Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation

  • Rachy Abraham
  • Robert Lyle McPherson
  • Easwaran Sreekumar
  • Anthony K. L. LeungEmail author
  • Diane E. GriffinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)


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 words

Alphavirus 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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Rachy Abraham
    • 1
  • Robert Lyle McPherson
    • 2
  • Easwaran Sreekumar
    • 1
    • 3
  • Anthony K. L. Leung
    • 2
    • 4
    • 5
    Email author
  • Diane E. Griffin
    • 1
    Email author
  1. 1.Department of Molecular Microbiology and ImmunologyBloomberg School of Public Health, Johns Hopkins UniversityBaltimoreUSA
  2. 2.Department of Biochemistry and Molecular BiologyBloomberg School of Public Health, Johns Hopkins UniversityBaltimoreUSA
  3. 3.Viral Disease Biology ProgramRajiv Gandhi Centre for Biotechnology (RGCB)ThiruvananthapuramIndia
  4. 4.Department of OncologySchool of Medicine, Johns Hopkins UniversityBaltimoreUSA
  5. 5.Department of Molecular Biology and GeneticsSchool of Medicine, Johns Hopkins UniversityBaltimoreUSA

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