title: Using click chemistry to measure the effect of viral infection on host-cell RNA synthesis.
abstract: Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as (3)H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity. The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization. This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.
authors: Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, USA. bkkalver@utmb.edu
authors: Birte
authors: B
authors: Kalveram
authors: Olga
authors: O
authors: Lihoradova
authors: Sabarish V
authors: SV
authors: Indran
authors: Jennifer A
authors: JA
authors: Head
authors: Tetsuro
authors: T
authors: Ikegami
mesh_terms: D057930:Click Chemistry / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D012295:Rift Valley Fever / Q000235:genetics* / Q000378:metabolism; D012296:Rift Valley fever virus / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D014764:Viral Proteins; D012313:RNA
references: Cytometry. 1993;14(4):455-8
references: 7685681
references: Curr Protoc Cytom. 2001 May;Chapter 4:Unit 4.2
references: 18770708
references: J Virol. 2006 Mar;80(6):2933-40
references: 16501102
references: Methods Achiev Exp Pathol. 1986;12:105-40
references: 2421137
references: Virology. 2013 Jan 20;435(2):415-24
references: 23063407
references: Prog Nucleic Acid Res Mol Biol. 1964;3:183-234
references: 4157816
references: J Vis Exp. 2012 May 05;(63):e4002
references: 22588480
references: J Org Chem. 2002 May 3;67(9):3057-64
references: 11975567
references: Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15779-84
references: 18840688
references: J Virol. 2013 Apr;87(7):3710-8
references: 23325696
references: PLoS One. 2012;7(9):e45730
references: 23029207
references: J Virol. 2011 Jul;85(13):6234-43
references: 21543505
references: PLoS Negl Trop Dis. 2013 Apr 18;7(4):e2181
references: 23638202
references: Cell. 2004 Feb 20;116(4):541-50
references: 14980221
references: Angew Chem Int Ed Engl. 2002 Jul 15;41(14):2596-9
references: 12203546
references: J Gen Virol. 1985 Oct;66 ( Pt 10):2271-7
references: 4045430