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

正如加州理工大学的物理学家理查德·费曼曾经解释的那样：“回答许多基本的生物学问题是非常容易的；你只要看着它就行了！”如果我们能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自然状态下相互作用，我们将极大地提高我们对病毒生命周期和细胞先天免疫反应的理解。事实上，通过成像技术的进步，病毒学和细胞生物学已经取得了几项关键的历史性突破。传统电子显微镜(EM)的发展导致了病毒的第一张详细图像，后来又产生了细胞细胞器的精细超微结构，如高尔基体和内质网。连续的技术进步使我们能够在接近原子分辨率的情况下对病毒进行成像，这一壮举得到了2017年诺贝尔化学奖的认可，因为它发展了冷冻电子显微镜，用于高分辨率确定溶液中的生物分子的结构。随着最新的电子冷冻断层扫描(Cryo-ET)技术的发展，我们正在接近可视化任何病毒的自然状态和细胞内环境的距离。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，到形态形成和出口，都可以在大分子分辨率上可视化。这是在这个领域工作的一个非常令人兴奋的时刻。这项新技术为我们提供了以前所未有的分辨率解决病毒蛋白在其自然栖息地中的结构的机会。为了了解使用冷冻技术的病毒与宿主的相互作用，我建议解决一些针对病毒目布尼亚病毒目的重要生物学问题。这一顺序的病毒是许多新出现的病毒的代表，这些病毒对人和动物的健康构成高风险。在低温冷冻中，样品被放入冷冻剂(液态乙烷，或乙烷和丙烷的混合物)中，保存在冷冻水合的、接近天然状态的状态下。然后，冷冻的样品在透射电子显微镜中成像，随着样品绕轴递增旋转，一系列二维投影图像被记录下来。这个所谓的“倾斜系列”然后被重建成三维“断层图”，其典型的分辨率足以分辨出大分子(~5 nm)的形状和排列。如果断层图像或一组断层图像包含感兴趣对象的结构同质副本，则可以通过计算提取、对准和组合包含对象的“断层图像子体积”，我们称之为“断层图像平均”(STA)以提高信噪比并澄清细节，通常将分辨率提高到~2-3 nm。对于特别有利的样品，如细胞或病毒上的伪晶蛋白质涂层，STA的分辨率可以提高到甚至3.1a，足以建立从头开始的原子模型。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，如冷冻-Clem，冷冻-FIB研磨，冷冻-ET和STA，以高分辨率原位可视化相关病毒TOSV和RVFV(Phenuiviridae)的感染。使用这些技术，我将寻找在免疫反应和触发细胞的抗病毒先天免疫反应期间发生的亲病毒和抗病毒过程的新的结构方面。更具体地说，我将研究(I)MxA限制因子对病毒RNPs的限制，以及(Ii)TOSV和RVFV宿主反应拮抗剂NSS丝在细胞核内的结构。识别病毒和宿主之间的新结构和关键相互作用将是理解病毒复制和宿主抗病毒反应的基本方面的一步变化，甚至可能允许对宿主靶向治疗的新见解。

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.