Visualizing Coronavirus RNA Synthesis in Time by Using Click Chemistry
Visualizing Coronavirus RNA Synthesis in Time by Using Click Chemistry
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DOI：
10.1128/jvi.07207-11
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发表时间：
2012-05-01
影响因子：
5.4
通讯作者：
de Haan, Cornelis A. M.
中科院分区：
文献类型：
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作者：
Hagemeijer, Marne C.;Vonk, Annelotte M.;de Haan, Cornelis A. M.
冠状病毒在感染细胞中诱导复制结构的形成，复制结构由双膜小泡(DMV)和卷曲的膜组成，病毒RNA合成可能发生在那里，非结构蛋白(NSP)定位在那里。双链RNA(DsRNA)是RNA合成的中间产物，定位于DMV内部。然而，由于尚未检测到连接DMV内部和细胞质的孔，因此尚不清楚RNA合成是否发生在这些相同的位置。在这里，我们通过用尿苷类似物喂养细胞来研究冠状病毒RNA的合成，然后用点击化学方法检测新生RNA。在感染早期，新生病毒RNA和NSP与dsRNA灶共定位或发生在dsRNA灶附近。在感染后期，dsRNA点之间的相关性不明显，然后发现分散在细胞质中，与NSP和新生RNA之间的相关性不明显。然而，新生的RNA的焦点总是被发现与nsp12编码的RNA依赖的RNA聚合酶共定位。这些结果证明了利用点击化学检测病毒RNA合成的可行性，并表明dsRNA斑点不一定与病毒RNA合成的活性部位相对应。相反，在感染后期，许多DMV可能含有dsRNA分子，这些分子不再作为RNA合成的中间体发挥作用。
Coronaviruses induce in infected cells the formation of replicative structures, consisting of double-membrane vesicles (DMVs) and convoluted membranes, where viral RNA synthesis supposedly takes place and to which the nonstructural proteins (nsp's) localize. Double-stranded RNA (dsRNA), the presumed intermediate in RNA synthesis, is localized to the DMV interior. However, as pores connecting the DMV interior with the cytoplasm have not been detected, it is unclear whether RNA synthesis occurs at these same sites. Here, we studied coronavirus RNA synthesis by feeding cells with a uridine analogue, after which nascent RNAs were detected using click chemistry. Early in infection, nascent viral RNA and nsp's colocalized with or occurred adjacent to dsRNA foci. Late in infection, the correlation between dsRNA dots, then found dispersed throughout the cytoplasm, and nsp's and nascent RNAs was less obvious. However, foci of nascent RNAs were always found to colocalize with the nsp12-encoded RNA-dependent RNA polymerase. These results demonstrate the feasibility of detecting viral RNA synthesis by using click chemistry and indicate that dsRNA dots do not necessarily correspond with sites of active viral RNA synthesis. Rather, late in infection many DMVs may harbor dsRNA molecules that are no longer functioning as intermediates in RNA synthesis.