p17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories

In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/ Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-MG-treated cells in a dose-dependent manner while the expression levels of viral proteins sigma A, sigma C, and sigma NS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of sigma C, sigma A, and sigma NS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins sigma NS and mu NS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein sigma C, inner core protein sigma A, and nonstructural protein sigma NS of ARV in viral factories. Depletion of Hsp90 inhibited sigma A, sigma C, and p17 proteins colocalized with sigma NS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins sigma A, sigma C, and p17 from colocalizing with sigma NS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins sigma A, sigma C, and sigma NS and facilitates accumulation of the outercapsid protein sigma C and inner core protein sigma A in viral factories for virus assembly.

Automated summary

This study determines the essential role of heat shock protein 90 (Hsp90) in avian reovirus (ARV) replication by chaperoning the ARV p17 protein. The Hsp90/Cdc37 chaperone complex protects p17 from degradation and enhances viral protein stability and virus production. Depletion of Hsp90 prevents viral proteins sigma A, sigma C, and p17 from colocalizing with sigma NS in viral factories.