Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation.
Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation.
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用于 ADP-核糖基化功能研究的重组甲病毒的制备。
DOI：
10.1007/978-1-4939-8588-3_21
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Griffin,DianeE
中科院分区：
文献类型：
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作者：
Abraham,Rachy;McPherson,RobertLyle;Sreekumar,Easwaran;Leung,AnthonyKL;Griffin,DianeE
最近，我们的特点是单（ADP-核糖基）水解酶（MAR水解酶）的活性的宏域的非结构蛋白3（nsP 3 MD）的基孔肯雅病毒。使用重组病毒与靶向突变的宏域，我们证明了水解酶功能是重要的病毒复制培养的神经元细胞和神经毒力在小鼠。在这里，我们描述了甲病毒的一般细胞培养和动物模型感染协议和技术细节的MAR水解酶活性的nsP 3 MD突变体的生化表征和重组病毒的制备，通过定点诱变的感染性cDNA病毒克隆纳入这些突变。
Recently we characterized the mono(ADP-ribosyl) hydrolase (MAR hydrolase) activity of the macrodomain of nonstructural protein 3 (nsP3MD) of chikungunya virus. Using recombinant viruses with targeted mutations in the macrodomain, we demonstrated that hydrolase function is important for viral replication in cultured neuronal cells and for neurovirulence in mice. Here, we describe the general cell culture and animal model infection protocols for alphaviruses and the technical details for biochemical characterization of the MAR hydrolase activity of nsP3MDmutants and the preparation of recombinant viruses incorporating those mutations through site-directed mutagenesis of an infectious cDNA virus clone.