Infectious bronchitis virus accessory protein 3a induces renal inflammatory injury by activating the NLRP3...

Infectious bronchitis virus (IBV) causes severe renal injury in chickens. Previous work showed that IBV promotes renal pathology by activating the NLRP3 (NOD-like receptor family pyrin domain-containing 3)-Caspase-1IL-1β (interleukin-1 beta) axis in collecting duct epithelial cells. Here, we identify viral determinants and upstream signaling pathways that drive this response. We find that the accessory protein 3a interacts with NLRP3, promotes its oligomerization and enhances inflammasome assembly, thereby acting as a key effector. We generated a 3a-knockout virus (rSD-Δ3a) and a start codon substitution mutant (rSD-3a-GTG) and rescued these recombinant viruses. Compared with the parental rSD strain, these mutants reduced Caspase-1 activity and IL-1β maturation and release in chicken embryonic kidney (CEK) cells. In vivo, IBV mutants lacking 3a expression significantly attenuated NLRP3 inflammasome activation and proinflammatory cytokine production in renal tissue, mitigated renal pathology, and improved survival. Mechanistically, IBV infection promotes K⁺ efflux and Ca²⁺ mobilization, which together facilitate NLRP3 inflammasome activation. Protein 3a colocalizes with NLRP3 at the endoplasmic reticulum (ER), enhances ER Ca²⁺ release, and drives mitochondrial Ca²⁺ overload and accumulation of mitochondrial reactive oxygen species (mtROS), both required for NLRP3 activation. Collectively, these findings establish 3a as a viral driver of NLRP3-mediated pathology, advance understanding of IBV renal pathogenesis, and support the rational design of attenuated IBV vaccine strains.IMPORTANCEInfectious bronchitis virus (IBV)-induced renal injury is associated with activation of the NLRP3 inflammasome in collecting duct epithelial cells, yet the mechanism by which IBV promotes inflammasome activation remains unclear. Here, we show that the accessory protein 3a interacts with NLRP3 and promotes its assembly, playing a pivotal role in activating the NLRP3 inflammasome and the subsequent renal injury. Specifically, 3a induces ER Ca²⁺ release and promotes accumulation of mitochondrial reactive oxygen species (mtROS), thereby enhancing NLRP3 inflammasome activation. These findings indicate that IBV 3a is a critical mediator of NLRP3-dependent inflammatory activation, advance our understanding of IBV renal pathogenesis, and provide a rationale and potential targets for the design of gene-deletion attenuated IBV vaccines.