Mechanism for hepatitis E virus exit from polarized hepatocytes

摘要 戊型肝炎病毒(HEV)感染是全世界急性肝炎的主要原因，并经常持续 免疫功能受损的人会导致严重的发病率和死亡率。目前，没有FDA- 美国批准的疫苗或HEV特异性治疗。我们的长期目标是阐明分子细节 了解HEV感染循环，以帮助合理设计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：该病毒以裸露颗粒的形式进入 但粪便在血液中以准包膜(EHEV)粒子的形式循环。这一领域的教条已经 EHEV介导病毒在感染宿主内传播，其生物发生需要病毒ORF3蛋白。 有人认为，ORF3篡夺了运输所需的细胞内体分选复合体(ESCRT) 从多囊泡体(MVB)获得包膜的机械。然而，在细胞培养中，HEV可以 传播与ORF3表达无关，携带ORF3起始密码子突变的临床分离株也已 描述。因此，HEV的释放机制可能比之前想象的更复杂。在混合动力汽车中- 感染极化的人肝细胞培养物和人肝嵌合小鼠，ORF3几乎只定位于 至根尖/小管膜。此外，我们最近发表的工作表明，ORF3是 HEV在极化的人肝癌细胞中的顶端释放，而不是基侧释放。此应用程序旨在 更好地确定HEV从极化肝细胞中释放的机制，并阐明ORF3在其中的作用 进程。我们的中心假设是，ORF3决定HEV的心尖释放，而不是基底外侧的必需 从极化的肝细胞中释放。我们提出了两个具体的目标来验证这一假设，使用体外和在 活体模型。目标1将使用高度极化的人肝癌细胞系来阐明HEV的机制 从人肝细胞顶端和基底外侧释放。我们将确定ORF3招募 HEV衣壳和ESCRT组件到心尖循环内小体(ARES)来源的转运囊泡到 促进HEV的包络和退出，独立于ORF3的机制负责HEV的释放 介导病毒传播的基底侧面。目标2将研究HEV的释放机制 新建立的戊型肝炎病毒感染大鼠模型。将调查ORF3、ESCRT、MVB和ARES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 极化人顶端和基底外侧释放的病毒粒子的特征和比较 肝癌细胞。这项工作的完成将为了解不寻常的混合动力汽车的生命周期提供新的线索，并澄清其作用 ORF3基因在HEV感染中的表达。从这项研究中获得的知识也将对 无包膜和包膜病毒的肝细胞生物学和非溶细胞性释放机制。

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.