Dynamics of HDV RNA Synthesis

项目摘要/摘要 丁型肝炎病毒(HDV)是一种人类病原体，可导致最严重的急性和慢性肝脏疾病。 疾病。这种病毒有一个独特的复制周期，目前还没有有效的特许治疗方法 很好理解。HDV基因组是一种环状单链rna，它利用宿主rna聚合酶II 复制。除了基因组外，感染细胞中还会产生两种抗原性RNA：环状 作为基因组合成模板的反基因组，以及编码核衣壳的mRNA- 就像一种叫做丁型肝炎抗原的蛋白质。基因组和反基因组是由一个滚动的圆合成的 聚合酶启动的机制，然后多次传递环状RNA模板。新开 合成的RNA被核酶切割成单位长度的单体，这些单体被宿主RNA连接成环 连接酶。这些过程决定了病毒RNA在感染细胞中的积累量，进而 影响病毒传播、持久性和宿主反应，但其机制和动态还不是很好 明白了。在最近的工作中，我们确定了基因组RNA合成的起始位置，我们 开发了跟踪这些过程的工具，通过跟踪滚动圆过程中产生的RNA的5‘端 复制--一个5‘端是起始点，另一个是核酶切割产生的5’端。基因组5‘ End本身的不同寻常之处在于，它以未模板化或以不寻常的方式模板化的G开头，例如 我们的方法为分析HDV RNA的早期阶段提供了新的方法 以不可能实现的方式进行合成。这项提议旨在利用这些新方法来 确定感染细胞中HDV RNA合成的动力学，进而确定HDV序列 以及影响起始和延伸的结构。有两个目标。在目标1中，我们将确定序列 以及决定HDV基因组RNA和mRNA合成的地点和效率的结构决定因素 提升者。我们将分析不同的HDV分离株和基因类型，包括最近发现的一种类似HDV的病毒。 在目标2中，我们将描述HDV基因组和反基因组rna合成过程中的动态。 细胞内的HDV复制。因此，我们将确定起始和伸长HDV的相对贡献 RNA水平并确定这些贡献的变化如何影响HDV RNA水平的变化 在细胞内复制。

Project Summary/Abstract Hepatitis delta virus (HDV) is a human pathogen that causes the most serious forms of acute and chronic liver disease. There is no effective licensed therapy for this virus, which has a unique replication cycle that is not well understood. The HDV genome is a circular single stranded RNA that uses host RNA polymerase II for replication. In addition to the genome, two antigenomesense RNAs are produced in infected cells: the circular antigenome, which serves as a template for genome synthesis, and an mRNA, which encodes a nucleocapsid- like protein called the hepatitis delta antigen. The genome and antigenome are synthesized by a rolling circle mechanism in which the polymerase initiates, then transits the circular RNA template numerous times. Newly made RNAs are cleaved by ribozymes into unit-length monomers that are ligated into circles by a host RNA ligase. These processes determine the amount of viral RNA that accumulates in infected cells, which in turn affect virus spread, persistence and host responses, but their mechanisms and dynamics are not well understood. In recent work, in which we identified the position at which synthesis of genome RNA initiates, we developed tools to track these processes by following the 5’ ends of RNAs generated during rolling circle replication – one 5’ end is the initiate site, the other is the 5’ end created by ribozyme cleavage. The genome 5’ end itself is unusual in that it starts with a G that is either untemplated or templated in an unusual way, such as by a U rather than C. Our methods provide new approaches to analyzing the early stages of HDV RNA synthesis in ways that have not been possible. This proposal is directed at exploiting these new approaches to determine the dynamics of HDV RNA synthesis in infected cells and, in turn, to determine the HDV sequences and structures that affect initiation and elongation. There are two aims. In Aim 1 we will identify the sequence and structural determinants that determine where and how efficiently HDV genome RNA and mRNA synthesis initiates. We will analyze different HDV isolates and genotypes, including a recently identified ”HDV-like” virus. In Aim 2 we will characterize the dynamics of HDV genome and antigenome RNA synthesis during the course of HDV replication in cells. We will thus determine the relative contributions of initiation and elongation HDV RNA levels and identify how changes in these contributions affect changes in HDV RNA levels during replication in cells.