Asymmetric reconstruction of the aquareovirus core at near-atomic resolution and mechanism of transcription initiation

The Reoviridae family of dsRNA viruses is characterized by its members’ capacity for endogenous transcription of their multipartite genomes within proteinaceous capsids of 1 to 3 layers. These viruses share inner core particles (ICPs) that conform to icosahedral, T=2*, symmetry, but differ in two major respects: first, the presence or absence of RNA-capping turrets at each icosahedral vertex; second, the number of additional host-specific capsid layers that are often lost upon cell entry. While the role of these additional layers in host infection is generally understood, the absence of asymmetric ICP structures from turreted, multilayered reoviruses has obfuscated our understanding of how successive removal of these external layers impact the structural organization of the ICP and transcription initiation. Here, we present the 3.3 Å resolution structure of the aquareovirus (ARV) ICP, and atomic models of the capsid proteins VP3 and VP6, transcriptional enzymatic complex (TEC) subunits VP2 and VP4, and RNA-capping turret protein VP1. These structures reveal significant differences when compared to those of the coated ARV, as well as their counterparts in single-layered cytoplasmic polyhedrosis virus (CPV). Compared to the double-layered ARV virion and infectious subvirion particle structures, the ARV ICP undergoes significant capsid expansion and widening of the nucleotide processing channels in its TEC and turret. Thus, the loss of outer capsid layers may regulate transcription initiation in ARV, unlike CPV which relies solely on allosteric regulation by binding transcriptional cofactors. These results shed new light on the mechanism of transcription initiation amongst turreted, multilayered members of Reoviridae . asymmetrically reconstructed dsRNA viruses, demonstrating the conformational difference This work provides the first near-atomic resolution asymmetric reconstruction of an ICP from a turreted, multilayered reovirus particle which exhibits subtle but functionally important conformational changes compared to coated ARV structures. These changes in the internal capsid volume, along-side a widening of TEC nucleotide channels and extension of the 5’-cap synthesizing turret proteins create the architectural environment conducive to endogenous transcription. As the ICP genome remains in the quiescent state, it is conceivable that transcription initiation requires factors such as S-adenosyl methionine and NTPs in addition the conformational changes observed here. In this manner, the highly specialized outer capsid layers serve as a useful transcriptional regulator, ensuring that transcription requires not only the presence of cofactors, but also loss of outer capsid layers which occurs upon cell entry. replication. Diagrammatic representation of an Aquareovirus in cross section and the proposed replication process. The identities of viral structural and enzymatic proteins are indicated using the nomenclature for


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Aquareovirus (ARV, Reoviridae) causes hemorrhagic disease in the economically important golden shiner and grass carp of America and China, respectively (Nason et al., 2000;Fang et al., 2005). Reoviridae members are characterized by endogenous transcription of their multipartite genomes within capsids of 1-3 layers and are further classified based on the presence (Spinareovirinae subfamily, 9 genera) or absence (Sedoreovirinae subfamily, 6 genera) of mRNA-capping turrets along the innermost layer (King et al., 2012). The innermost layer of reoviruses is always an icosahedral, T = 2*, inner capsid particle (ICP) or core, which is transcriptionally competent (Farsetta et al., 2000). Among turreted reoviruses, cytoplasmic polyhedrosis virus (CPV) has a single-layered capsid, which is equivalent to the ICP within double-or triple-layered reoviruses (Hill et al., 1999;Zhang et al., 1999;Yu et al., 2011). This simple structural organization makes CPV an attractive model to study turreted reoviruses, but renders it inadequate to describe possible impacts of shedding the external layers from the numerous, multi-layered Spinareovirinae members (Zhang et al., 2022). Here we used a sequential symmetry expansion and relaxation approach to resolve the first asymmetric reconstruction of the ARV ICP by cryoEM to 3.3 Å (Fig. S2). Comparison with existing ARV virion and infectious subvirion particle (ISVP) structures (Ding et al., 2018) reveals expansion of the ICP and concomitant conformational changes to the transcription related proteins.
Lacking the outer capsid proteins VP5 and VP7, the ARV ICP retains the icosahedral, T = 2*, inner capsid shell composed of 60 asymmetric dimers of the 1214-residue, wedge-shaped, VP3 capsid shell proteins (CSPs) and 120 symmetrically arranged copies of the clamp protein (VP6) which form the ICP frame and provide support, respectively (Fig. 1A). VP3 dimers (containing conformers VP3 A and VP3 B ) encircle each 5-fold vertex; with VP3 A conformers seated around the 5-fold vertex center, creating pores adjacent to each transcriptional enzymatic complex's (TEC's) template exit channel for direct transcript capping and release via VP1 turret proteins (TPs) ( Fig. 1C and 1D). VP3 B conformers partially intercalate between VP3 A monomers, and form 3-fold vertices with neighboring decameric assemblies (Fig.  1D). Absent conspicuous VP3 rearrangement, the internal volume of the ICP increased from 5.51 × 10 7 Å 3 to 6.02 × 10 7 Å 3 , or about 9.3% relative to the grass carp reovirus (GCRV) virion and ISVP with which golden shiner reovirus (GSRV) shares 96%-100% a.a. sequence identity ( Fig. 1B) (McEntire et al., 2003;Ding et al., 2018;Wang et al., 2018). By reducing the packaging density and thus viscosity of the genome, the enlarged ICP provides the rigid dsRNA segments greater freedom of movement and presumably reduces the energy required to initiate transcription (Demidenko and Nibert, 2009). The changes undergone by individual decamers includes a 10 Å rise away from the virion origin and subtle expansion of the CSPs (Movie S2). The observed expansion can be attributed to a non-uniform elongation of the CSP monomers approximately 6 Å radially from the icosahedral 5-fold (I5) vertices, relative to their coated counterparts and differs between VP3 conformers (Movies S1 and S3).
