Small interfering RNA targeted to stem-loop II of the 5' untranslated region effectively inhibits expression of six HCV genotypes.
Small interfering RNA targeted to stem-loop II of the 5' untranslated region effectively inhibits expression of six HCV genotypes.
复制标题
DOI：
10.1186/1743-422x-3-100
复制
发表时间：
2006-11-27
期刊：
影响因子：
4.8
通讯作者：
Dash S
中科院分区：
文献类型：
--
作者：
Prabhu R;Garry RF;Dash S
干扰素的抗病毒作用是针对丙型肝炎病毒的5‘非翻译区(UTR)，通过内部核糖体进入位点(IRES)机制翻译蛋白质。虽然这个序列在不同的临床菌株中高度保守，但大约一半的慢性丙型肝炎患者对干扰素治疗没有反应。因此，开发针对5‘非编码区的小干扰RNA(SiRNA)来抑制IRES介导的翻译可能是一种绕过干扰素耐药性问题的替代方法。制备了四种不同的质粒载体，用于靶向丙型肝炎病毒5‘非编码区茎环II-III的siRNAs的细胞内递送。利用绿色荧光蛋白(GFP)基因与6种不同丙型肝炎病毒IRES序列之间的嵌合克隆，研究了siRNA的产生对IRES介导的翻译的影响。针对茎环II的siRNA有效地介导了6种不同的丙型肝炎病毒IRES mRNA的降解，并抑制了GFP的表达，而针对茎环III的siRNA则没有。此外，胞质内siRNA的表达可有效降解丙型肝炎病毒基因组RNA，并抑制丙型肝炎病毒1a株和1b株感染性克隆的核心蛋白表达。这些体外研究表明，针对茎环II的siRNA可以高效地抑制IRES介导的主要丙型肝炎病毒的翻译。茎环II siRNA可能是开发基于细胞内免疫策略的抗病毒治疗的良好靶点，以抑制未被干扰素抑制的丙型肝炎病毒株。
The antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.