Jiang Wang, Ma Pengjuan, Deng Libin, Liu Zhi, Wang Xu, Liu Xiyu, Long Gang
Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Institut Pasteur of Shanghai, University of Chinese Academy of Sciences, Beijing, China.
J Extracell Vesicles. 2020 Jan 22;9(1):1716513. doi: 10.1080/20013078.2020.1716513. eCollection 2020.
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.
甲型肝炎病毒（HAV）是一种典型的无包膜病毒，最近发现它主要通过劫持宿主内体转运所需的分选复合物（ESCRT）以准包膜HAV（eHAV）的形式释放。与无包膜病毒粒子不同，eHAV在HAV衣壳表面含有病毒蛋白pX作为VP1的延伸。HAV衣壳如何获得宿主包膜以及pX蛋白是否参与这一过程此前尚不清楚。在此，我们分析了pX在外泌体样细胞外囊泡（EVs）中外源蛋白分泌以及eHAV形成中的作用。pX与eGFP融合通过将eGFP导向多泡体（MVBs）将eGFP引导至外泌体样EVs中，并且凋亡相关基因2相互作用蛋白X（ALIX）的释放显著增强。免疫共沉淀（co-IP）证明了pX与ALIX V结构域之间的相互作用。去除pX的C端一半消除了eHAV的释放并减少了HAV病毒粒子与ALIX之间的相互作用。最后，单独的pX C端一半通过与ALIX相互作用足以将eGFP加载到EVs中。总之，pX的C端部分对eHAV的产生很重要，并且可能具有将大蛋白复合物加载到外泌体样EVs中用于治疗目的的潜力。
