Abstract
Upon influenza A virus (IAV) infection, the IAV progeny ribonucleoprotein complex, with a defective viral genome, is sensed by DNA-dependent activator of interferon-regulatory factor (DAI). DAI initiates the recruitment of an array of proteins to form a multiprotein platform (PANoptosome), which triggers apoptosis, necroptosis, and pyroptosis during IAV infection. However, the mechanisms mediating the assembly of the PANoptosome are unclear. Here, we identified a scaffold protein, sperm-associated antigen 9 (SPAG9), which could interact with DAI to promote cell death during IAV infection. We further demonstrated that the cell death enhanced by SPAG9 was achieved through the DAI/SPAG9/c-Jun N-terminal kinase (JNK) axis, which could promote IAV-induced DAI-mediated cell death, including apoptosis, necroptosis, and pyroptosis. Our data further showed that the DAI/SPAG9/JNK signaling pathway enhanced the interactions among receptor-interacting serine/threonine kinase 1 (RIPK1), RIPK3, and DAI, thereby promoting IAV-induced PANoptosome formation. Overall, our study for the first time revealed a feed-forward circuit signaling pathway that enhanced IAV-induced DAI-mediated cell death, provided insights into the molecular mechanisms of cell death, and established therapeutic targets to address infectious and inflammatory diseases. IMPORTANCE Upon influenza A virus (IAV) infection, DAI is activated, recruits downstream proteins to assemble a multiprotein platform (PANoptosome), and then triggers cell death. Until now, the protein composition and assembly mechanism of the PANoptosome during IAV infection had not been elucidated. Using proximity labeling and mass spectrometry technology, we identified SPAG9 as a novel component of the PANoptosome and confirmed that SPAG9 promotes IAV-induced cell death by enhancing the interaction among RIPK1, RIPK3, and DAI. Our study will broaden the knowledge of the molecular mechanisms of cell death.