Using click chemistry to measure the effect of viral infection on host-cell RNA synthesis

Birte Kalveram, Olga Lihoradova, Sabarish V. Indran, Jennifer A. Head, Tetsuro Ikegami

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number78
DOIs
StatePublished - 2013

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Click Chemistry
Virus Diseases
RNA
Viruses
Azides
Uridine
Transcription
Rift Valley fever virus
Scintillation Counting
Triazoles
Alkynes
Flow cytometry
Fluorophores
Fluorescence microscopy
RNA Viruses
Scintillation
Viral Proteins
Autoradiography
Fluorescence Microscopy
Nucleosides

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Using click chemistry to measure the effect of viral infection on host-cell RNA synthesis. / Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Head, Jennifer A.; Ikegami, Tetsuro.

In: Journal of visualized experiments : JoVE, No. 78, 2013.

Research output: Contribution to journalArticle

Kalveram, Birte ; Lihoradova, Olga ; Indran, Sabarish V. ; Head, Jennifer A. ; Ikegami, Tetsuro. / Using click chemistry to measure the effect of viral infection on host-cell RNA synthesis. In: Journal of visualized experiments : JoVE. 2013 ; No. 78.
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