Three-Dimensional Architecture of Tick-Borne Encephalitis Virus Replication Sites and Trafficking of the Replicated RNA
Three-Dimensional Architecture of Tick-Borne Encephalitis Virus Replication Sites and Trafficking of the Replicated RNA
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DOI：
10.1128/jvi.03456-12
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发表时间：
2013-06-01
影响因子：
5.4
通讯作者：
Marcello, Alessandro
中科院分区：
文献类型：
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作者：
Miorin, Lisa;Romero-Brey, Ines;Marcello, Alessandro
黄病毒复制伴随着细胞膜的重排，这可能促进病毒基因组复制并保护病毒成分免受宿主细胞反应。病毒复制位点的拓扑结构和复制的病毒 RNA 的命运尚不完全清楚。我们利用电子显微镜绘制了受感染细胞和转染复制子的细胞中蜱传脑炎病毒（TBEV）复制区室的组织图。在这两种条件下，在内质网 (ER) 的管腔内都可以看到 80 nm 的囊泡，而在受感染的细胞中也含有病毒颗粒。通过电子断层扫描，这些囊泡表现为内质网膜的内陷，显示出一个孔，可以将新合成的病毒 RNA 释放到细胞质中。为了追踪 TBEV RNA 的命运，我们利用最近开发的病毒 RNA 荧光标记方法与漂白技术相结合进行活细胞成像。在病毒诱导的囊泡外部发现了 TBEV RNA，该囊泡要么与内质网膜相关，要么在并置的内质网池的限定区域内自由移动。根据我们的结果，我们提出了黄病毒复制区室可能的拓扑组织的生物学相关模型，该复制区室由复制囊泡和保留复制病毒RNA的有限囊外空间组成。因此，TBEV 改变了 ER 膜结构，为病毒复制提供受保护的环境，并维持新复制的 RNA 可用于病毒生命周期的后续步骤。
Flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replication and protect viral components from host cell responses. The topological organization of viral replication sites and the fate of replicated viral RNA are not fully understood. We exploited electron microscopy to map the organization of tick-borne encephalitis virus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. Under both conditions, 80-nm vesicles were seen within the lumen of the endoplasmic reticulum (ER) that in infected cells also contained virions. By electron tomography, the vesicles appeared as invaginations of the ER membrane, displaying a pore that could enable release of newly synthesized viral RNA into the cytoplasm. To track the fate of TBEV RNA, we took advantage of our recently developed method of viral RNA fluorescent tagging for live-cell imaging combined with bleaching techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER membranes or free to move within a defined area of juxtaposed ER cisternae. From our results, we propose a biologically relevant model of the possible topological organization of flavivirus replication compartments composed of replication vesicles and a confined extravesicular space where replicated viral RNA is retained. Hence, TBEV modifies the ER membrane architecture to provide a protected environment for viral replication and for the maintenance of newly replicated RNA available for subsequent steps of the virus life cycle.