HEPATOCYTE REMODELING BY HEPATITIS C VIRUS

 描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)重塑肝细胞内质网(ER)膜以建立受保护的复制部位。这些结构被认为可以保护病毒RNA免受胞浆细胞核酸酶和先天免疫传感器的影响。细胞膜重组的机制还知之甚少。尽管病毒的NS4B蛋白在历史上与这一功能有关，但最近的证据表明，NS5A在膜重塑中起主要作用。我们和其他人以前确定细胞脂蛋白激酶磷脂酰肌醇-4激酶-3(PI4KA)是丙型肝炎病毒复制所必需的。我们发现病毒NS5A蛋白与PI4KA结合，将其招募到病毒复制部位，并刺激其活性。在缺乏PI4KA表达的细胞中，丙型肝炎病毒的复制物崩溃，导致复制酶聚集而不起作用。PI4KA的酶活性是丙型肝炎病毒感染所必需的，提示其产物PI(4)P在丙型肝炎病毒复制中的确切功能尚不清楚，但已被认为在招募具有PI(4)P结合域的蛋白质，如Rab GTP酶和脂转移蛋白方面发挥作用。这些蛋白质可能会改变内质网的脂质微环境，以促进其重塑。此外，PI4KA-NS5A的相互作用对于调节NS5A的磷酸化是重要的，这对NS5A在丙型肝炎病毒复制中的作用至关重要。在描述NS5A-PI4KA相互作用在丙型肝炎病毒复制中的作用时，我们假设一类目前进入第三阶段临床试验的令人兴奋的丙型肝炎药物，称为NS5A直接作用抗病毒药物(DAA)可能针对这种相互作用。这类药物的作用机制 尚不清楚；然而，它被认为是针对丙型肝炎病毒NS5A蛋白的，因为病毒NS5A基因中积累了耐药突变[1]。事实上，我们发现NS5A DAA的原型BMS-790052(BMS)可以阻止丙型肝炎病毒对PI4KA活性的刺激。此外，BMS产生类似于沉默PI4KA的表型，因为病毒复制酶折叠和聚集。BMS不直接抑制PI4KA，也不抑制NS5A与PI4KA的相互作用。它明显阻止了NS5A在初始结合后对PI4KA的激活。我们建议表征NS5A-PI4KA相互作用在建立受保护复制位点中的功能，同时表征NS5A DAA的作用机制。考虑到这些目的在生物学上是重叠的，并且NS5A DAA将成为研究NS5A-PI4KA功能的宝贵工具，这种方法是合乎逻辑的。其具体目的是：1.明确PI4KA在改变丙型肝炎病毒复制的脂质微环境中的作用。2.明确PI4KA在调节NS5A磷酸化和功能中的作用。3.确定原型NS5A DAA在调节NS5A-PI4KA相互作用中的作用机制。

 DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) remodels hepatocyte endoplasmic reticulum (ER) membranes to establish protected sites of replication. These structures are thought to shield the viral RNAs from cytosolic cellular nucleases and innate immune sensors. The mechanism of cellular membrane reorganization is poorly understood. Although the viral NS4B protein has been historically linked to this function, more recent evidence points to a primary role for NS5A in membrane remodeling. We, and others, previously identified the cellular lipid kinase phosphatidylinositol-4 kinase-III (PI4KA) as a requirement or HCV replication. We found that the viral NS5A protein binds PI4KA, recruits it to sites of viral replication, and stimulates its activity. In cells deficient for PI4KA expression, the HCV replicas collapses, resulting in aggregates of replicase that are non-functional. The enzymatic activity of PI4KA is required for HCV infection, suggesting a role for its product, PI(4)P. The exact function of PI4KA and PI(4)P in HCV replication is unclear but it has been suggested by play a role in recruiting proteins with PI(4)P binding domains, such as rab GTPases and lipid transfer proteins. These proteins may modify the lipid micro-environment of the ER to facilitate its remodeling. Additionally, the PI4KA-NS5A interaction is important for regulating the phosphorylation of NS5A, which is critical to NS5A function in HCV replication. While characterizing the role of the NS5A-PI4KA interaction in HCV replication, we hypothesized that an exciting class of HCV drugs currently entering phase III clinical trials called NS5A direct-acting antivirals (DAAs) might target this interaction. The mechanism of action for this drug class is unknown; however, it is thought to target the HCV NS5A protein since drug resistant mutations accumulate in the viral NS5A gene [1]. Indeed, we found that a prototype NS5A DAA, BMS-790052 (BMS), prevents the stimulation of PI4KA activity by HCV. Additionally, BMS produces a phenotype similar to silencing PI4KA in that the viral replicase collapses and aggregates. BMS does not inhibit PI4KA directly, nor does it inhibit the interaction of NS5A with PI4KA. It apparently prevents the activation of PI4KA by NS5A after initial binding. We propose to characterize the function of the NS5A-PI4KA interaction in establishing protected sites of replication while, in parallel, characterizing the mechanism of action of NS5A DAAs. This approach is logical given that these aims overlap biologically and that the NS5A DAAs will be an invaluable tool in studying NS5A- PI4KA function. The specific aims are: 1. Define the function of PI4KA in modifying the lipid microenvironment for HCV replication. 2. Define the role of PI4KA in regulating NS5A phosphorylation and function. 3. Define the mechanism of action of a prototype NS5A DAA in modulating the NS5A-PI4KA interaction.