Supplementary file 1_Megavirus baoshanense Mb0671 modulates host translation and increases viral fitness.docx

Amoeba giant viruses encode many translation-related proteins, but the function of these proteins remains obscure. In the current work, we studied the potential eukaryotic translation initiation factor 4A (eIF4A, Mb0671) encoded by Megavirus baoshanense, a member of the family Mimiviridae. The protein was shown to possesse ATPase activity and RNA-binding capacity, localize in the cytoplasm of infected cells, and present in mature virions. Interactome analysis showed that Mb0671 interacted primarily with ribosomal proteins and translation-related proteins. Specifically, Mb0671 was found to interact indirectly with host eIF4A, suggesting that it was associated with the translation apparatus. Proteomic analysis revealed that the protein profile of Acanthamoeba castellanii cells stably expressing Mb0671 was altered significantly compared to wild-type cells. The cellular proteins that were significantly upregulated included those in the pathways of spliceosome, amino acids biosynthesis, ribosome biogenesis, vesicular transportation, mTOR signaling pathway, etc. Both Mb0671 overexpression or siRNA-mediated reduction of its expression level significantly affected the synthesis of viral proteins. Furthermore, overexpressing Mb0671 accelerated cell growth and virus replication, whereas reduction of Mb0671 expression by siRNA delayed virus replication. These results suggested that Mb0671 altered cellular translation, possibly through its association with the host translation machinery, and played an important role in enhancing virus adaptability.

变形虫巨型病毒（Amoeba giant viruses）编码诸多与翻译过程相关的蛋白质，但其功能至今仍未明确。本研究针对宝山巨型病毒（Megavirus baoshanense，拟菌病毒科（Mimiviridae）成员）所编码的真核翻译起始因子4A（eukaryotic translation initiation factor 4A，eIF4A，基因标识为Mb0671）展开了系统性探究。实验结果表明，该蛋白兼具ATP酶活性与RNA结合能力，定位于受感染宿主细胞的细胞质中，且可存在于成熟病毒粒子内。相互作用组分析显示，Mb0671主要与核糖体蛋白及翻译相关蛋白发生相互作用；尤为关键的是，Mb0671可与宿主eIF4A间接结合，提示其与宿主翻译装置存在紧密关联。蛋白质组学分析结果显示，稳定表达Mb0671的棘阿米巴（Acanthamoeba castellanii）细胞的蛋白表达谱与野生型细胞相比发生了显著改变。其中显著上调的细胞蛋白涉及剪接体通路、氨基酸生物合成、核糖体生物发生、囊泡运输以及mTOR信号通路等多个生物学过程。无论是过表达Mb0671，还是通过小干扰RNA（small interfering RNA，siRNA）下调其表达水平，均会对病毒蛋白的合成产生显著影响。进一步实验发现，过表达Mb0671可加速宿主细胞生长与病毒复制进程，而通过siRNA降低Mb0671的表达则会延迟病毒复制。上述研究结果表明，Mb0671可通过结合宿主翻译机器改变细胞翻译过程，在增强病毒适应性方面发挥着重要作用。