Structure Analysis of Viral Assembly Mechanisms

病毒组装机制的结构分析

• 批准号: 8269842
• 负责人: Barbie K. Ganser-Pornillos
• 金额: $ 40.4万
• 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/8269842
• 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; Mutagenesis; N-terminal; Pathogenesis; Peptide Hydrolases; Preparation; Process; Protein Engineering; Proteins; RNA; Relative (related person); Resolution; Retinal Cone; Retroviridae; Roentgen Rays; 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

DESCRIPTION (provided by applicant): 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-E 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 3 specific aims: (1) We will devote 40% 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 40% 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. (3) We will devote 20% effort to explore the structures of possible maturation intermediates during assembly. We have generated multiple 2D crystal forms of a Gag-like construct of murine leukemia virus that display subnanometer diffraction, and our working assumption is that analysis of these varying crystal forms may reveal structural rearrangements that occur during maturation. 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.

描述（由申请方提供）：未成熟逆转录病毒从感染细胞中释放后，经历成熟过程，其中Gag被病毒蛋白酶切割成MA、CA和NC，引发大的形态学变化并产生感染性病毒体。在成熟病毒粒子内，MA仍然与病毒包膜结合，而CA组装为具有约250个CA六聚体和12个CA五聚体的富勒烯锥，其包围与NC复合的RNA基因组。在上一个资助周期中，我们通过二维晶体的电子晶体学确定了全长HIV-1 CA的9-E分辨率cryoEM密度图。N-末端结构域（NTD）和C-末端结构域（CTD）的高分辨率结构的对接产生了引导稳定NTD六聚体的二硫键插入的分子模型。进一步的诱变使连接相邻六聚体的CTD二聚体不稳定，从而能够溶解和3D结晶。所得的原子分辨率X射线结构显示，CA六聚体由相对刚性的NTD亚基内环组成，周围环绕着移动的CTD亚基带。CTD带的流动性可能是在富勒烯锥中产生连续弯曲的衣壳晶格的潜在机制。然后使用相同的二硫化物策略来产生稳定的CA五聚体，我们正在完成第一个高分辨率的X射线结构。在下一个资助周期，我们将追求3个具体目标：（1）我们将投入40%的精力继续我们对成熟衣壳晶格的结构研究。除了完成五聚体的X射线结构，我们将确定具有可变直径的CA管的亚纳米cryoEM重建。利用六聚体和五聚体的高分辨率结构，并在CA管中的六聚体相互作用的指导下，我们将使用计算方法来建立锥形衣壳的原子模型。(2)我们将投入40%的精力来研究未成熟的Gag晶格的结构。我们已经产生了显示螺旋衍射的Gag突变体，并作为未成熟晶格的体外模拟物。通过与我们对成熟晶格的研究类比，cryoEM和分子对接将产生一个模型，该模型将指导用于cryoEM和X射线晶体学研究的可溶性Gag六聚体的工程。(3)我们将花费20%的努力来探索组装过程中可能的成熟中间体的结构。我们已经产生了多种2D晶体形式的一个GAG样构建的小鼠白血病病毒，显示亚纳米衍射，我们的工作假设是，这些不同的晶体形式的分析可能会揭示结构重排，发生在成熟过程中。我们希望，我们的结构研究将继续提供深入了解逆转录病毒组装的原则，这将是重要的设计新的治疗策略。