ADP Ribosylation Factor 1 Plays an Essential Role in the Replication of a Plant RNA Virus
ADP Ribosylation Factor 1 Plays an Essential Role in the Replication of a Plant RNA Virus
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DOI：
10.1128/jvi.02383-12
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发表时间：
2013-01-01
影响因子：
5.4
通讯作者：
Okuno, Tetsuro
中科院分区：
文献类型：
--
作者：
Hyodo, Kiwamu;Mine, Akira;Okuno, Tetsuro
真核正链 RNA 病毒使用膜结合复制酶复合物进行复制，该复合物含有多种病毒和宿主成分。病毒感染诱导细胞内膜的重塑。病毒诱导的膜结构被认为增加了复制所需成分的局部浓度，并为束缚复制酶复合物提供了支架。然而，人们对病毒诱导的膜重塑的机制知之甚少。红三叶草坏死花叶病毒 (RCNMV) 是一种正链 RNA 植物病毒，其 RNA 复制与内质网 (ER) 膜相关，并且在 RCNMV 感染的细胞中，ER 形态受到干扰。在这里，我们在亲和纯化的 RCNMV RNA 依赖性 RNA 聚合酶组分中鉴定出 ADP 核糖基化因子 1 (Arf1)。 Arf1 是一种高度保守、普遍存在的小型 GTP 酶，参与高尔基体膜上外壳蛋白复合物 I (COPI) 囊泡的形成。通过体外 Pulldown 和双分子荧光互补分析，我们发现 Arf1 与病毒诱导的 ER 膜大点状结构内的病毒 p27 复制蛋白相互作用。我们发现使用抑制剂布雷菲德菌素 A (BFA) 抑制 Arf1 的核苷酸交换活性会破坏病毒复制酶复合物的组装和 p27 介导的 ER 重塑。我们还表明，BFA 处理和显性失活 Arf1 突变体的表达会损害原生质体中 RCNMV RNA 的复制。有趣的是，Sar1 的显性失活突变体（ER 出口位点 COPII 囊泡生物发生的关键调节因子）的表达也会损害 RCNMV RNA 复制。这些结果表明 RCNMV 的复制依赖于宿主膜交通机制。
Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.