Preparation of recombinant alphaviruses for functional studies of ADP-ribosylation

Rachy Abraham, Robert Lyle McPherson, Easwaran Sreekumar, Anthony K.L. Leung, Diane E. 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 (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.

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

Publication series

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

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

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