Rotaviruses (RV) are classified into nine species (A–D and F–J). Species A (RVA) is the best studied and primarily infects infants and young animals, while non-RVA species infect adults, various mammals, and birds. However, research on non-RVA species has been limited by the lack of suitable cell systems and molecular tools, leaving their replication mechanisms largely unexplored.

In RVA, replication occurs in cytoplasmic inclusions called viroplasms, composed mainly of NSP5, NSP2, and VP2. In uninfected cells, co-expression of NSP5 with either NSP2 or VP2 can generate viroplasm-like structures (VLSs) that mimic genuine viroplasms but do not produce viral progeny. These VLSs provide valuable models for investigating the molecular basis of viroplasm formation.

We investigated whether NSP5, NSP2, and VP2 from non-RVA species can form VLSs. NSP5 co-expressed with NSP2 produced globular VLSs in RVA, RVB, RVD, RVF, RVG, and RVI, but not in RVC, RVH, or RVJ. In contrast, VP2-induced VLSs formed across all species, with RVH and RVJ also recruiting their respective NSP2 proteins. All NSP5 proteins self-oligomerize, with their C-terminal “tail” regions required for interaction with NSP2 and VP2 and for VLS formation. Interestingly, VLSs formed more readily between closely related species.

Replacing the NSP5 tail in RVH and RVJ with that of RVA restored VLS formation, while mutations in conserved VP2 residues disrupted it. Moreover, swapping the VP2 N-terminus between RVB and RVG also supported VLS formation. These findings reveal conserved principles of viroplasm assembly that could guide strategies to prevent rotavirus infections across species A–J.