Background Replication and assembly of vertebrate reoviruses occur in specific intracellular compartments known as viral factories. Recently, NS88 and NS80, the nonstructural proteins from aquareoviruses, have been proposed to share common traits with μNS from orthoreoviruses, which are involved in the formation of viral factories. Methodology/Principal Findings In this study, the NS80 characteristics and its interactions with other viral components were investigated. We observed that the NS80 structure ensured its self-aggregation and selective recruitment of viral proteins to viral factories like structures (VFLS). The minimum amino acids (aa) of NS80 required for VFLS formation included 193 aa at the C-terminal. However, this truncated protein only contained one aa coil and located in the nucleus. Its N-terminal residual regions, aa 1–55 and aa 55–85, were required for recruiting viral nonstructural protein NS38 and structural protein VP3, respectively. A conserved N-terminal region of NS38, which was responsible for the interaction with NS80, was also identified. Moreover, the minimal region of C-terminal residues, aa 506–742 (Δ505), required for NS80 self-aggregation in the cytoplasm, and aa 550–742 (Δ549), which are sufficient for recruiting viral structure proteins VP1, VP2, and VP4 were also identified. Conclusions/Significance The present study shows detailed interactions between NS80 and NS38 or other viral proteins. Sequence and structure characteristics of NS80 ensures its self-aggregation to form VFLS (either in the cytoplasm or nucleus) and recruitment of viral structural or nonstructural proteins.
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