. c Mercator projection of the core density illustrating the organization of ten TECs. The two TEC-absent vertices (red circles at positions 5 and 8) are occupied by three RNA duplexes. d Slice view of the boxed region in (b), highlighting the five concentric layers of RNA duplexes and virus proteins. e Illustration of TEC organization of MRV, cytoplasmic polyhedrosis virus (CPV), and aquareovirus (ARV). The vertices are aligned for ease of comparison. The TEC-absent vertices are highlighted in red. Particle…

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  Fig. 2. In situ structure of the TEC. a Ribbon representation of TEC, which contains NTPase μ2 and RdRp λ3, with domains distinguished by different colors. b Ribbon representation of NTPase μ2 and its three domains, RNA-binding domain (RBD), in green, NTPase domain, in cyan, and C-terminal domain (CTD), in orange. The unmodelled residues are shown in dashed lines colored as domains. The vertical dotted line on the RBD is the divider between the “main” and “tip” subdomain. c–e Ribbon representation of μ2 domains: CTD (c), NTPase domain (d), and RBD (e), reoriented to provide a clear view of the secondary structure elements (labeled). The inset in (d) shows the atomic model (red sticks) and density (mesh) of the bound γ-phosphate group. f Ribbon representation of RdRp λ3 structure and its five domains: N-terminal domain (yellow), thumb (green), fingers (blue), palm (red), and C-terminal bracelet domain (purple). g Superpositions of the ribbon representations of our in situ cryoEM structure [colored as in f] and the crystal structure structures (gray) of λ3. h Right-hand illustration and ribbon representation of the RdRp domains, highlighting the entrances of NTP and template RNA as well as…

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  Fig. 3. The interface between RdRp λ3 and NTPase μ2 and identification of key interacting residues. a, b Ribbon representation of TEC (a), and NTPase μ2 and RdRp λ3 rotated 90° in opposite directions, exposing the interface and the interacting residues between the two proteins (b). c, d Same view as (a, b), with positive, neutral, and negative electrostatic potentials indicated in blue, white, and red, respectively. e, f Same view as (a, b), with hydrophilic, neutral, and hydrophobic area, indicated in cyan, white, and gold, respectively.

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  Fig. 4. Molecular interactions between TEC and RNA. a Surface representation of TEC, with surrounding RNA strands in surface representation and duplexes in both surface and ribbon representation. b 90° rotated view of (a), with ribbon representation of TEC and RNA duplexes. c–g Zoom-in views of the boxed regions in (b), detailing hydrogen bond interactions between TEC and surrounding RNA duplexes. h, i Surface representation of TEC and unwound terminal dsRNA with transcription fork (h), and the 90° rotated view (i). j The zoom-in view of the boxed region in (h), showing the atomic models of initial template RNA and priming loop and the density of putative transcript within the RdRp active site, with RdRp in the ribbon representation and template RNA in stick representation. k Superposition of mesh and stick representation of the template RNA with mesh representation for the putative RNA transcript density. l Zoom-in view of the boxed region in i. Top panel, electrostatic surfaces representation of the RdRp, with positive, neutral and negative Coulomb potentials indicated in blue, white, and red, respectively. Bottom panel, ribbon representation of the RdRp and stick representation of the…

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  Fig. 5. N-terminal fragments and loops of λ1 involved in TEC and RNA interactions. a Surface representation of λ11–5, colored in red, orange, yellow, green, and blue respectively, and TEC, colored as in Fig. 4, viewed from inside of ISVP. b Ribbon representation of λ1, TEC, and surrounding RNA duplexes. Main bodies of λ1A1–5 and λ1B1–5 represented in light and dark gray, respectively. Loops 581–590 of λ1A1–5, the N-terminal fragment of λ1A1–5, and promiscuous loops (PL) 562–571 of λ1B1–5 are colored as in (a). Eye symbols indicate different view directions. c Superposition of ribbon representation of λ11–5 pairs for easy comparison of different conformations, colored as in (b). d–f Ribbon representation of N-terminal fragments of λ1A1–5 from the view indicated by the eye symbols in (b). g Ribbon representation of promiscuous loops of λ1B1–5 from the view indicated by the eye symbols in (b).

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  Fig. 6. Autolytically cleaved penetration protein μ1 with myristoyl groups and interaction loops. a Density map view of ISVP facing the 2-fold axis. λ2 is colored in green, while μ1.Q, S, R, and T trimers are colored in purple, yellow, blue, and red, respectively. White pentagons, triangle, and oval represent the icosahedral 5-fold, 3-fold, and 2-fold symmetry axes, respectively. Black triangle, connecting white symmetry points, represents an asymmetric unit. b Ribbon representation of μ1 trimer, with one μ1 monomer colored by domain (domain I, blue; domain II, green; domain III, yellow; domain IV, magenta). c Ribbon representation of isolated μ1 monomer, showing autolytic cleavage between residues 42 and 43 in the boxed region. d Zoom-in surface representation of μ1 hexagon from (a), formed by μ1 S1, S2, Q2, R2, T, and R1 trimers. e Zoom-in view of R1’s myristoyl group (stick representation) and hydrophobic pocket (ribbon representation), as well as neighboring S1 (ribbon representation) from black box in (d). f Zoom-in view of Q1’s myristoyl group (stick representation) and hydrophobic pocket (ribbon representation), as well as neighboring λ2 (ribbon representation) from blue box in…

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  Fig. 7. Hydrogen bonding between symmetry-mismatched receptor-binding protein σ1 trimer and capping enzyme λ2 pentamer. a, b Surface representation of receptor binding protein σ1, mRNA capping protein λ2, and surrounding μ1. c Ribbon representation of σ1, λ2 and a surrounding μ1 trimer Q1 as viewed from the top of (a). d Superposition of the ribbon representation of our in situ λ2 structure (colored as in a) and X-ray λ2 structure (gray). The stick representation of Q1’s myristoyl group and the ribbon representation of the hydrophobic pocket is also indicated. e Rotated zoom-in view of the dotted box in (d), highlighting the conformational difference of loop 581–588 between in situ and crystal structure. f Clipped view of the dotted pentagon in (c), detailing the interactions between σ1 and λ2. The interacting residues are represented in stick style. g Schematic illustration of symmetry mismatch between σ1 (C3) and λ2 (C5). The two hydrogen bond areas are indicated in transparent light blue.