The Biophysics of Virus Capsid Assembly

描述（由申请人提供）：在感染的细胞中，乙型肝炎病毒（HBV）核心在细胞质中组装。这些核心由含有逆转录酶-前基因组RNA复合物和相关伴侣蛋白的核心蛋白（Cp）的二十面体衣壳组成。我们表明，二聚Cp具有很强的倾向，在体外自组装，并会结合任何核酸。在错误的时间或地点进行组装，使错误的核酸或蛋白质互补体发生突变，将导致非生产性感染。初步数据支持我们的假设，即组装可以被变构触发。我们将使用生物物理和结构的方法来检查组装在体外。在目标1中，我们将检查二聚体内界面（Cp二聚体内）的变构转换证据。该界面处的突变对组装具有显著影响，尽管它们不参与将衣壳保持在一起的二聚体间接触。在目标2中，我们将剖析二聚体间的接触。理论研究表明，紧密结合将导致动力学陷阱，从而干扰衣壳组装。我们将研究增强和抑制组装的突变体，体外定量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E. coli，HBV Cp包装随机细菌核酸。通过研究随机和病毒核酸上的调节组装和组装的机制，我们解决了是否存在体内特异性的内在机制。这些实验将导致检查的物理和结构基础的Cp组装。通过将Cp视为分子机器，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的靶点，从而干扰HBV生命周期。

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.