Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation

Rachy Abraham; Robert Lyle McPherson; Easwaran Sreekumar; Anthony K L Leung; Diane E Griffin

doi:10.1007/978-1-4939-8588-3_21

Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation

Methods Mol Biol. 2018:1813:297-316. doi: 10.1007/978-1-4939-8588-3_21.

Authors

Rachy Abraham¹, Robert Lyle McPherson², Easwaran Sreekumar^{1

3}, Anthony K L Leung^{4

5

6}, Diane E Griffin⁷

Affiliations

¹ Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
² Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
³ Viral Disease Biology Program, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, Kerala, India.
⁴ Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA. [email protected].
⁵ Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA. [email protected].
⁶ Department of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, MD, USA. [email protected].
⁷ Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA. [email protected].

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.

Keywords: Alphavirus; Chikungunya virus; In vitro transcription; Infectious cDNA clone; MAR hydrolase assay; Reverse genetics; Sindbis virus; Site-directed mutagenesis.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

ADP-Ribosylation / genetics*
Alphavirus / genetics*
Alphavirus / pathogenicity
Alphavirus Infections / genetics
Alphavirus Infections / virology
Animals
Cell Culture Techniques / methods
Cell Line
Disease Models, Animal
Mice
Molecular Biology / methods*
Mutagenesis, Site-Directed / methods
Neurons / virology
Viral Nonstructural Proteins / chemistry*
Viral Nonstructural Proteins / genetics
Virus Replication / genetics

Substances

Viral Nonstructural Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding