A critical role of a cellular membrane traffic protein in poliovirus RNA replication.
A critical role of a cellular membrane traffic protein in poliovirus RNA replication.
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DOI：
10.1371/journal.ppat.1000216
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发表时间：
2008-11
期刊：
影响因子：
6.7
通讯作者：
Ehrenfeld E
中科院分区：
文献类型：
--
作者：
Belov GA;Feng Q;Nikovics K;Jackson CL;Ehrenfeld E
许多RNA病毒的复制伴随着细胞内膜的广泛重塑。在脊髓灰质炎病毒感染的细胞中，ER和高尔基体堆叠消失，而新的囊泡样结构簇形成病毒RNA合成的位点。布雷菲德菌素A（Brefeldin A，BFA）可抑制病毒复制，表明病毒生长中细胞分泌途径的某些组分。病毒蛋白表达诱导的特征性囊泡的形成不受BFA的抑制，但它们在功能上有缺陷。GBF 1是小细胞GTP酶Arf的鸟嘌呤核苷酸交换因子，负责病毒感染对BFA的敏感性，并且是病毒复制所需的。GBF 1表达的敲低抑制了病毒复制，这是由具有完整N-末端序列的催化活性蛋白拯救的。我们确定了GBF 1中的一个突变，该突变允许脊髓灰质炎病毒在BFA存在下生长。GBF 1和病毒蛋白3A之间的相互作用决定了BFA存在下的感染结果。所有的正链RNA病毒复制它们的基因组与膜结构相关联，所述膜结构在感染后通过重塑预先存在的细胞器而形成。膜的作用和病毒蛋白质利用的机制来协调病毒膜复制复合物的形成和功能在很大程度上是未知的。脊髓灰质炎病毒的复制受到布雷菲德菌素A（BFA）的严重抑制，布雷菲德菌素A是一种众所周知的细胞分泌途径抑制剂。三种细胞蛋白（GBF 1，BIG 1和BIG 2）激活小GTP酶称为Arfs，其活性是分泌途径正常运作所必需的，是BFA的已知靶点。在这里，我们证明，脊髓灰质炎病毒利用GBF 1依赖的Arf激活途径进行复制。我们的数据解释了BFA抑制脊髓灰质炎病毒复制的机制，证明病毒蛋白3A结合并招募GBF 1支持病毒RNA合成的膜。BFA灭活GBF 1可阻止Arf的激活和募集，并阻止功能性复制复合物的形成。令人惊讶的是，在BFA的存在下，与病毒复制复合物形态相似的膜结构的形成发生，尽管这些结构在病毒RNA的合成中不起作用。已知其他正链RNA病毒对BFA表现出敏感性，我们的数据表明，劫持Arf激活途径可能是不同病毒组共有的共同特征。
Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA), implicating some components(s) of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA. All positive strand RNA viruses replicate their genomes in association with membranous structures that are formed after infection by remodeling pre-existing cellular organelles. The role of membranes and the mechanisms exploited by viral proteins to orchestrate the formation and functioning of viral membranous replication complexes are largely unknown. Poliovirus replication is severely suppressed by brefeldin A (BFA), a well-known inhibitor of the cellular secretory pathway. Three cellular proteins (GBF1, BIG1 and BIG2) that activate small GTPases called Arfs, whose activity is necessary for normal functioning of the secretory pathway, are known targets of BFA. Here we demonstrate that poliovirus utilizes the GBF1-dependent Arf activation pathway for its replication. Our data explain the mechanism of BFA inhibition of poliovirus replication by demonstrating that viral protein 3A binds and recruits GBF1 to membranes that support viral RNA synthesis. Inactivation of GBF1 by BFA prevents Arf activation and recruitment, and prevents formation of functional replication complexes. Surprisingly, formation of membranous structures morphologically similar to viral replication complexes occurs in the presence of BFA, although these structures do not function in the synthesis of viral RNA. Other plus strand RNA viruses are known to exhibit sensitivity to BFA and our data suggest that hijacking of the Arf activation pathway may be a common feature shared by diverse groups of viruses.
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