Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: stability and interactions with the core RNAi machinery.
Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: stability and interactions with the core RNAi machinery.
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DOI：
10.1016/j.jinsphys.2014.03.002
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发表时间：
2014-05
影响因子：
2.2
通讯作者：
L. Swevers;Anna Kolliopoulou;Zheng Li;Maria G Daskalaki;Frédéric Verret;K. Kalantidis;G. Smagghe;Jingchen Sun
中科院分区：
文献类型：
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作者：
L. Swevers;Anna Kolliopoulou;Zheng Li;Maria G Daskalaki;Frédéric Verret;K. Kalantidis;G. Smagghe;Jingchen Sun
虽然已经进行了几项研究来研究dsRNA在细胞外介质（血淋巴、肠内容物、唾液）中的稳定性，但是关于dsRNA一旦被引入细胞中的持久性知之甚少。在这里，我们调查的稳定性的家蚕moricytoplasmic多角体病毒（BmCPV）的基因组dsRNA片段转染到家蚕衍生的Bm 5细胞后。使用RT-PCR作为检测方法，我们发现dsRNA可以在细胞内环境中持续很长时间（长达8天）。虽然BmCPV基因组dsRNA被RNAi机制加工，但它的存在对其他RNAi过程没有影响，例如dsLuc沉默荧光素酶报告基因。我们还发现，BmCPV基因组dsRNA的转染不能在Bm 5细胞中建立病毒感染，即使当与靶向Dicer和Argonaute基因的dsRNA进行共转染时，这表明RNAi的中和作用在体外培养系统的建立中不起作用。本文还讨论了dsRNA在Bm 5细胞中稳定性的机理，以及对建立BmCPV体外培养体系的意义。
While several studies have been conducted to investigate the stability of dsRNA in the extracellular medium (hemolymph, gut content, saliva), little is known regarding the persistence of dsRNA once it has been introduced into the cell. Here, we investigate the stability ofBombyx moricytoplasmic polyhedrosis virus (BmCPV) genomic dsRNA fragments after transfection intoBombyx-derived Bm5 cells. Using RT-PCR as a detection method, we found that dsRNA could persist for long periods (up to 8 days) in the intracellular environment. While the BmCPV genomic dsRNA was processed by the RNAi machinery, its presence had no effects on other RNAi processes, such as the silencing of a luciferase reporter by dsLuc. We also found that transfection of BmCPV genomic dsRNA could not establish a viral infection in the Bm5 cells, even when co-transfections were carried out with dsRNAs targeting Dicer and Argonaute genes, suggesting that the neutralization by RNAi does not play a role in the establishment of anin vitroculture system. The mechanism of the dsRNA stability in Bm5 cells is discussed, as well as the implications for the establishment for anin vitroculture system for BmCPV.