Rinderine suppresses African swine fever virus replication in porcine alveolar macrophages by dual targeting of membrane-associated profilin 1 and phosphatidylethanolamine

African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV), posing a serious threat to the global pig industry. Natural small molecules have been reported to exhibit anti-ASFV potential. This study utilized the previously established high-throughput platform for screening ASFV inhibitors to identify rinderine (RIN), a pyrrolizidine alkaloid that not only significantly inhibits ASFV replication but also maintains sustained anti-ASFV activity at 24, 48, and 72 hours postinfection. Moreover, RIN mainly suppresses the early stages of ASFV replication, particularly by inhibiting ASFV attachment to primary porcine alveolar macrophages (PAMs). Mechanistically, mass spectrometry-based cellular thermal shift assay, further experimental validation and molecular docking analysis indicate that profilin 1 (PFN1) is a key target protein mediating the inhibitory effects of RIN on ASFV attachment to PAMs. Structural prediction analysis, co-immunoprecipitation assay, and confocal microscopy demonstrate that RIN disrupts the interaction between PFN1 and actin, thereby decreasing the actin remodelling. Notably, untargeted metabolomics profiling and further experiments reveal that RIN reduces the phosphatidylethanolamine level derived from the glycerophospholipid metabolism pathway and decreases the membrane permeability of PAMs, contributing to its anti-ASFV activity. In conclusion, integrative proteomics and untargeted metabolomics analyses demonstrate that RIN exerts the antiviral effects against ASFV primarily by targeting proteins and metabolites associated with cell membrane formation and function, providing a novel insight into the prevention and control of ASF.