Quantitative mRNA expression comparison of Hepatitis C Virus replicon (2a) on Huh7.5 cell lines. Homo sapiens

We developed transcriptome expression assisted non-directed proteome profiling (TEAnDPP) method to investigate host-pathogen interaction. Analysis of HCV replicon induced host-cell metabolism perturbation at gene expression level. Gene enrichment analysis on DEG revealed disulfide formation related genes were significantly enriched. Based on this observation, we addminitrated thiol reactive chemical probes to visualize reactive thiol profile in live cell, and observed unique reactivity profile. Using SILAC-based quantitative profiling method, we identified 26 proteins that are labeled by iodoacetamide probes. Among these proteins, we discovered t-plastin was upregulated in APC140 cells, and its knock-down experiment showed significant HCV replication inhibition effect. In short, TEAnDPP strategy demonstrated its usefulness in host-pathogen interaction study for HCV infection. Overall design: Total RNA obtained from APC140 (stable cell line expressing HCV2a replicon) was compared to vehicle cell line (Huh7.5).

本研究开发了转录组表达辅助非定向蛋白质组分析（transcriptome expression assisted non-directed proteome profiling, TEAnDPP）方法，用于探究宿主-病原体相互作用。针对丙型肝炎病毒（Hepatitis C virus, HCV）复制子在基因表达水平上诱导的宿主细胞代谢扰动开展分析。对差异表达基因（Differentially Expressed Genes, DEG）的富集分析结果显示，二硫键形成相关基因呈现显著富集。基于上述观测结果，我们施用巯基反应性化学探针以可视化活细胞内的反应性巯基谱，并观测到独特的反应性特征。采用细胞培养中氨基酸稳定同位素标记（Stable Isotope Labeling by Amino acids in Cell culture, SILAC）的定量分析方法，我们鉴定出26种可被碘乙酰胺探针标记的蛋白质。在这些蛋白质中，我们发现t-plastin在APC140细胞中呈上调表达，且其敲低实验显示出显著的HCV复制抑制效果。简言之，TEAnDPP策略在HCV感染相关的宿主-病原体相互作用研究中展现出应用价值。实验整体设计：将从APC140（表达HCV2a复制子的稳定细胞系）中提取的总RNA，与空载体细胞系（Huh7.5）的总RNA进行对比分析。