Modification of host lipid raft proteome upon hepatitis C virus replication
Modification of host lipid raft proteome upon hepatitis C virus replication
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DOI：
10.1074/mcp.m600121-mcp200
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发表时间：
2006-12-01
影响因子：
7
通讯作者：
Beretta, Laura
中科院分区：
文献类型：
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作者：
Mannova, Petra;Fang, Ruihua;Beretta, Laura
丙型肝炎病毒 (HCV) 复制复合体驻留在去污剂不溶性的亚细胞结构域或脂筏中。我们使用两种蛋白质组学方法来表征从 Huh7 细胞中分离的脂筏的蛋白质含量及其对 HCV 复制的修饰。使用二维电泳和质谱分析，我们在分离的脂筏中鉴定出类似的100个蛋白质点；其中，与对照细胞系相比，HCV复制子细胞系中有39个被可重复地修饰。我们还使用细胞培养中的氨基酸稳定同位素标记 (SILAC) 结合一维电泳分离和质谱分析。使用这种方法，我们根据至少 95% 置信度选择的肽鉴定了 1036 个单独的蛋白质；其中，413种蛋白质被鉴定出至少含有两种肽。定量分析发现，与对照相比，HCV 复制细胞中的 150 种蛋白质修饰至少为 2.5 倍（110 种上调和 40 种下调）。两种蛋白质组学方法获得的蛋白质鉴定和定量基本一致。调节的蛋白质包括参与囊泡和蛋白质运输以及细胞信号传导的大部分蛋白质。值得注意的是，对于大量蛋白质来说，它们在 HCV 复制子细胞脂筏中的上调是由于它们的重新定位。通过使用针对调节的小 GTPase Cdc42 和 RhoA 的小干扰 RNA，我们观察到 HCV 复制增加，而突触蛋白 7 表达的减少导致 HCV 复制减少。我们的研究结果表明，蛋白质亚细胞重定位发生在含有 HCV 的细胞中，可直接影响 HCV 复制。
Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified similar to 100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.