Sub-headings HCV uses CMA and eMI for facilitating viral propagation
Aims We previously reported that HCV NS5A binds to the chaperone protein HSC70, thereby promoting HSC70 recruitment to the KFERQ-motif on the target protein and causing protein degradation via Chaperone-mediated autophagy (CMA) or endosomal microautophagy (eMI). To elucidate the regulatory mechanisms of CMA and eMI in HCV infection, we investigated the interaction between HCV NS5A and HSC70 and its role in viral infection.
Methods To examine a role of HSC70 in HCV proliferation, we analyzed the levels of HCV RNA and HCV proteins in shHSC70-mediated knockdown cells. As the interaction of HCV NS5A with HSC70 is essential for the enhanced recruitment of HSC70 to the target proteins, we examined its interaction with NS5A using HSC70 dominant-negative mutants, which lacks ATPase activity. To examine the effect of HSC70 function on HCV infection, HCV-infected cells were treated with the HSC70 inhibitor.
Results The levels of HCV RNA and HCV protein were markedly reduced in HSC70-knockdown cells, suggesting that HSC70 is essential for HCV proliferation. HSC70 K71M mutant increased interaction with HCV NS5A. Moreover, NS5A protein levels, but not HCV RNA levels, were decreased by HSC70 K71M mutant. HCV NS5A protein levels were decreased with the treatment of VER155008. These results suggest that HSC70 with ATPase activity tightly associates with target proteins and weakly associates with HCV NS5A, resulting in stabilization of NS5A protein. On the other hand, HSC70 dominant negative mutant tightly associates with NS5A and affects NS5A structure, resulting in the decrease of NS5A protein levels. Taken together, HSC70 ATPase activity is crucial for HCV proliferation.
Conclusions We provide evidence suggesting that HSC70 ATPase activity is crucial for acceleration of HCV proliferation. We are currently investigating the details of the HCV strategy via the CMA and eMI pathways in viral infection.