Identification of GRP78 as a novel host factor that facilitates zoonotic porcine deltacoronavirus internalization and replication via clathrin-mediated endocytosis

Porcine deltacoronavirus (PDCoV) infects a wide range of hosts and can spread across species. Moreover, it is capable of infecting pAPN-knockout cell lines and pigs, suggesting that other important host factors facilitate PDCoV infection. To identify these important host factors, in this study, we employed co-immunoprecipitation (Co-IP) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 19 significantly upregulated host membrane proteins that interact with the PDCoV S1 protein. Among these, glucose-regulated protein 78 (GRP78) was found to positively regulate PDCoV attachment and internalization, as validated by knockdown, blocking, and overexpression assays. Affinity assays confirmed a strong interaction between GRP78 protein and PDCoV S1 protein. This regulatory function of GRP78 operates in a pAPN-independent way. Mechanistically, we demonstrated that GRP78 interacts with the PDCoV S1 protein via its substrate-binding domain (SBD) and facilitates viral entry through clathrin-mediated endocytosis. Notably, the role of GRP78 in promoting viral entry and replication has also been applied by other coronaviruses, underscoring its potential as a conserved host factor in coronavirus infection. Together, these findings reveal a novel mechanism of PDCoV-host interaction that centers on GRP78-mediated viral attachment and internalization via clathrin-dependent endocytosis.IMPORTANCEPorcine deltacoronavirus (PDCoV) represents a significant zoonotic threat with pandemic potential, exhibiting a broad tissue tropism that underscores its capacity for cross-species spread. Current understanding of PDCoV entry mechanisms, however, remains largely limited to the porcine aminopeptidase N (pAPN), whose knockout fails to fully block infection-highlighting the critical need to identify alternative host entry factors. In this study, we identified the cell membrane protein GRP78 as a novel host factor that binds the C-terminal domain (CTD) of the PDCoV S1 protein via its substrate-binding domain (SBD), thereby mediating viral adsorption and internalization. Furthermore, GRP78 engages clathrin to facilitate viral internalization through endocytosis. Notably, GRP78-mediated entry operates independently of pAPN and demonstrates a degree of broad-spectrum activity relevant to other coronaviruses. Collectively, these findings provide new insights into the early entry mechanisms of PDCoV and identify GRP78 as a potential broad-spectrum target for antiviral intervention.