Nasheri Neda, Joyce Michael, Rouleau Yanouchka, Yang Pengyu, Yao Shao, Tyrrell D Lorne, Pezacki John Paul
Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
Chem Biol. 2013 Apr 18;20(4):570-82. doi: 10.1016/j.chembiol.2013.03.014.
The hepatitis C virus (HCV) induces alterations of host cells to facilitate its life cycle. Fatty acid synthase (FASN) is a multidomain enzyme that plays a key role in the biosynthesis of fatty acids and is upregulated during HCV infection. Herein, we applied activity-based protein profiling (ABPP) that allows for the identification of differentially active enzymes in complex proteomic samples, to study the changes in activity of FASN during HCV replication. For this purpose, we used an activity-based probe based on the FASN inhibitor Orlistat, and observed an increase in the activity of FASN in the presence of a subgenomic and a genomic HCV replicon as well as in chimeric SCID/Alb-uPA mice infected with HCV genotype 1a. To study the molecular basis for this increase in FASN activity, we overexpressed individual HCV proteins in Huh7 cells and observed increased expression and activity of FASN in the presence of core and NS4B, as measured by western blots and ABPP, respectively. Triglyceride levels were also elevated in accordance with FASN expression and activity. Lastly, immunofluorescence and ABPP imaging analyses demonstrated that while the abundance and activity of FASN increases significantly in the presence of HCV, its localization does not change. Together these data suggest that the HCV-induced production of fatty acids and neutral lipids is provided by an increase in FASN abundance and activity that is sufficient to allow HCV propagation without transporting FASN to the replication complexes.
丙型肝炎病毒（HCV）会诱导宿主细胞发生改变以促进其生命周期。脂肪酸合酶（FASN）是一种多结构域酶，在脂肪酸生物合成中起关键作用，且在HCV感染期间上调。在此，我们应用基于活性的蛋白质谱分析（ABPP），其可用于鉴定复杂蛋白质组样品中差异活性的酶，以研究HCV复制过程中FASN活性的变化。为此，我们使用了一种基于FASN抑制剂奥利司他的基于活性的探针，并观察到在存在亚基因组和基因组HCV复制子的情况下以及在感染HCV 1a基因型的嵌合SCID/Alb-uPA小鼠中FASN活性增加。为了研究FASN活性增加的分子基础，我们在Huh7细胞中过表达单个HCV蛋白，并分别通过蛋白质印迹和ABPP检测，观察到在存在核心蛋白和NS4B的情况下FASN的表达和活性增加。甘油三酯水平也根据FASN的表达和活性而升高。最后，免疫荧光和ABPP成像分析表明，虽然在存在HCV的情况下FASN的丰度和活性显著增加，但其定位没有改变。这些数据共同表明，HCV诱导的脂肪酸和中性脂质的产生是由FASN丰度和活性的增加提供的，这足以使HCV繁殖而无需将FASN转运至复制复合体。
