viruses Review Captivating Perplexities of Spinareovirinae 50 RNA Caps Justine Kniert †, Qi Feng Lin † and Maya Shmulevitz * Department of Medical Microbiology & Immunology, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada; [email protected] (J.K.); [email protected] (Q.F.L.) * Correspondence: [email protected]; Tel.: +1-780-492-0623 † These authors contributed equally to this work. Abstract: RNAs with methylated cap structures are present throughout multiple domains of life. Given that cap structures play a myriad of important roles beyond translation, such as stability and immune recognition, it is not surprising that viruses have adopted RNA capping processes for their own benefit throughout co-evolution with their hosts. In fact, that RNAs are capped was first discovered in a member of the Spinareovirinae family, Cypovirus, before these findings were translated to other domains of life. This review revisits long-past knowledge and recent studies on RNA capping among members of Spinareovirinae to help elucidate the perplex processes of RNA capping and functions of RNA cap structures during Spinareovirinae infection. The review brings to light the many uncertainties that remain about the precise capping status, enzymes that facilitate specific steps of capping, and the functions of RNA caps during Spinareovirinae replication. Keywords: Spinareovirinae; reovirus; capping; RNA; Orthoreovirus; Aquareovirus; Cypovirus; transcrip- tion; nucleotide; virus 1. Introduction Citation: Kniert, J.; Lin, Q.F.; Shmulevitz, M. Captivating Although this review centers on RNA capping by members of the Spinareovirinae Perplexities of Spinareovirinae 50 RNA subfamily, we will first provide a brief introduction to the virus family phylogeny, basic Caps. Viruses 2021, 13, 294. https:// structure, and replication cycle. doi.org/10.3390/v13020294 Phylogeny. Reoviridae is a family of segmented, dsRNA viruses divided into two subfamilies based on the presence of “turrets/spikes” at the vertices of the viral capsid [1]. Academic Editor: John S. L. Parker The non-turreted members of the Reoviridae family form the Sedoreovirinae subfamily, while the turreted viruses form the Spinareovirinae subfamily, the focus of this review. The Received: 31 December 2020 Spinareovirinae subfamily is composed of nine genera: Orthoreovirus, Aquareovirus, Coltivirus, Accepted: 8 February 2021 Mycoreovirus, Cypovirus, Fijivirus, Dinovernavirus, Idnoreovirus, and Oryzavirus, with a wide Published: 13 February 2021 host range including vertebrates, invertebrates, plants, and fungi. Phylogenetic trees based on the amino acid (aa) sequences of the RNA-dependent RNA polymerases (RdRps) have Publisher’s Note: MDPI stays neutral been built to visualize the relationship between each virus, as depicted in Figure1. Though with regard to jurisdictional claims in structurally conserved, when comparing aa sequences of homologous proteins among published maps and institutional affil- the genera, there is often less than 26% sequence similarity. However, protein regions iations. with structural and/or enzymatic function are typically more similar than the rest of the protein [1]. Structure. All Spinareovirinae members share the same, basic virion structure: concen- tric layers of proteins with icosahedral symmetry encapsidating the segmented dsRNA Copyright: © 2021 by the authors. genomes (Figure2)[ 1]. The number of layers varies among the genera, as depicted in Licensee MDPI, Basel, Switzerland. Figure1. All Spinareovirinae members consist of a core, or innermost particle, which is This article is an open access article transcriptionally active with T = 1 symmetry. The core particle is formed by 60 asymmetric distributed under the terms and homodimers of the major-core structural protein, λ1 of Mammalian Orthoreovirus (MRV, conditions of the Creative Commons or “reovirus” henceforth) [2,3]. All proteins discussed hereafter will be given the MRV Attribution (CC BY) license (https:// designation; however, it is important to recognize that homologous proteins of the other creativecommons.org/licenses/by/ genera may have different names. Enforcing this backbone structure are the “clamp” σ2 4.0/). Viruses 2021, 13, 294. https://doi.org/10.3390/v13020294 https://www.mdpi.com/journal/viruses Viruses 2021, 13, x FOR PEER REVIEW 2 of 27 distributed about the 5-, 3-, and 2-fold axes [2]. At the twelve 5-fold axes sits the pentameric “turrets”, formed by the λ2 protein [2,5]. These turrets form hollow cylinders and possess both methyl- and guanylyl-transferase activity (MTase and GTase, respectively), discussed in detail in later sections. Among all the genera, there are always 12 turrets; however, their shapes and configurations vary. For example, Oryzavirus