Mammalian reoviruses contain a genome of 10 segments of double-stranded RNA (dsRNA). Reovirus replication and assembly occur within distinct structures called viral inclusions, which form in the cytoplasm of infected cells. Viral nonstructural proteins muNS and sigmaNS and core protein mu2 play key roles in forming viral inclusions and recruiting other viral proteins and RNA to these structures for replication and assembly. However, the precise functions of these proteins in viral replication are poorly defined. Therefore, to better understand the functions of reovirus proteins associated with formation of viral inclusions, we used plasmid-based vectors to establish 293T cell lines stably expressing small interfering RNAs (siRNAs) specific for transcripts encoding the mu2, muNS, and sigmaNS proteins of strain type 3 Dearing (T3D). Infectivity assays revealed that yields of T3D, but not those of strain type 1 Lang, were significantly decreased in 293T cells stably expressing mu2, muNS, or sigmaNS siRNA. Stable expression of siRNAs specific for any one of these proteins substantially diminished viral dsRNA, protein synthesis, and inclusion formation, indicating that each is a critical component of the viral replication machinery. Using cell lines stably expressing muNS siRNA, we developed a complementation system to rescue viral replication by transient transfection with recombinant T3D muNS in which silent mutations were introduced into the sequence targeted by the muNS siRNA. Furthermore, we demonstrated that muNSC, which lacks the first 40 amino residues of muNS, is incapable of restoring reovirus growth in the complementation system. These results reveal interdependent functions for viral inclusion proteins and indicate that cell lines stably expressing reovirus siRNAs are useful tools for the study of viral protein structure-function relationships.