Role of Lipid Droplets in Hepatitis C Virus Infection

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)流行是一个全球性的健康问题，影响全世界约1.7亿人。在约80%的病例中，病毒感染变为慢性，使丙型肝炎病毒成为肝脏相关发病率和死亡率的主要原因。慢性丙型肝炎病毒感染的一个常见症状是脂肪变性，即肝脏中脂滴的异常堆积。约55%的慢性丙型肝炎患者可观察到脂肪变性，脂肪变性是肝纤维化和癌症发展的重要危险因素。在转基因小鼠的肝脏中表达的病毒核衣壳核心概括了这种情况；核心本身也定位于脂滴(LDS)的表面，这是一个对后代病毒粒子在LDS附近的膜上组装至关重要的过程。我们最近发现甘油三酯合成酶DGAT1是一种新的丙型肝炎病毒组装的宿主因子(Herker等人，NAT。地中海医院。2010)。在缺乏DGAT1或经DGAT1抑制剂处理的细胞中，CORE不能定位于LDS，也不能将病毒RNA募集到邻近的内质网膜上进行囊化。其结果是，丙型肝炎病毒颗粒的产生严重受损。我们最近发表了另外三项研究，证明1)另一种丙型肝炎病毒蛋白NS5A与DGAT1相互作用，需要DGAT1进行LD定位(Camus等人，J.Biol)。化学。2013)，2)LDS表面核心减少这些LDS的脂解，从而引起脂肪变性(Harris，Herker等，J.Biol。化学。3)通过LD相关蛋白TIP47和NS5A之间的新的相互作用，LDS在丙型肝炎病毒感染中发挥了更大的作用，从而参与了丙型肝炎病毒的RNA复制(Vogt等，PLoS pathog)。2013)。我们试图从分子上详细研究LDS在丙型肝炎病毒生命周期中作为关键宿主细胞器的作用。我们提出了三个实验。1)明确DGAT1在向LDS募集丙型肝炎病毒蛋白中的作用。我们将检验这样的假设，即DGAT1、NS5A和CORE的三方复合体已经进化为将CORE和NS5A共同招募到DGAT1生成的LDS中。我们还将确定NS5A和CORE是否为DGAT1酰基转移酶活性的新蛋白质靶点。2)确定丙型肝炎病毒核心蛋白如何抑制脂解作用。我们将测试LDS表面的核心是否干扰甘油三酯脂肪酶ATGL/PNPLA2和PNPLA3的招募或活性，从而抑制脂解。由于感染不同基因型丙型肝炎病毒的患者发生脂肪变性的风险不同，我们将比较不同基因型病毒核心蛋白的抗脂代谢活性。3)探讨NS5A-TIP47相互作用如何调控丙型肝炎病毒RNA复制。我们将确定低密度脂蛋白在丙型肝炎病毒感染中是否起双重作用：一个是丙型肝炎病毒RNA复制的膜和能量来源，另一个是组装平台。我们将确定NS5A与TIP47的结合是否在这一过程中发挥重要作用。总之，我们提出的研究将为LDS在丙型肝炎病毒生命周期中的作用带来新的分子洞察力，并可能发现治疗慢性丙型肝炎疾病的潜在新的治疗策略。

DESCRIPTION (provided by applicant): The hepatitis C virus (HCV) epidemic is a global health problem and affects ~170 million people worldwide. In ~80% of cases, viral infection becomes chronic, rendering HCV a leading cause of liver-related morbidity and mortality. A common symptom of chronic HCV infection is steatosis, the abnormal accumulation of lipid droplets in the liver. Steatosis is observed in ~55% of chronically HCV-infected patients and represents an important risk factor for the development of liver fibrosis and cancer. The viral nucleocapsid core expressed in livers of transgenic mice recapitulates this condition; core itself also localizes to the surface of lipid droplets (LDs), a process critical for the assembly of progeny virions at membranes in close proximity to LDs. We recently identified the triglyceride-synthesizing enzyme DGAT1 as a novel host factor for HCV assembly (Herker et al, Nat. Med. 2010). In cells lacking DGAT1 or treated with a DGAT1 inhibitor, core cannot localize to LDs and cannot recruit viral RNA to neighboring endoplasmic reticulum membranes for encapsidation. As a consequence, HCV particle production is severely impaired. We recently published three additional studies that demonstrate 1) that a second HCV protein, NS5A, interacts with DGAT1 and requires DGAT1 for LD localization (Camus et al, J. Biol. Chem. 2013), 2) that core at the surface of LDs decreases the lipolysis of these LDs, thereby causing steatosis (Harris, Herker et al, J. Biol. Chem. 2012), and 3) that LDs play a larger role in HCV infection with a new involvement in HCV RNA replication via a new interaction between the LD- associated protein TIP47 and NS5A (Vogt et al, PLoS Pathog. 2013). We seek to study in molecular detail the role of LDs as critical host organelles in the HCV lifecycle. We propose three experiments. 1) To define the role of DGAT1 in recruiting HCV proteins to LDs. We will test the hypothesis that a tripartite complex of DGAT1, NS5A and core has evolved to co-recruit core and NS5A to DGAT1-generated LDs. We will also determine if NS5A and core are new protein targets for the acyltransferase activity of DGAT1. 2) To determine how the HCV core protein inhibits lipolysis. We will test whether core at the surface of LDs interferes with the recruitment or activity of the triglyceride lipases ATGL/PNPLA2 and PNPLA3, thus inhibiting lipolysis. Because patients infected with different HCV genotypes are at different risks to develop steatosis, we will compare anti-lipolytic activities of core proteins from different viral genotypes. 3) To explore how the NS5A-TIP47 interaction regulates HCV RNA replication. We will determine if LDs serve a dual role in HCV infection: one as membrane and energy sources for HCV RNA replication and another as assembly platforms. We will determine whether binding of NS5A to TIP47 plays an important role in this process. Collectively, our proposed studies will bring new molecular insight into the role of LDs in the HCV lifecycle and may uncover potential novel therapeutic strategies to treat chronic HCV disease.