Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.
Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.
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DOI：
10.1099/vir.0.015909-0
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发表时间：
2010-01
期刊：
影响因子：
--
通讯作者：
Patel AH
中科院分区：
文献类型：
--
作者：
Angus AG;Dalrymple D;Boulant S;McGivern DR;Clayton RF;Scott MJ;Adair R;Graham S;Owsianka AM;Targett-Adams P;Li K;Wakita T;McLauchlan J;Lemon SM;Patel AH
细胞死亡盒蛋白DDX 3最近被证明是丙型肝炎病毒（HCV）复制所必需的。在此之前，我们已经报道了HCV核心在酵母双杂交和瞬时转染试验中与DDX 3结合。在这里，我们通过免疫共沉淀证实这种相互作用发生在复制JFH 1病毒的细胞中。与该结果一致，感染细胞的免疫荧光染色显示细胞质DDX 3通过核心蛋白到脂滴周围的病毒组装位点的显著再分布。鉴于DDX 3与核心和脂滴的这种密切联系，以及其参与病毒复制，我们研究了这种宿主因子在病毒生命周期中的重要性。突变研究定位在JFH 1核心的N-末端结构域中的单个氨基酸，当其改变为丙氨酸时，显著废除了这种相互作用。令人惊讶的是，这种突变没有改变感染性病毒的产生和RNA复制，表明核心-DDX 3相互作用在HCV生命周期中是不稳定的。与之前的研究一致，siRNA诱导的DDX 3敲除降低了WT JFH 1和无法结合DDX 3的突变病毒的病毒产量和RNA复制水平。因此，我们的研究首次表明，HCV复制对DDX 3的需求与其与病毒核心蛋白的相互作用无关。
The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core–DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.
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