Cosic, A.; Lee, M.; Tobler, K.; Aguilar, C.; Fraefel, C.; Eichwald, C.

Rotavirus (RV) is classified into nine species, A-D and F-J, with RV species A (RVA) being the most extensively studied. While RVA infects infants and young animals, non-RVA species infect adult humans, various mammals, and birds. However, the lack of appropriate research tools has limited our understanding of non-RVA life cycles. RVA replication and assembly occur in cytosolic inclusions termed viroplasms. We recently identified viroplasm-like structures (VLS) composed of NSP5 and NSP2, designated as VLS(NSP2)i, in non-RVA. In this context, globular VLS(NSP2)i formed in RVA, RVB, RVD, RVF, RVG, and RVI, but not in RVC, RVH, and RVJ. Additionally, in RVA, VLS can also be formed through co-expression of NSP5 with VP2, referred to as VLS(VP2)i. Here, we report VLS(VP2)i formation in all non-RVA species except RVB, with notable VLS formation in RVH and RVJ. Moreover, NSP2 RVH or RVJ are recruited into VLS(VP2)i. The NSP5 C-terminal region in non-RVA is required for association with VP2 and forming VLS(VP2)i. Mutation of conserved VP2-L141 in RVA to alanine disrupts viroplasm formation, impairing RV replication. Equivalent residues within the same predicted VP2 region disrupt VLS formation across non-RVA. We also observed interspecies VLS formation, particularly between closely related RVA and RVC, RVH and RVJ, and RVD and RVF. Interestingly, substituting the N-terminal region of VP2 RVB with that of the closely related VP2 RVG restores its ability to form VLS with NSP5 RVB. Elucidating the formation of viroplasms is essential for developing strategies to halt infection across RV species A to J.