Abraham Rachy, McPherson Robert Lyle, Sreekumar Easwaran, Leung Anthony K L, Griffin Diane E
Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
Methods Mol Biol. 2018;1813:297-316. doi: 10.1007/978-1-4939-8588-3_21.
Recently we characterized the mono(ADP-ribosyl) hydrolase (MAR hydrolase) activity of the macrodomain of nonstructural protein 3 (nsP3) 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 nsP3 mutants and the preparation of recombinant viruses incorporating those mutations through site-directed mutagenesis of an infectious cDNA virus clone.
最近，我们对基孔肯雅病毒非结构蛋白3（nsP3）的巨结构域的单（ADP-核糖基）水解酶（MAR水解酶）活性进行了表征。利用在巨结构域中具有靶向突变的重组病毒，我们证明水解酶功能对于在培养的神经元细胞中的病毒复制以及对小鼠的神经毒力很重要。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方案，以及nsP3突变体的MAR水解酶活性生化表征的技术细节，以及通过对感染性cDNA病毒克隆进行定点诱变来制备包含这些突变的重组病毒的方法。
