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目的对1b型丙型肝炎病毒Con1株的NS5A结构域Ⅰ宿主相互作用蛋白进行鉴定分析,为研究NS5A功能及其参与病毒复制机制提供理论基础。方法构建pGEX-4T-1-Con1-NS5A-35-215质粒,诱导表达GST-Con1-NS5A-35-215融合蛋白并纯化。采用GST pull-down联合液相色谱-串联质谱(LC-MS/MS)对其相互作用蛋白进行鉴定,并使用OmicsBean在线软件对Con1-NS5A-35-215相互作用蛋白进行GO功能富集分析和信号通路分析。结果在Huh7.5.1细胞裂解液中鉴定到547个潜在相互作用蛋白。GO功能富集分析发现相互作用蛋白主要参与细胞运输、应激反应、凋亡、蛋白泛素化等生物学过程。信号通路分析发现相互作用蛋白主要参与蛋白酶体、糖代谢、氨基酸合成、RNA转运等通路。通过免疫印迹验证了Con1-NS5A-35-215可与ACBD3和Vps35结合。结论 NS5A结构域Ⅰ通过其相互作用蛋白参与多种生物学过程,如调控细胞内运输、应激反应、蛋白稳态等过程。与ACBD3和Vps35相互作用的NS5A的片段缩短为35-215。
Objective To identify and analyze the NS5A domain Ⅰ interacting protein of type 1b hepatitis C virus Con1 strain and provide a theoretical basis for studying the function of NS5A and its involvement in viral replication mechanism. Methods The pGEX-4T-1-Con1-NS5A-35-215 plasmid was constructed and the GST-Con1-NS5A-35-215 fusion protein was induced and purified. The interaction proteins were identified by GST pull-down combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the Con1-NS5A-35-215 interacting protein was enriched by OmicsBean online software Signal path analysis. Results 547 potential interacting proteins were identified in Huh7.5.1 cell lysate. GO enrichment analysis revealed that the interacting proteins are mainly involved in biological processes such as cell transport, stress response, apoptosis and protein ubiquitination. Signal pathway analysis found that the major interacting proteins involved in proteasome, glucose metabolism, amino acid synthesis, RNA transport and other pathways. Con1-NS5A-35-215 was demonstrated to bind to ACBD3 and Vps35 by immunoblotting. Conclusion NS5A domain Ⅰ is involved in many biological processes through its interacting proteins, such as regulation of intracellular transport, stress response and protein homeostasis. The fragment of NS5A that interacts with ACBD3 and Vps35 is shortened to 35-215.