Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET).
Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET).
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DOI：
10.1016/j.bbrc.2008.01.094
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发表时间：
2008-04
影响因子：
3.1
通讯作者：
Na Li;Zongqiang Cui;Ji-Kai Wen;Zhi-ping Zhang;H. Wei;Yafeng Zhou;Xian-En Zhang
中科院分区：
文献类型：
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作者：
Na Li;Zongqiang Cui;Ji-Kai Wen;Zhi-ping Zhang;H. Wei;Yafeng Zhou;Xian-En Zhang
脊髓灰质炎病毒RNA复制由膜囊泡的玫瑰花状排列上的复制复合物指导。来自脊髓灰质炎病毒基因组p3区的蛋白质，如3D、3AB和3B（VPg），在复制复合物的形成和功能中发挥关键作用。在本研究中，通过使用受体光漂白协议的荧光共振能量转移（FRET）成像，我们可视化的3D，3AB，和VPg在活细胞中的相互作用。通过活细胞FRET分析确定3AB-VPg的相互作用。定量分析显示，在病毒复制复合物形成和发挥功能的细胞质中，3AB-3D、VPg-3D和3AB-VPg的FRET效率分别为3.9±0.4%（n=36）、4.5±0.4%（n=39）和8.3±0.6%（n=44）。脊髓灰质炎病毒感染增强了VPg-3D和3AB-3D的蛋白质相互作用，病毒感染细胞中的FRET效率分别为10.7±1.1%（n=39）和9.0±0.9%（n=37）。这种活细胞分析脊髓灰质炎病毒RNA复制复合物中蛋白质相互作用的方法为进一步了解脊髓灰质炎病毒RNA复制的实时过程奠定了基础。
Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB–VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB–3D, VPg–3D, and 3AB–VPg were 3.9±0.4% (n=36), 4.5±0.4% (n=39), and 8.3±0.6% (n=44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg–3D and 3AB–3D, with FRET efficiencies in the virus-infected cells of 10.7±1.1% (n=39) and 9.0±0.9% (n=37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.