The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication

Abstract

Rotavirus (RV) 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 serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), 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 negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

The Role of the Host Cytoskeleton in the Formation and Dynamics of Rotavirus Viroplasms

“…So far, only RVA has been shown experimentally to induce viroplasm formation; their formation in other species remains to be demonstrated. The viroplasms comprise NSP5, NSP4, NSP2, VP6, VP1, VP2, VP3, virus single-and double-stranded RNA, and host components such as tubulin, perilipin, the host proteasome, and cellular chaperonins [28,[43][44][45][46][47][48]. Finally, viroplasms are adjacent to the ER, enriched in VP4 and VP7 [43,49].…”

“…cellular chaperonins [28,[43][44][45][46][47][48]. Finally, viroplasms are adjacent to the ER, enriched in V and VP7 [43,49].…”

“…Additionally, the globular morphology of the viroplasms seems dependent on the phosphorylation of NSP5, since unphosphorylated NSP5 leads to aberrant viroplasms [68]. However, VP2 also plays a role in the morphology of viroplasms, in which the inhibition of TRiC chaperonin leads to defective VLS composed of NSP5 and VP2 without affecting VLS composed of NSP5 and VP2, suggesting that the proper folding of VP2 is required for viroplasm structure [48]. Similarly, the expression of VP2 harboring L124 mutated to alanine leads to defective viroplasm formation [84].…”

Emergence of a Novel G4P[6] Porcine Rotavirus with Unique Sequence Duplication in NSP5 Gene in China

“…RV finishes its replication in the viroplasms. Several viral proteins form the viroplasms; among these viral proteins, NSP5 acts as the main building block, hence, it was considered to be an essential component for RV replication [ 15 ]. NSP5 affects viroplasm formation and virus replication through interacting with NSP2, NSP6, and VP2 [ 16 ].…”