GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

ABSTRACT Hepatitis E virus (HEV) is a viral hepatitis pathogen that poses a significant threat to global human health, representing a serious yet long-overlooked public health concern. In this study, we identified glucose-regulated protein 75 (GRP75) as an interaction partner of HEV-ORF2 using recombinant ORF2 truncation as bait. The substrate-binding domain of GRP75 interacted with HEV-ORF2 and inhibited HEV replication by facilitating HEV-ORF2 degradation. Further analysis revealed that HEV-ORF2 contains three KFERQ-like motifs, the key signature sequence required for chaperone-mediated autophagy (CMA). Our data demonstrated that GRP75-mediated degradation of HEV-ORF2 was heat-shock cognate protein 70 (HSC70)-dependent, although no direct interaction between HSC70 and ORF2 was detected. Instead, GRP75, together with HEV-ORF2 and HSC70, formed a complex that mediated CMA-dependent degradation of HEV-ORF2, whereas deletion of all three KFERQ-like motifs from ORF2 conferred resistance to such processes. Additionally, GRP75 blocked mitochondrial transport of HEV-ORF2, potentially mitigating ORF2’s function as an interferon (IFN) induction antagonist. Furthermore, GRP75 enhanced the interaction between mitochondrial antiviral signaling protein (MAVS) and TANK-binding kinase 1 (TBK1), promoting IFN-β production and ultimately inhibiting HEV infection. In conclusion, our findings identify GRP75 as a novel restriction factor for HEV infection and provide new insights into its role in CMA and antiviral innate immunity. IMPORTANCE Due to the lack of an effective in vitro model, the viral-host interaction of HEV remains largely elusive. This study uncovers a novel mechanism by which GRP75 inhibits HEV infection. On one hand, the GRP75 protein facilitates the degradation of HEV-ORF2 through the lysosome-associated, chaperone-mediated autophagy by recognizing KFERQ-like motif presented on HEV-ORF2. On the other hand, GRP75 enhances the production of IFN-β by promoting interaction between MAVS and TBK1, thereby establishing an antiviral state and suppressing HEV infection. This research expands our current understanding of host resistance to HEV and provides a new function of GRP75, suggesting that GRP75 might be a novel antiviral factor against virus infection.