Comparative analysis of NSP5/VP2-induced viroplasm-like structures in rotavirus species A to J

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 in non-RVA. In this context, globular VLS induced by NSP2 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 the co-expression of NSP5 with VP2. Here, we report that VP2-induced VLS formed in RV species A to J, with notable formation in RVH and RVJ, where NSP2 RVH or RVJ was also recruited into VLSs. The NSP5 C-terminal region in non-RVA is required for association with VP2 and forming VLS. Mutation of conserved VP2-L124 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, most notably between the closely related pairs RVA–RVC, RVH–RVJ, and RVD–RVF. Interestingly, substituting the N-terminal region of VP2 from RVB with that of VP2 from RVG supported VLS formation with NSP5 from RVB in avian cells. Elucidating the formation of viroplasms is essential for developing strategies to halt infection across RV species A to J. IMPORTANCE Rotaviruses (RV) are a group of viruses classified into species A through J, with species A being the best understood. Other RV species infecting animals and humans are less studied due to limited research tools. In RVA, the virus replicates in specialized compartments called viroplasms formed in the cytoplasm by viral proteins, including NSP5, NSP2, and VP2. In this study, we explored how similar structures, termed viroplasm-like structures (VLS), are formed by proteins of RV species A–J. We found that for all tested RV species, NSP5 and VP2 form VLSs. We also identified key regions in the VP2 protein that are essential for forming these structures. Understanding how viroplasms form across different RV species may help develop new strategies to block infection in humans and animals.