viruses Article Polycistronic Genome Segment Evolution and Gain and Loss of FAST Protein Function during Fusogenic Orthoreovirus Speciation Yiming Yang 1, Gerard Gaspard 1, Nichole McMullen 1 and Roy Duncan 1,2,* 1 Department of Microbiology and Immunology, Dalhousie University, Halifax, NS B3H 4R2, Canada; [email protected] (Y.Y.); [email protected] (G.G.); [email protected] (N.M.) 2 Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada * Correspondence: [email protected] Received: 29 March 2020; Accepted: 25 June 2020; Published: 29 June 2020 Abstract: The Reoviridae family is the only non-enveloped virus family with members that use syncytium formation to promote cell–cell virus transmission. Syncytiogenesis is mediated by a fusion-associated small transmembrane (FAST) protein, a novel family of viral membrane fusion proteins. Previous evidence suggested the fusogenic reoviruses arose from an ancestral non-fusogenic virus, with the preponderance of fusogenic species suggesting positive evolutionary pressure to acquire and maintain the fusion phenotype. New phylogenetic analyses that included the atypical waterfowl subgroup of avian reoviruses and recently identified new orthoreovirus species indicate a more complex relationship between reovirus speciation and fusogenic capacity, with numerous predicted internal indels and 5’-terminal extensions driving the evolution of the orthoreovirus’ polycistronic genome segments and their encoded FAST and fiber proteins. These inferred recombination events generated bi- and tricistronic genome segments with diverse gene constellations, they occurred pre- and post-orthoreovirus speciation, and they directly contributed to the evolution of the four extant orthoreovirus FAST proteins by driving both the gain and loss of fusion capability. We further show that two distinct post-speciation genetic events led to the loss of fusion in the waterfowl isolates of avian reovirus, a recombination event that replaced the p10 FAST protein with a heterologous, non-fusogenic protein and point substitutions in a conserved motif that destroyed the p10 assembly into multimeric fusion platforms. Keywords: orthoreovirus; FAST proteins; syncytium formation; recombination; virus evolution 1. Introduction The fusogenic reoviruses are rare examples of non-enveloped viruses that induce cell–cell fusion and syncytium formation [1]. This diverse group of non-enveloped viruses, with segmented, dsRNA genomes, are members of two distinct but related genera in the Reoviridae family: the Orthoreovirus genus, whose members infect a wide range of vertebrate hosts, and the Aquareovirus genus, whose hosts are restricted to freshwater and saltwater fish [2]. Three of the five recognized and sequenced species in the Aquareovirus genus are fusogenic. Of the seven currently recognized species of orthoreoviruses, only Mammalian orthoreovirus (MRV) and Piscine orthoreovirus (PRV) lack isolates that are fusogenic. Isolates of Reptilian orthoreovirus (RRV), Mahlapitsi orthoreovirus, (MaRV), Nelson Bay orthoreovirus (NBV), Baboon orthoreovirus (BRV), and Avian orthoreovirus (ARV) all induce syncytium formation [3,4]. Furthermore, three additional orthoreovirus species have been proposed, all of which are fusogenic: Broome orthoreovirus (BrRV) from a bat, Neoavian orthoreovirus (ARVN) from wild birds, and Testudine orthoreovirus (RRVT) from a tortoise (Figure1A). The preponderance of fusogenic over Viruses 2020, 12, 702; doi:10.3390/v12070702 www.mdpi.com/journal/viruses Viruses 2020, 12, 702 2 of 20 Viruses 2020, 12, x FOR PEER REVIEW 2 of 20 non-fusogenicnon-fusogenic reoviruses reoviruses suggests suggests there there isis positivepositive evolutionary evolutionary pressure pressure to obtain to obtain and/or and maintain/or maintain the fusogenicthe fusogenic phenotype. phenotype. FigureFigure 1. The 1. The diversity diversity of of orthoreovirus orthoreovirus species,species, their their polycistronic polycistronic genome genome segments, segments, and andtheir their fusion-associatedfusion-associated small small transmembrane transmembrane (FAST) (FAST) proteins. proteins. (A) Maximum (A) Maximum likelihood likelihood unrooted unrooted phylogram basedphylogram on 23 concatenated based on 23 amino concatenated acid sequences amino ofacid the sequences seven consistently of the seven homologous consistently structural homologous proteins (corestructural shell, core proteins RdRp, (core core turret,shell, core core RdRp, NTPase, core coreturret, clamp, core NTPase, outer-shell, core outer-clamp)clamp, outer-shell, encoded outer- by the indicatedclamp) 10 encoded species by or the proposed indicated species 10 species of orthoreoviruses or proposed species (in boldface).of orthoreoviruses