Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.
Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.
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DOI：
10.1371/journal.ppat.1004501
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发表时间：
2014-10
期刊：
影响因子：
6.7
通讯作者：
Moradpour D
中科院分区：
文献类型：
--
作者：
Gouttenoire J;Montserret R;Paul D;Castillo R;Meister S;Bartenschlager R;Penin F;Moradpour D
非结构蛋白4B(NS4B)是丙型肝炎病毒复制复合体形成的关键组织者。在与其他非结构蛋白的协同作用下，它诱导特定的膜重排，被称为膜网，作为丙型肝炎病毒复制酶的支架。NS4B的N-末端部分包括一个预测的和一个结构上可分辨的两亲性α-螺旋，分别命名为AH1和AH2。在这里，我们报告了NS4B AH1的详细结构和功能分析。圆二色谱和核磁共振结构分析表明，AH1折叠成一个从NS4B氨基酸4延伸到32位的两亲性α-螺旋，螺旋两侧带有正电荷残基。这些残基在肝炎病毒中是保守的。对假逆转体的诱变和筛选揭示了这些残基通过影响组成膜网的双膜小泡的生物发生在RNA复制中的重要作用。此外，螺旋亲水侧保守的酸性残基的丙氨酸取代降低了感染性，但没有显著影响RNA复制，表明AH1也参与了病毒的生产。对含有NS4B AH1和AH2表位标签的功能性复制子的选择性膜透过性和免疫荧光显微镜分析表明，在丙型肝炎病毒RNA复制过程中，NS4B的N末端部分具有双膜拓扑结构。腔易位不受引入AH1的突变的影响，但被引入AH2的突变所废除。总之，我们的研究报告了丙型肝炎病毒NS4B的AH1的三维结构，并强调了这个两亲性α-螺旋两侧的正电荷氨基酸残基在膜网形成和RNA复制中的重要性。此外，我们还证明了AH1在RNA复制和病毒生产中具有双重作用，可能受NS4B N末端部分不同拓扑的支配。据估计，有1.8亿慢性感染者，丙型肝炎病毒(丙型肝炎病毒)是全球慢性肝炎、肝硬变和肝细胞癌的主要原因。丙型肝炎病毒是一种正链RNA病毒，它在重新排列的细胞膜上建立复制复合体，称为膜网。丙型肝炎病毒非结构蛋白4B(NS4B)是丙型肝炎病毒膜网络和复制复合体形成的关键组织者。在这里，我们提供了一个NS4B的N末端两亲性α-螺旋的详细结构和功能分析，命名为AH1，并证明它在塑造膜网络以及病毒生产中发挥关键作用。我们还表明，NS4B的N末端部分在复制环境中采用了双膜拓扑结构，这可能反映了该蛋白在病毒生命周期中的不同作用。
Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B. With an estimated 180 million chronically infected individuals, hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. HCV is a positive-strand RNA virus that builds its replication complex on rearranged intracellular membranes, designated as membranous web. HCV nonstructural protein 4B (NS4B) is a key organizer of HCV membranous web and replication complex formation. Here, we provide a detailed structure-function analysis of an N-terminal amphipathic α-helix of NS4B, named AH1, and demonstrate that it plays key roles in shaping the membranous web as well as in virus production. We also show that the N-terminal part of NS4B adopts a dual membrane topology in a replicative context, possibly reflecting the different roles of this protein in the viral life cycle.
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