Mechanism for hepatitis E virus exit from polarized hepatocytes

戊型肝炎病毒从极化肝细胞中退出的机制

• 批准号: 10578386
• 负责人: Zongdi Feng
• 金额: $ 51.8万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-14 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10578386
• 关键词: Acute Hepatitis; Apical; Bile fluid; Biliary; Biogenesis; Biology; Blood Circulation; Capsid; Cell Culture Techniques; Cell Line; Cell Membrane Permeability; Cell Polarity; Cells; Clinical; Complex; Data; Endosomes; Enteral; FDA approved; Feces; Goals; HepG2; Hepatitis B; Hepatitis E virus; Hepatocyte; Human; Immunocompromised Host; In Vitro; Individual; Infection; Initiator Codon; Knowledge; Life Cycle Stages; Liver; Liver diseases; Macaca; Mediating; Membrane; Modeling; Molecular; Morbidity - disease rate; Morphology; Multivesicular Body; Mus; Mutation; Outcome; Play; Prevention; Process; Proteins; Publishing; RNA Viruses; Rattus; Recycling; Research; Risk; Role; Side; Sorting; Surface; Testing; Vaccines; Viral; Viral Pathogenesis; Viremia; Virion; Virus; Virus Diseases; Work; hepatoma cell; in vivo Model; insight; mortality; mutant; novel; palmitoylation; particle; rational design; recruit; trafficking; transmission process; vesicle transport

Summary Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide and can often persist in immunocompromised individuals leading to significant morbidity and mortality. Currently, there are no FDA- approved vaccines or HEV-specific therapy in the U.S. Our long-term goal is to elucidate the molecular details of the HEV infectious cycle to aid in the rational design of HEV-specific prevention and treatment. HEV is an enterically transmitted positive-strand RNA virus with a unique life cycle: the virus is shed as naked particles into the feces but circulates as quasi-enveloped (eHEV) particles in the bloodstream. The dogma in the field has been that eHEV mediates virus spread in the infected host, and its biogenesis requires the viral ORF3 protein. It is proposed that ORF3 usurps the cellular endosomal sorting complex required for transport (ESCRT) machinery to acquire an envelope from the multivesicular bodies (MVBs). However, in cell culture, HEV can spread regardless of ORF3 expression, and clinical isolates bearing ORF3 start codon mutations have also been described. Thus, the release mechanism for HEV is likely to be more complex than previously thought. In HEV- infected polarized human hepatocyte cultures and human liver chimeric mice, ORF3 almost exclusively localizes to the apical/canalicular membrane. Moreover, our recently published work shows that ORF3 is required for apical but not the basolateral release of HEV from polarized human hepatoma cells. This application seeks to better define the mechanism for HEV release from polarized hepatocytes and to clarify the role of ORF3 in this process. Our central hypothesis is that ORF3 dictates apical release of HEV but is not required for basolateral release from polarized hepatocytes. We propose two specific aims to test this hypothesis using in vitro and in vivo models. Aim 1 will use a highly polarized human hepatoma cell line to elucidate the mechanism for HEV release from both the apical and basolateral sides of human hepatocytes. We will determine that ORF3 recruits HEV capsid and ESCRT components to the apical recycling endosomes (AREs)-derived transport vesicles to facilitate HEV envelopment and exit, and an ORF3-independent mechanism is responsible for HEV release at the basolateral surface that mediates virus spread. Aim 2 will investigate the HEV release mechanisms in a newly developed rat model of HEV infection. The role of ORF3, ESCRT, MVBs and AREs will be investigated using various viral mutants. The morphology and protein composition of HEV virions in the bloodstream will be characterized and compared to virions released from the apical and basolateral sides of polarized human hepatoma cells. The completion of this work will shed new light on the unusual HEV life cycle and clarify the role of ORF3 in HEV infection. The knowledge gained from this research will also have broad implications for hepatocyte biology and noncytolytic release mechanisms for both nonenveloped and enveloped viruses.

概括 戊型肝炎病毒 (HEV) 感染是全世界急性肝炎的主要原因，并且通常会持续存在 免疫功能低下的个体导致显着的发病率和死亡率。目前，FDA还没有 在美国批准的疫苗或 HEV 特异性疗法。我们的长期目标是阐明分子细节 HEV 感染周期的研究有助于合理设计 HEV 特异性预防和治疗。混合动力汽车是一种 肠道传播的正链 RNA 病毒，具有独特的生命周期：病毒以裸露颗粒的形式排出到体内 粪便中，但作为准包膜 (eHEV) 颗粒在血流中循环。该领域的教条有 eHEV 介导病毒在受感染宿主中传播，其生物发生需要病毒 ORF3 蛋白。 有人提出 ORF3 篡夺了运输所需的细胞内体分选复合体 (ESCRT) 从多泡体（MVB）获取包膜的机器。然而，在细胞培养中，HEV 可以 无论 ORF3 表达如何，病毒都会传播，携带 ORF3 起始密码子突变的临床分离株也已被 描述的。因此，HEV 的释放机制可能比之前想象的更加复杂。在混合动力汽车中- 感染极化人肝细胞培养物和人肝嵌合小鼠后，ORF3几乎完全定位 到心尖/小管膜。此外，我们最近发表的工作表明 ORF3 是 HEV 从极化的人肝癌细胞的顶端释放，但不是基底外侧释放。该应用程序旨在 更好地定义极化肝细胞释放 HEV 的机制，并阐明 ORF3 在此过程中的作用 过程。我们的中心假设是 ORF3 决定 HEV 的顶端释放，但基底外侧不需要 从极化的肝细胞中释放。我们提出了两个具体目标来使用体外和体内测试这一假设 体内模型。目标 1 将使用高度极化的人肝癌细胞系来阐明 HEV 的机制 从人肝细胞的顶端和基底外侧释放。我们将确定 ORF3 招募 HEV 衣壳和 ESCRT 成分到顶端回收内体 (ARE) 衍生的运输囊泡 促进 HEV 包封和退出，并且独立于 ORF3 的机制负责 HEV 在 介导病毒传播的基底外侧表面。目标 2 将研究混合动力汽车的释放机制 新开发的HEV感染大鼠模型。将研究 ORF3、ESCRT、MVB 和 ARE 的作用 使用各种病毒突变体。血液中 HEV 病毒粒子的形态和蛋白质组成将是 表征并与从极化人类的顶端和基底外侧释放的病毒颗粒进行比较 肝癌细胞。这项工作的完成将为非同寻常的混合动力汽车生命周期提供新的视角，并阐明其作用 ORF3在HEV感染中的作用。从这项研究中获得的知识也将产生广泛的影响 无包膜病毒和包膜病毒的肝细胞生物学和非溶细胞释放机制。