Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication
Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication
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DOI：
10.1073/pnas.0902693106
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发表时间：
2009-05-05
影响因子：
11.1
通讯作者：
Randall, Glenn
中科院分区：
文献类型：
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作者：
Berger, Kristi L.;Cooper, Jacob D.;Randall, Glenn
丙型肝炎病毒（HCV）重组细胞膜以建立复制位点。所需的宿主途径和细胞膜重组的机制尚不清楚。因此，我们研究了一个定制的小干扰RNA（siRNA）文库，该文库针对140个宿主膜转运基因，以鉴定HCV亚基因组复制和感染性病毒产生所需的基因。我们确定了7个宿主病毒复制辅助因子，包括Cdc42和Rock2（肌动蛋白聚合）、EEA1和Rab5A（早期核内体）、Rab7L1、pi3激酶C2gamma和pi4激酶iii α（磷脂代谢）。药物抑制剂的研究表明，肌动蛋白聚合和磷脂激酶活性是HCV复制所必需的。我们发现HCV复制酶标记NS5A和双链RNA与Rab5A广泛共定位，与Rab7L1部分共定位。pi4k - iii α除了存在于含有NS5A的耐洗涤剂膜中外，还与NS5A和双链RNA共定位。在II型和III型pi4激酶的比较中，HCV进入不需要PI4Ks，而HCV复制只需要pi4k -III α。尽管pi4k - iii - alpha sirna可以降低HCV复制和病毒产生几乎100%，但它们对初始HCV RNA翻译没有影响，这表明pi4k - iii - alpha在翻译后阶段起作用。在HCV感染的细胞中，电子显微镜鉴定出膜状网的存在，这被认为是HCV复制的部位。pi4k - iii α sirna预处理大大减少了hcv感染细胞中这些膜状网状结构的积累。我们提出pi4k - iii α在导致HCV复制复合体形成的膜改变中起重要作用。
Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA(siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha co-localized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.