RNA in Viral DNA Packaging

病毒 DNA 包装中的 RNA

• 批准号: 8071051
• 负责人: SHELLEY GRIMES
• 金额: $ 35.84万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8071051
• 关键词: ATP phosphohydrolase; Adenoviruses; Antiviral Agents; Bacteriophages; Base Pairing; Binding; Capsid; Complex; Cryoelectron Microscopy; DNA; DNA Packaging; Docking; Double Stranded DNA Virus; Enzymes; Genomics; Goals; Head; Health; Herpesviridae; Hydroxyl Radical; Infection; Left; Maps; Modeling; Molecular Motors; Motor; Neck; Nucleotides; Pharmaceutical Preparations; Process; RNA; Research; Role; Site; Solutions; Structure; Tail; Viral; Virion; Virus Diseases; X-Ray Crystallography; base; design; laser tweezer; mutant; novel; reconstruction; single molecule; viral DNA

DESCRIPTION (provided by applicant): The 174-base bacteriophage ?29 prohead RNA (pRNA) is an essential component of the motor that packages the 19-kilobase pair genomic DNA-gp3 complex (DNA-gp3) into the viral precursor capsid (prohead). This motor is one of the strongest molecular motors known, generating ~110 pN of force. pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules. This multimer binds to the head-tail connector of the prohead, where it appears as a pentameric ring by cryoEM-3D reconstruction. A multimer of the ATPase gp16 then binds to the pRNA oligomer to constitute the ATP-hydrolyzing subunits of the motor. pRNA is hypothesized to function in docking of the DNA-gp3 on the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and in assembly and enzyme function of the DNA translocating ATPase. pRNA exits the DNA-filled head during neck and tail assembly, and it is not a part of the mature virion. Study of the structure and function of this RNA-dependent DNA packaging motor may have general significance in uncovering targets for antiviral agents. The ultimate goal of the research is to determine the structural and functional roles of pRNA in the mechanism of DNA translocation. The current aims are: 1) X-ray crystallography will be used to determine atomic structures of monomeric and oligomeric pRNAs; 2) NMR will be used to determine the structure of the pRNA intermolecular pseudoknot and the essential CCA bulge of the A-helix; 3) Solution and site-directed hydroxyl radical probing will be used to map pRNA nucleotides in prohead/pRNA complexes with and without the ATPase gp16; 4) Site-directed and random pRNA mutants will be isolated to interrogate function of the pRNA in DNA translocation; and 5) Single-molecule laser tweezers studies of pRNA mutants having partial function will probe the roles of pRNA in ?29 DNA translocation. PUBLIC HEALTH RELEVANCE: There is a paucity of antiviral agents, and the design of new drugs depends upon a thorough understanding of the basic mechanisms of viral infection and assembly. The infection and assembly mechanisms of the bacteriophage ?29 serve as models for understanding the infection and assembly mechanisms of dsDNA viruses such as herpesvirus and adenovirus, which have significant commonalities to ?29. The assembly processes of ?29 studied here, such as DNA packaging, are targets for antiviral agents.

描述（由申请人提供）： 174 个碱基的噬菌体 ?29 前头 RNA (pRNA) 是将 19 千碱基对基因组 DNA-gp3 复合物 (DNA-gp3) 包装到病毒前体衣壳（前头）中的马达的重要组成部分。 ）。该电机是已知最强的分子电机之一，可产生约 110 pN 的力。 pRNA 通过相同分子的分子间碱基配对形成新型环状六聚体。该多聚体与前头的头尾连接器结合，通过 CryoEM-3D 重建显示为五聚体环。然后 ATP 酶 gp16 的多聚体与 pRNA 寡聚体结合，构成马达的 ATP 水解亚基。假设 pRNA 在 DNA-gp3 与前头的对接中发挥作用，识别 DNA-gp3 的左端以启动包装，并在 DNA 易位 ATP 酶的组装和酶功能中发挥作用。 pRNA 在颈部和尾部组装过程中离开充满 DNA 的头部，并且它不是成熟病毒体的一部分。研究这种依赖于 RNA 的 DNA 包装马达的结构和功能对于揭示抗病毒药物的靶点可能具有普遍意义。该研究的最终目标是确定pRNA在DNA易位机制中的结构和功能作用。目前的目标是：1）X射线晶体学将用于确定单体和寡聚pRNA的原子结构； 2) NMR将用于确定pRNA分子间假结的结构和A螺旋的基本CCA凸起； 3) 溶液和定点羟基自由基探测将用于在有和没有 ATPase gp16 的情况下绘制 prohead/pRNA 复合物中的 pRNA 核苷酸图谱； 4) 分离定点和随机 pRNA 突变体，以探究 pRNA 在 DNA 易位中的功能； 5) 对具有部分功能的 pRNA 突变体的单分子激光镊研究将探讨 pRNA 在 ?29 DNA 易位中的作用。公共卫生相关性：抗病毒药物缺乏，新药的设计取决于对病毒感染和组装基本机制的透彻理解。噬菌体α29的感染和组装机制可以作为理解双链DNA病毒（例如疱疹病毒和腺病毒）的感染和组装机制的模型，这些病毒与α29有显着的共性。这里研究的 ?29 组装过程，例如 DNA 包装，是抗病毒药物的目标。