Replication Elements in the 5' End of the HCV Genome

描述（由申请方提供）：丙型肝炎病毒（HCV）是黄病毒科中肝炎病毒属的单一成员。 基因组由单个正链RNA分子组成。HCV基因组与许多其他RNA病毒的不同之处在于，它既不具有帽结构，也不具有聚（A）尾。 复制复合物识别病毒基因组的正链和负链拷贝需要非常高的特异性。 模板特异性被认为是通过位于病毒基因组末端的顺式作用RNA信号发生的，尽管内部定位的复制信号也已被鉴定。 现在已经显示几种病毒的5' NTR参与翻译、负链和正链合成。 本提案的总体目标是确定位于病毒基因组5'端的哪些RNA序列和结构元件参与HCV RNA合成，并确定这些信号是否在正链或负链合成中发挥作用。 我们推测HCV开放阅读框的5'端NTR和C-E1-E2-NS 2区含有调节HCV RNA合成所必需的元件。 我们将使用一种改良的复制子系统，该系统将5' NTR的翻译功能与其在复制中的作用分开。 将进行系统的突变分析以定位5' NTR中影响复制效率的区域。 C-E1-E2-NS 2编码序列中调节复制的区域将通过一系列缺失突变体来描绘。 通过荧光素酶表达检测正链蓄积，通过定量RT-PCR测定负链水平。 将分析这两种RNA的水平和比率，作为复制受损发生步骤的指标。 我们将设计特定的突变，并利用修饰的反义寡核苷酸，以特异性地阻断RNA元件的正链或负链方向。 该结果将导致更好地理解位于基因组5'端的参与模板特异性的信号，并且可以导致更有效的全长复制子系统的开发，并且将有助于干扰复制信号形成的化合物的开发。

DESCRIPTION (provided by applicant): Hepatitic C virus (HCV) is the single member of the genus hepacivirus in the family Flaviviridae. The genome consists of a single positive-stranded RNA molecule. The HCV genome differs from many other RNA viruses in that it possess neither a cap structure not a poly (A)-tail. The recognition of the positive and negative strand copies of the viral genome by the replication complex required an extraordinary level of specificity. The template specificity is thought to occur through cis-acting RNA signals located at the termini of the viral genome, although internally located replication signals have also been identified. The 5' NTR of several viruses has now been shown to be involved in translation, minus-strand, and plus-strand synthesis. The overall goal of this proposal is to determine which RNA sequence, and structural elements, located in the 5' end of the viral genome are involved in HCV RNA synthesis and determine if these signals play a role in plus- or minus-strand synthesis. We hypothesize that the 5' NTR and the C-E1-E2-NS2 region of the HCV open-reading frame contain elements that are essential, for modulating HCV RNA synthesis. We will use a modified replicon system that separates the translational functions of the 5' NTR from its role in replication. A systematic mutational analysis will be performed to localize regions in the 5' NTR that affect replication efficiency. The regions in the C-E1-E2-NS2 coding sequence that modulate replication will be delineated by a series of deletion mutants. Positive-strand accumulation will be detected by luciferase expression and negative strand levels will be determined by quantitative RT-PCR. The level and ratio of these two RNAs will be analyzed as an indicator to the step at which the impairment of replication occurs. We will design specific mutations and utilize modified anti-sense oligo nucleotides to specifically block RNA elements in either plus- or minus-strand orientation. The results will lead to a better understanding of the signals located at the 5' end of the genome involved in template specificity and can lead to the development of more efficient full length replicon systems and will aid the development of compounds that interfere with the formation of replication signals.