Recruitment of TRiC chaperonin in rotavirus viroplasms directly associates with virus replication.

Rotavirus replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell followed by 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. To investigate the relationship between rotavirus proteins and host components of 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 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 triple-layered particles (TLPs) but harbored empty DLPs. Through sequence-specific direct RNA nanopore sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly (-)ssRNA. Moreover, TRiC associates and regulates the folding of VP2, a cofactor allowing dsRNA synthesis. This study provides in-cell culture evidence of the regulatory mechanism by which dsRNA genome segment replication is controlled and coordinated in the rotavirus viroplasms. ImportanceThe replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the eleven double-stranded RNA genome segments are synthesized and packaged individually into the newly generated virus particles. In this study, we show for the first time that the TRiC complex, a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral (-)ssRNA. Specifically, TRiC interacts and assists in folding VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited in viroplasm to assist replication.