In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins
In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins
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DOI：
10.1128/jvi.76.1.269-279.2002
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发表时间：
2002-01-01
影响因子：
5.4
通讯作者：
Wang, XT
中科院分区：
文献类型：
--
作者：
Hu, JM;Toft, D;Wang, XT
嗜肝DNA病毒（乙型肝炎病毒）中逆转录的启动取决于病毒逆转录酶（RT）将RNA信号（包装信号，epsilon）特异性结合到前基因组RNA模板上，并由RT本身引发（蛋白质引发）。我们之前已经证明 RT-E 相互作用和蛋白质启动需要细胞热休克蛋白 Hsp90。然而，这些反应所需的其他宿主因素仍有待确定。我们现在报道，五种细胞伴侣蛋白（所有已知的 Hsp90 辅助因子）足以在体外重建具有 epsilon 结合和蛋白启动活性的鸭乙型肝炎病毒 RT 活性。 RT 活性的重建需要四种蛋白质：Hsp90、Hsp70、Hsp40 和 Hop，第五种蛋白质 p23 进一步增强了重建动力学。伴侣蛋白的 RT 激活是一个依赖于 ATP 水解和 Hsp90 ATP 酶活性的动态过程。因此，我们的结果定义了 RT 激活所需且充分的宿主因子的最小补充。此外，这种定义的体外重建系统现已为未来的生化和结构研究铺平了道路，以阐明 RT 激活和伴侣功能的机制。
Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-E interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in epsilon binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.