Shaping the lipid landscape of the membraneous viral replication organelles by Hepatitis C virus

丙型肝炎病毒（Hepatitis C virus，HCV）是黄病毒科的一种正链RNA病毒，是引起人类严重肝病的重要病原体。我们最近鉴定了脂质激酶磷脂酰肌醇4-磷酸激酶III α（PI4KIIIa，PI4KA）作为HCV复制的重要宿主因子。PI4KA将磷脂酰肌醇转化为磷脂酰肌醇4-磷酸（PI4P）。PI4KA的酶活性被HCV激活，导致细胞内PI4P水平强烈升高。在最近的资助期间，我们发现Huh 7肝癌细胞中PI 4 KA的激活对大多数HCV分离株有害，因为PI 4 P过量，这解释了细胞培养物中患者来源病毒的复制效率低下。令人惊讶的是，我们可以证明，复制增强适应性突变实际上依赖于功能机制的丧失，废除PI4KA的激活以补偿与原代肝细胞相比肝癌细胞中的高PI4KA表达水平。基于这一发现，我们可以建立一种基于PI4KA/酪蛋白激酶I α（CKI α）治疗的方案，允许HCV wt分离株在细胞培养物中有效复制。使用这种技术，我们确定了一种新的HCV基因型1b野生型分离株，称为GLT 1，在细胞培养中复制到非常高的水平。通过PI4KA/CKI α处理或Sec14L2（一种在肝细胞中表达但不在Huh 7中表达的脂质转运蛋白）表达，HCV GLT 1的复制可被刺激高达4个数量级，这表明病毒复制细胞器的脂质组成具有关键作用。Sec14L2最近被其他人鉴定为刺激HCV wt分离株的复制，但其机制仍然不清楚，并且迄今为止对所有测试的分离株的作用都非常温和。Sec14L2或PI4KA/CKI α抑制对GLT 1复制的显著刺激现在为在分子水平上了解细胞培养中HCV复制的要求以及确定Sec14L2对脂质和蛋白质组成的影响以及真实HCV复制细胞器的形态提供了独特的机会。因此，这项更新建议遵循三个主要目标：1。通过与相关的gt1b分离株产生嵌合体来了解GLT 1有效复制的机制。我们还将尝试使GLT 1分离株适应有效的病毒体生产，以获得gt1b.2的第一个全复制周期培养模型。我们将追求脂质组学和蛋白质组学比较纯化的复制细胞器中存在和不存在的Sec14L2使用复制酶表达模型和实验验证各自的候选脂质和蛋白质。3.在存在和不存在Sec14L2的情况下，病毒复制细胞器的超微结构比较将确定有利于或不利于病毒复制的结构。将通过超分辨率显微镜和活细胞成像分析候选脂质相对于病毒复制囊泡的分布。

Hepatitis C virus (HCV) is a positive strand RNA virus belonging to the family of Flaviviridae and an important human pathogen causing severe liver disease. We have recently identified the lipid kinase phosphatidylinositol 4-phosphate kinase IIIα (PI4KIIIa, PI4KA) as an essential host factor of HCV replication. PI4KA converts phosphatidylinositol to phosphatidylinositol 4-phosphate (PI4P). The enzymatic activity of PI4KA is activated by HCV, resulting in strongly elevated intracellular PI4P levels. During the recent funding period we found that activation of PI4KA in Huh7 hepatoma cells is deleterious for most HCV isolates due to an excess of PI4P, explaining the inefficient replication of patient derived viruses in cell culture. Surprisingly, we could demonstrate that replication enhancing adaptive mutations in fact rely on a loss of function mechanism, abrogating activation of PI4KA to compensate for high PI4KA expression levels in hepatoma cells compared to primary hepatocytes. Based on this finding we could establish a regimen based on PI4KA/Casein Kinase Iα (CKIα) treatment, allowing efficient replication of HCV wt isolates in cell culture. Using this technology we identified a new HCV genotype 1b wt isolate, termed GLT1, replicating to very high levels in cell culture. Replication of HCV GLT1 can be stimulated by up to 4 orders of magnitude either by PI4KA/CKIα treatment or by expression of Sec14L2, a lipid transporter protein expressed in hepatocytes but not in Huh7, arguing for a critical role of the lipid composition of the viral replication organelles. Sec14L2 was recently identified by others to stimulate replication of HCV wt isolates, but the mechanism is still poorly defined and the effect was very moderate for all isolates tested so far. The dramatic stimulation of GLT1 replication by Sec14L2 or PI4KA/CKIα inhibition now provides the unique opportunity to understand the requirements of HCV replication in cell culture at a molecular level and to define the impact of Sec14L2 on the lipid and protein composition, as well as the morphology of authentic HCV replication organelles. Therefore this renewal proposal follows three major aims: 1. Understanding the mechanisms underlying efficient GLT1 replication by generating chimeras with a related gt1b isolate. We will also try to adapt the GLT1 isolate to efficient virion production to obtain the first full-replication cycle culture model for gt1b.2. We will pursue lipidomic and proteomic comparisons of purified replication organelles in presence and absence of Sec14L2 using a replicase expression model and experimentally validate respective candidate lipids and proteins. 3. An ultrastructural comparison of viral replication organelles in presence and absence of Sec14L2 will identify structures favorable or unfavorable for viral replication. The distribution of candidate lipids relative to viral replication vesicles will be analyzed by super-resolution microscopy and live cell imaging.