HEPATOCYTE REMODELING BY HEPATITIS C VIRUS

 描述（由申请方提供）：丙型肝炎病毒（HCV）重塑肝细胞内质网（ER）膜，以建立受保护的复制位点。这些结构被认为是保护病毒RNA免受胞质细胞核酸酶和先天免疫传感器的影响。细胞膜重组的机制知之甚少。尽管病毒NS 4 B蛋白在历史上与该功能有关，但最近的证据表明NS 5A在膜重塑中的主要作用。我们和其他人先前确定细胞脂质激酶磷脂酰肌醇-4激酶-III激酶（PI 4KA）是HCV复制的必要条件。我们发现病毒NS 5A蛋白结合PI 4KA，将其招募到病毒复制的位点，并刺激其活性。在PI 4KA表达缺陷的细胞中，HCV复制物崩溃，导致无功能的复制酶聚集体。PI 4KA的酶活性是HCV感染所需的，表明其产物PI（4）P的作用。PI 4KA和PI（4）P在HCV复制中的确切功能尚不清楚，但已表明其在募集具有PI（4）P结合结构域的蛋白质（如rab GTP酶和脂质转移蛋白）中发挥作用。这些蛋白质可以改变ER的脂质微环境以促进其重塑。此外，PI 4KA-NS 5A相互作用对于调节NS 5A的磷酸化是重要的，这对HCV复制中的NS 5A功能至关重要。 在描述NS 5A-PI 4KA相互作用在HCV复制中的作用的同时，我们假设目前进入III期临床试验的一类令人兴奋的HCV药物称为NS 5A直接作用抗病毒药物（DAA）可能靶向这种相互作用。该类药物的作用机制 目前尚不清楚;然而，由于病毒NS 5A基因中积累了耐药突变，因此认为其靶向HCV NS 5A蛋白[1]。事实上，我们发现原型NS 5A DAA BMS-790052（BMS）阻止HCV对PI 4KA活性的刺激。此外，BMS产生与沉默PI 4KA相似的表型，因为病毒复制酶崩溃和聚集。BMS不直接抑制PI 4KA，也不抑制NS 5A与PI 4KA的相互作用。在初始结合后，它明显阻止NS 5A对PI 4KA的活化。 我们建议的NS 5A-PI 4KA相互作用的功能的特点，在建立复制的保护位点，同时，在平行，表征NS 5A DAA的作用机制。这种方法是合乎逻辑的，因为这些目标在生物学上重叠，并且NS 5A DAA将是研究NS 5A-PI 4KA功能的宝贵工具。具体目标是：1.定义PI 4KA在改变HCV复制的脂质微环境中的功能。2.定义PI 4KA在调节NS 5A磷酸化和功能中的作用。3.定义原型NS 5A DAA在调节NS 5A-PI 4KA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.