viruses Review Trans-Acting RNA–RNA Interactions in Segmented RNA Viruses Laura R. Newburn and K. Andrew White * Department of Biology, York University, Toronto, ON M3J 1P3, Canada * Correspondence: [email protected] Received: 18 July 2019; Accepted: 11 August 2019; Published: 14 August 2019 Abstract: RNA viruses represent a large and important group of pathogens that infect a broad range of hosts. Segmented RNA viruses are a subclass of this group that encode their genomes in two or more molecules and package all of their RNA segments in a single virus particle. These divided genomes come in different forms, including double-stranded RNA, coding-sense single-stranded RNA, and noncoding single-stranded RNA. Genera that possess these genome types include, respectively, Orbivirus (e.g., Bluetongue virus), Dianthovirus (e.g., Red clover necrotic mosaic virus) and Alphainfluenzavirus (e.g., Influenza A virus). Despite their distinct genomic features and diverse host ranges (i.e., animals, plants, and humans, respectively) each of these viruses uses trans-acting RNA–RNA interactions (tRRIs) to facilitate co-packaging of their segmented genome. The tRRIs occur between different viral genome segments and direct the selective packaging of a complete genome complement. Here we explore the current state of understanding of tRRI-mediated co-packaging in the abovementioned viruses and examine other known and potential functions for this class of RNA–RNA interaction. Keywords: RNA structure; RNA–RNA interactions; RNA virus; RNA packaging; segmented virus; influenza virus; bluetongue virus; reovirus; red clover necrotic mosaic virus 1. Introduction RNA viruses are important pathogens that infect a wide variety of hosts. The RNA genomes of these viruses can exist in different forms [1]. Those that are single-stranded (ss) can be either positive-sense [(+) RNA, i.e., messenger-sensed] or negative-sense [( )RNA, i.e., complementary to the − coding strand]. Another class of RNA virus includes those that utilize double-stranded (ds) RNA for their genomes. All of these viruses can be further categorized by the number of genome segments they possess. Those with a single genome segment are referred to as non-segmented, while those with a genome divided into two or more segments are defined according to the scheme by which their genomes are packaged. Viruses that package all of their genome segments into a single particle are called segmented viruses, whereas those that package their genome segments into two or more particles are referred to as multipartite viruses [2,3]. Segmented RNA viruses include all three of the abovementioned RNA genome types and examples of those with ( )RNA, dsRNA, and (+)RNA genomes include, respectively, alphainfluenzaviruses, − orbiviruses, and dianthoviruses. Although these viruses benefit from their ability to reassort genome segments, which allows for replacement of defective segments and rapid evolution, there are also drawbacks associated with partitioning a genome [4]. For instance, these viruses face the task of packaging two or more genome segments into a single virion. This represents a daunting challenge for viruses like alphainfluenzaviruses, which contain eight genome segments, [5]. Clearly, such events must involve some form of communication between the different segments to achieve incorporation of one copy of each into a single particle. The required crosstalk could be mediated by viral or Viruses 2019, 11, 751; doi:10.3390/v11080751 www.mdpi.com/journal/viruses Viruses 2019, 11, 751 2 of 12 Viruses 2019, 11, x FOR PEER REVIEW 2 of 12 mediated by viral or cellular factors, such as proteins. Alternatively, direct interactions between cellular factors, such as proteins. Alternatively, direct interactions between genome components, genome components, i.e., intermolecular or trans-acting RNA–RNA interactions (tRRIs), could also i.e., intermolecular or trans-acting RNA–RNA interactions (tRRIs), could also facilitate co-packaging. facilitate co-packaging. Indeed, the latter strategy appears to be involved in this process for a number Indeed, the latter strategy appears to be involved in this process for a number of segmented RNA of segmented RNA viruses [6–8]. Additionally, there is at least one clear example where the function viruses [6–8]. Additionally, there is at least one clear example where the function of a tRRI extends of a tRRI extends beyond packaging [8]. Accordingly, the potential for this class of interaction to beyond packaging [8]. Accordingly, the potential for this class of interaction to mediate different mediate different viral processes may not yet be fully appreciated. Herein, we examine and discuss viral processes may not yet be fully appreciated. Herein, we examine and discuss the roles of the roles of tRRIs in the reproductive cycles of certain segmented RNA viruses that possess (−)RNA, tRRIs in the reproductive cycles of certain segmented RNA viruses that possess ( )RNA, dsRNA, dsRNA, and (+)RNA genomes. − and (+)RNA genomes. 