An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.
An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.
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DOI：
10.1371/journal.ppat.1000351
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发表时间：
2009-03
期刊：
影响因子：
6.7
通讯作者：
Ahlquist P
中科院分区：
文献类型：
--
作者：
Liu L;Westler WM;den Boon JA;Wang X;Diaz A;Steinberg HA;Ahlquist P
雀麦花叶病毒(BMV)蛋白1a在病毒RNA复制中具有多种关键作用。1A作为一种外周膜蛋白定位于核周内质网(ER)膜，诱导ER膜内陷，形成RNA复制复合体，并在这些位置招募和稳定BMV 2a聚合酶(2aPol)和RNA复制模板，建立活性复制复合体。在复制过程中，1a提供RNA封顶、NTPase和可能的RNA解旋酶功能。在这里，我们在BMV 1a中鉴定了一个两亲性α-螺旋A，并使用核磁共振分析来确定其结构和插入疏水胶束的倾向。我们表明，螺旋A对于有效的1a-ER膜结合和正常的核周ER定位是必不可少的，并且螺旋A的缺失或突变会取消RNA复制。引人注目的是，螺旋A的突变导致了两种截然相反的1a功能表型，这意味着螺旋A作为分子开关调节可分离的1a功能之间的复杂平衡。一类螺旋A缺失和氨基酸替换显著抑制1a膜结合，取消ER膜内陷、病毒RNA模板募集和复制，但使1a介导的2aPol积累增加一倍。第二类螺旋A突变不仅保持了有效的1a膜结合，而且使1a诱导的膜内陷的数量增加了5-8倍，并增强了病毒RNA模板的募集，但未能刺激2aPol的积累。这些结果为RNA复制复合体的组装提供了新的见解，并表明螺旋A对病毒RNA复制复合体的组装和功能至关重要，包括它在靶向复制组件和控制1a作用模式方面的中心作用。正链RNA病毒(占所有病毒属的三分之一)以信使核糖核酸的形式在宿主细胞之间转移它们的遗传物质，这些核糖核酸在进入宿主细胞后立即转化为蛋白质。这些蛋白质的一个直接功能是在细胞内膜上建立RNA复制隔间，以复制传入的病毒RNA。虽然人们对这种复制复合体中的病毒蛋白和RNA成分了解很多，但对复制复合体组装所需的多个蛋白质-膜-RNA相互作用是如何调节的了解很少。为了研究这一点，我们使用了一个成熟的模型病毒，它只编码两种复制蛋白：一种是复制病毒RNA的RNA聚合酶，另一种是引导细胞内膜重排形成复制间隔并将病毒RNA模板和聚合酶招募到这些位置的组装协调蛋白。我们在这个引导复制蛋白中发现了一个小螺旋，它对于正确的细胞膜类型的有效结合和重排以及调节复制引导蛋白至少两个不同功能状态之间的转换是必不可少的。这个小螺旋上的突变干扰了可分离的引导蛋白功能，揭示了正链RNA病毒RNA复制复合体形成的顺序步骤。
Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2aPol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic α-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action. Positive-strand RNA viruses (one-third of all virus genera) transfer their genetic material between host cells as RNA of mRNA polarity, which are translated into proteins immediately upon entry. One immediate function of these proteins is to establish RNA replication compartments on intracellular membranes to copy the incoming viral RNA. Although much is known about the viral protein and RNA components in such replication complexes, little is understood about how the multiple protein–membrane–RNA interactions required for replication complex assembly are regulated. To study this, we used a well-established model virus that encodes only two replication proteins: an RNA polymerase enzyme that copies the viral RNA and an assembly-coordinating protein that guides the rearrangement of intracellular membranes to form replication compartments and recruits the viral RNA template and polymerase to these sites. We identified a small helix in this guiding replication protein that is essential for efficient association with and rearrangement of the correct intracellular membrane type and for regulating a switch between at least two different functional states of the replication guide protein. Mutations in this small helix interfere with separable guide protein functions, revealing new insights into the sequential steps in positive-strand RNA virus RNA replication complex formation.
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