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

丙型肝炎病毒是黄病毒科的一种正链RNA病毒，是引起严重肝病的重要人类病原体。我们最近发现脂蛋白激酶磷脂酰肌醇-4-磷酸激酶IIIα(PI4KIIIa，PI4KA)是丙型肝炎病毒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)。丙型肝炎病毒激活PI4KA的酶活性，导致细胞内PI4P水平显著升高。在最近的资助期间，我们发现在Huh7肝癌细胞中PI4KA的激活对大多数丙型肝炎病毒分离株是有害的，这是由于PI4P过量，解释了患者来源的病毒在细胞培养中复制效率低下的原因。令人惊讶的是，我们可以证明复制促进适应性突变实际上依赖于功能机制的丧失，取消PI4KA的激活来补偿肝癌细胞中PI4KA的高表达水平。基于这一发现，我们可以建立一种基于PI4KA/酪蛋白激酶Iα(CKIα)的治疗方案，允许在细胞培养中高效复制丙型肝炎病毒wt分离株。利用这项技术，我们鉴定了一种新的丙型肝炎病毒1bwt分离株，命名为GLT1，在细胞培养中复制到非常高的水平。PI4KA/CKIα处理或表达Sec14L2可刺激HCVGLT1复制高达4个数量级，Sec14L2是一种在肝细胞中表达而在HuH7中不表达的脂转运蛋白，这表明病毒复制细胞器的脂组成起着关键作用。Sec14L2最近被其他人发现可以刺激丙型肝炎病毒wt分离株的复制，但其机制仍然不清楚，到目前为止对所有测试的分离株的效果都非常温和。SEC14L2或PI4KA/CKIα抑制对GLT1复制的显著刺激为在分子水平上了解细胞培养中丙型肝炎病毒复制的要求、确定SEC14L2对脂质和蛋白质组成的影响以及正品丙型肝炎病毒复制细胞器的形态提供了独特的机会。因此，这一更新建议遵循三个主要目的：1.通过与相关gt1b分离物产生嵌合体来了解GLT1高效复制的机制。我们还将尝试使GLT1分离株适应高效的病毒粒子生产，以获得第一个gt1b的全复制周期培养模型。我们将使用复制酶表达模型对纯化的复制细胞器在存在和不存在Sec14L2的情况下进行脂质学和蛋白质组学比较，并通过实验验证各自的候选脂质和蛋白质。3.比较有无Sec14L2存在和不存在的病毒复制细胞器的超微结构，将发现有利于或不利于病毒复制的结构。候选脂质相对于病毒复制囊泡的分布将通过超分辨率显微镜和活细胞成像进行分析。

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.