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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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
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author Abraham, Rachy
McPherson, Robert Lyle
Sreekumar, Easwaran
Leung, Anthony K. L.
Griffin, Diane E.
author_facet Abraham, Rachy
McPherson, Robert Lyle
Sreekumar, Easwaran
Leung, Anthony K. L.
Griffin, Diane E.
author_sort Abraham, Rachy
collection PubMed
description 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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spelling pubmed-63824692019-02-20 Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation Abraham, Rachy McPherson, Robert Lyle Sreekumar, Easwaran Leung, Anthony K. L. Griffin, Diane E. ADP-ribosylation and NAD+ Utilizing Enzymes Article 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. 2018-04-27 /pmc/articles/PMC6382469/ /pubmed/30097877 http://dx.doi.org/10.1007/978-1-4939-8588-3_21 Text en © Springer Science+Business Media, LLC, part of Springer Nature 2018 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
spellingShingle Article
Abraham, Rachy
McPherson, Robert Lyle
Sreekumar, Easwaran
Leung, Anthony K. L.
Griffin, Diane E.
Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation
title Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation
title_full Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation
title_fullStr Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation
title_full_unstemmed Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation
title_short Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation
title_sort preparation of recombinant alphaviruses for functional studies of adp-ribosylation
topic Article
url 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
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