Opening new windows into viruses inside the cell by electron cryo-tomography (cryo-ET)

通过电子冷冻断层扫描 (cryo-ET) 打开了解细胞内病毒的新窗口

• 批准号: MR/W010690/1
• 负责人: Stephen Carter
• 金额: $ 166.88万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FW010690%2F1
• 关键词: Opening; new; windows; into; viruses

As the Caltech physicist Richard Feynman once explained, "It is very easy to answer many fundamental biological questions; you just look at the thing!". If we could simply look at a virus inside a eukaryotic cell and observe all the host and virus molecules interacting with one another in their native state, we would vastly improve our understanding of the virus life cycle and the cellular innate immune responses. In fact, several key historical breakthroughs in virology and cellular biology have been made through advances in imaging technologies. The development of traditional electron microscopy (EM) led to the first detailed pictures of a virus and later the fine ultrastructure of cellular organelles such as the Golgi apparatus and endoplasmic reticulum. Successive technological advances have brought us to the point that we are able to image viruses at close to atomic resolution, a feat recognized by the 2017 Nobel Prize in Chemistry 'for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution". With recent electron cryo-tomography (cryo-ET) technology developments, we are in touching distance of visualizing any virus in its native state and context within the cell. For the first time, all stages of a viral replication cycle from attachment and entry through genome replication to morphogenesis and egress may be visualized at macromolecular resolution. This is an exceedingly exciting moment to work in this field. This new technology affords us the opportunity to solve the structure of viral proteins in their natural habitat with unprecedented resolution. To understand virus-host interactions using cryo-ET technology I am proposing to address a number of important biological questions targeting the virus order Bunyavirales. Viruses in this order are representative of many emerging viruses which pose a high-risk to human and animal health. In cryo-ET, samples are plunged into a cryogen (liquid ethane, or a mixture of ethane and propane), preserving them in a frozen-hydrated, near-native state. The frozen samples are then imaged in a transmission electron microscope, and a series of 2-D projection images are recorded as the sample is rotated incrementally around an axis. This so-called "tilt-series" is then reconstructed into a 3-D "tomogram", with typical resolution sufficient to make out the shapes and arrangement of large macromolecules (~5 nm). If the tomogram, or a set of tomograms, contains structurally homogeneous copies of an object of interest, "tomogram subvolumes" containing the objects can be computationally extracted, aligned and combined, a process we call "subtomogram averaging" (STA) to improve the signal-to-noise ratio and clarify details, typically improving the resolution to ~2-3 nm. For exceptionally favorable samples such as pseudo-crystalline protein coats on cells or viruses, the resolution of STA can be pushed to even 3.1A, sufficient to build atomic models de novo. To learn about important aspects of Bunyavirales biology, my research will apply state of the art technologies, such as cryo-CLEM, cryo-FIB milling, cryo-ET and STA to visualize infection of the related viruses TOSV and RVFV (Phenuiviridae) in situ at high-resolution. Using these techniques, I will look for novel structural aspects of both the pro- and antiviral processes that take place during the immune response and the triggering of the antiviral innate immune response of the cell. More specifically I will investigate (i) restriction of the virus RNPs by the MxA restriction factor, and (ii) the architecture of TOSV and RVFV host response antagonist NSs filaments inside the nucleus. Identifying new structures and key interactions between the virus and the host will be a step change in understanding fundamental aspects of viral replication and the host antiviral responses and might even allow for new insights into host targeted therapeutics.

正如加州理工学院物理学家理查德·费曼（Richard Feynman）曾经解释的那样：“回答许多基本的生物学问题非常容易；你只需看看这个东西！”。如果我们能够简单地观察真核细胞内的病毒，并观察所有宿主和病毒分子在其天然状态下相互作用，我们将极大地提高对病毒生命周期和细胞先天免疫反应的理解。事实上，病毒学和细胞生物学的几个关键历史性突破都是通过成像技术的进步取得的。传统电子显微镜 (EM) 的发展带来了病毒的第一张详细图像，以及后来的高尔基体和内质网等细胞器的精细超微结构。连续的技术进步使我们能够以接近原子分辨率对病毒进行成像，这一壮举得到了 2017 年诺贝尔化学奖的认可，“因为它开发了冷冻电子显微镜，用于溶液中生物分子的高分辨率结构测定”。随着最近电子冷冻断层扫描 (cryo-ET) 技术的发展，我们已经距离可视化任何病毒的天然状态和细胞内环境已经很近了。 第一次，病毒复制周期的所有阶段从附着和进入到基因组复制再到形态发生和流出都可以在大分子分辨率下可视化。对于这一领域的工作来说，这是一个非常激动人心的时刻。这项新技术使我们有机会以前所未有的分辨率解析自然栖息地中的病毒蛋白结构。为了使用冷冻电子断层扫描技术了解病毒与宿主的相互作用，我建议解决一些问题 针对布尼亚病毒目病毒的重要生物学问题。此顺序的病毒是许多对人类和动物健康构成高风险的新兴病毒的代表。在冷冻电子断层扫描中，样品被浸入冷冻剂（液体乙烷，或乙烷和丙烷的混合物）中，将其保存在冷冻水合、接近天然的状态。然后将冷冻样品在透射电子显微镜中成像，并 当样品绕轴增量旋转时，会记录一系列二维投影图像。然后，这种所谓的“倾斜系列”被重建为 3D“断层图”，其典型分辨率足以辨别大分子的形状和排列（~5 nm）。如果断层图或一组断层图包含感兴趣对象的结构同质副本，则“断层图 包含对象的“子体积”可以通过计算提取、对齐和组合，我们将这一过程称为“次断层图平均”(STA)，以提高信噪比并澄清细节，通常将分辨率提高到约 2-3 nm。对于特别有利的样品，例如细胞或病毒上的伪晶体蛋白涂层，STA 的分辨率甚至可以提高到 3.1A， 足以从头构建原子模型。为了了解布尼亚病毒目生物学的重要方面，我的研究将应用最先进的技术，例如冷冻 CLEM、冷冻 FIB 研磨、冷冻 ET 和 STA，以高分辨率在原位可视化相关病毒 TOSV 和 RVFV（苯鸟病毒科）的感染。使用这些技术，我将寻找亲和的新颖结构方面 在免疫反应和触发细胞抗病毒先天免疫反应期间发生的抗病毒过程。更具体地说，我将研究 (i) MxA 限制因子对病毒 RNP 的限制，以及 (ii) TOSV 和 RVFV 宿主反应拮抗剂 NS 细丝在细胞核内的结构。识别病毒与宿主之间的新结构和关键相互作用将是理解上的一步改变 病毒复制和宿主抗病毒反应的基本方面，甚至可能为宿主靶向治疗提供新的见解。

项目成果

Structure and Function of the Dot/Icm T4SS

• DOI: 10.1101/2023.03.22.533729
• 发表时间: 2023-03
• 期刊: bioRxiv
• 作者: Przemysław Dutka;Yuxi Liu;S. Maggi;D. Ghosal;Jue Wang;S. Carter;Wei Zhao;Sukhithasri Vijayrajratnam;J. Vogel;G. Jensen