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 (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 nsP3MD mutants and the preparation of recombinant viruses incorporating those mutations through site-directed mutagenesis of an infectious cDNA virus clone.
Key wordsAlphavirus Chikungunya virus Sindbis virus MAR hydrolase assay Infectious cDNA clone Reverse genetics Site-directed mutagenesis In vitro transcription
This work was supported by a Johns Hopkins Catalyst Award (AKLL) and research grants from the Johns Hopkins University School of Medicine Sherrilyn and Ken Fisher Center for Environmental Infectious Disease (DEG and AKLL). This work is also in part supported by R01GM104135S1 (AKLL and RLM) and T32CA009110 (RLM) from the U.S. National Institutes of Health.
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