Viral and Host Determinants of Endosomal Trafficking during HBV Infection

项目总结 乙肝病毒是一种DNA病毒，属于庚型核糖核酸病毒家族，与慢性肝炎有关 感染了全球超过2.5亿人，导致每年近100万人死于严重的肝脏 肝硬变和肝细胞癌等疾病。不幸的是，抗病毒治疗未能治愈乙肝病毒 由于作为病毒模板的共价闭合环状DNA(CccDNA)的韧性而引起的感染 转录和随后翻译建立和维持乙肝病毒复制所需的所有病毒产物。 关于cccDNA产生后的乙肝病毒复制事件，人们了解得很多，但对 最初生产cccDNA以建立生产性感染所需的细胞内转运步骤。 我的博士论文研究旨在确定病毒衣壳在乙肝病毒内吞转运中的作用 感染(目标1)。我们已经鉴定出一系列具有独特表型的HBV衣壳突变体 在感染期间，它们不会导致产生性感染(即不形成cccDNA)，但cccDNA可以形成 在细胞内扩增的过程中，这绕过了感染过程中的病毒进入步骤，这表明 在感染变异病毒的过程中，病毒进入的步骤被阻止，这在 转染法。我们计划监测入境后WT和变异乙肝病毒的贩运事件(目标1.1)，并将进一步 通过使用衣壳抑制物来确定病毒衣壳在引导内体转运中的作用 与我们的衣壳突变体相似的效果(目标1.2)。这些衣壳蛋白抑制剂正在积极的临床开发中； 因此，至关重要的是，我们要了解哪些进入步骤受到它们的影响。回答相关问题 通过对病毒进入受体hNTCP的鉴定，使进入乙肝病毒成为可能，然而其 在HEK293细胞中表达不能增加对乙肝病毒感染的易感性，而这可以支持cccDNA的形成 但在感染过程中不支持cccDNA的形成，表现为复制乙肝病毒的影响 衣壳突变和抑制物。这个项目的第二部分将集中在定义宿主细胞在 在感染期间传播乙肝病毒(目标2)。我们将解决HEK293-hNTCP细胞中乙肝病毒的胞内转运问题 识别感染中的阻断(S)发生的位置，使这些细胞不敏感(目标2.1)。以帮助确定 我们设计的宿主决定因素，在进入受体hNTCP之外，可能在乙肝病毒进入中发挥作用 一种基于GFP的裂解乙肝病毒报告系统，将用于分离HepG2-hNTCP细胞亚群， 对乙肝病毒感染高度易感(目标2.2)，并进行RNA测序分析以区分 在敏感细胞和非敏感细胞之间表达基因。这些拟议的研究将增加我们的 了解乙肝病毒进入，并确定导致生产性感染的必要的内吞运输步骤。 总之，我的论文的成功完成将对(1)理解产生深远的影响 乙肝病毒内体转运和进入的基本事件及(2)确定乙肝病毒衣壳和新宿主 对乙肝病毒进入和生产性感染至关重要的决定因素。

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.