HCV INTERNAL RIBOSOME ENTRY SITE AS TARGET FOR THERAPY

我们实验室的长期目标是确定病毒RNA中结构和功能的独特区域，使它们能够在不遭受严重细胞损伤的情况下受到攻击。在美国和国外有数百万慢性HCV感染患者，但目前治疗的有效性是一个难题。 因此，HCV是基于靶向病毒的独特RNA序列、结构和功能的新型抗病毒疗法的主要候选者。 由于这种治疗在HCV基因组的保守序列中效果最好，我们的项目将集中在病毒RNA的5'末端结构域，其中包括内部核糖体进入位点（IRES），这是HCV和其他几种RNA病毒发出蛋白质合成起始信号的不寻常结构，与细胞mRNA分子形成鲜明对比，细胞mRNA分子采用传统的7-甲基鸟苷帽结构。该建议的具体目标是：（i）绘制HCV基因组保守的5'末端400个碱基的保守序列、二级结构和局部三级结构元件的区域，强调结合所有三个元件的独特区域。 我们已经使用的技术，包括寡核苷酸结合，凝胶转移，RNA酶H-定向切割，RNA酶III消化和RNA-RNA交联的紫外光都将被采用。 (ii)我们将采用核糖体结合和蛋白质合成测定来鉴定IRES功能所需的确切RNA结构域，并将测试修饰和突变序列以绘制其精细结构。 我们采用网织红细胞裂解物中RNA酶保护的直接核糖体结合位点分离方法将用于精确定位这些IRES序列及其在目的（i）中衍生的结构。 (iii)我们将设计核酶靶向序列和其他试剂，这些试剂将针对目标（i）和（ii）中表征的关键功能结构域。 包括肝细胞中人RNase P的外部指导序列（EGS）的设计和测试。

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.