The Biophysics of Virus Capsid Assembly

描述(申请人提供)：在受感染的细胞中，乙肝病毒(乙肝)核心在细胞质中聚集。这些核心由一个二十面体的核心蛋白(CP)衣壳组成，其中包含一个逆转录酶-前基因组RNA复合体和相关的伴侣蛋白。我们发现，二聚体CP在体外有很强的自组装倾向，可以与任何核酸结合。在错误的时间或地点组装，包装错误的核酸或蛋白质补体，将导致非生产性感染。初步数据支持我们的假设，即组装可以通过变构触发。我们将使用生物物理和结构方法来检查体外组装。在目标1中，我们将检查内部二聚体界面(在CP二聚体内)，以寻找变构转变的证据。这个界面的突变对组装有戏剧性的影响，尽管它们不参与将衣壳连接在一起的二聚体之间的接触。在目标2中，我们将剖析二聚体间接触。理论研究表明，紧密结合会导致动力学陷阱，从而干扰衣壳的组装。我们将检查增强和抑制组装的突变体，量化体外组装，并观察培养细胞的表型。在目标3中，我们将研究组装的调节如何在RNA填充衣壳的形成中发挥作用。当在大肠杆菌中过表达时，HBVCP包装随机的细菌核酸。通过研究随机核和病毒核上调控组装和组装的机制，我们解决了体内是否存在特异性的内在机制。这些实验将导致对CP组装的物理和结构基础的检查。通过将CP视为一台分子机器，我们将能够识别结构中的调控元件，这些元件可能是干扰组装从而干扰乙肝病毒生命周期的目标。

DESCRIPTION (provided by applicant): In an infected cell, Hepatitis B virus (HBV) cores assemble in the cytoplasm. These cores are comprised of an icosahedral capsid of core protein (Cp) containing a reverse transcriptase-pregenomic RNA complex and associated chaperonins. We show that the dimeric Cp has a strong propensity to self-assemble in vitro and will bind any nucleic acid. Assembly at the wrong time or place, encapsidating the wrong nucleic acid or complement of proteins, would lead to non-productive infections. Preliminary data support our hypothesis that assembly can be allosterically triggered. We will use biophysical and structural approaches to examine assembly in vitro. In aim 1, we will examine the intra-dimer interface (within a Cp dimer) for evidence of allosteric transitions. Mutations at this interface have dramatic effects on assembly though they are not involved in the inter-dimer contacts that hold a capsid together. In aim 2, we will dissect that inter-dimer contact. Theoretical studies indicate that tight binding will lead to kinetic traps and thus interfere with capsid assembly. We will examine mutants that enhance and inhibit assembly, quantifying assembly in vitro and observing phenotype in cultured cells. In aim 3, we will examine how regulation of assembly plays a role in the formation of RNA-filled capsids. When over-expressed in E. coli, HBV Cp packages random bacterial nucleic acid. By examining mechanisms of regulating assembly and assembly on random and viral nucleic, we address whether there is an intrinsic mechanism for specificity in vivo. These experiments will lead to examination of the physical and structural basis of Cp assembly. By considering Cp as a molecular machine, we will be able to identify regulatory elements in the structure that may be targets for interfering with assembly and thus the HBV lifecycle.