The oncolytic avian reovirus p17 protein triggers chaperone-mediated autophagy by modulating Hsp90 and the T-complex protein-1 ring complex chaperones and co-chaperones to activate the IKK/NF-κB signaling

This study is the first to reveal that oncolytic avian reovirus (ARV) modulates the IKK/NF-κB signaling through the Hsp90-Cdc37, T-complex protein-1 ring complex (TRiC)/Hsc70, and TRiC-phosducin-like protein 1 (PhLP1) chaperone complexes, thereby inducing chaperone-mediated Autophagy in mammalian and human Cancer lines (referred to as Vero and A549). Specifically, the ARV p17 protein elevates the binding of co-chaperone PhLP1 to TRiC, thereby reducing the complex formation of TRiC-Hsc70 and leading to IκB degradation, suggesting that these two co-chaperones exhibit a competitive relationship. The p17 protein of ARV plays a crucial role in enhancing the formation of the TRiC/PhLP1 complex to protect Viral Proteins from ubiquitin-proteasome-mediated degradation. Interestingly, we found that ARV p17 enhances the interaction between the HSP90/Cdc37 chaperone complex and IKK through activation of Casein Kinase 2 (CK2), which in turn activates NF-κB. Moreover, ARV p17 transcriptionally upregulates Beclin1 and increases the formation of Beclin1-PtdIns3K complex through the CK2-Hsp90/Cdc37 pathway. Immunofluorescence staining reveals that ARV p17 promotes the formation of GFP-LC3 puncta in both Vero and A549 cell lines, while a significant reduction in GFP-LC3 puncta was observed in CK2 and HSP90 knockdown cells. Interestingly, in situ proximity ligation assay indicated that ARV p17 promotes the interaction between LC3-II and the cytosolic chaperonin complex 2, triggering chaperone-mediated Autophagy. This study provides novel insights into ARV-modulated suppression of IκB by regulating co-chaperone PhLP1 and Hsc70 binding to TRiC and activation of the CK2/HSP90/Cdc37/IKK/NF-κB pathway to induce chaperone-mediated Autophagy.

Importance: This study reveals that the oncolytic avian reovirus (ARV) p17 activates chaperone-mediated Autophagy in Vero and A549 cells by modulating the HSP90/Cdc37, T-complex protein-1 ring complex (TRiC)/Hsc70, and TRiC/phosducin-like protein 1 (PhLP1) chaperone complexes. ARV p17 enhances PhLP1 binding to TRiC, reducing TRiC/Hsc70 complex formation, which promotes IκB degradation and activates the IKK/NF-κB pathway. Additionally, p17 increases Casein Kinase 2 (CK2)-mediated phosphorylation, strengthening the HSP90/Cdc37/IKK interaction and transcriptionally upregulating Beclin1, forming the Beclin1/PtdIns3K complex to further induce Autophagy. Immunofluorescence shows p17-induced GFP-LC3 puncta, which decreases upon CK2 and HSP90 knockdown. In situ proximity ligation assay revealed that p17 promotes LC3-II and CCT2 interactions, confirming chaperone-mediated Autophagy activation. This study provides novel insights into ARV-modulated suppression of IκB by modulating co-chaperone PhLP1 and Hsc70 binding to TRiC and activation of the CK2/HSP90/Cdc37/IKK/NF-κB pathway to induce chaperone-mediated Autophagy. This work expands our understanding of the role of ARV in regulating host cell Autophagy pathways and viral replication. It also provides a new avenue for understanding viral modulation of host cellular processes in the context of oncolytic virotherapy.