An integrative approach to deciphering the entry process in Herpesviruses

细胞过程是由被称为蛋白质和核酸的生物大分子的复杂的协调和动力学控制的。它们不是孤立地起作用，而是相互作用并聚集形成细胞复合物。了解复合体的结构不仅是理解基本细胞生物学的重要一步，也是理解疾病的重要一步，因为这种复合体也会在入侵病原体（如病毒）的蛋白质和宿主细胞蛋白质之间形成。事实上，要确定病毒如何发挥作用，不仅需要了解病毒自身蛋白质的分子排列，还需要了解它们在病毒生命周期中与宿主细胞的相互作用。特别有趣的是进入过程，周期中感染的最早阶段，当病毒第一次接触宿主细胞并将病毒物质引入细胞时。我们项目的目标是获得最大和最复杂的感染人类的病毒家族之一——疱疹病毒的进入过程的结构视图。这些病毒引起的疾病的严重程度从唇疱疹、生殖器溃疡和水疱到失明和危及生命的疾病，包括致命的脑炎、脑膜炎和癌症。由于其在世界范围内流行、易于传播和相关症状的严重性，该家族构成了一个主要的公共卫生问题。为了实现这一目标，我们提出了一种集成计算和实验方法的多学科方法。帮助这个项目的领域是结构生物学。它通过使用x射线晶体学和核磁共振等实验技术提供大分子复合物及其组分的“图片”，每种技术都有其自身的局限性，这取决于所调查样品的大小和纯度、制备条件以及所含复合物的均匀性。在过去的十年中，低温电子显微镜和断层扫描也成为观察生物复合物的重要技术。利用这些技术，使用低温液体快速冷冻样品，然后用电子轰击，产生许多二维样品的图像，这些图像可以组合成更清晰的三维图像。在断层扫描中，这样的图像可以提供细胞和组织的整体组织，并可以在细胞入侵期间捕获病原体。它们含有许多不同的大分子复合物，可以在它们的天然环境中检测到。尽管这些技术带来了许多有趣的发现，但也有局限性，通常不会产生近原子图像。在这个项目中，我们将研究人类疱疹病毒的进入过程。为此，我们将开发一种计算方法，将来自各种实验技术的信息整合在一起，以构建一个更清晰、更完整的描述最初由低温电子断层扫描成像的复合物结构。这种方法将有能力结合哪些蛋白质与哪些蛋白质相互作用的信息（或者它们彼此之间的距离有多近）。这些信息可以来自于各种各样的技术，通常统称为“蛋白质组学”。在一起，我们将把所有不同的信息片段像拼图一样组合起来，创造出更高分辨率的可视化复合体的图像。获得选定复合物（在病毒包膜上的蛋白质之间以及它们与宿主细胞中相互作用的蛋白质之间形成）的结构将代表我们对疱疹病毒发病机制的分子和机制细节的理解取得重大进展。这将使我们能够改进目前的进入过程模型，这是确定药物靶点的关键一步。我们的新方法将适用于许多病毒系统，并将为其他病原体的类似研究打开大门。

Cellular processes are governed by the intricate coordination and dynamics of biological macromolecules called proteins and nucleic acids. These do not act in isolation but rather interact with each other and assemble to form cellular complexes. Understanding the structures of complexes can be an important step not only in understanding basic cell biology but also disease, as such complexes are also formed between the proteins of invading pathogens like viruses and the host cell proteins. Indeed, to determine how a virus functions, knowledge is needed not only about the molecular arrangement of its own proteins but also about their interactions with the host cell over the course of the viral life cycle. Particularly interesting is the entry process, the earliest stage of infection in the cycle, when the virus comes into first contact with the host cell and introduces viral material into the cell. The goal of our project is to gain a structural view of the entry process in one of the largest and most complex families of viruses that infect humans - the Herpesviruses. The severity of conditions caused by these viruses ranges from cold sores, genital ulcers, and blisters to blindness and life-threatening conditions including fatal encephalitis, meningitis and cancer. This family constitutes a major public health concern due to their worldwide prevalence, ease of spread, and severity of the associated symptoms. To achieve this, we propose a multi-disciplinary approach that integrates computational and experimental methods.The field that aids this project is Structural Biology. It provides 'pictures' of macromolecular complexes and their components through the use of experimental techniques such as X-ray crystallography and nuclear magnetic resonance, each of which has its own limitations to what it can accomplish, depending on the size and purity of sample under investigation, the conditions in which its prepared, and the homogeneity of the complex it contains. In the last decade, cryo electron microscopy and tomography have also become important techniques for observing biological complexes. With these techniques, samples are rapidly frozen using cryogenic liquids and then bombarded with electrons, yielding many images of the 2-dimensional sample that can be combined into a clearer 3-dimensional picture. In tomography, such pictures can provide the overall organization of cells and tissues, and can capture pathogens during cell invasion. They contain many different macromolecular complexes that can be detected in their native environment. Though these techniques have led to many interesting discoveries, here too there are limitations, typically not resulting in near-atomic pictures.In this project, we will study the entry process in human herpesviruses. To this end, we will develop a computational approach that pulls together information from a variety of experimental techniques to construct a clearer and more complete description of structures of complexes imaged initially by cryo electron tomography. The method will have the capability of incorporating information about which protein interacts with which (or how close they are to each other). Such information could come from a variety of techniques, often grouped under the name 'proteomics'. Together, we will fit all the different pieces of information like a jigsaw puzzle, creating a higher resolution picture of the visualised complexes. Obtaining the structures of selected complexes (formed between the proteins placed on the envelope of the virus and between them and their interacting proteins from the host cell) will represent a major advance in our understanding of the molecular and mechanistic details of herpesvirus pathogenesis. This will allow us to improve current models of the entry process, a crucial step towards identifying drug targets. Our novel approach will be applicable to many viral systems and will open the door for similar studies on other pathogens.