Mammalian reoviruses are nonenveloped particles containing a genome of 10 double-stranded RNA (dsRNA) gene segments. Reovirus replication occurs within viral inclusions, which are specialized nonmembranous cytoplasmic organelles formed by viral nonstructural and structural proteins. Although these structures serve as sites for several major events in the reovirus life cycle, including dsRNA synthesis, gene segment assortment, and genome encapsidation, biochemical mechanisms of virion morphogenesis within inclusions have not been elucidated because much remains unknown about inclusion anatomy and functional organization. To better understand how inclusions support viral replication, we have used RNA interference (RNAi) and reverse genetics to define functional domains in two inclusion-associated proteins, muNS and mu2, which are interacting partners essential for inclusion development and viral replication. Removal of muNS N-terminal sequences required for association with mu2 or another muNS-binding protein, sigmaNS, prevented the capacity of muNS to support viral replication without affecting inclusion formation, indicating that muNS-mu2 and muNS-sigmaNS interactions are necessary for inclusion function but not establishment. In contrast, introduction of changes into the muNS C-terminal region, including sequences that form a putative oligomerization domain, precluded inclusion formation as well as viral replication. Mutational analysis of mu2 revealed a critical dependence of viral replication on an intact nucleotide/RNA triphosphatase domain and an N-terminal cluster of basic amino acid residues conforming to a nuclear localization motif. Another domain in mu2 governs the capacity of viral inclusions to affiliate with microtubules and thereby modulates inclusion morphology, either globular or filamentous. However, viral variants altered in inclusion morphology displayed equivalent replication efficiency. These studies reveal a modular functional organization of inclusion proteins muNS and mu2, define the importance of specific amino acid sequences and motifs in these proteins for viral replication, and demonstrate the utility of complementary RNAi-based and reverse genetic approaches for studies of reovirus replication proteins.