Hepatitis C Virus interaction with the innate immune system in HepG2-HFL cells

描述（由申请人提供）：丙型肝炎病毒是西半球肝脏疾病的主要原因。目前的HCV治疗是不充分的，因为它与严重的副作用有关，而且往往无效。大多数HCV感染是持续的、终生的慢性感染，缓慢地诱发发病机制。此外，HCV似乎并不直接诱导肝脏疾病，而是通过刺激细胞因子的产生和炎症间接诱导，这表明它对先天免疫途径的损害不是绝对的。支持持续HCV复制和慢性感染的病毒-宿主相互作用和分子机制，以及最终诱发肝脏疾病的机制尚不清楚。我们假设先天细胞内免疫防御在调节HCV复制中起重要作用，并且病毒的持久性与HCV对先天防御过程的控制有关。几乎所有关于这一主题的研究都是在Huh-7细胞或衍生物中进行的，它们表现出严重受损的先天免疫反应。我们最近发现，如果肝脏特异性microRNA miR-122与缺失的进入因子CD-81 （HepG2- hfl细胞）一起表达，肝癌来源的HepG2细胞有效地支持整个HCV生命周期。虽然含有这两种因子的HepG2细胞可以复制HCV的整个生命周期，但我们发现，与先前建立的Huh-7.5细胞系相比，这些细胞支持感染和传播的能力存在显著差异。同时，我们发现这些细胞的先天免疫反应对HCV感染的反应更为活跃。因此，我们假设HepG2-HFL细胞将忠实地再现肝细胞中存在的先天免疫反应，从而为理解HCV与先天免疫系统之间的相互作用提供更好的模型。我们的最终目标是更好地了解丙型肝炎病毒如何在激活炎症途径的同时破坏抗病毒反应。我们建议通过实验比较HepG2-HFL细胞与原代人肝细胞和Huh-7细胞的先天免疫途径，特别关注细胞病毒RNA传感器RIG-I、MDA5和TLR3诱导的应答。接下来，我们将研究HCV与HepG2-HFL细胞先天免疫途径之间的相互作用，以确定HCV是否激活先天免疫反应，从而有效地对抗感染，并通过病毒蛋白的作用抑制这种反应。我们预计这些研究的结果将为HCV和先天免疫系统之间的相互作用提供新的见解，尽管它在相关疾病的发病机制中很重要，但由于缺乏强大的模型系统，人们对HCV和先天免疫系统的相互作用仍然知之甚少。

DESCRIPTION (provided by applicant): Hepatitis C virus is the leading cause of liver disease in the Western hemisphere. The current HCV therapy is inadequate, as it is associated with severe side effects and often not effective. The majority of HCV infections are persistent, life-long chronic infections that slowly induce pathogenesis. Furthermore, HCV does not appear to directly induce liver disease, rather it does so indirectly by stimulating cytokine production and inflammation, which suggests that its impairment of innate immune pathways is not absolute. The virus-host interactions and molecular mechanisms that support persistent HCV replication and chronic infection, and those that eventually induce liver disease are not understood. We hypothesize that innate intracellular immune defenses play an essential role in regulating HCV replication and that viral persistence is linked to HCV control of innate defense processes. Nearly all the studies of this topic have been conducted in Huh-7 cells or derivatives, which exhibit severely impaired innate immune responses. We recently showed that hepatoma-derived HepG2 cells efficiently support the entire HCV life cycle if the liver-specific microRNA miR-122 is expressed along with the missing entry factor CD-81 (HepG2-HFL cells). While HepG2 cells containing these two factors can replicate the entire life cycle of HCV, we found that there was a striking difference in these cells' ability to support infection and spread when compared to the previously established Huh-7.5 cell line. At the same time, we found that the innate immune response in these cells was more active in response to HCV infection. Therefore, we hypothesize that HepG2-HFL cells will faithfully recapitulate the innate immune responses present in hepatocytes, and thus provide a better model through which to understand the interactions between HCV and the innate immune system. Our ultimate goal is to better understand how HCV can subvert the antiviral response while allowing for activation of inflammatory pathways. We propose experiments to compare innate immune pathways in HepG2-HFL cells with those of primary human hepatocytes and Huh-7 cells, with a particular focus on the responses induced by the cellular viral RNA sensors RIG-I, MDA5, and TLR3. We will next examine the interaction between HCV and innate immune pathways in HepG2-HFL cells to determine whether HCV activates innate immune responses that can effectively combat infection as well as inhibit such responses through the action of viral proteins. We anticipate tha the results of these studies will provide novel insights into the interaction between HCV and the innate immune system, which remains poorly understood due to a lack of robust model systems, despite its importance in the pathogenesis of associated disease.