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bioRxiv · 10.1101/2025.06.09.658619

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

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

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. ImportanceRotaviruses (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 other RV species. We found that in most species, NSP5 and VP2 form VLSs. One exception was RVB, where VLS formation was observed only when the VP2 protein was mutated to resemble that of a related species. 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.

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Cosic, A., Lee, M., Tobler, K., Aguilar, C., Fraefel, C., Eichwald, C.. 2025-06-10. Comparative analysis of NSP5/VP2-induced viroplasm-like structures in rotavirus species A to J. https://doi.org/10.1101/2025.06.09.658619

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