Dynamics of HDV RNA Synthesis

HDV RNA 合成动力学

基本信息

批准号：
10646632
负责人：
JOHN L CASEY
金额：
$ 22.06万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10646632
关键词：
Acute Affect Catalytic RNA Cells DNA Polymerase II DNA-Directed RNA Polymerase Disease Elements Genome Genomics Genotype Hepatitis Delta Virus Hepatitis delta Antigens Human Immune response Infection Knowledge Laboratories Length Licensing Life Cycle Stages Ligation Liver diseases Messenger RNA Methods Modeling Modification Mutation Nucleocapsid Pathogenesis Polymerase Positioning Attribute Process Proteins RNA RNA Ligase (ATP)RNA Polymerase II RNA Sequences RNA Viruses RNA chemical synthesis RNA replication RNA-Directed RNA Polymerase Replication-Associated Process Reporting Role Series Site Structure System Testing Therapeutic Time Transfection Variant Viral Genome Virus Virus Replication Woodchuck Work cell type chronic liver disease circular RNA design effective therapy experimental study human pathogen insight monomer novel strategies structural determinants therapy development tool viral RNA

项目摘要

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.

项目概要/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病这种病毒没有有效的许可疗法，它具有独特的复制周期。 HDV 基因组是一种环状单链 RNA，使用宿主 RNA 聚合酶 II。除了基因组之外，受感染的细胞中还会产生两种反义RNA：环状RNA。反基因组，作为基因组合成的模板，以及 mRNA，编码核衣壳- 像称为丁型肝炎抗原的蛋白质一样，基因组和反基因组是通过滚环合成的。聚合酶启动，然后多次穿过环状 RNA 模板的机制。生成的 RNA 被核酶切割成单位长度的单体，并通过宿主 RNA 连接成环状这些过程决定了受感染细胞中积累的病毒 RNA 的量，而这反过来又决定了病毒 RNA 的量。影响病毒传播、持久性和宿主反应，但其机制和动态尚不清楚在最近的工作中，我们确定了基因组 RNA 合成的起始位置。开发了通过追踪滚环过程中产生的 RNA 5' 端来跟踪这些过程的工具复制 – 一个 5' 末端是起始位点，另一个是核酶切割产生的 5' 末端基因组 5'。 end 本身很不寻常，因为它以 G 开头，该 G 要么是未模板化的，要么是以不寻常的方式模板化的，例如由 U 而不是 C。我们的方法提供了分析 HDV RNA 早期阶段的新方法该提案旨在利用这些新方法来实现不可能的合成。确定受感染细胞中 HDV RNA 合成的动态，进而确定 HDV 序列在目标 1 中，我们将确定影响起始和伸长的结构。决定 HDV 基因组 RNA 和 mRNA 合成地点和效率的结构决定因素我们将分析不同的 HDV 分离株和基因型，包括最近发现的“HDV 样”病毒。在目标 2 中，我们将描述课程期间 HDV 基因组和反基因组 RNA 合成的动态特征因此，我们将确定 HDV 起始和延伸的相对贡献。 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。