The Biophysics of Virus Capsid Assembly

6.总结 在受感染的细胞中，乙肝病毒(乙肝)核心聚集在细胞质中。这些核心由以下部分组成 核心蛋白(CP)的二十面体衣壳，包含逆转录酶-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，并且 会与任何核酸结合。在错误的时间或地点组装，包装错误的核酸或 蛋白质的补充，会导致非生产性感染。初步数据支持我们的假设 这种集合体可以变构触发。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)，以寻找证据 变构转变。这个界面的突变对组装有很大的影响，尽管它们不是 参与了将衣壳连接在一起的二聚体间接触。在目标2中，我们将剖析中间二聚体 联系。理论研究表明，紧密结合会导致动力学陷阱，从而干扰衣壳 集合。我们将检查增强和抑制组装的突变体，量化体外组装和 观察培养细胞的表型。在目标3中，我们将研究对装配的调节如何在 形成充满核糖核酸的衣壳。当在大肠杆菌中过表达时，HBVCP包装随机细菌 核酸。通过研究调控随机核和病毒核上的组装和组装的机制， 我们讨论是否存在体内特异性的内在机制。 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， 可能是干扰组装从而干扰乙肝病毒生命周期的靶标。

6. Summary 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.