HCV INTERNAL RIBOSOME ENTRY SITE AS TARGET FOR THERAPY

我们实验室的广泛和长期目标是定义病毒RNA中独特的结构和功能区域，使它们能够在不遭受严重细胞损害的情况下受到攻击。在美国和国外有数以百万计的慢性丙型肝炎患者，但目前的治疗方法的有效性是一个难题。因此，基于针对病毒独特的RNA序列、结构和功能的新的抗病毒疗法，丙型肝炎病毒是领先的候选药物。由于这种治疗在丙型肝炎病毒基因组的保守序列中效果最好，我们的项目将专注于病毒RNA的5‘末端区域，其中包括内部核糖体进入位点(IRES)，这是一种不寻常的结构，丙型肝炎病毒和其他几种RNA病毒利用这种结构来发出蛋白质合成开始的信号，这与细胞mRNA分子使用传统的7-甲基鸟苷帽子结构形成了鲜明对比。这项建议的具体目的是：(I)为丙型肝炎病毒基因组保守的5‘端400碱基绘制保守序列、二级结构和局部三级结构元素的区域，强调结合所有三种元素的独特区域。我们已经使用的技术，包括寡核苷酸结合、凝胶移位、RNaseH-定向切割、RNaseIII酶切和RNA-RNA紫外光交联都将被使用。(Ii)我们将使用核糖体结合和蛋白质合成分析来确定IRES功能所需的确切RNA结构域，并将测试修饰和突变的序列以绘制其精细结构图。我们的直接核糖体结合位点分离方法利用网织红细胞裂解物中的RNA酶保护，将被用来精确定位这些IRES序列及其在AIM(I)中衍生的结构。(Iii)我们将针对AIMS(I)和(II)中描述的关键功能结构域设计核酶靶向序列和其他试剂。包括设计和测试肝细胞中人核糖核酸酶P的外部引导序列(EGSS)。

The broad, long-term objectives of our laboratory are to define unique regions of structure and function in viral RNAs, allowing them to be attacked without sustaining heavy cellular damage. There are millions of patients with chronic HCV infection in the United States and abroad, but the efficiency of present therapies is a difficult problem. HCV is thus a leading candidate for new antiviral therapies based on targeting unique RNA sequences, structures and functions of the virus. Since such treatments will work best in conserved sequences of the HCV genome, our project will focus on the 5' terminal domain of the viral RNA, which includes the Internal Ribosome Entry Site (IRES), the unusual structure with which HCV and several other RNA viruses signal protein synthesis initiation, in striking contrast to cellular mRNA molecules, which employ a conventional 7-methyl guanosine cap structure. The specific aims of this proposal are: (i) To map the conserved 5' terminal 400 bases of the HCV genome for regions of conserved sequence, secondary structure, and local tertiary structural elements, emphasizing unique regions combining all three elements. Techniques we already use, including oligonucleotide binding, gel shift, RNase H-directed cleavage, RNase III digestion and RNA-RNA crosslinking by UV light will all be employed. (ii) We will employ ribosome binding and protein synthesis assays to identify the exact RNA domains needed for IRES function, and will test modified and mutant sequences to map their fine structure. Our direct ribosome binding site isolation method employing RNAase protection in the reticulocyte lysate will be employed to pinpoint these IRES sequences and their structures derived in aim (i). (iii) We will design ribozyme targeting sequences and other agents which will be directed against key functional domains characterized in aims (i) and (ii). Included will be design and testing of External Guide Sequences (EGSs) for human RNase P in liver cells.