Francesca Arnoldi. aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and Echinomycin NS38. Further co-immunoprecipitation analysis confirmed the conversation between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1C471) of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for conversation with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that Echinomycin N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus contamination. Introduction Aquareoviruses, the isolates from aquatic animals, are members of the genus in the family [1]. Grass carp reovirus (GCRV) has been recognized as the most pathogenic among the isolated aquareoviruses [2]. The particle of GCRV is usually non-enveloped with icosahedral symmetry enclosing a segmented double-stranded RNA genome Mmp11 in its central core. The eleven genomic segments encode seven structural proteins (VP1 to VP7) and five nonstructural proteins (NS80, NS38, NS31, NS26 and NS16) [3, 4]. Like other reoviruses, the outer-capsid proteins VP5 and VP7 are required for viral entry into host cells during contamination, whilst other proteins VP1-VP4 and VP6 compose the inner core of aquareovirus, which play an important role in viral replication [5C8]. Similar to other viruses, the replication and assembly of reoviruses take place in specific intracellular compartments called viral inclusion bodies (VIBs), viral factories (VFs) or virioplasms [9C12]. Previous studies have exhibited that the nonstructural protein NS of mammalian orthoreoviruses (MRV) and avian orthoreoviruses (ARV) formed inclusion bodies when expressed alone in cells or during viral contamination [9, 13C16]. And also NS could retain the nonstructural protein NS and inner-capsid proteins within viral factories by interacting with these proteins [9, 14, 17C20]. Moreover, host ribosomal subunits and related proteins involved in translation were found to colocalize with inclusion bodies in MRV [11]. Besides, nonstructural protein NSP5 of rotavirus was able to form virioplasms when expressed alone in cells [21, 22]. Further investigation indicated that rotavirus inner-capsid proteins VP1, VP2, VP3 and VP6 could be also recruited into its inclusions by interacting with NSP5 or NSP2 [23C27]. In addition to the viral proteins, newly synthesized viral RNAs were also located within viral inclusion bodies [18, 28C30]. The nonstructural protein NS80 of aquareovirus, encoded by genome segment S4, is usually consisted of 742 amino acids (aa) with a molecular weight of about 80 kDa [31]. Previous study in our lab has exhibited that NS80 can form viral inclusion bodies in singly expressed or infected cells, and these virioplasms have no colocalization with poly-ubiquitin in infected and transfected cells, indicating that NS80-derived inclusion bodies in cells are not induced by misfolding proteins. And the C-terminal regions including His569 and Cys571 in the intercoil region of NS80 were identified to be crucial for viral inclusions formation [32]. In addition, NS80 is also found to associate with aquareovirus inner-capsid proteins (VP1-VP4, VP6), the putative single-stranded RNA (ssRNA) binding protein NS38 and newly synthesized viral RNAs in both transfected and infected cells [32, Echinomycin 33]. More recently, a report indicated that NS80 was able to coordinate the expression of viral structural proteins and viral replication [33]. To further understand the role of NS80 played in viral replication and assembly, it is necessary to identify functional regions of NS80 that interacted with viral proteins during contamination. In this present study, the functional Echinomycin regions of NS80 associated Echinomycin with proteins VP1, VP4, VP6 and NS38 was defined using RV NSP5-based protein association platform by immunofluorescence assays. And the conversation regions between NS80 and VP1, VP4, VP6, or NS38 were confirmed.