Downloaded from www.microbiologyresearch.org by IP: 54.167.195.90 On: Sat, 18 Jun 2016 01:06:58 Avian and mammalian reoviruses use different molecular mechanisms to synthesize their mNS isoforms Lisa K. Busch, 1 Javier Rodrı ´guez-Grille, 1 J. Ignacio Casal, 2 Jose ´ Martı ´nez-Costas 1 and Javier Benavente 1 Correspondence Javier Benavente franciscojavier.benavente@usc.es Received 24 May 2011 Accepted 27 July 2011 1 Departamento de Bioquı ´mica y Biologı ´a Molecular, Facultad de Farmacia, y Centro de Investigacio ´n en Quı ´mica Biolo ´ gica y Materiales Moleculares, Universidad de Santiago de Compostela, Santiago de Compostela, Spain 2 Departamento de Medicina Celular y Molecular, Centro de Investigaciones Biolo ´ gicas, Consejo Superior de Investigaciones Cientı ´ficas, Madrid, Spain Previous reports revealed that the M3 gene of both avian and mammalian reoviruses express two isoforms of the non-structural protein mNS in infected cells. The larger isoforms initiate translation at the AUG codon closest to the 59 end of their respective m3 mRNAs, and were therefore designated mNS. In this study we have performed experiments to identify the molecular mechanisms by which the smaller mNS isoforms are generated. The results of this study confirmed the previous findings indicating that the smaller mammalian reovirus mNS isoform is a primary translation product, the translation of which is initiated at the internal AUG-41 codon of mammalian reovirus m3 mRNA. Our results further revealed that the smaller avian reovirus mNS isoform originates from a specific post-translational cleavage site near the amino terminus of mNS. This cleavage produces a 55 kDa carboxy-terminal protein, termed mNSC, and a 17 kDa amino-terminal polypeptide, designated mNSN. These results allowed us to extend the known avian reovirus protein-encoding capacity to 18 proteins, 12 of which are structural proteins and six of which are non-structural proteins. Our finding that avian and mammalian reoviruses use different mechanisms to express their mNSC isoforms suggests that these isoforms are important for reovirus replication. INTRODUCTION Mammalian reovirus (MRV) and avian reovirus (ARV) are species groups of the genus Orthoreovirus (Chappel et al., 2005), which differ in host range, degree of pathogenicity, genome coding capacity and in some biological properties. Mammalian reovirus is the prototype of the non-fusogenic orthoreoviruses and avian reovirus is the prototype of the fusogenic orthoreoviruses (Benavente & Martı ´nez-Costas, 2007; Nibert & Schiff, 2001; Zhang et al., 2005). They are non-enveloped viruses that contain a genome formed by ten dsRNA segments enclosed within a double protein-capsid shell. The reoviral genes are transcribed by a virion- associated RNA polymerase to generate mRNAs that are identical to the positive-sense strand of the dsRNA segments encoding them. Viral transcripts, which are designated with lower-case letters, possess a type-1 cap at their 59 ends and lack a polyadenylated 39 tail (Banerjee & Shatkin, 1970; Furuichi et al., 1975; Martinez-Costas et al., 1995). The replication and assembly of reoviruses takes place in distinctive cytoplasmic inclusions called viral factories (Fields et al., 1971), which contain structural and non-structural viral proteins but lack membranes and cellular organelles. The observation that the M3-encoded non-structural mNS protein of either MRV or ARV is the only viral protein that forms factory-like inclusions when expressed in transfected cells suggests that mNS forms the framework of the viral factories in infected cells (Becker et al., 2003; Brandariz- Nun ˜ez et al., 2010; Broering et al., 2002; Tourı ´s-Otero et al., 2004b). Furthermore, it has been shown that viral core proteins and the non-structural protein sNS associate with mNS inclusions in co-transfected cells, thus suggesting that mNS recruits these proteins to the viral factories of infected cells (Miller et al., 2003, 2010; Tourı ´s-Otero et al., 2004a). The M3 genes of MRV and ARV have been reported to express two protein isoforms in infected cells (Tourı´s-Otero et al., 2004b; Wiener et al., 1989). The smaller isoforms could originate from post-translational cleavage or by secondary initiation at in-frame, downstream AUG codons of their respective m3 mRNAs. Compelling experimental evidence suggests that the smaller MRV mNS isoform is a primary translation product that originates by initiation at an internal AUG codon of the MRV m3 mRNA: (i) pulse–chase analysis of MRV-infected cells failed to demonstrate a precursor– product relationship between the two MRV mNS isoforms Journal of General Virology (2011), 92, 2566–2574 DOI 10.1099/vir.0.036459-0 2566 036459 G 2011 SGM Printed in Great Britain