Each ARV CSP is divided into three distinct domains which include the apical "tip" nearest the I5 vertices, the large carapace domain, and the small β-sheet rich dimerization domains (Fig.  1D). The local shifts of CSP dimers reveal a non-uniform elongation of the apical and carapace domains, with the alpha helices migrating away from the I5 vertices and towards the icosahedral two-fold and three-fold (I2 and I3) vertices for VP3 A and VP3 B , respectively (Movie S2). In the apical domain helices 13 and 14 and their conjoining loop (a.a. 490-518), which line the I5 transcript exit channel at the luminal side of the capsid, are largely unperturbed by the capsid shifts and help to maintain a consistent pore diameter. By contrast, the other I5 adjacent elements of the apical domains move away from the I5 channel, and the helices 13 and 14 of VP3 B migrate in a similar manner as the other secondary structural elements. Closer inspection reveals striking  conservation of VP3 A residues adjacent to the I5 pore (a.a. 490-518, RMSD 464 Å) when compared to the quiescent virion and ISVP structures (Ding et al., 2018;Wang et al., 2018). Despite exhibiting outward movement, the secondary structural elements of CSPs which interface with the clamp and turret proteins interfaces appear to move as rigid bodies, presumably constrained by interactions with the essential clamp and turret proteins (Movie S2).
Interactions of ARV inner and outer capsid proteins (VP3-VP5) are mediated through the clamp protein VP6, making it important for both stabilization against the genome and outer shell association. Previous work has shown the related mammalian orthoreovirus (MRV) ICPs can be recoated to form ISVPs (Farsetta et al., 2000), suggesting the ARV clamps remain in a VP5 receptive state following uncoating. Superimposition of ICP and virion clamp proteins reveal significant conformational similarity (RMSD 0.667 Å) despite their migration away from the I5 vertices in ICPs. While inconsequential for parental ARV particles, which irreversibly cleave VP5 during entry, this may provide a platform onto which VP5 can bind, compress nascent core particles, and halt transcription inside ARV progeny.
Within ICPs, several dsRNA segments interact with each TEC and these interactions stabilize segments, enabling improved visualization of their major and minor grooves as observed in other RNA viruses (Fig. 1E) (Pan et al., 2021). Five major dsRNA segments are observed adjacent to each TEC and are labeled based on their positions relative to the TEC, with a 6th segment observed adjacent to the template entrance in polar vertices (Fig.  1E). The VP2 RNA-dependent RNA polymerase (RdRp) is organized into the N-terminal domain (NTD), C-terminal bracelet (CTB), and RdRp core which is further differentiated into the thumb, fingers, and palm subdomains (Figs. 1F and S4) (Ding et al., 2018;Wang et al., 2018). The fingers house the NTP entry channel and, with the thumb, facilitate elongation and proofreading while the palm catalyzes phosphodiester bond formation between new NTPs and growing strands via the highly conserved D591, D740, and D741 residues (Fig. S4B). The polymerase possesses several channels to funnel RNA templates and transcription products while the CTB of the inactive ICP does not occlude the template exit channel as in quiescent CPV ( Fig. S4D and Movie S3) (Ding et al., 2018).
Our RdRp structure reveals several local architectural changes within these RdRp channels when compared to the ISVP ( Fig. 1F-H, and Movie S3). Viewed down the template entry channel, a positively charged finger domain loop extends into the template entry channel in the ICP, widening to accommodate nucleic acids (Fig. 1G, Movie S3). From within the channel, the priming loop-thought to separate template and transcript strands-shifts away from the transcript exit channel and orients the catalytically important serine residues towards the would-be incoming template (Fig. 1H, Movie S3). This migration away from the CTB widens the mRNA exit channel, likely promoting exit through the adjacent I5 pores and turrets. From the external TEC view, the RdRp expansion along the capsid lumen appears linked to the radial expansion of the capsid beneath it (Movie S3). This contrasts with the VP4 NTPase which undergoes a unidirectional shift, consistent with the radial expansion of its associated CSP monomers (Movie S3). These movements may be linked to the asymmetric association of the TEC along the expanding decameric subunit. As VP4 is situated primarily atop the VP3 1 dimer it moves along with VP3 1 dimer elongation, whereas the VP2, seated atop VP3 1-4 , is drawn in several directions based on uniform expansion of the capsid and the proportion of RdRp associated with each conformer pair ( Fig. 2A and 2B). As uncoating is necessary to synthesize complete viral transcripts (Farsetta et al., 2000), these subtle conformational changes in the TECs described here may be essential to carrying out efficient viral transcription.
