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.

非洲猪瘟（African Swine Fever, ASF）是由非洲猪瘟病毒（African Swine Fever Virus, ASFV）引发的高传染性疫病，对全球养猪业构成严重威胁。已有研究报道天然小分子具备抗ASFV的潜力。本研究依托此前建立的高通量ASFV抑制剂筛选平台，筛选得到吡咯里西啶类生物碱（pyrrolizidine alkaloid）瑞替林（rinderine, RIN），该物质不仅可显著抑制ASFV复制，且在感染后24、48及72小时均能维持持久的抗ASFV活性。此外，RIN主要抑制ASFV复制的早期阶段，尤其是通过阻断ASFV对猪原代肺泡巨噬细胞（primary porcine alveolar macrophages, PAMs）的吸附。从机制层面来看，基于质谱的细胞热位移测定、后续实验验证及分子对接分析均表明，前纤维蛋白1（profilin 1, PFN1）是介导RIN抑制ASFV吸附PAMs的关键靶蛋白。结构预测分析、免疫共沉淀实验及共聚焦显微镜成像结果证实，RIN会破坏PFN1与肌动蛋白的相互作用，进而削弱肌动蛋白重塑过程。值得注意的是，非靶向代谢组学分析与后续实验显示，RIN可降低甘油磷脂代谢通路衍生的磷脂酰乙醇胺水平，并降低PAMs的细胞膜通透性，这一作用有助于其发挥抗ASFV活性。综上，整合蛋白质组学与非靶向代谢组学分析结果表明，RIN主要通过靶向与细胞膜形成及功能相关的蛋白和代谢物来发挥抗ASFV作用，为ASF的防控提供了全新的研究思路。