Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation

Recently we characterized the mono(ADP-ribosyl) hydrolase (MAR hydrolase) activity of the macrodomain of nonstructural protein 3 (nsP3(MD)) of chikungunya virus. Using recombinant viruses with targeted mutations in the macrodomain, we demonstrated that hydrolase function is important for viral repli...

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Detalles Bibliográficos
Autores principales: Abraham, Rachy, McPherson, Robert Lyle, Sreekumar, Easwaran, Leung, Anthony K. L., Griffin, Diane E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2018
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382469/
https://www.ncbi.nlm.nih.gov/pubmed/30097877
http://dx.doi.org/10.1007/978-1-4939-8588-3_21
Descripción
Sumario:Recently we characterized the mono(ADP-ribosyl) hydrolase (MAR hydrolase) activity of the macrodomain of nonstructural protein 3 (nsP3(MD)) 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(MD) mutants and the preparation of recombinant viruses incorporating those mutations through site-directed mutagenesis of an infectious cDNA virus clone.

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