Inclusion Bodies Are a Site of Ebolavirus Replication
Inclusion Bodies Are a Site of Ebolavirus Replication
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DOI：
10.1128/jvi.01525-12
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发表时间：
2012-11-01
影响因子：
5.4
通讯作者：
Feldmann, Heinz
中科院分区：
文献类型：
--
作者：
Hoenen, Thomas;Shabman, Reed S.;Feldmann, Heinz
包涵体是埃博拉病毒感染细胞的特征。它们含有大量预先形成的核衣壳，但它们的生物学意义一直存在争议，它们被认为是病毒蛋白质的聚集体，没有任何进一步的生物学功能。然而，最近的数据表明，其他病毒产生类似的结构，包涵体可能参与基因组复制和转录。为了研究丝状病毒包涵体，我们将mCherry与包涵体中发现的埃博拉病毒聚合酶L融合。得到的L-mCherry融合蛋白在微型基因组测定中是功能性的，并掺入病毒样颗粒中。重要的是，转染细胞中的L-mCherry荧光很容易检测到，并以包涵体特有的点状模式分布。拯救了编码L-mCherry而不是L的重组埃博拉病毒，并显示出与野生型病毒几乎相同的生长动力学和终点滴度。使用这种病毒，我们表明，包涵体形成的开始对应于病毒基因组复制的开始，但病毒转录发生在包涵体形成之前。活细胞成像进一步表明，包涵体是高度动态的结构，它们可以在细胞分裂过程中进行戏剧性的重组。最后，通过使用点击技术标记新生RNA，我们表明包涵体确实是病毒RNA合成的位点。基于这些数据，我们得出结论，埃博拉病毒包涵体不是核衣壳的惰性聚集体，而是复杂和动态的结构，是病毒RNA复制发生的重要位点。
Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.