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To assemble, or not to assemble: The initiation of retroviral capsid assembly.

机译：组装或不组装：逆转录病毒衣壳组装的启动。

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摘要

During the complex multistep process of retroviral maturation the immature Gag lattice is cleaved apart prior to the formation of the mature capsid shell. Proper maturation and capsid assembly are required for infectivity. The capsid protein (CA) contains two domains [N-terminal domain (NTD) and C-terminal domain (CTD)] and self-assembles to enclose the genomic RNA and other viral proteins. Retroviral capsids are highly polymorphic, exhibiting a wide-range of structures, and were previously modeled as a lattice consisting of 12 CA pentamers unequally distributed among 150--300 hexameric rings. Three CA-CA interfaces form between assembled subunits in the mature capsid: NTD-NTD, NTD-CTD, and CTD-CTD. Very little is known about the mechanism and pathway of assembly that form the mature capsid despite being attractive targets for antiviral therapy. A full understanding of events occurring during maturation, including capsid assembly will require further advances such as: (1) establishing how the cleavage products of Gag reorganize to stimulate capsid assembly during maturation, (2) defining how the CA subunits self-assemble, (3) characterizing the CA pentamer and its interaction with the previously characterized hexamer, and (4) visualizing the capsid lattice at an atomic resolution to guide inhibitor development. Understanding capsid assembly and structure will help to elucidate how the capsid interacts with other viral components to provide necessary functions during the early stages of viral replication.;Ancillary information suggests that the assembly of the capsid is nucleated at a specific location in the maturing virion following Gag cleavage. The presumptive nucleation event is explored in greater detail in this thesis using protein from Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) and a novel in vitro system that, at least for RSV, recapitulates many features of capsid assembly occurring in the virus. Results clearly demonstrate that RSV CA assembles via a nucleation-driven mechanism in vitro. Assembly was demonstrated to be electrostatically controlled and multivalent anionic salts were demonstrated to induce CA to form structures resembling authentic mature capsids. A complex as small as a CA dimer acted as a seed for assembly was demonstrated by isolating intermediates of the capsid assembly pathway. Biochemical evidence combined with genetic analyses supports the proposal that a similar nucleation step happens in the maturing virion.;The conserved major homology region (MHR) in the CTD was shown to be required for proper initiation of capsid assembly. Lethal MHR mutations were suppressed by secondary substitutions located in the NTD and CTD that increased the propensity of CA to assemble in vitro. The results are consistent with CA undergoing structural transformations during maturation. Proteolytic cleavage of Gag results in the accumulation of a transient CA-SP (spacer peptide, C-terminal of CA) intermediate. CA-SP enhanced the kinetics of in vitro assembly. The data suggest that CA-SP participates in nucleation of capsid assembly and the MHR participates in an early step of assembly.;Identifying icosahedral particles formed by multimerization of RSV CA protein permitted the development of a pseudo-atomic model for the retroviral CA pentamer. The data are the first description of all three mature CA-CA interactions (NTD-NTD, NTD-CTD, CTD-CTD) forming in pentamers and hexamers. Although further research is needed to understand the polymorphic nature of retroviral capsids, the results provide a major step forward in understanding the architecture of mature capsids at the atomic level. The data further support a model whereby initiation of capsid assembly involves a tripartite interaction between two NTDs and one CTD. The model invokes a critical participatory role not previously described for the interdomain NTD-CTD interaction in forming a functional retroviral capsid.
机译：在逆转录病毒成熟的复杂的多步骤过程中，未成熟的Gag晶格在形成成熟的衣壳壳之前被切开。感染性需要适当的成熟和衣壳组装。衣壳蛋白（CA）包含两个结构域[N端结构域（NTD）和C端结构域（CTD）]，并自组装以包围基因组RNA和其他病毒蛋白。逆转录病毒衣壳是高度多态的，具有广泛的结构，以前被建模为由12个CA五聚体组成的晶格，这些五聚体在150--300个六聚环之间不均匀分布。成熟衣壳中组装的亚基之间形成了三个CA-CA接口：NTD-NTD，NTD-CTD和CTD-CTD。尽管是抗病毒治疗的诱人靶标，但对于形成成熟衣壳的组装机制和途径知之甚少。对成熟过程中发生的事件（包括衣壳组装）的全面理解将需要进一步的发展，例如：（1）确定Gag的裂解产物如何重组以在成熟过程中刺激衣壳组装，（2）定义CA亚基如何自我组装，（ 3）表征CA五聚体及其与先前表征的六聚体的相互作用，以及（4）以原子分辨率可视化衣壳晶格以指导抑制剂的发展。了解衣壳的组装和结构将有助于阐明衣壳如何在病毒复制的早期阶段与其他病毒组分相互作用以提供必要的功能。；辅助信息表明，在随后的成熟病毒体的特定位置，衣壳的组装成核堵嘴分裂。本论文使用劳斯肉瘤病毒（RSV）和人免疫缺陷病毒（HIV）的蛋白质以及一种新颖的体外系统（至少对于RSV而言，概括了在壳肉中发生的衣壳装配的许多特征），对推测的成核事件进行了更详细的研究。病毒。结果清楚地表明，RSV CA在体外通过成核驱动机制组装。组装被证明是静电控制的，多价阴离子盐被证明诱导CA形成类似于真实成熟衣壳的结构。通过分离衣壳装配途径的中间体，证明了与CA二聚体一样小的复合物可作为装配的种子。生化证据与遗传分析相结合，支持了在成熟病毒体中发生类似成核步骤的提议。; CTD中保守的主要同源区域（MHR）被证明是衣壳装配正确启动所必需的。致命的MHR突变被位于NTD和CTD中的二级取代抑制，这增加了CA在体外组装的倾向。结果与CA在成熟过程中经历结构转变是一致的。 Gag的蛋白水解裂解导致瞬时CA-SP（间隔肽，CA的C端）中间体的积累。 CA-SP增强了体外组装的动力学。数据表明，CA-SP参与了衣壳组装的成核作用，而MHR参与了组装的早期步骤。鉴定RSV CA蛋白多聚形成的二十面体颗粒，可以为逆转录病毒CA五聚体建立假原子模型。数据是在五聚体和六聚体中形成的所有三个成熟的CA-CA相互作用（NTD-NTD，NTD-CTD，CTD-CTD）的第一个描述。尽管需要进一步研究以了解逆转录病毒衣壳的多态性，但结果为在原子水平上理解成熟衣壳的结构提供了重要的一步。数据进一步支持了一种模型，其中衣壳组装的启动涉及两个NTD和一个CTD之间的三方相互作用。该模型在形成功能性逆转录病毒衣壳中调用了域间NTD-CTD相互作用之前未描述的关键参与角色。

著录项

  • 作者

    Purdy, John Gerard.;

  • 作者单位

    The Pennsylvania State University.;

  • 授予单位 The Pennsylvania State University.;
  • 学科 Chemistry Biochemistry.;Biology Virology.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 310 p.
  • 总页数 310
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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