Structure Analysis of Viral Assembly Mechanisms

病毒组装机制的结构分析

• 批准号: 8142303
• 负责人: Barbie K. Ganser-Pornillos
• 金额: $ 40.28万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8142303
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Architecture; Binding; Biochemical; C-terminal; Caliber; Capsid; Cell membrane; Cells; Cleaved cell; Complex; Computer Analysis; Computer Simulation; Computing Methodologies; Conflict (Psychology); Cryoelectron Microscopy; Crystallization; DNA Sequence Rearrangement; Disulfides; Docking; Electron Microscopy; Electrons; Encapsulated; Engineering; Fullerenes; Funding; Gagging; Genome; HIV-1; Image Analysis; In Vitro; Length; Life; Link; Maps; Membrane; Methods; Modeling; Molecular; Molecular Models; Molecular Probes; Murine leukemia virus; Mutagenesis; N-terminal; Pathogenesis; Peptide Hydrolases; Preparation; Process; Protein Engineering; Proteins; RNA; Reaction; Regulation; Relative (related person); Resolution; Retinal Cone; Retroviridae; Roentgen Rays; Solutions; Staging; Structural Models; Structure; System; Techniques; Tube; Variant; Viral; Virion; Virus; Work; X-Ray Crystallography; density; design; dimer; disulfide bond; drug development; electron crystallography; gag Gene Products; image reconstruction; inhibitor/antagonist; insight; molecular modeling; mutant; novel; novel therapeutic intervention; novel therapeutics; particle; reconstruction; small molecule; viral RNA

Upon release from infected cells, immature retroviruses undergo a maturation process in which Gag is cleaved by the viral protease into MA, CA, and NC, triggering large morphological changes and producing infectious virions. Within the mature virion, MA remains associated with the viral envelope, while CA assembles as a fullerene cone with ~250 CA hexamers and 12 CA pentamers, which encloses the RNA genome complexed with NC. In the last funding cycle we determined a 9-¿ resolution cryoEM density map of full-length HIV-1 CA by electron crystallography of 2D crystals. Docking of high- resolution structures of the N-terminal domain (NTD) and C-terminal domain (CTD) yielded a molecular model that guided the insertion of disulfide bonds that stabilized the NTD hexamer. Further mutagenesis destabilized the CTD dimers that link adjacent hexamers, thereby enabling solubilization and 3D crystallization. The resulting atomic- resolution X-ray structures revealed that the CA hexamer is composed of a relatively rigid inner ring of NTD subunits, surrounded by a mobile belt of CTD subunits. Mobility of the CTD belt is likely to be an underlying mechanism for generating the continuously curved capsid lattice in the fullerene cone. The same disulfide strategy was then used to generate stable CA pentamers, and we are completing the first high-resolution X-ray structure. For the next funding cycle we will pursue 2 specific aims: (1) We will devote 50% effort to continue our structural studies of the mature capsid lattice. In addition to completing X-ray structures of the pentamer, we will determine subnanometer cryoEM reconstructions of CA tubes with variable diameters. With high-resolution structures of the hexamer and pentamer, and guided by the hexamer interactions in the CA tubes, we will use computational methods to build an atomic model for the conical capsid. (2) We will devote 50% effort to structural studies of the immature Gag lattice. We have generated Gag mutants that display helical diffraction and serve as an in vitro mimic of the immature lattice. By analogy with our studies of the mature lattice, cryoEM and molecular docking will yield a model that will guide the engineering of soluble Gag hexamers for cryoEM and X-ray crystallographic studies. We are hopeful that our structural studies will continue to provide insight into principles of retrovirus assembly that will be important for the design of new therapeutic strategies.

从感染细胞释放后，未成熟的逆转录病毒在 病毒蛋白酶裂解到MA，CA和NC，触发大的堵嘴 形态变化并产生传染病。在成熟的病毒中，马 仍然与病毒信封相关 〜250 ca六聚体和12个Ca五聚体，它们封闭了RNA基因组复合的 与NC。在上一个融资周期中，我们确定了9-€的分辨率冷冻密度图 通过2D晶体的电子晶体学的全长HIV-1 Ca。对接高级 N末端结构域（NTD）和C末端结构域（CTD）的分辨率结构 产生了一个分子模型，该模型指导插入二硫键，以稳定 NTD Hexamer。进一步的诱变破坏了连接相邻的CTD二聚体 六聚体，从而实现溶解和3D结晶。由此产生的原子 - 分辨率X射线结构揭示了CA六聚体由相对组成 NTD亚基的刚性内环，周围是CTD亚基的移动带。流动性 CTD带可能是产生连续的基本机制 在富勒烯锥中弯曲的衣壳晶格。然后使用相同的二硫键策略 产生稳定的Ca Pentamer，我们正在完成第一个高分辨率X射线 结构。对于下一个资金周期，我们将追求2个具体目标：（1）我们将部署 50％的努力继续我们对成熟的衣壳晶格的结构研究。此外 完成Pentamer的X射线结构，我们将确定亚纳米冷冻 具有可变直径的Ca管的重建。具有高分辨率结构 六聚体和五角星，并在CA管中的己酸酯相互作用的指导下，我们 将使用计算方法来构建锥形衣壳的原子模型。 （2）我们 将花50％的精力用于未成熟插科打晶格的结构研究。我们有 产生的插科打突变体，显示螺旋衍射，并作为体外模仿 未成熟的晶格。通过类似于我们对成熟晶格，冷冻和 分子对接将产生一个模型，该模型将指导固体堵嘴的工程 冷冻和X射线晶体学研究的六聚体。我们希望我们的 结构研究将继续深入了解逆转录病毒组装原理 这对于设计新的治疗策略很重要。