2. Alphainfluenzaviruses Alphainfluenzaviruses The genus AlphainfluenzavirusAlphainfluenzavirus belongs to the family Orthomyxoviridae and includes important pathogens of animals and humans [5]. [5]. Influenza Influenza A A virus virus (IAV), (IAV), the the type type genus genus of of the the family, family, has a − ( )RNA)RNA genome composedcomposed ofof eight eight linear linear segments segments that that range range from from 2.3 2.3 to 0.9to 0.9 kb inkb length in length (Figure (Figure1A). − 1A).Viral Viral genome genome replication replication occurs occurs in the nucleusin the nu viacleus a (+ via)RNA a (+)RNA antigenome antigenome intermediate, intermediate, and progeny and − progeny( )RNA genomes( )RNA genomes generated generated are transported are transported as viral as ribonucleoprotein viral ribonucleoprotein (vRNP) (vRNP) complexes complexes to the − tocytoplasm the cytoplasm where where they bud they from bud the from plasma the plasma membrane membrane [5]. Within [5]. Within virions, virions, the terminal the terminal regions regions of each ofsegment each segment are associated are associated with RNA with replication RNA replication and transcription and transcription proteins proteins (PB2, PA (PB2, and PB1, PA and the latterPB1, thesubunit latter being subunit the RNA-dependentbeing the RNA-dependent RNA polymerase RNA polymerase or RdRp). The or RdRp). remaining The portions remaining of the portions genomes of theare coatedgenomes with are nucleocapsid coated with protein nucleocapsid (NP) (Figure protei1B).n (NP) Eight (Figure di fferent 1B). vRNPs Eight are different encased vRNPs by a matrix are encasedthat is then by a covered matrix bythat an is outer then lipidcovered envelope by an ou toter create lipid a completeenvelope IAVto create virion. a complete IAV virion. Figure 1. Genome-related structures for Influenza A virus. (A) Linear representation of influenza A Figure 1. Genome-related structures for Influenza A virus. (A) Linear representation of influenza A virus (IAV) genome segments, labeled 1 to 8 (left), along with corresponding proteins encoded by each virus (IAV) genome segments, labeled 1 to 8 (left), along with corresponding proteins encoded by (right). (B) Schematic representation of a generic IAV vRNP complex with relevant structural features each (right). (B) Schematic representation of a generic IAV vRNP complex with relevant structural labelled. Not to scale. (C) Axial view of 7+1 arrangement of vRNPs depicting their parallel alignment features labelled. Not to scale. (C) Axial view of 7+1 arrangement of vRNPs depicting their parallel inside IAV particles. (D) Predicted RNA secondary structures of interacting RNA regions in vRNP 8 alignment inside IAV particles. (D) Predicted RNA secondary structures of interacting RNA regions and vRNP 2 (left) and their in trans RNA–RNA interaction (right) that mediates vRNP association [9]. in vRNP 8 and vRNP 2 (left) and their in trans RNA–RNA interaction (right) that mediates vRNP The local RNA secondary structures (left) are predicted to extend from their respective vRNPs (as association [9]. The local RNA secondary structures (left) are predicted to extend from their respective shown in B, labeled, RNA secondary structure), thereby facilitating nucleation the interaction. vRNPs (as shown in B, labeled, RNA secondary structure), thereby facilitating nucleation the interaction.Portions of the terminal regions of each genome segment are complementary and base paired, and RNA regions located more internally interact with NP to form a double helical structure (Figure1B). Portions of the terminal regions of each genome segment are complementary and base paired, and RNA regions located more internally interact with NP to form a double helical structure (Figure Viruses 2019, 11, 751 3 of 12 NP-bound regions were originally believed to be uniformly coated, with NP associating with the viral RNA in a consistent manner. However, recent studies have revealed that NP, which interacts with ~12 nt long segments of sequence, is spread unevenly on viral RNA, exhibiting regions of high and low density [10–12]. This unequal distribution is likely dictated primarily by RNA sequences and/or structures that influence NP binding, however the specific structural features guiding the differences in concentration remain to be defined. It is also possible that NP binding may be able to remodel or direct the folding of RNA secondary structure, which could, in turn, influence accessibility for tRRI formation. Additionally,