CALR-dependent endoplasmic reticulum N-glycoprotein maturation promotes Japanese encephalitis virus entry and replication

The endoplasmic reticulum (ER) chaperone machinery participates in multiple stages of viral infection, from entry through assembly and release. However, how ER-resident quality-control mechanisms shape cellular permissiveness to Japanese encephalitis virus (JEV) remains unclear. Here, we identify the ER-resident chaperone calreticulin (CALR) as a key host factor that promotes cellular permissiveness to JEV infection. CRISPR/Cas9-mediated knockout of CALR markedly reduces JEV attachment, internalization, and productive replication. Mechanistically, CALR depletion disrupts N-linked glycosylation homeostasis and selectively reduces the abundance of the integrin subunit ITGA3 through an ER glycan-dependent maturation checkpoint. Glycomic analysis revealed accumulation of immature N-glycan precursors, indicating compromised N-glycoprotein maturation. In parallel, CALR deficiency markedly reduced the abundance of the viral envelope (E) glycoprotein, suggesting that CALR plays a dual role in shaping host permissiveness and supporting viral glycoprotein biogenesis. Together, these results identify CALR as a central regulator of a replication-permissive ER glycoprotein maturation state that supports both ITGA3-associated cellular adhesion and efficient viral E protein biogenesis, thereby supporting JEV replication. Our findings highlight the calnexin/calreticulin (CANX/CALR)-dependent N-glycosylation axis as a potential host-directed target for antiviral strategies.