Preparation of recombinant alphaviruses for functional studies of ADP-ribosylation

Rachy Abraham, Robert Lyle McPherson, Easwaran Sreekumar, Anthony Kar Lun Leung, Diane Griffin

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages297-316
Number of pages20
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1813
ISSN (Print)1064-3745

Fingerprint

Alphavirus
Hydrolases
Adenosine Diphosphate
Viruses
Alphavirus Infections
Chikungunya virus
Mutation
Site-Directed Mutagenesis
Cultured Cells
Animal Models
Complementary DNA
Clone Cells
Cell Culture Techniques
Proteins

Keywords

  • Alphavirus
  • Chikungunya virus
  • In vitro transcription
  • Infectious cDNA clone
  • MAR hydrolase assay
  • Reverse genetics
  • Sindbis virus
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Abraham, R., McPherson, R. L., Sreekumar, E., Leung, A. K. L., & Griffin, D. (2018). Preparation of recombinant alphaviruses for functional studies of ADP-ribosylation. In Methods in Molecular Biology (pp. 297-316). (Methods in Molecular Biology; Vol. 1813). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8588-3_21

Preparation of recombinant alphaviruses for functional studies of ADP-ribosylation. / Abraham, Rachy; McPherson, Robert Lyle; Sreekumar, Easwaran; Leung, Anthony Kar Lun; Griffin, Diane.

Methods in Molecular Biology. Humana Press Inc., 2018. p. 297-316 (Methods in Molecular Biology; Vol. 1813).

Research output: Chapter in Book/Report/Conference proceedingChapter

Abraham, R, McPherson, RL, Sreekumar, E, Leung, AKL & Griffin, D 2018, Preparation of recombinant alphaviruses for functional studies of ADP-ribosylation. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1813, Humana Press Inc., pp. 297-316. https://doi.org/10.1007/978-1-4939-8588-3_21
Abraham R, McPherson RL, Sreekumar E, Leung AKL, Griffin D. Preparation of recombinant alphaviruses for functional studies of ADP-ribosylation. In Methods in Molecular Biology. Humana Press Inc. 2018. p. 297-316. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8588-3_21
Abraham, Rachy ; McPherson, Robert Lyle ; Sreekumar, Easwaran ; Leung, Anthony Kar Lun ; Griffin, Diane. / Preparation of recombinant alphaviruses for functional studies of ADP-ribosylation. Methods in Molecular Biology. Humana Press Inc., 2018. pp. 297-316 (Methods in Molecular Biology).
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