Affinity capture and identification of host cell factors associated with hepatitis C virus (+) strand subgenomic RNA.
Affinity capture and identification of host cell factors associated with hepatitis C virus (+) strand subgenomic RNA.
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与丙型肝炎病毒 ( ) 链亚基因组 RNA 相关的宿主细胞因子的亲和捕获和鉴定。
DOI：
10.1074/mcp.m112.017020
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
Pandey,VirendraN
中科院分区：
文献类型：
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作者：
Upadhyay,Alok;Dixit,Updesh;Manvar,Dinesh;Chaturvedi,Nootan;Pandey,VirendraN
丙型肝炎病毒（HCV）感染导致慢性肝炎是导致肝硬化、肝细胞癌和肝功能衰竭的主要因素。这个过程可能涉及各种宿主细胞因子的相互作用，以及这些因子与病毒RNA和蛋白质的相互作用。我们报道了一种新的策略，利用序列特异性的生物素化肽核酸（PNA）-新霉胺偶联物靶向HCV RNA，用于在MH 14宿主细胞中原位捕获亚基因组HCV（+）RNA，沿着与其相关的细胞因子和病毒因子。使用这种亲和捕获系统结合LC/MS/MS，我们已经确定了83个细胞因子和三个病毒蛋白（NS 5 B，NS 5A和NS 3 -4a蛋白酶解旋酶）与病毒基因组。捕获是非常具体的。这些蛋白质没有用没有HCV复制子的治愈的MH 14细胞评分，因为细胞中不存在PNA-新霉胺探针的靶序列，还因为与寡聚DNA不同，细胞蛋白质对PNA没有亲和力。所鉴定的细胞因子属于不同的功能组，包括信号传导、致癌、伴侣蛋白、转录调节因子和RNA解旋酶以及DEAD盒蛋白、核糖体蛋白、翻译调节因子/因子和代谢酶，其代表了与HCV RNA基因组相关的多种细胞因子。小干扰RNA介导的沉默的HCV复制子细胞系中的不同类别的选定的蛋白质增强或抑制HCV复制/翻译，这表明这些细胞因子在HCV复制中具有调节作用。
Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for thein situcapture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3–4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication.