Cytopathic BVDV engages VDAC1 through NS3 to trigger mitochondrial permeability transition-dependent intrinsic apoptosis.

Cytopathic (CP) and non-cytopathic (NCP) biotypes of bovine viral diarrhea virus (BVDV) exhibit strikingly different effects on host cell survival, yet the mechanism that confers cytopathogenicity to CP strains remains unresolved. Here we demonstrate that the nonstructural protein 3 (NS3), uniquely produced by CP viruses, acts as a mitochondrial death trigger by engaging the outer membrane channel voltage-dependent anion channel 1 (VDAC1) to drive Ca²⁺-dependent permeability transition. CP infection, but not NCP infection, provoked mitochondrial Ca²⁺ overload, membrane depolarization, mPTP opening, and caspase-3 activation. Expression of NS3 was sufficient to reproduce these defects, whereas the NS2-3 precursor failed to trigger these events. NS3 partially localized to mitochondria and was enriched at mitochondria-associated ER membranes, where it directly associated with VDAC1. Disruption of the NS3-VDAC1 interface by deletion of residues 618-664 abrogated mitochondrial Ca²⁺ accumulation, prevented mPTP opening, and abolished apoptotic execution. Moreover, pharmacological inhibition of mPTP attenuated NS3-induced mitochondrial dysfunction, supporting a functional requirement for permeability transition. Collectively, our findings establish an NS3-VDAC1-Ca²⁺ axis that links CP-specific NS3 expression to mitochondrial permeability transition and intrinsic apoptosis, thereby providing a mechanistic framework for BVDV cytopathogenicity and providing a molecular explanation for the severe tissue injury associated with mucosal disease.