Hepatitis A virus (HAV), a classic nonenveloped virus, has recently been found to be released mainly in the form of quasi-enveloped HAV (eHAV) by hijacking host endosomal sorting complexes required for transport (ESCRT) complexes. Unlike the nonenveloped virion, eHAV contains the viral protein pX on the surface of the HAV capsid as an extension of VP1. How HAV capsids acquire the host envelope and whether the pX protein is involved in this process were previously unknown. Here, we analyse the role of pX in foreign protein secretion in exosome-like extracellular vesicles (EVs) and the formation of eHAV. Fusion of pX to eGFP guided eGFP into exosome-like EVs through directing eGFP into multivesicular bodies (MVBs), and apoptosis-linked gene 2-interacting protein X (ALIX) release was significantly enhanced. Coimmunoprecipitation (co-IP) demonstrated the interaction between pX and the ALIX V domain. Removal of the C-terminal half of pX abolished eHAV release and reduced the interaction between the HAV virion and ALIX. Finally, the C-terminal half of pX alone was sufficient for loading eGFP into EVs by interacting with ALIX. In conclusion, the C-terminal part of pX is important for eHAV production and may have potential for large protein complex loading into exosome-like EVs for therapeutic purposes.

中文翻译：

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甲型肝炎病毒结构蛋白pX与ALIX相互作用，并通过外泌体样小泡促进病毒体和外源蛋白的分泌。

甲型肝炎病毒（HAV）是一种经典的非包膜病毒，最近被发现通过劫持运输所需的宿主内体分选复合物（ESCRT）复合物，以半包膜HAV（eHAV）的形式释放。与非包膜病毒体不同，eHAV在HAV衣壳表面含有病毒蛋白pX作为VP1的延伸。以前尚不清楚HAV衣壳如何获得宿主包膜以及pX蛋白是否参与该过程。在这里，我们分析外泌体样细胞外囊泡（EVs）和eHAV形成中外源蛋白分泌中pX的作用。pX与eGFP的融合通过指导eGFP进入多囊泡体（MVB）引导eGFP进入外泌体样EV，并且显着增强了凋亡相关基因2相互作用蛋白X（ALIX）的释放。免疫共沉淀（co-IP）证明了pX和ALIX V结构域之间的相互作用。去除pX的C末端部分消除了eHAV释放，并减少了HAV病毒体与ALIX之间的相互作用。最后，仅通过pX的C端一半就足以通过与ALIX相互作用将eGFP加载到EV中。总之，pX的C端部分对于eHAV的生产很重要，并且可能具有将大量蛋白复合物装载到外泌体样EV中以进行治疗的潜力。