The B169L protein of African swine fever virus functions as a viroporin that activates the calcium-mediated inflammasome.

African swine fever (ASF) is a highly contagious and often fatal viral disease caused by African swine fever virus (ASFV), which poses a significant economic burden on the global pig industry. ASFV infection triggers a robust production of proinflammatory cytokines, leading to severe inflammation that contributes significantly to the high mortality rate associated with ASF. However, the underlying mechanisms remain incompletely understood. Here, we identified the ASFV B169L protein (pB169L) as a viroporin that exerts dual functions in viral replication and proinflammatory responses. We demonstrated that pB169L formed oligomeric calcium (Ca2+)-permeable channels in vitro by bilayer lipid membrane assay. The ectopically expressed pB169L significantly altered Ca2+ homeostasis in cells and induced robust proinflammatory responses. Mutagenesis revealed critical residues-including P29, K55, and K57-that are indispensable for channel function and proinflammatory signaling. Importantly, the B169L gene knockdown during ASFV infection reduced inflammasome activation and viral replication, highlighting its dual role as both a structural component of virus and an inflammatory mediator. These findings provide the first direct evidence that ASFV encodes a functional viroporin and uncover a novel mechanism by which ASFV manipulates Ca2+ homeostasis to drive inflammasome activation, offering new insights into ASFV pathogenesis and potential antiviral targets.