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.

项目总结/摘要 丁型肝炎病毒（HDV）是一种人类病原体，可引起最严重的急性和慢性肝病 疾病目前还没有有效的许可治疗这种病毒，它有一个独特的复制周期， 很好理解。HDV基因组是环状单链RNA，其使用宿主RNA聚合酶II进行RNA聚合。 复制的除了基因组外，在感染的细胞中还产生两种抗原基因组RNA： 反基因组，其用作基因组合成的模板，和mRNA，其编码核衣壳， 比如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圈合成的 聚合酶启动的机制，然后通过环状RNA模板无数次。新 合成的RNA被核酶切割成单位长度的单体，单体被宿主RNA连接成环状 连接酶这些过程决定了在感染细胞中积累的病毒RNA的数量， 影响病毒的传播、持久性和寄主反应，但其作用机制和动态尚不清楚 明白在最近的工作中，我们确定了基因组RNA合成起始的位置， 通过跟踪滚环过程中产生的RNA的5'端， 复制-一个5'端是起始位点，另一个是由核酶切割产生的5'端。基因组5' end本身是不寻常的，因为它以一个G开头，这个G要么是未模板化的，要么是以一种不寻常的方式模板化的，例如 由U而不是C。我们的方法为分析HDV RNA的早期阶段提供了新的方法， 以一种不可能的方式进行合成。本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 以及影响引发和延伸的结构。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合成的位置和效率的结构决定因素 新成员。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HDV基因组和反基因组RNA合成过程中的动态 HDV在细胞中复制的过程。因此，我们将确定引发和延长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。