Identification and Characterization of the 480-Kilodalton Template-Specific RNA-Dependent RNA Polymerase Complex of Red Clover Necrotic Mosaic Virus
Identification and Characterization of the 480-Kilodalton Template-Specific RNA-Dependent RNA Polymerase Complex of Red Clover Necrotic Mosaic Virus
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DOI：
10.1128/jvi.00054-10
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发表时间：
2010-06-01
影响因子：
5.4
通讯作者：
Okuno, Tetsuro
中科院分区：
文献类型：
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作者：
Mine, Akira;Takeda, Atsushi;Okuno, Tetsuro
正链RNA病毒的复制是通过与膜相关的病毒RNA复制复合体的组装进行的，该复合体包括病毒复制酶蛋白、病毒RNA模板和宿主蛋白。红三叶坏死花叶病毒(RCNMV)是一种正链RNA植物病毒，基因组由RNA1和RNA2组成。RNA1编码的两个蛋白质，一个是27 kDa的蛋白质(P27)，一个是含有依赖RNA的RNA聚合酶(RdRP)基序的88 kDa的蛋白质(P88)，是RCNMV RNA复制所必需的。为了分析RCNMV RNA复制复合体，我们使用了蓝色天然聚丙烯酰胺凝胶电泳法(BN/PAGE)，这使得我们能够分析洗涤剂溶解的大分子膜蛋白复合体。P27和P88在RCNMV侵染植株中形成480 kDa的复合体。作为蔗糖梯度沉淀的结果，480 kDa的复合体同时具有内源模板结合和外源模板依赖的RdRP活性。480 kDa复合体的量与外源模板依赖的RdRP的活性相对应，后者通过特异性识别RCNMV RNA的3‘端核心启动子序列而产生RNA片段，但与内源模板结合的RdRP的活性不对应，内源模板结合的RdRP在不添加RNA模板的情况下产生基因组大小的RNA。这些结果表明，480 kDa的复合体有助于模板依赖的RdRP活性。我们对这些RdRP络合物进行了亲和纯化，并用双向BN/十二烷基硫酸钠-PAGE(BN/十二烷基硫酸钠-PAGE)和质谱仪分析了它们的组成。480 kDa复合体包含p27、P88和可能的宿主蛋白，原始亲和纯化的RdRP制剂包含HSP70、HSP90和一些在480 kDa复合体中未检测到的核糖体蛋白。提出了RCNMV RNA复制复合体的形成模型。
Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3'-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.