Structural characterization of native HBV capsids and virions from human cells

项目总结 乙肝病毒感染是一个全球性的公共卫生问题。尽管有效的疫苗可以预防这种情况 对于这种疾病，目前批准的治疗方法很少能完全治愈。有一种未得到满足的医疗需求 开发新的治疗乙肝病毒感染的方法，可以导致持续的反应。靶向乙肝病毒衣壳 组装工艺已成为开发新的抗病毒药物治疗乙肝病毒的一种新兴策略。然而， 通过使用在大肠杆菌中表达的不同衣壳蛋白(CP)结构来进行许多努力 模仿或重建原生的病毒颗粒。然而，这些结构不能正确地代表本地的乙肝病毒 由于缺乏CP、病毒基因组和病毒酶的核酸结合域而形成的构象 它们是病毒复制所必需的。此外，缺乏对CP的翻译后修改也 阻碍了观察到的CP结构之间对从哺乳动物获得的生物医学数据的解释 细胞培养系统或实验动物。到目前为止还没有可用的高分辨率天然乙肝病毒结构， 这是对乙肝病毒领域知识的一大空白。 在这项提议中，我们的目标是使用冷冻电子显微镜(Cryo-EM)来直接表征 来自人类细胞的天然的乙肝病毒衣壳和病毒粒子。在目标1中，我们将确定的高分辨率结构 纯化的含有不同类型病毒基因组的细胞内乙肝病毒衣壳。我们将解决关键问题 关于基因组成熟过程中乙肝病毒衣壳结构的动态变化。我们还将确定 乙肝病毒逆转录酶(RT)的结构及其在逆转录过程中的位置 它的动作模式(RT是静止的还是移动的)。在目标2中，我们将研究高分辨率结构 分泌出的乙肝病毒粒子。这一目标将解决有关乙肝病毒衣壳如何与病毒相互作用的问题。 包膜蛋白质。最后，这两个目标的实验结果将结合起来阐明衣壳。 乙肝病毒复制过程中的动力学和阐明乙肝病毒包膜的分子决定因素(S)。 这一提议有望解决4种类型的细胞内乙肝病毒衣壳结构(空的、充满RNA的、单细胞的). 充满DNA的链状病毒和成熟的部分双链DNA填充的衣壳)和3个分泌型包膜乙肝病毒 病毒粒子结构(空的、成熟的和过早分泌的病毒粒子)使用冷冻-EM来定义构象 病毒复制期间衣壳的变化，特别是在不同的病毒基因组形式和 衣壳和表面蛋白之间的相互作用。这种方法利用了CP的适当突变 和RT，以确保获得如上所述的各种类型的均质颗粒，这可以进一步 经过计算分类，以最大限度地减少交叉污染。 了解乙肝病毒的天然结构将为乙肝生物学提供有价值的新信息，并指导 未来新型抗病毒药物的设计。该项目预计将影响从乙肝病毒到 分子病毒学、抗病毒药物开发、大分子结构和功能。

PROJECT SUMMARY Hepatitis B virus (HBV) infection is a global public health concern. Despite effective vaccines to prevent this disease, current approved treatment rarely leads to a complete cure. There is an unmet medical need for developing new therapeutics for HBV infection that can lead to a sustained response. Targeting HBV capsid assembly process has become an emerging strategy for developing new antiviral treatment for HBV. However, many efforts have been made by using different capsid protein (Cp) constructs expressed in Escherichia coli to mimic or reconstitute native-like viral particles. Yet, these structures cannot correctly represent the native HBV conformations due to the lacks of nucleic acid binding domain of the Cp, viral genome, and viral enzymes, all of which are required for viral replication. Furthermore, the lack of post translational modifications of the Cp also hampers the interpretation between observed Cp structures to the biomedical data obtained from the mammalian cell culture system or experimental animals. To date there is no available high-resolution native HBV structures, which is a major gap in knowledge of the HBV field. In this proposal, we aim to use cryo-electron microscope (cryo-EM) to directly characterize the structures of native HBV capsids and virions from human cells. In Aim 1, we will determine the high-resolution structures of purified intracellular HBV capsids with different types of viral genome. We will address the key questions concerning the structural dynamics of HBV capsids during genome maturation. We will also determine the structure of the HBV reverse transcriptase (RT) and its location during reverse transcription to help understand its mode of action (whether the RT is static or moves). In Aim 2, we will investigate the high-resolution structures of secreted HBV virions. This aim will address the questions concerning how HBV capsids interact with the viral envelop proteins. Finally, experimental findings from these two Aims will be integrated to elucidate capsid dynamics during HBV replication and illuminate the molecular determinant(s) of HBV envelopment. This proposal is expected to solve 4 types of intracellular HBV capsid structures (empty, RNA-filled, single- stranded DNA-filled, and mature partially double-stranded DNA-filled capsids) and 3 secreted enveloped HBV virion structures (empty, mature, and prematurely secreted virions) using cryo-EM to define the conformational changes of the capsid during viral replication, particularly in the context of different viral genome forms and interactions between the capsid and surface proteins. The methodology exploits appropriate mutations of Cp and RT to ensure obtaining homogenous particles of the various types as described above, which can be further computationally classified to minimize cross-contamination. Understanding the native HBV structures will provide valuable new information for HBV biology and guide the design of novel antiviral drugs in the future. The project is anticipated to impact fields ranging from HBV, molecular virology, antiviral drug development, and macromolecular structure and function.