Captivating Perplexities of Spinareovirinae 5′ RNA Caps

Figures

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  Figure 1. Spinareovirinae phylogenetic tree. A phylogenetic tree (neighbour joining) of the Reoviridae subfamily, Spinareovirinae. Clustering is determined by sequence homology between the RNA-dependent RNA polymerase protein sequences. Capsid layers found among each genus are indicated. *Oryzavirus contains a complete core particle, and a partial outercapsid more closely resembling intermediate subviral particles (ISVPs) seen with Mammalian Orthoreovirus (MRV), Figure 2. Figure generated using BLOSUM62 and re-illustrated using Biorender.com. Accession numbers: (AKG65873, ALK02203 for Orthoreovirus), (AGR34045, AAM92745, AAL31497 for Aquareovirus), (AAM18342, 690891, AAK00595 for Coltivirus), (001936004, BAC98431 for Mycoreovirus), (AEC32904, AAC36456 for Oryzavirus), (AAN46860, AAK73087 for Cypovirus), (Q8JYK1, BAA08542 for Fijivirus), (ABB17205 for Idnoreovirus) and (AAZ94068 for Dinovernavirus).

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  Figure 2. Structures of Mammalian Orthoreovirus (MRV) 3. Left: Transcriptionally active MRV cores composed of the λ1 shell (grey) held together by the σ2 clamps (green). The λ2 turrets (red) are found at each vertex, below which the RNA-dependent RNA polymerase (RdRp) λ3 (maroon) and μ2 (purple) are predicted to sit; Middle: Intermediate subviral particles (ISVPs) are generated through proteolytical cleavage within endosomes after cell-entry. The σ3 outercapsid protein is completely cleaved away, while μ1 (light blue) is cleaved to δ. The σ1 attachment protein (yellow) is also cleaved at the N-terminus. ISVPs may also be generated in the natural niche, which for MRV is the intestinal tract; Right: Transcriptionally inactive whole virions are generated from core particles becoming decorated with σ3 (dark blue) and μ1 heterohexamers, forming the outercapsid. Additionally, complete σ1 proteins also attach to the λ2 pentamers. Figure generated using Biorender.com.

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  Figure 3. Replication cycle of Mammalian Orthoreovirus 3. (1) Receptor-mediated attachment of complete virions via σ1 to junctional adhesion molecule A (JAM-A) and/or sialic acid (SA) residues on the mammalian cell surface; (2) Following membrane attachment, virions enter the cell via clathrin-mediated endocytosis; (3) Endosomal acid-dependent proteases digest the MRV outercapsid, generating ISVPs before the eventual release of core particles into the cytoplasm; (4) Now-transcriptionally active core particles produce mRNAs (initial replication) using the viral λ3 polymerase; (5) After being secreted through the λ2 channels, mRNAs are translated into proteins. Viral factories are formed which serve as sites for viral replication, and (6) assemble into more core particles; (7) These newly generated cores then transcribe more mRNA, which in turn is translated into more proteins and assembles into more cores; (8) Outercapsid proteins assemble onto cores, halting transcription and producing progeny fully-infectious virions; (9) Mature infectious virions egress from the cell and disseminate. Figure generated using Biorender.com.

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  Figure 4. mRNA capping by Spinareovirinae. (A) A depiction of the cap structures on 5′ termini of a dsRNA segment; (B) Proposed stepwise enzymatic generation of cap structures found within Spinareovirinae RNA. An RdRp catalyzes the first step of the reaction, production of (+)RNA. Secondly, RTPase-mediated hydrolysis of the γ phosphate produces diphosphate RNA 5′ ends. In the third step, guanylyltransferase activity mediates the addition of guanosine via a reverse 5′ to 5′ triphosphate linkage. Methyltransferases then mediate methylation at the N7 position of the guanosine cap (step 4), and at the 2′O position of the adjacent first mRNA nucleotide (step 5), producing cap0 and cap1 structures sequentially. While it is debatable whether reovirus RNAs have additional methylation of the second RNA nucleotide to produce cap2 structures, such an activity would also require a methyltransferase; (C) Depictions of the molecular structures of RNA cap0, cap1, and cap2 with key methylated residues. Figure generated using Biorender.com.

