Host Partners and Virus Assembly

描述（由申请人提供）：在感染细胞中，乙型肝炎病毒（HBV）核在细胞质中聚集。该核心是由一个逆转录-基因组前RNA复合物和包含在核心蛋白（Cp）二十面体衣壳中的相关伴侣蛋白组成。RNA逆转录后，现在充满dna的核心被引导到细胞核以维持HBV感染或进入ER以分泌。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。运送到细胞核的dna填充的核被磷酸化，而分泌的病毒粒子被去磷酸化，这表明激酶和磷酸酶的作用。无论是哪种命运，充满dna的核都必须主动运输。我们假设HBV衣壳是一个高度动态的结构，在响应内部和外部刺激时，会短暂地显示闭塞的片段（磷酸化位点、核定位信号、晚期结构域）。在本R21应用中，我们将研究HBV Cp与两种被认为对细胞内活性至关重要的宿主蛋白的相互作用。Ser-Arg定向蛋白激酶SRPK已被证明可以磷酸化衣壳内部的Cp rna结合域。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NEDD4的相互作用，NEDD4是一种E3泛素连接酶，对调节HBV颗粒的运输很重要。NEDD4通过结合PPxY序列与大多数蛋白相互作用；在HBV衣壳中，这一序列隐藏在蛋白-蛋白界面，但在衣壳呼吸时短暂暴露。为了这两个目的，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，从而在病毒成熟和运输之间建立联系。这些实验将导致检查其他宿主伴侣以及调节其HBV活动的物理和结构基础。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上抗病毒治疗的新靶点。公共卫生相关性：慢性乙型肝炎病毒（HBV）感染是肝硬化和肝细胞癌的主要危险因素，每年造成100万人死亡。像所有病毒一样，HBV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HBV如何与两种宿主蛋白相互作用，这两种宿主蛋白对HBV复制至关重要，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.