has both wider and taller turrets compared to MRV [4]. The shape of the λ2 turrets may reflect differences in infection and transcriptional mechanisms; when the outercapsid of MRV is cleaved away, the λ2 turrets take on a more “open” conformation, potentially facilitating the release of mRNA into the host cytoplasm [6]. Viruses 2021, 13, 294 2 of 26 Some Spinareovirinae members, such as Oryzavirus, Cypovirus, and Dinovernavirus, do not have additional layers beyond the core particle. In contrast, Orthoreovirus, Aquareovirus, Coltivirus, Mycoreovirus, Fijivirus, and Idnoreovirus have layers of outercapsid proteins with considerable structuralproteins, similarity which are that only surro presentund the among innermostSpinareovirinae core particle,[2,4]. forming MRV contains a T = 150 copies of 13 symmetrical thestructure. clamp protein,(Figure 2) distributed [1]. Two major about theoutercapsid 5-, 3-, and proteins, 2-fold axes μ1 [and2]. Atσ3, the form twelve 5-fold axes heterohexamerssits (200 the units pentameric for MRV) “turrets”, that attach formed onto the by core the λparticles,2 protein forming [2,5]. These the T turrets = 13 form hollow lattice [3,7]. Thecylinders μ1 proteins and found possess within both thes methyl-e complexes and guanylyl-transferase contact both the σ2 activityclamps and (MTase and GTase, λ2 turrets to anchorrespectively), the outercapsid discussed onto in the detail core in[2,3]. later In sections.the case of Among MRV, allthere the is genera, an addi- there are always tional outercapsid12 turrets;receptor however, binding pr theirotein shapes that is and absent configurations among the other vary. Spinareovirinae For example, Oryzavirus has λ genera, σ1, knownboth to wider bind andboth taller junctional turrets adhesion compared molecule to MRV A [4 ].(JAM-A) The shape and of sialic the acids2 turrets may reflect (SAs) on the surfacedifferences of mammalian in infection cells and [1,8]. transcriptional mechanisms; when the outercapsid of MRV is cleaved away, the λ2 turrets take on a more “open” conformation, potentially facilitating the release of mRNA into the host cytoplasm [6]. Figure 1. Spinareovirinae phylogenetic tree. A phylogenetic tree (neighbour joining) of the Reoviridae Figure 1. Spinareovirinae phylogenetic tree. A phylogenetic tree (neighbour joining) of the Reoviridae subfamily, Spinareoviri- subfamily, Spinareovirinae. Clustering is determined by sequence homology between the RNA-de- nae. Clustering is determined by sequence homology between the RNA-dependent RNA polymerase protein sequences. pendent RNA polymerase protein sequences. Capsid layers found among each genus are indicated. Capsid layers*Oryzavirus found among contains each a complete genus are core indicated. particle, *Oryzavirus and a partialcontains outercapsid a complete more closely core particle, resembling and a inter- partial outercap- sid more closelymediate resembling subviral particles intermediate (ISVPs) subviral seen with particles Mammalian (ISVPs) Orthoreovirus seen with (MRV),Mammalian Figure Orthoreovirus2. Figure generated(MRV), Figure2. Figure generatedusing BLOSUM62 using BLOSUM62 and re-illustrated and re-illustrated using Biorende usingr.com. Biorender.com. Accession Accessionnumbers: (AKG65873, numbers: (AKG65873, ALK02203 ALK02203 for Orthoreovirusfor Orthoreovirus), (AGR34045,), (AGR34045, AAM92745, AAM92745, AAL31497 AAL31497 for Aquareovirus for Aquareovirus), (AAM18342,), (AAM18342, 690891, 690891, AAK00595 AAK00595 for Coltivirus), (001936004,for BAC98431 Coltivirus for), (001936004,Mycoreovirus BAC98431), (AEC32904, for Mycoreovirus AAC36456 for), (AEC32904,Oryzavirus), AAC36456 (AAN46860, for AAK73087 Oryzavirus for), Cypovirus), (Q8JYK1, BAA08542(AAN46860, for AAK73087Fijivirus), (ABB17205 for Cypovirus for),Idnoreovirus (Q8JYK1, BAA08542) and (AAZ94068 for Fijivirus for Dinovernavirus), (ABB17205 ).for Idnoreovi- rus) and (AAZ94068 for Dinovernavirus). Some Spinareovirinae members, such as Oryzavirus, Cypovirus, and Dinovernavirus, do not have additional layers beyond the core particle. In contrast, Orthoreovirus, Aquareovirus, Coltivirus, Mycoreovirus, Fijivirus, and Idnoreovirus have layers of outercapsid proteins with considerable structural similarity that surround the innermost core particle, forming a T = 13 symmetrical structure. (Figure2)[ 1]. Two major outercapsid proteins, µ1 and σ3, form heterohexamers (200 units for MRV) that attach onto the core particles, forming the T = 13 lattice [3,7]. The µ1 proteins found within these complexes contact both the σ2 clamps and λ2 turrets to anchor the outercapsid onto the core [2,3]. In the case of MRV, there is an additional outercapsid receptor binding protein that is absent among the other Spinareovirinae