China-Japan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China.
Cytometry A. 2011 Aug;79(8):653-60. doi: 10.1002/cyto.a.21094. Epub 2011 Jun 27.
The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5'-end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs.
丙型肝炎病毒（HCV）的翻译起始于其基因组 RNA 5' 端的内部核糖体进入位点（IRES）。为了研究 HCV IRES 的功能，我们构建了一个报告质粒，该质粒可生成编码两种荧光蛋白的双顺反子 mRNA：依赖 cap 的 DsRed2 和依赖 IRES 的 Azami Green（AG）。我们将该质粒转染入 Huh7.5.1 和 HEK293 细胞，并通过流式细胞术测量单个细胞中 AG 信号与 DsRed2 信号的比值来测定 IRES 的相对活性。为了比较我们的方法和传统的生化方法，我们构建了一个结构相似的报告质粒，其中 Renilla 和 Firefly 荧光素酶分别替代了 DsRed2 和 AG。使用这些系统，我们发现 IRES 的 A164G 取代降低了其活性，干扰素 α以细胞类型特异性的方式影响 IRES 活性，并且针对 IRES 的合成 micro-RNA 能够抑制基因表达。总之，在研究 IRES 突变和宿主细胞类型时，这两种方法的灵敏度相当。我们讨论了我们发现的意义以及细胞测量法的潜在优势：可应用于 HCV 翻译的分子研究以及抗 IRES 药物的筛选。
