Host Partners and Virus 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。核心由逆转录酶-前基因组RNA复合体和相关的伴侣蛋白组成，包含在核心蛋白(CP)的二十面体衣壳中。在RNA被逆转录后，现在充满DNA的核心被引导到细胞核以维持乙肝病毒的感染或被引导到内质网进行分泌。所有这些步骤都需要与宿主蛋白相互作用。为了包装正确的RNA，CP必须被磷酸化。运输到细胞核的充满DNA的核心被磷酸化，而分泌的病毒粒子被去磷酸化，这表明了激酶和磷酸酶的作用。无论是哪种命运，填充了DNA的核心都必须积极运输。我们假设，乙肝病毒衣壳是一种高度动态的结构，它会对内外刺激做出反应，瞬间显示闭塞的片段(磷酸化位点、核定位信号、晚期结构域)。在R21的应用中，我们将研究HBVCP与两种宿主蛋白的相互作用，这两种宿主蛋白被认为是细胞内活性的关键。Ser-Arg导向的蛋白激酶SRPK被证明能够磷酸化位于衣壳内部的CP RNA结合域。在目标1中，我们将研究SRPK与空的和充满核酸的衣壳的相互作用。在目标2中，我们将研究衣壳与NEDD4的相互作用，NEDD4是一种E3泛素连接酶，对调节乙肝病毒颗粒的运输非常重要。NEDD4通过与PPxY序列结合而与大多数蛋白质相互作用；在乙肝病毒衣壳中，该序列被埋在蛋白质-蛋白质界面上，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、柱分析和表面等离子体共振来检查结合。SRPK和NEDD4都与衣壳结合可能需要衣壳呼吸和扭曲衣壳四级结构。我们将研究衣壳的稳定性和内容物如何与结合活性有关，从而在病毒成熟和贩运之间建立联系。这些实验将导致对其他东道主伙伴的检查，以及调节他们与乙肝病毒活动的物理和结构基础。通过识别宿主伙伴及其结合机制，我们确定了宿主细胞内和病毒核心上抗病毒治疗的新靶点。公共卫生相关性：慢性乙肝病毒(乙肝)感染是导致肝硬变和肝细胞癌的主要危险因素，每年导致100万人死亡。像所有的病毒一样，乙肝病毒劫持宿主细胞的成分来调节病毒的组装、成熟和分泌。我们将研究乙肝病毒如何与两种宿主蛋白相互作用，这两种宿主蛋白对乙肝病毒复制至关重要，并可能成为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.