Structural Characterization of Archaeal Virus Assembly and Host Interactions

描述（由申请人提供）：病毒篡夺宿主细胞的功能并修复细胞以优化其病毒复制。它们通过重定向宿主蛋白质并在必要时引入自己的蛋白质来实现这一目标。为了理解潜在的机制，在细胞背景下描述病毒事件并确定参与这些过程的病毒和宿主因子是很重要的。我们已经能够应用全细胞电子冷冻断层扫描（ECT）来跟踪在完整的古细菌Sulfolobus细胞中处于类似生命状态的Sulfolobus塔状二十面体病毒（STIV）的组装和成熟。由于细胞厚度的限制，整个细胞ECT方法在大多数系统中是有限的，这使得STIV/ Sulfolobus成为少数模型系统之一，可以在低纳米分辨率下将病毒感染导致的整个细胞中的结构变化联系起来。我们建议结合ECT、单颗粒低温重建、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)表征感染诱导的金字塔结构介导的新的病毒释放机制。细胞断层扫描研究揭示了充满dna的STIV病毒粒子、不含dna的原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Sulfolobus细胞质中共同组装。组装后的原壳被DNA包裹，成熟为病毒粒子。在具体目标1，我们建议确定专门的包装顶点和表征结构变化伴随包装。我们还发现，STIV病毒粒子倾向于在细胞内形成准晶体阵列，而原壳体大多分散在阵列的外部或边缘。我们假设这些颗粒在包装基因组时被组织成病毒阵列。我们建议通过解耦衣壳组装和DNA包装来验证这一假设。该模型预测，阻断dna包装将阻断阵列的形成。此外，我们发现病毒感染诱导金字塔状突起，具有明确的切面和顶点。形成这些金字塔需要对细胞膜进行大量的重塑和破坏表面蛋白质层，从而对这种新的病毒释放机制有了深入的了解。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。这一建议将有助于我们对古细菌病毒发病机制和PRD1-Adeno谱系病毒的细胞生物学的理解。

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.