Structural Characterization of Archaeal Virus Assembly and Host Interactions

古菌病毒组装和宿主相互作用的结构表征

• 批准号: 8165138
• 负责人: Chi-yu Fu
• 金额: $ 8.8万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8165138
• 关键词: ATP phosphohydrolase; Agriculture; Archaea; Archaeal Viruses; Area; Biochemical; Biological Models; Capsid; Cell physiology; Cell surface; Cells; Cellular Membrane; Cellular biology; Coupled; Cryoelectron Microscopy; Cytolysis; Cytoplasm; DNA; DNA Packaging; Electrons; Event; Genes; Genetic; Genome; Growth; Infection; Integration Host Factors; Life; Life Cycle Stages; Mediating; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Morphogenesis; Organelles; Plaque Assay; Process; Proteins; Reaction; Resolution; Role; Rupture; Staging; Structure; Sulfolobus; Sulfolobus solfataricus; Surface; System; Testing; Thick; Tomogram; Viral; Viral Genes; Viral Genome; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; expression cloning; human disease; in vivo; insight; nanometer; novel; particle; prevent; reconstruction; response; three dimensional structure; tomography; virus pathogenesis

DESCRIPTION (provided by applicant): Viruses usurp host cell functions and recondition the cell to optimize it for viral replication. They do this by redirecting host proteins and by bringing in their own proteins as necessary. To understand the underlying mechanisms, it is important to characterize viral events in the context of the cell and identify viral and host factors involved in these process. We have been able to apply whole cell electron cryotomography (ECT) to follow the assembly and maturation of Sulfolobus turreted icosahedral virus (STIV) in a life-like state in intact archaeon Sulfolobus cells. The whole cell ECT approach, which is limited in most systems due to the cell thickness, makes STIV/ Sulfolobus one of the few model systems to correlate the structural changes that occur in an entire cell as a consequence of virus infection in 3D at low-nm resolution. We propose to combine ECT, single particle CryoEM reconstruction, genetic and biochemical approaches to (1) determine the morphogenesis of STIV, to (2) dissect the factors contributing to the distinct cellular distribution of the particles at different maturation stages, and to (3) characterize a new viral release mechanism mediated by infection-induced pyramid structures. Cellular tomography studies have revealed DNA-filled STIV virions, DNA-free procapsids and partially assembled particles and showed that the capsid and inner membrane co-assemble in the cytoplasm of Sulfolobus that lacks membrane-containing organelles. The assembled procapsids are packaged with DNA and mature to virions. In Specific Aim 1, we propose to identify the specialized packaging vertex and characterize the structural changes accompanying packaging. We also found that STIV virions tend to form quasi-crystalline arrays in the cell while procapsids are mostly scattered outside or on the edges of the arrays. We hypothesize that the particles are organized to form viral arrays when packaging the genome. We propose to test the hypothesis by uncoupling capsid assembly and DNA packaging. The model predicts that blocking DNA-packaging will block array formation. Furthermore, we found that viral infection induced pyramid-like protrusions, which have sharply defined facets and apexes. Forming these pyramids requires substantial remodeling of the cellular membrane and destruction of the surface protein layer, leading to insights about this new virus-release mechanism. We will characterize the role of viral protein C92 (a major pyramid protein) in pyramid formation and pyramid-mediated viral release. The proposal will contribute significantly to our understanding of the cell biology of archaeal virus pathogenesis and viruses in the PRD1-Adeno lineage in general. PUBLIC HEALTH RELEVANCE: Viruses cause many human diseases and agricultural loses. To understand how viruses replicate and hijack hosts, it is important to follow viral events in vivo and characterize viral-host interactions involved in various steps of viral life cycle. The proposal will contribute significantly to our understanding of the cell biology of Sulfolobus turreted icosahedral virus infection and viruses in the PRD1-Adeno lineage in general.

描述（由申请人提供）：病毒侵占宿主细胞功能并修复细胞以优化其病毒复制。他们通过重定向宿主蛋白质并根据需要引入自己的蛋白质来做到这一点。为了了解潜在的机制，重要的是表征细胞背景下的病毒事件并识别参与这些过程的病毒和宿主因素。 我们已经能够应用全细胞电子冷冻断层扫描（ECT）来追踪硫化叶菌转塔二十面体病毒（STIV）在完整古菌硫化叶菌细胞中以逼真状态的组装和成熟。全细胞 ECT 方法由于细胞厚度而在大多数系统中受到限制，这使得 STIV/Sulfolobus 成为少数几个模型系统之一，可以将由于 3D 病毒感染而在整个细胞中发生的结构变化关联起来。纳米分辨率。我们建议结合 ECT、单颗粒 CryoEM 重建、遗传和生化方法来 (1) 确定 STIV 的形态发生，(2) 剖析导致不同成熟阶段颗粒不同细胞分布的因素，以及 (3 ）描述了由感染诱导的金字塔结构介导的新病毒释放机制。 细胞断层扫描研究揭示了充满 DNA 的 STIV 病毒粒子、无 DNA 的衣壳和部分组装的颗粒，并表明衣壳和内膜在缺乏含膜细胞器的硫化叶菌的细胞质中共同组装。组装好的衣壳原体与 DNA 一起包装并成熟为病毒体。在具体目标 1 中，我们建议识别专门的包装顶点并描述包装伴随的结构变化。 我们还发现 STIV 病毒颗粒倾向于在细胞内形成准晶体阵列，而原衣壳大多分散在阵列外部或边缘。我们假设在包装基因组时，颗粒被组织形成病毒阵列。我们建议通过解偶联衣壳组装和 DNA 包装来检验这一假设。该模型预测，阻断 DNA 包装将阻断阵列形成。 此外，我们发现病毒感染会引起金字塔状的突起，这些突起具有清晰的刻面和顶点。形成这些金字塔需要对细胞膜进行大量重塑并破坏表面蛋白层，从而深入了解这种新的病毒释放机制。我们将描述病毒蛋白 C92（一种主要的金字塔蛋白）在金字塔形成和金字塔介导的病毒释放中的作用。该提案将极大地促进我们对古菌病毒发病机制和 PRD1-Adeno 谱系病毒的细胞生物学的理解。 公共卫生相关性：病毒会导致许多人类疾病和农业损失。为了了解病毒如何复制和劫持宿主，重要的是跟踪体内的病毒事件并表征病毒生命周期各个步骤中涉及的病毒与宿主的相互作用。该提案将极大地促进我们对硫化叶菌转塔二十面体病毒感染和 PRD1-腺病毒谱系病毒的细胞生物学的理解。