Host Partners and Virus Assembly

描述（由申请人提供）：在感染的细胞中，B肝炎病毒（HBV）核心在细胞质中组装。核心由逆转录酶-前基因组RNA复合物和包含在核心蛋白（Cp）的二十面体衣壳中的相关伴侣蛋白组成。在RNA被逆转录后，现在DNA填充的核心被引导到细胞核以维持HBV感染或被分泌到ER。所有这些步骤都需要与宿主蛋白质相互作用。为了包装正确的RNA，Cp必须被磷酸化。被运输到细胞核的DNA填充的核心被磷酸化，而分泌的病毒体被去磷酸化，表明激酶和磷酸酶的作用。对于任何一种命运，DNA填充的核心必须积极运输。我们假设，HBV衣壳是一个高度动态的结构，瞬时显示闭塞段（磷酸化位点，核定位信号，后期结构域），以响应内部和外部刺激。在这个R21应用程序中，我们将研究HBV Cp与两个宿主蛋白质的相互作用，这两个蛋白质被认为是细胞内活性的关键。Ser-Arg指导的蛋白激酶SRPK已显示磷酸化位于衣壳内部的Cp RNA结合结构域。在目的1中，我们将研究SRPK与空衣壳和核酸填充衣壳的相互作用。在目标2中，我们将研究衣壳与NEDD 4的相互作用，NEDD 4是一种E3泛素连接酶，对调节HBV颗粒的运输很重要。NEDD 4通过结合PPxY序列与大多数蛋白质相互作用;在HBV衣壳中，该序列被埋在蛋白质-蛋白质界面处，但通过衣壳呼吸短暂暴露。对于这两个目标，我们将使用酶活性，柱测定和表面等离子体共振检查结合。SRPK和NEDD 4与衣壳的结合可能需要衣壳呼吸和衣壳四级结构的扭曲。我们将研究衣壳稳定性和内容物如何与结合活性相关，从而在病毒成熟和贩运之间建立联系。这些实验将导致检查其他主机的合作伙伴和物理和结构的基础上，调节他们的活动与HBV。通过鉴定宿主伴侣及其结合机制，我们确定了宿主细胞内和病毒核心的抗病毒治疗的新靶点。公共卫生相关性：慢性B型肝炎病毒（HBV）感染是肝硬化和肝细胞癌的主要危险因素，每年导致100万人死亡。像所有病毒一样，HBV劫持宿主细胞的组分以调节病毒组装、成熟和分泌。我们将研究HBV如何与两种对HBV复制至关重要的宿主蛋白相互作用，并可能代表抗病毒治疗的新靶点。

DESCRIPTION (provided by applicant): In an infected cell, Hepatitis B virus (HBV) cores assemble in the cytoplasm. The cores are comprised of a reverse transcriptase-pregenomic RNA complex and associated chaperonins contained in an icosahedral capsid of core protein (Cp). After the RNA is reverse transcribed, the now DNA-filled cores are directed to either the nucleus to maintain HBV infection or to the ER to be secreted. All of these steps require interaction with host proteins. To package the correct RNA, the Cp must be phosphorylated. DNA-filled cores that are transported to the nucleus are phosphorylated while secreted virions are dephosphorylated, indicating roles for kinases and phosphatases. For either fate, DNA-filled cores must be actively transported. We hypothesize that the HBV capsid is a highly dynamic structure that transiently displays occluded segments (phosphorylation sites, nuclear localization signals, late domains) in response to internal and external stimuli. In this R21 application we will investigate HBV Cp interaction with two host proteins believed to be critical for intracellular activity. The Ser-Arg directed protein kinase SRPK has been shown to phosphorylate the Cp RNA-binding domain, which is on the interior of capsids. In aim 1, we will investigate the interaction of SRPK with empty and nucleic acid-filled capsid. In aim 2, we will examine interaction of capsids with NEDD4, an E3 ubiquitin ligase that is important for regulating trafficking of HBV particles. NEDD4 interacts with most proteins by binding to PPxY sequences; in HBV capsids this sequence is buried at a protein-protein interface but is transiently exposed by capsid breathing. For both aims, we will examine binding using enzymatic activity, column assays, and surface plasmon resonance. Binding of both SRPK and NEDD4 to capsids is likely to require capsid breathing and distortion of capsid quaternary structure. We will examine how capsid stability and contents relate to binding activity, making a connection between virus maturation and trafficking. These experiments will lead to examination of other host partners and the physical and structural basis of regulating their activities with HBV. By identifying host partners and their mechanism of binding, we identify new targets for antiviral therapeutics within the host cell and on the core of the virus. PUBLIC HEALTH RELEVANCE: Chronic Hepatitis B virus (HBV) infection is a major risk factor for cirrhosis of the liver and hepatocellular carcinoma, contributing to 1 million deaths per year. Like all viruses, HBV hijacks components of host cells to regulate virus assembly, maturation, and secretion. We will investigate how HBV interacts with two host proteins that are critical to HBV replication and may represent new targets for antiviral therapeutics.