See Supplementary (in boldface). See Table S1 Supplementary Table S1 for a list of viruses, proteins, and accession numbers. Lowercase labels for a list of viruses, proteins, and accession numbers. Lowercase labels denote isolates within a species: denote isolates within a species: Gal and Ans are landfowl and waterfowl isolates of avian Gal and Ans are landfowl and waterfowl isolates of avian orthoreovirus (ARV); Bu and Co are bulbul and orthoreovirus (ARV); Bu and Co are bulbul and corvid isolates of neoavian orthoreovirus (ARVN). corvidBootstrap isolates values of neoavian (500 replicates) orthoreovirus are shown (ARVN). for Bootstrapbranch points values where (500 the replicates) associated are taxa shown clustered for branch pointstogether where in the less associated than 100% taxa of the clustered replicate togethertrees. The in tree less with than the 100% highest of thelog replicatelikelihood trees.is shown. The tree withBranch the highest lengths log are likelihood to scale and is measured shown. Branch by the number lengths of are substitutions to scale and per measured site. (B) Diagrams by the numberof the of substitutionspolycistronic per site.S1 and (B) DiagramsS4 genome of segments the polycistronic from the S1indicated and S4 genomeorthoreovirus segments species, from drawn the indicated to orthoreovirusapproximate species, scale. Open drawn reading to approximate frames are scale.depicted Open by rectangles, reading frames labeled are to indicate depicted the by protein rectangles, labeledproduct to indicate and color-coded the protein to productdepict functional and color-coded or evolutionary to depict protein functional relationships: or evolutionary yellow—FAST protein relationships:protein; blue—fiber yellow—FAST protein; protein; green—potential blue—fiber ce protein;ll cycle green—potential inhibitory protein; cell purple—homologous cycle inhibitory protein; purple—homologousproteins with no defined proteins functi withon; no pink—nucleocytoplasmic defined function; pink—nucleocytoplasmic shuttling protein. Numbers shuttling refer protein.to mRNA length. (C) Diagrams of the orthoreovirus FAST proteins, depicting their topology in the Numbers refer to mRNA length. (C) Diagrams of the orthoreovirus FAST proteins, depicting their plasma membrane. Structural motifs contained within their N-terminal ectodomains and C-terminal topology in the plasma membrane. Structural motifs contained within their N-terminal ectodomains cytoplasmic endodomains are color-coded as described in the legend at the bottom. Numbers indicate and C-terminal cytoplasmic endodomains are color-coded as described in the legend at the bottom. the number of residues in each protein. Numbers indicate the number of residues in each protein. Orthoreoviruses are members of the Spinareovirinae subfamily, whose virions have large turrets Spinareovirinae Orthoreovirusesat the icosahedral vertices. are members The doub ofle-layered the capsid containssubfamily, eight structural whose virionsproteins, have five of large which turrets at thecomprise icosahedral the transcriptionally vertices. The double-layeredactive core particle capsid containing contains the eight viral structuralRNA-dependent proteins, RNA five of whichpolymerase comprise (RdRp) the transcriptionally [5,6]. Two of the active three core rema particleining proteins, containing the theouter viral clamp RNA-dependent and outer shell RNA polymeraseproteins, (RdRp)assemble [5 ,into6]. heterohe Two ofxameric the three complexes remaining and proteins,form the theouter outer capsid clamp shell and[7]. During outer shell proteins,reovirus assemble endocytic into cell heterohexameric entry, cathepsins proteoly complexestically and degrade form the the outer outer clamp capsid protein shell to[ 7expose]. During reovirusthe underlying endocytic cellouter entry, shell cathepsins protein that proteolytically mediates the exit degrade of the the core outer particle clamp from protein the endocytic to expose the underlyingpathway outer into shellthe cytoplasm protein that[8]. The mediates RdRp functions the exit of from the corewithin particle the core from particle the to endocytic transcribe pathway mostly into the cytoplasm [8]. The RdRp functions from within the core particle to transcribe mostly monocistronic mRNAs from the 10 genome segments that are capped but not polyadenylated by the turret protein during the exit from the core particle [9,10]. At some stage, interactions between the viral RdRp, Viruses 2020, 12, 702 3 of 20 packaging signals in the viral mRNA, and the core shell protein result in the coordinated replication of the mRNA and the encapsidation of the 10 dsRNA genome segments inside the progeny virus core particles [11,12]. These