RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase in human cells reveals requirements for de novo initiation and protein-protein interaction.
RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase in human cells reveals requirements for de novo initiation and protein-protein interaction.
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人体细胞中雀麦花叶病毒 RNA 依赖性 RNA 聚合酶的 RNA 合成揭示了从头起始和蛋白质-蛋白质相互作用的要求。
DOI：
10.1128/jvi.00069-12
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发表时间：
2012
影响因子：
5.4
通讯作者：
Kao,CCheng
中科院分区：
文献类型：
--
作者：
Subba-Reddy,ChennareddyV;Tragesser,Brady;Xu,Zhili;Stein,Barry;Ranjith-Kumar,CT;Kao,CCheng
雀麦花叶病毒（BMV）是一种模式正链RNA病毒，其复制已在许多替代宿主中进行了研究。在瞬时转染的人类细胞中，BMV聚合酶2a激活了先天免疫受体RIG-I的信号传导，该受体识别新启动的非自身RNA。2a中的活性位点突变废除了RIG-I激活，BMV 1a蛋白的共表达刺激了2a的活性。以前显示的突变，取消1a和2a的相互作用，防止1a依赖的增强2a的活动。对1a-2a相互作用的新认识包括发现1a的解旋酶活性位点是增强2a聚合酶活性所必需的，并且2a的位置110和120之间的带负电荷的氨基酸残基有助于与1a解旋酶样结构域的相互作用，但不有助于内在的聚合酶活性。共聚焦荧光显微镜观察发现BMV 1a和2a共定位于人细胞核周区。然而，没有核周球样结构，在人类细胞中检测到免疫电镜。用RIG-I免疫共沉淀的RNA的测序揭示了2a合成的短RNA来源于用于翻译2a的信息。也就是说，2a对BMV RNA 2表现出强烈的顺式偏好。引人注目的是，2a RNA产物的起始序列（5′-GUAAA-3′）与BMV基因组RNA 2和RNA 3的5′序列相同。这些结果表明，BMV 2a聚合酶不需要其他BMV蛋白来启动RNA合成，但1a解旋酶结构域和可能的解旋酶活性可以影响2a的RNA合成。
Brome mosaic virus (BMV) is a model positive-strand RNA virus whose replication has been studied in a number of surrogate hosts. In transiently transfected human cells, the BMV polymerase 2a activated signaling by the innate immune receptor RIG-I, which recognizesde novo-initiated non-self-RNAs. Active-site mutations in 2a abolished RIG-I activation, and coexpression of the BMV 1a protein stimulated 2a activity. Mutations previously shown to abolish 1a and 2a interaction prevented the 1a-dependent enhancement of 2a activity. New insights into 1a-2a interaction include the findings that helicase active site of 1a is required to enhance 2a polymerase activity and that negatively charged amino acid residues between positions 110 and 120 of 2a contribute to interaction with the 1a helicase-like domain but not to the intrinsic polymerase activity. Confocal fluorescence microscopy revealed that the BMV 1a and 2a colocalized to perinuclear region in human cells. However, no perinuclear spherule-like structures were detected in human cells by immunoelectron microscopy. Sequencing of the RNAs coimmunoprecipitated with RIG-I revealed that the 2a-synthesized short RNAs are derived from the message used to translate 2a. That is, 2a exhibits a strongcispreference for BMV RNA2. Strikingly, the 2a RNA products had initiation sequences (5′-GUAAA-3′) identical to those from the 5′ sequence of the BMV genomic RNA2 and RNA3. These results show that the BMV 2a polymerase does not require other BMV proteins to initiate RNA synthesis but that the 1a helicase domain, and likely helicase activity, can affect RNA synthesis by 2a.