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  Figure 5. Structure of the Spinareovirinae RNA-dependent RNA polymerase. (A) The MRV λ3 RdRp (pink) (PDB accession #1MUK) depicted in approximate location relative to a single capsid unit (λ2 (red), σ2 (green), and λ1 (grey)) (PDB accession #6XF8); (B) Structure of the MRV λ3 protein with each domain denoted by a different colour: N-terminus in orange, Thumb in green, Palm in red, Fingers in blue, and C-terminus in pink (PDB accession #1MUK); (C) Structure of grass carp reovirus (GCV) VP2 (λ3 homolog) in complex with VP4 (μ2 homolog) (PDB accession #6M99). Black spheres on VP4 represent the conserved NTPase motifs. Structure was rotated in order to visualize the different channels within the λ3 complex. Figure generated using Biorender.com.

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  Figure 6. λ1 contact sites. (A) Structural depiction of a reovirus vertex. λ1 dimers are light-colored grey/pink/blue/green and derived from MRV core structure PDB 6M99 after superimposed onto the GCV VP3 λ1-homologue from PDB 1EJ6. The first resolvable amino acids at amino ends of the five vertex-proximal λ1 monomers are colored pink, representing the location of disordered N-termini proposed to have NTPase activity for MRV. The VP2 of GCRV (MRV λ3 RdRp homolog) and VP4 of GCRV (MRV μ2 homolog) are colored orange and yellow, respectively, and obtained from PDB 6M99; (B) Multiple sequence alignments of MRV λ1 homologs among Orthoreoviruses and Aquareoviruses acquired from ClustalOmega and visualized using Jalview 2.11.1.3. Accession numbers are provided in the figure. Conserved motifs (Walker A, Walker B, and helicase-like) are boxed in red. MRV, Mammalian orthoreovirus; PorcRV, Porcine orthoreovirus, BRV, Baboon orthoreovirus; ARV, Avian orthoreovirus; MDRV, Muscovy duck reovirus; GCRV, Grass carp reovirus; MERV, Marbled eel reovirus; FCRV, Fall chinook aquareovirus.

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  Figure 7. Conserved residues among μ2 homologs. Multiple sequence alignments for μ2 homologs among Orthoreoviruses and Aquareoviruses acquired using ClustalOmega and viewed using Jalview 2.11.1.3. Accession numbers are provided in the figure. Sequences of interest resembling the A (residues 426–437) and B (residues 462–465) motifs of ATPases are boxed in red. MRV3, Mammalian orthoreovirus serotype 3; ARV, Avian orthoreovirus; BRV, Baboon orthoreovirus; FCRV, Fall chinook aquareovirus; GCRV, Grass carp reovirus; MERV, Marbled eel reovirus.

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  Figure 8. Proposed models for mRNA transcription mechanisms. Three proposed models for the RTP/NTPase accessing the 5′ terminus during transcription: (A) The nascent RNA exits the polymerase and undergoes 5′phosphohydrolysis before then returning to the polymerase for elongation followed by ejection; (B) The RTP/NTPase generates diphosphate primers that are then used by the polymerase for nascent strand generation; (C) Nascent RNA becomes the next (+) genomic RNA template that is then dephosphorylated after passing through the template exit portal. Figure generated using Biorender.com.

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  Figure 9. Spinareovirinae turret protein structure (MRV) and sequence alignments. (A) The λ2 monomer (PDB 1EJ6) with highlighted domains labelled. The moving loops (red) within the MTase 1 domain are highlighted using arrows. Lysine residues at position 171 and 190 are also highlighted using arrows (cyan) within the active site of the GTase domain. Figure generated using Biorender.com; (B) Multiple sequence alignment of λ2 homologs among Orthoreoviruses and Aquareoviruses acquired using ClustalOmega and viewed using Jalview 2.11.1.3. Accession numbers are provided in the figure. Conserved lysine residues 181 and 201, corresponding to MRV λ2 K171 and K190, are denoted by arrowheads. Conserved histidines 234 and 243, corresponding to MRV λ2 H223 and H232, are denoted with stars. Lysine 226 found in certain Orthoreoviruses is denoted with a diamond. Conserved aspartic acid 846 and glycine 848, corresponding to MRV λ2 D827 and G829, are denoted with arrows; (C) Multiple sequence alignment of λ2 homologs among all Spinareovirinae genera acquired using ClustalOmega and viewed using Jalview 2.11.1.3. MRV, Mammalian orthoreovirus; PorcRV, Porcine orthoreovirus, PiscRV, Piscine orthoreovirus; CRV,…