Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation
Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation
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DOI：
10.1128/jvi.01921-06
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发表时间：
2007-03-01
影响因子：
5.4
通讯作者：
Ishikawa, Masayuki
中科院分区：
文献类型：
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作者：
Komoda, Keisuke;Mawatari, Natsuki;Ishikawa, Masayuki
真核正链RNA病毒基因组的复制发生在膜结合的RNA复制复合体中。以前，我们发现抽提的烟草BY-2原生质体提取物(BYL)能够支持包括番茄花叶病毒(TOMY)在内的植物正链RNA病毒基因组RNA的翻译和随后的复制。在这里，为了剖析ToMV RNA复制复合体形成之前的过程，我们制备了膜耗竭BYL(MdBYL)，其中膜被离心法去除。在mdBYL中，ToMV RNA被翻译成130 kDa和180 kDa的复制蛋白，但没有合成任何与ToMV相关的RNA。当用嘌呤霉素终止翻译后，将BYL膜加回到ToNIV RNA翻译的mdBYL中时，ToMV RNA被复制。使用编码标记的180 kDa复制蛋白的具有复制能力的ToMV衍生物，亲和纯化表明，翻译到mdBYL后，形成了包含130 kDa和180 kDa蛋白与ToNW基因组RNA的复合体。当该复合体与BYL膜混合时，ToMV RNA被复制，这表明该核糖核蛋白复合体是TowRNA复制复合体形成的中间产物。我们将这种核糖核蛋白复合体命名为“前膜靶向复合体”。我们的数据表明，前膜靶向复合体的形成与ToNIV RNA的翻译相耦合，而翻译后添加的外源180-kDa蛋白和复制模板分别有助于复制和复制。基于这些结果，我们讨论了ToMV RNA复制复合体的形成机制。
The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMY). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToNIV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToNW genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToW RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToNIV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.