RNA in Viral DNA Packaging

病毒 DNA 包装中的 RNA

• 批准号: 7821384
• 负责人: SHELLEY GRIMES
• 金额: $ 38.38万
• 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/7821384
• 关键词: ATP Hydrolysis; 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; Herpesviridae; Hydroxyl Radical; Infection; Lasers; 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; mutant; novel; public health relevance; 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碱基噬菌体？29 prohead RNA（PRNA）是将19千洛碱基对基因组DNA-GP3复合物（DNA-GP3）包装包装到病毒前体capsid（Prohead）中的电动机的重要组成部分。该电动机是已知最强的分子电动机之一，产生了约110 pn的力。 PRNA通过相同分子的分子间碱基对形成一种新型的环状六聚体。该多聚机结合了Prohead的头尾连接器，在该连接器中，它通过Cryoem-3D重建作为五聚会环。然后，ATPase GP16的多聚体与PRNA低聚物结合，构成电动机的ATP氢化亚基。假设PRNA可以在Prohead上的DNA-GP3对接，以识别DNA-GP3的左端以启动包装，并在DNA转移ATPase的组装和酶功能中发挥包装。 PRNA在颈部和尾部组装过程中退出了DNA填充的头部，它不是成熟病毒体的一部分。研究该RNA依赖性DNA包装电机的结构和功能可能在发现抗病毒剂的靶标方面具有一般意义。该研究的最终目标是确定PRNA在DNA易位机理中的结构和功能作用。目前的目的是：1）X射线晶体学用于确定单体和低聚PRNA的原子结构； 2）NMR将用于确定PRNA间分子假单胞菌的结构和A螺旋的必需CCA凸起； 3）溶液和定位的羟基自由基探测将用于绘制带有ATPase GP16的Prohead/PRNA复合物中的PRNA核苷酸； 4）将分离位置定向和随机PRNA突变体，以询问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，将探测PRNA在29个DNA易位中的作用。公共卫生相关性：抗病毒药很少，新药的设计取决于对病毒感染和组装的基本机制的透彻理解。噬菌体的感染和组装机制是理解DSDNA病毒的感染和组装机制的模型，例如疱疹病毒和腺病毒，它们具有重要的共同点？29。在这里研究的29个组装过程，例如DNA包装，是抗病毒剂的靶标。