Foot-and-mouth disease virus non-structural protein 3D induces necroptosis through interaction with RIPK3 in PK-15 and BHK-21 cells.

INTRODUCTION: Foot-and-mouth disease (FMD) is a highly contagious infectious disease caused by foot-and-mouth disease virus (FMDV). FMDV replicates not only in epithelial cells, where it induces vesicular lesions, but also in myocardial and skeletal muscle tissues, leading to myocarditis and even fatal outcomes in infected animals. RIPK3/MLKL-mediated necroptosis is a proinflammatory cell death process that plays a significant role in viral infections. However, whether FMDV infection can trigger necroptosis in host cells remains unclear. METHODS: We assessed necroptosis induced by FMDV infection through activation of the RIPK3/MLKL pathway in PK-15 and BHK-21 cells. Specific inhibitors GSK'872 (targeting RIPK3 phosphorylation) and NSA (targeting MLKL phosphorylation) were applied to examine the impact of this pathway on viral replication. Additionally, Co-immunoprecipitation (Co-IP) was used to analyze the interactions between FMDV-encoded proteins and RIPK3/MLKL, and AlphaFold3 was used to predict the interaction interface between the 3D protein and the RHIM-containing region of RIPK3. RESULTS: FMDV infection activated the RIPK3/MLKL pathway and induced necroptosis in both PK-15 and BHK-21 cells. Inhibitor assays revealed that blocking RIPK3 or MLKL phosphorylation significantly reduced FMDV VP1 expression and viral titers, indicating that the necroptosis pathway favors FMDV replication. Further investigation revealed that the FMDV non-structural protein 3D is the key protein responsible for activating RIPK3/MLKL pathway. Moreover, we confirmed that the 3D protein interacts and colocalizes with RIPK3. AlphaFold3 structural prediction suggested that 3D can directly associate with the RHIM-containing region of RIPK3, with residues R320, H322, E324, and Y346 of 3D interacting with residues N466, V469, and K471 of RIPK3 at the interface. DISCUSSION: Our results demonstrate that FMDV infection induces necroptosis in host cells. This finding expands the modes of host cell death caused by FMDV infection, advances the understanding of the role of necroptosis in viral infections, and may help elucidate the pathogenic mechanisms of FMDV.