DNAJ Homolog Subfamily C Member 11 Stabilizes SARS-CoV-2 NSP3 to Promote Double-Membrane Vesicle Formation.

Coronaviruses, particularly those classified as highly pathogenic species, pose a significant threat to global health. These viruses hijack host cellular membranes and proteins to facilitate their replication, primarily through the formation of replication organelles (ROs). However, the precise regulatory mechanisms underlying RO formation remain poorly understood. To elucidate these mechanisms, we conducted mass spectrometry analyses, identifying interactions between the host protein DnaJ homolog subfamily C member 11 (DNAJC11) and the SARS-CoV-2 nonstructural protein 3 (NSP3) protein. Notably, results showed that DNAJC11 depletion reduces SARS-CoV-2 infection, indicating possible positive regulatory involvement. But the ectopic expression of DNAJC11 did not lead to marked alterations in immune or inflammatory responses. DNAJC11 enhanced NSP3 expression stability through endogenous apoptosis pathways and facilitated its interaction with NSP4, thereby promoting the formation of double-membrane vesicles (DMVs). Knockdown of DNAJC11 reduced DMV number and size, accompanied by dysregulation of the endoplasmic reticulum and mitochondria. However, supplementation with DNAJC11 restored both DMV number and size. These findings provide novel insights into the role of DNAJC11 as a host factor that modulates DMV formation and supports SARS-CoV-2 replication by targeting the NSP3 protein. This study advances our understanding of the molecular interactions between host and viral components and highlights DNAJC11 as a potential target for antiviral interventions.