Rotavirus replication takes place in the viroplasms, which are cytosolic inclusions allowing the synthesis of virus genome segments, followed by their encapsidation in the core shell and the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which is the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. Aiming to investigate the relationship between rotavirus proteins and host components in the viroplasms, we performed a pull-down assay of lysates from rotavirus-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the TRiC complex, a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our validated results show that TriC is recruited in viroplasms and there specifically surrounds newly formed double-layered particles (DLPs). Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Interestingly, TRiC-inhibited RV-infected cells lacked TLPs but harbored empty DLPs. Using sequence-specific direct RNA nanopore sequencing, we demonstrate that TRiC is essential for RV replication since it modulates dsRNA genome segment synthesis, specifically (-)RNA. Moreover, TRiC is associated with RNA-dependent RNA polymerase, VP1, and regulates the folding of VP2, a cofactor allowing dsRNA synthesis. This study provides in vivo evidence of the regulatory mechanism by which dsRNA genome segment replication is controlled and coordinated in the rotavirus viroplasms.
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