Viral and Host Determinants of Endosomal Trafficking during HBV Infection

项目摘要 B型肝炎病毒（HBV）是属于嗜肝DNA病毒家族的DNA病毒， 感染全球超过2.5亿人，每年因严重肝病导致近百万人死亡。 肝硬化和肝细胞癌等疾病。不幸的是，抗病毒治疗未能治愈HBV 由于共价闭合环状DNA（cccDNA）的韧性，其是病毒感染的模板， 转录和随后的所有病毒产物的翻译建立和维持HBV复制。 关于cccDNA产生后的HBV复制事件了解得很多，然而关于HBV复制的机制知之甚少。 细胞内运输步骤，这是初始cccDNA产生所需的，以建立生产性感染。 我的博士论文研究的目的是确定的作用，病毒衣壳内吞运输过程中的HBV 感染（目标1）。我们已经鉴定了一系列具有独特表型的HBV衣壳突变体， 在感染期间，它们不会导致生产性感染（即，无cccDNA形成），但cccDNA可形成 在通过细胞内扩增转染期间，其绕过了感染期间的病毒进入步骤，表明 在感染突变病毒期间，病毒进入步骤中存在阻断， 转染我们计划监测WT和突变型HBV的入境后贩运事件（目标1.1），并将进一步 定义病毒衣壳在通过使用衣壳抑制剂引导内体运输中的作用， 与我们的衣壳突变体类似的效果（Aim 1.2）。这些衣壳抑制剂正在积极的临床开发中; 因此，至关重要的是，我们要了解它们调节了哪些进入步骤。处理与下列问题有关的问题： 通过病毒进入受体hNTCP的鉴定，已经使HBV进入成为可能，但是其 表达不能赋予HEK293细胞对HBV感染的易感性，这可以支持cccDNA形成 但不能支持感染期间cccDNA的形成，表型模仿HBV的作用， 衣壳突变和抑制剂。本项目的第二部分将集中于定义宿主细胞在 感染期间的HBV运输（目的2）。我们将研究HBV在HEK293-hNTCP细胞中的内吞转运 以确定在感染中发生阻滞的位置，使这些细胞不敏感（目标2.1）。来帮助识别 我们设计了一个宿主决定簇，除了进入受体hNTCP，可能在HBV进入中发挥作用， 一种基于GFP的HBV报告系统，用于分离HepG2-hNTCP细胞亚群， 对HBV感染高度敏感（目标2.2），并进行RNA测序分析，以确定差异 在易感细胞和非易感细胞之间表达基因。这些研究将增加我们的 了解HBV进入并确定导致生产性感染的基本内吞运输步骤。 总之，我的论文的成功完成将产生深远的影响（1）理解 HBV内体运输和进入的基本事件和（2）鉴定HBV衣壳和新宿主 决定因素是HBV进入和生产性感染的关键。

PROJECT SUMMARY Hepatitis B virus (HBV) is a DNA virus belonging to the hepadnavirus family and is responsible for chronically infecting over 250 million individuals worldwide, resulting in nearly a million annual deaths due to severe liver diseases such as cirrhosis and hepatocellular carcinoma. Unfortunately, anti-viral treatments fail to cure HBV infection due to the tenacity of covalently closed circular DNA (cccDNA), which is the template for viral transcription and subsequent translation of all viral products necessary to establish and sustain HBV replication. Much is learnt about HBV replication events post cccDNA production, however little is known about the intracellular trafficking steps that are required for the initial cccDNA production to establish productive infection. My PhD dissertation research aims to define the role of the viral capsid in endocytic trafficking during HBV infection (Aim 1). We have identified a series of HBV capsid mutants which have a unique phenotype in that during infection, they do not lead to productive infection (i.e., no cccDNA formation), however cccDNA can form during transfection via intracellular amplification, which bypasses the viral entry steps during infection, indicating that there is a block in the viral entry steps during infection with the mutant viruses that are not required during transfection. We plan to monitor post-entry trafficking events of WT and mutant HBV (Aim 1.1) and will further define the role of the viral capsid in directing endosomal trafficking through use of capsid inhibitors that induce a similar effect to our capsid mutants (Aim 1.2). These capsid inhibitors are in active clinical development; therefore, it is crucial that we understand what entry steps are modulated by them. Addressing questions related to HBV entry has been made possible by the identification of the viral entry receptor, hNTCP, however its expression cannot confer susceptibility to HBV infection in HEK293 cells, which can support cccDNA formation during transfection but cannot support cccDNA formation during infection, phenocopying the effects of the HBV capsid mutations and inhibitors. The second portion of this project will focus on defining the role of host cells in HBV trafficking during infection (Aim 2). We will address the endocytic trafficking of HBV in HEK293-hNTCP cells to identify where the block(s) in infection occurs, rendering these cells non-susceptible (Aim 2.1). To help identify the host determinants, beyond the entry receptor hNTCP, that may play a role in HBV entry, we have designed a split GFP-based HBV reporter system that will be used to isolate a subpopulation of HepG2-hNTCP cells that are highly susceptible to HBV infection (Aim 2.2) and perform RNA sequencing analysis to identify differentially expressed genes between susceptible vs. non-susceptible cells. These proposed studies will increase our understanding of HBV entry and identify the essential endocytic trafficking steps that lead to productive infection. Altogether, the successful accomplishment of my dissertation will have profound impacts on (1) understanding the essential events of HBV endosomal trafficking and entry and (2) identifying the HBV capsid and novel host determinants that are critical for HBV entry and productive infection.