title: Electroporation of Alphavirus RNA Translational Reporters into Fibroblastic and Myeloid Cells as a Tool to Study the Innate Immune System.
abstract: The ability to transfect synthetic mRNAs into cells to measure processes such as translation efficiency or mRNA decay has been an invaluable tool in cell biology. The use of electroporation over other methods of transfection is an easy, inexpensive, highly efficient, and scalable method to introduce synthetic mRNA into a wide range of cell types. More recently, coupling of noncoding RNA sequences or protein coding regions from viral pathogens to fluorescent or bioluminescence proteins in RNA "reporters" has permitted study of host-pathogen interactions. These can range from virus infection of cells to translation of the viral genome, replication and stability of viral RNAs, or the efficacy of host antiviral responses. In this chapter, we describe a method for electroporating viral RNA reporters into both fibroblastic and myeloid cells that encode firefly or Renilla luciferase, whose reaction with specific substrates and light emitting activity is a measure of viral RNA translation efficiency. We have used this method to examine host interferon-dependent responses that inhibit viral translation along with identifying secondary structures in the 5' nontranslated region (NTR) and microRNA binding sites in the 3' NTR that are responsible for antagonizing the host innate immune responses and restricting viral cell tropism.
authors.affiliation: Department of Microbiology and Molecular Genetics, Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA, USA. clg50@pitt.edu.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Gardner
authors.affiliation: Department of Microbiology and Molecular Genetics, Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Derek W
authors.initials: DW
authors.lastname: Trobaugh
authors.affiliation: Department of Microbiology and Molecular Genetics, Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Kate D
authors.initials: KD
authors.lastname: Ryman
authors.affiliation: Department of Microbiology and Molecular Genetics, Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: William B
authors.initials: WB
authors.lastname: Klimstra
mesh_terms: D000524:Alphavirus / Q000235:genetics / Q000276:immunology*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D018274:Electroporation / Q000379:methods*; D005347:Fibroblasts / Q000276:immunology*; D007113:Immunity, Innate; D007372:Interferons / Q000378:metabolism; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D022423:Myeloid Cells / Q000276:immunology*; D012367:RNA, Viral / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D012367:RNA, Viral; D011994:Recombinant Proteins; D007372:Interferons; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly
keywords: Alphavirus; Electroporation; Luciferase; Neon transfection; Translation