Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle
Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle
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DOI：
10.1128/jvi.03257-15
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发表时间：
2016-05-01
影响因子：
5.4
通讯作者：
Becker, Stephan
中科院分区：
文献类型：
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作者：
Biedenkopf, Nadine;Lier, Clemens;Becker, Stephan
埃博拉病毒是在人类和非人类灵长类动物中造成高致死率的严重发热的病原体。埃博拉病毒转录和复制的调控机制目前还不清楚。调控病毒转录的一个重要因子是VP30，这是一种与病毒核衣壳相关的埃博拉病毒特异性转录因子。先前的研究表明，VP30的磷酸化状态影响病毒转录。与NP、L和聚合酶辅助因子VP35一起，非磷酸化VP30支持病毒转录。在VP30磷酸化后，病毒转录停止。磷酸化削弱了VP30与聚合酶辅因子VP35和/或病毒RNA之间的相互作用。因此，VP30被排除在病毒转录复合体之外，同时导致病毒复制增加，这是由NP、L和VP35单独支持的。在这里，我们使用感染性病毒样颗粒试验和重组病毒来证明VP30的动态磷酸化对于VP30与核衣壳共同运输到病毒RNA合成位点至关重要，而VP30是启动病毒初级转录所必需的。我们进一步证明，在第29个氨基酸位置的单个丝氨酸残基足以使VP30在初级转录中具有活性，并产生具有与野生型病毒相似特征的重组病毒。相比之下，在VP30的30号位置有一个单一丝氨酸的重组病毒的拯救是失败的。我们的研究结果表明VP30的磷酸化和去磷酸化在病毒生命周期中起着关键作用。目前在西非爆发的埃博拉病毒已造成28,000多例病例和11,000人死亡。关于埃博拉病毒如何转录和复制其基因组的分子机制，人们知之甚少。先前的研究表明，VP30的转录支持活性在VP30去磷酸化后被激活。目前的研究表明，情况更为复杂，重组埃博拉病毒的初级转录和拯救也需要VP30的短暂磷酸化。VP30编码6个n -近端丝氨酸残基作为磷酸化受体位点。目前的研究表明，仅29位丝氨酸的动态磷酸化就足以激活初级病毒转录。我们的研究结果表明，一系列磷酸化/去磷酸化事件触发了与核衣壳和转录复合物的结合和释放，这对于VP30的充分活性是必不可少的。
Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle.IMPORTANCEThe current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that the situation is more complex and that primary transcription as well as the rescue of recombinant Ebola virus also requires the transient phosphorylation of VP30. VP30 encodes six N-proximal serine residues that serve as phosphorylation acceptor sites. The present study shows that the dynamic phosphorylation of serine at position 29 alone is sufficient to activate primary viral transcription. Our results indicate a series of phosphorylation/dephosphorylation events that trigger binding to and release from the nucleocapsid and transcription complex to be essential for the full activity of VP30.