Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Summary

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

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 ³H-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.

Introduction

Traditionally, transcriptional activity has been measured through incorporation of either radioactive (³H-uridine)¹ or halogenated (BrU)² nucleosides into nascent RNA. These uridine analogs are taken up by cells, converted into uridine-triphosphate (UTP) through the nucleoside salvage pathway, and subsequently incorporated into newly synthesized RNA. ³H-uridine can be detected by autoradiography, which can be time-consuming, or scintillation counting, which requires specialized equipment. Furthermore, the use of radioactive isotopes may not be practical in a number of laboratory settings, including high containment biosafety....

Protocol

Analysis of transcription by fluorescence microscopy

1. Infect Cells with MP-12 and Label Nascent RNA with EU

1. Place 12-mm round coverslips into 12-well tissue culture plate, one coverslip per well.

Note: If cells have trouble adhering to coverslips, coat with poly-L-lysine (MW ≥ 70,000) before placing in wells. To this aim, submerge coverslips in a sterile 0.1 mg/ml solution in H₂O of poly-L-lysine for 5 min. Rinse coverslips with sterile H₂O and air-dry for at least 2 hr.

2. To seed 293 cells onto coverslips, add 5 x 10⁴ ce....

Results

The NSs protein of RVFV (family Bunyaviridae, genus Phlebovirus) ¹¹ inhibits host cell general transcription through two distinct mechanisms: (i) by sequestering the p44 subunit of the basal transcription factor TFIIH¹¹ and (ii) by promoting the proteasomal degradation of the p62 subunit of TFIIH ⁶. The RVFV MP-12 vaccine strain ¹³ has been used for the experiments described in this protocol since MP-12 strain can be handled in a biosafety level 2 laboratory a.......

Discussion

The provided protocol describes a method to measure the effect of viral infection on host cell transcription via incorporation of the uridine analog EU into nascent RNA. This method has several advantages over previous methods: it is fast, sensitive, and it does not rely on the use of radioactive isotopes. Furthermore, the method can be adapted to yield qualitative data through fluorescence microscopy or quantitative data via flow cytometry using essentially the same reagents. When combined with immunofluorescence staini.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-ethynyl-uridine	Berry Associates	PY 7563	dissolve in DMSO for 100 mM stock
12-mm round coverslips	Fisherbrand	12-545-82
5 ml polystyrene tubes	BD Biosciences	352058
Actinomycin D (ActD)	Sigma	9415	dissolve in DMSO for 10 mM stock
Alexa Fluor 488 goat anti-mouse IgG	Invitrogen	A-11029
Bovine Serum Albumin (BSA)	Santa Cruz	sc-2323
CuSO4	Sigma	C-8027	dissolve in H2O for 100 mM stock
DAPI	Sigma	D-9542	dissolve in H2O for 5 mg/ml stock
DMEM	Invitrogen	11965092
FBS	Invitrogen	16000044
fluorescent azide (Alexa Fluor 594-coupled)	Invitrogen	A10270
fluorescent azide (Alexa Fluor 657-coupled)	Invitrogen	A10277
Fluoromount-G	Southern Biotech	0100-01
L-ascorbic acid	Sigma	A-5960	dissolve in H2O for 500 mM
paper filter	VWRbrand	28333-087
paraformaldehyde	Sigma	158127
Penicillin-Streptomycin	Invitrogen	15140122
poly-L-lysine (MW ≥ 70000)	Sigma	P1274
Triton X-100	Sigma	T-8787
Trizma base	Sigma	T-1503	dissolve in H2O for 1.5 M stock, pH 8.5
Trypsin-EDTA	Invitrogen	25200056
Fluorescence Microscope			e.g. Olympus IX71
Flow Cytometer			e.g. BD Biosciences LSRII Fortessa