Structural Characterization of Archaeal Virus Assembly and Host Interactions

描述（由申请方提供）：病毒篡夺宿主细胞功能并重新调节细胞以优化其病毒复制。它们通过改变宿主蛋白质的方向并在必要时引入自己的蛋白质来做到这一点。为了了解潜在的机制，重要的是要在细胞的背景下表征病毒事件，并确定参与这些过程的病毒和宿主因子。 我们已经能够应用全细胞电子冷冻断层扫描（ECT），以遵循组装和成熟的硫化叶菌塔形二十面体病毒（STIV）在一个完整的古菌硫化叶菌细胞中的生活样状态。全细胞ECT方法由于细胞厚度而在大多数系统中受到限制，使得STIV/ Sulfolobus成为为数不多的模型系统之一，以低nm分辨率在3D中关联由于病毒感染而在整个细胞中发生的结构变化。我们建议结合联合收割机ECT、单颗粒CryoEM重建、遗传学和生物化学方法来（1）确定STIV的形态发生，（2）剖析导致颗粒在不同成熟阶段的不同细胞分布的因素，以及（3）表征由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DNA填充的STIV病毒粒子，无DNA的原衣壳和部分组装的颗粒，并表明衣壳和内膜共组装在细胞质中的硫化叶菌缺乏含膜细胞器。组装的原衣壳与DNA包装在一起并成熟为病毒体。在具体目标1中，我们建议识别专门的包装顶点，并描述伴随包装的结构变化。 我们还发现，STIV病毒粒子倾向于在细胞内形成准晶体阵列，而前衣壳大多分散在阵列的外部或边缘。我们假设，当包装基因组时，颗粒被组织成病毒阵列。我们建议通过解偶联衣壳组装和DNA包装来验证这一假设。该模型预测，阻断DNA包装将阻断阵列形成。 此外，我们发现，病毒感染诱导的神经元样突起，其中有明确的小平面和顶点。形成这些金字塔需要大量重塑细胞膜和破坏表面蛋白层，从而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）在金字塔形成和病毒介导的病毒释放中的作用。该建议将有助于我们理解古细菌病毒发病机制和PRD 1-Adeno谱系病毒的细胞生物学。 公共卫生相关性：病毒引起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，重要的是要在体内跟踪病毒事件并表征病毒生命周期各个步骤中涉及的病毒-宿主相互作用。该建议将有助于我们理解硫化叶菌转塔二十面体病毒感染的细胞生物学和PRD 1-Adeno谱系的病毒。

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.