Situated beneath 11 of 12 I5 vertices in ARV are TECs, heterodimers of RdRp and NTPase (Figs. 1A and S3A). NTPase has an RNAbinding domain (RBD) with its "tip" and "main" subdomains, an NTPase domain, and a C-terminal domain (CTD) (Figs. 1I and S5) (Ding et al., 2018). The previously missing tip and much of the main subdomains are now observed extending away from the TEC core towards the template exit channel (Fig. S5). This separation may accommodate dynamic RNA interactions throughout transcription. The newly modeled N-terminal residues also reveal a flexible region homologous to that of ARV's MRV cousin but of a distinct fold (Pan et al., 2021), with extensive genome segment interactions (Fig. 1J).
The N-terminal residues of VP3 A conformers were shown to associate with and lie along the exterior of the TEC, on both RdRp and NTPase (Ding et al., 2018), and were suggested to anchor the TEC into place. The newly modeled VP3 A N-terminal residues include the previously unresolved residues 108-152 (VP3 A1-4 ) which contain Zn-finger domains (a.a. 116-141) ( Fig. 2B and 2E), and conform to a traditional Cys 2 His 2 nucleic acid binding motif ( Fig. 2E) (Yu et al., 2011) Here four newly resolved Zn-fingers (VP3 A1-4 ) lie along the TEC, with VP3 A2-4 situated along an RdRp cleft opposite the NTPase, and VP3 A1 seated along the VP4 NTPase domain forming a four-pronged setting that anchors the TEC complex to the capsid shell (Fig. 2D). The VP3 A3 Zn-finger also contacts the rear bottom genome segment (Fig. 2E), which suggests involvement in transcription initiation as observed in rotavirus (Ding et al., 2019). ARV VP3s are thus multifunctional, promoting TEC assembly and stability while maintaining genome organization in the quiescent particles. Despite expansion of the capsid shell, the Zn-fingers are positioned as in ISVP, suggesting the Zn-fingers function independent of the capsid shell. This may be enabled by the flexible linker within the CSP N-terminal domains (a.a. , which extend to accommodate capsid expansion, while maintaining their TEC association and without altering genome organization or TEC activation state (Movie S3).
Atop each I5 vertex sits a pentameric turret, composed of five copies of VP1 TPs (Figs. 1A and 2F) with their axial channels reaching the nascent mRNA translocating pores of the TEC (Reinisch et al., 2000). When ARV ICP and virion turrets are compared, we observe an axial extension of the TPs and widening of the axial channels ( Fig. 2G and 2H). Local alignment of the 5 TP domains reveals the enzymatic guanylyltransferase (GTase) and methyltransferase I & II (MTase I & II) domains remain stable (RMSD 0.842 Å and 0.907 Å respectively) while domain separation occurs along the flexible linker regions between the GTase, MTase, and flap domains (Fig.  2H). This separation is enabled by the absence of the VP5 layer that would otherwise clash with the ICP turret conformation (Fig. 2G). Despite MRV possessing a similar double layered architecture, analogous TP shifts are not observed when the MRV ICP and complete virions are compared (Reinisch et al., 2000;Pan et al., 2021).
CPV and MRV virions both have spike proteins which occupy the turret channel and serve dual roles, mediating cell entry, and preventing premature transcript escape (Fig. 2F) (Pan et al., 2021;Zhang et al., 2022). As ARV lacks a spike protein homologue, we investigated the flap domains that line the turret exit channel. From virion to ICP, the two C-terminal IG domains undergo an outward shift of 14 Å and counterclockwise twist when viewed from within the I5 channel and originates at the flexible linker region (a.a. 1,129) (Fig. 2I). This finding, coupled with the changes to capsid and TEC architecture, suggests a regulatory mechanism of transcription wherein uncoating yields a particle whose capsid better accommodates genome transcription and whose capping enzymes are widened, or primed, for transcript export. In summary, this work provides the first near-atomic resolution asymmetric reconstruction of an ICP from a turreted, multi-layered reovirus particle. The structure reveals subtle but functionally important conformational changes compared to the structures of coated ARV. These changes in the internal capsid volume, along-side a widening of TEC nucleotide channels and extension of the 5ʹ-cap synthesizing turret proteins create an architectural environment conducive to endogenous transcription. Therefore, the specialized outer capsid layers serve as a useful transcriptional regulator, ensuring transcription requires not only the presence of cofactors, but also loss of outer capsid layers which occurs upon cell entry.