Dynamics of HDV RNA Synthesis

HDV RNA 合成动力学

• 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
• 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

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.

项目摘要/摘要 肝炎三角洲病毒（HDV）是一种人类病原体，导致最严重的急性和慢性肝脏形式 疾病。该病毒没有有效的许可疗法，该病毒的独特复制周期不是 理解。 HDV基因组是一种圆形的单链RNA，使用宿主RNA聚合酶II用于 复制。除基因组外，在感染细胞中还产生了两个抗原素RNA：圆形 作为基因组合成模板的抗原组和mRNA，该模板编码了核蛋白酶。 就像蛋白质称为肝炎三角洲抗原。基因组和抗原组由滚动圆合成 聚合酶启动的机制，然后多次转移圆形RNA模板。新 将RNA通过核酶裂解成单位长的单体，这些单体被宿主RNA连接到电路中 连接酶。这些过程决定了在感染细胞中积累的病毒RNA的量，从而 影响病毒传播，持久性和宿主反应，但它们的机制和动态不好 理解。在最近的工作中，我们确定了基因组RNA启动的合成的位置，我们 开发的工具可以通过遵循滚动圆中生成的RNA的5'端来跟踪这些过程 复制 - 一个5'端是启动位点，另一个是由核酶裂解产生的5'端。基因组5' 结束本身是不寻常的，因为它以g的g进行了，该g是以不寻常的方式进行模板或模板的 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 以不可能的方式综合。该建议旨在利用这些新方法 确定感染细胞中HDV RNA合成的动力学，进而确定HDV序列 以及影响主动性和伸长率的结构。有两个目标。在AIM 1中，我们将确定序列 以及确定HDV基因组RNA和mRNA合成的何处以及如何有效的结构决定剂 发起。我们将分析不同的HDV分离株和基因型，包括最近鉴定出的“ HDV样”病毒。 在AIM 2中，我们将表征HDV基因组和抗原组RNA合成过程中的动力学 细胞中的HDV复制。因此，我们将确定主动性和伸长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 细胞中的复制。