Members of the genus Orthoreovirus replicate in cytoplasmic inclusions termed viral factories. Compelling evidence suggests that the nonstructural protein NS forms the matrix of the factories and recruits specific viral proteins to these structures. In the first part of this study, we analyzed the properties of avian reovirus factories and NS-derived inclusions and found that they are nonaggresome cytoplasmic globular structures not associated with the cytoskeleton which do not require an intact microtubule network for formation and maturation. We next investigated the capacity of avian reovirus NS to form inclusions in transfected and baculovirus-infected cells. Our results showed that NS is the main component of the inclusions formed by recombinant baculovirus expression. This, and the fact that NS is able to self-associate inside the cell, suggests that NS monomers contain all the interacting domains required for inclusion formation. Examination of the inclusion-forming capacities of truncated NS versions allowed us to identify the region spanning residues 448 to 635 of NS as the smallest that was inclusion competent, although residues within the region 140 to 380 seem to be involved in inclusion maturation. Finally, we investigated the roles that four different motifs present in NS(448-635) play in inclusion formation, and the results suggest that the C-terminal tail domain is a key determinant in dictating the initial orientation of monomer-to-monomer contacts to form basal oligomers that control inclusion shape and inclusion-forming efficiency. Our results contribute to an understanding of the generation of structured protein aggregates that escape the cellular mechanisms of protein recycling.Most viruses replicate within cellular compartments that are termed viroplasms, viral factories, or virus inclusion bodies. These compartments, which are held together by protein-protein interactions, are thought to concentrate those viral components required to increase the overall efficiency of the replication process (22,23). Factories made by cytoplasmic viruses are large inclusions that usually form at pericentriolar sites close to the microtubule-organizing center (MTOC) and resemble in many ways the aggresomes formed in cells in response to protein misfolding/aggregation (14,32,33). Misfolded/aggregated proteins initially form small globular structures that are delivered to the MTOC by dynein-mediated retrograde transport along microtubules for subsequent degradation by proteasomes. When the degradative capacity of the proteasome is exceeded, these aggregates coalesce to form aggresomes, which require microtubules in order to form but not to be maintained. These phase-dense structures located in the perinuclear region are often ubiquitinated, contain dynein, and are surrounded by a vimentin cage to facilitate their positioning near the MTOC.Avian reoviruses (ARVs) are members of the genus Orthoreovirus, one of the 12 genera of the family Reoviridae (1, 20). These viruses have been described as the etiological a...