Duck Tembusu virus NS3 protein induces apoptosis by activating the PERK/PKR pathway and mitochondrial pathway

ABSTRACT Duck Tembusu virus (DTMUV) infection causes severe infectious diseases in poultry and can induce apoptosis in host cells. In this study, the mechanisms underlying DTMUV-induced apoptosis were investigated. First, DTMUV infection can activate the endoplasmic reticulum stress (ERS), and administration of the ERS inhibitor 4-phenylbutyric acid can protect cells from DTMUV-induced apoptosis, indicating that ERS is involved in DTMUV-induced apoptosis. Interestingly, suppression of either apoptosis or ERS led to impaired DTMUV proliferation. Then, we found that DTMUV activated three branches of unfolded protein response signaling [RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1, and activating transcription factor 6] in duck embryo fibroblasts. Further study revealed that activation of PERK-eukaryotic initiation factor 2α up-regulated CCAAT/enhancer-binding protein homologous protein and DNA damage-inducible protein 34, which subsequently promoted apoptosis. Moreover, we found that among the DTMUV viral proteins, the nonstructural protein 3 (NS3) is the main inducer of apoptosis. On the one hand, the PERK/PKR pathway is involved in the NS3-mediated mitochondrial apoptosis pathway. On the other hand, NS3 interacts with voltage-dependent anion channel 2 (VDAC2) and inhibits the anti-apoptotic protein VDAC2 to induce apoptosis, which is accompanied by the depolarization of mitochondrial membrane potential and accumulation of intracellular reactive oxygen species. This study provides a theoretical basis for revealing the pathogenic mechanism of DTMUV infection and lays a foundation for finding antiviral targets. IMPORTANCE Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses.