We thank R.B. Tesh for providing the mouse polyclonal anti-RVFV antiserum and M. Griffin of the UTMB Flow Cytometry Core Facility for help with flow cytometry. This work was supported by 5 U54 AI057156 through the Western Regional Center of Excellence, NIH grant R01 AI08764301, and funding from the Sealy Center for Vaccine Development at UTMB.B.K. was supported by the James W. McLaughlin Fellowship Fund at UTMB.O.L. was supported by a Maurice R. Hilleman Early-Stage Career Investigator Award.

Analysis of transcription by fluorescence microscopy
1. Infect Cells with MP-12 and Label Nascent RNA with EU
<ol>
	<li>Place 12-mm round coverslips into 12-well tissue culture plate, one coverslip per well.</li>
</ol>
Note: If cells have trouble adhering to coverslips, coat with poly-L-lysine (MW &ge; 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.
<ol start="2">
	<li>To seed 293 cells onto coverslips, add 5 x 104 cells in 1 ml growth medium (DMEM containing 10% fetal bovine serum, 100 U/ml Penicillin and 100 mg/ml Streptomycin) per well. Incubate in a humidified incubator at 37 &deg;C and 5% CO2 O/N.</li>
	<li>Infect cells with MP-12 at a multiplicity of infection (m.o.i.) of 3. Include at least two uninfected wells: one of the two wells will serve as the uninfected control, the other will be treated with actinomycin D (ActD) at step 1.5 as a control for transcriptional suppression. Remove growth medium and dilute the virus stock so that the total volume added to each well is 200 ml. Incubate for 1 hr in a humidified incubator at 37 &deg;C and 5% CO2.</li>
</ol>
Note: Instead of infecting cells, cells can also be transfected with plasmid DNA or in vitro synthesized RNA encoding a specific viral protein. To this aim, transfect cells with an appropriate chemical transfection reagent according to the manufacturer's instructions and proceed to step 1.5 at 16 hr post transfection. It is advisable to optimize the transfection conditions and incubation time before RNA labeling and fixation.
<ol start="4">
	<li>Remove inoculum and add 1 ml fresh growth medium per well. Incubate at 37 &deg;C for 15 hr.</li>
</ol>
Note: If adapting this protocol for use with another virus, it is advisable to perform a time course experiment to determine the optimal time point for RNA labeling and fixation. Set up this time course so that the infection times are staggered and all samples can be labeled, fixed, and stained at the same time.
<ol start="5">
	<li>At 15 hr post infection (h.p.i.), replace growth medium with medium containing 1 mM EU. To one of the mock infected wells, also add 5 &mu;g/ml ActD. This will serve as the control for transcriptional suppression, as ActD inhibits DNA-dependent RNA synthesis. Return cells to the incubator.</li>
	<li>At 16 h.p.i., remove the medium and wash the coverslips once with 1 ml PBS (KCl 0.20 g/L, KH2PO4 0.20 g/L, NaCl 8.00 g/L, Na2HPO4 1.15 g/L, pH 7.4) per well. Fix cells by adding 1 ml of 4% paraformaldehyde (PFA) per well and incubating for 30 min at RT.</li>
</ol>
Note: To prepare 4% PFA fixation solution, dissolve 10 g paraformaldehyde in 150 ml H2O heated to 60 &deg;C on a heated magnetic stirrer. Add 500 &mu;l of 1M NaOH and continue to stir until solution becomes clear. Filter PFA solution through a paper filter to remove particulates and add 62.5 ml phosphate buffer (77 mM NaH2PO4, 287 mM Na2HPO4, pH 7.4). Add H2O up to a total volume of 250 ml. Freeze at -20 &deg;C in single-use aliquots.
<ol start="7">
	<li>Wash once with 1 ml PBS per well. No incubation time is required for this and all subsequent wash steps. Proceed immediately to step 2.</li>
</ol>
Note: It is important to immediately proceed with the click reaction, as the RNA degenerates if kept at this stage for prolonged periods of time; a loss of the RNA fluorescence signal can already be observed if cells are kept for 1 hr at 4 &deg;C. Similarly, if performing a time course experiment, be sure to label, fix, and stain all samples at the same time.
2. Click Reaction to Detect Labeled RNA
<ol>
	<li>Permeabilize cells by adding 1 ml 0.2% Triton X-100 in PBS. Incubate for 10 min at RT.</li>
</ol>
Note: All solutions used in steps 2.1 to 2.3 need to be nuclease free.
<ol start="2">
	<li>Wash three times with 1 ml PBS per well.</li>
	<li>Remove coverslips from 12-well plate and transfer into humid chamber. Cover each coverslip with 50 - 100 &mu;l click staining solution (100 mM Tris pH 8.5, 1 mM CuSO4, 20 &mu;M fluorescent azide [excitation/emission maximum: 590/617 nm], 100 mM ascorbic acid) each. Incubate for 1 hr at RT in the dark.</li>
</ol>
Note: To assemble the humid chamber, line the bottom of a cell culture or petri dish with a 10 cm diameter with plastic paraffin film. Line the inside of the edge of the dish with damp paper towels. Place the coverslips onto the plastic paraffin film.
Note: Be careful not to let the coverslips dry out during this or any other step in the protocol. It is best to add the staining solution to each coverslip directly after it has been placed in the humid chamber.
Note: Prepare the click staining solution fresh immediately prior to this step. Stock solutions of 1.5 M Tris pH 8.5, 100 mM CuSO4, and 500 mM ascorbic acid may be stored at 4 &deg;C. However, discard any ascorbic acid stock solution that has turned yellow.
Note: Be sure to select a fluorophore that matches the filters on your fluorescent microscope.
<ol start="4">
	<li>Remove coverslips from humid chamber and place into a fresh 12-well plate and wash three times with 1 ml PBS per well.</li>
</ol>
Note: If no detection of viral proteins is desired, coverslips can be mounted and imaged after this step (proceed to step 3.3).
3. Immunofluorescence to Detect Expression of Viral Proteins
<ol>
	<li>Add 1 ml of blocking buffer (3% (w/v) bovine serum albumin (BSA) in PBS) to each well and incubate for 30 min at RT in the dark.</li>
	<li>Remove coverslips from 12-well plate, transfer into humid chamber and cover with 50 - 100 &mu;l of primary antibody (anti-RVFV, a kind gift from Dr. R.B. Tesh, UTMB) diluted in blocking buffer (1:500). Incubate for 1 hr at RT in the dark.</li>
</ol>
Note: When adapting this protocol for use with a different virus, determine the optimal dilution for your primary antibody first.
Note: Alternatively, this step can be performed O/N at 4 &deg;C in the dark.
<ol start="3">
	<li>Remove coverslips from humid chamber and place back into 12-well plate and wash three times with 1 ml PBS per well.</li>
	<li>Remove coverslips from 12-well place, place into humid chamber and cover with 50 - 100 &mu;l of secondary antibody (anti-mouse IgG conjugated to fluorescent dye [excitation/emission maximum: 495/519 nm]) diluted in blocking buffer (1:500). Incubate for 1 hr at RT in the dark.</li>
</ol>
Note: Be sure to select a secondary antibody that can recognize your primary antibody and matches the filters on your fluorescent microscope.
Note: If desired, 200 ng/ml DAPI can be added to the secondary antibody dilution to visualize nuclei.
<ol start="5">
	<li>Remove coverslips from humid chamber and place back into 12-well plate and wash three times with 1 ml PBS per well.</li>
	<li>Mount coverslips by placing one drop (ca. 15 &mu;l) of Fluoromount-G onto a microscope slide. Dip the coverslip into H2O, remove excess H2O by touching the side against a paper towel, and place cell-side down into the drop of Fluoromount-G. Air dry for 5 min.</li>
</ol>
Note: If imaging on an inverted microscope, allow Fluoromount-G to solidify at 4 &deg;C O/N or affix the coverslips to the microscope slide by sealing the edge with transparent nail polish.
Note: Slides can be imaged immediately or stored at 4 &deg;C in the dark for up to a week.
4. Acquisition of Data
<ol>
	<li>Image using a fluorescent microscope.</li>
</ol>
Note: Be sure to select appropriate fluorophores which can be visualized using your microscope. For reference, the following are the fluorophores and filter sets used to acquire the images shown in this publication.
DAPI : 
Excitation filter: BP 330 - 385 
Dichromatic mirror: DM 400 
Emission filter: LP 420
Fluorophore with an excitation/emission maximum of 495/519: 
Excitation filter: BP 460 - 490 
Dichromatic mirror: DM 505 
Emission filter: LP 510
Fluorophore with an excitation/emission maximum of 590/617: 
Excitation filter: BP 545 - 580 
Dichromatic mirror: DM 600 
Emission filter: LP 610
Analysis of transcription by flow cytometry
5. Infect Cells with MP-12 and Label Nascent RNA with EU
<ol>
	<li>Seed 293 cells into 6-well tissue culture plate in growth medium (DMEM containing 10% fetal bovine serum, 100 U/ml Penicillin, 100 &mu;g/ml Streptomycin). Incubate in a humidified incubator at 37 &deg;C and 5% CO2 until cells have reached 80 - 100% confluency.</li>
	<li>Infect cells with MP-12 at an m.o.i. of 3. Include at least two uninfected wells: one of the two wells will serve as the uninfected control, the other will be treated with ActD at step 5.4. Remove growth medium and dilute the virus stock so that the total volume added to each well is 400 &mu;l. Incubate for 1 hr in a humidified incubator at 37 &deg;C and 5% CO2.</li>
	<li>Remove inoculum and add 2 ml fresh growth medium per well. Incubate at 37 &deg;C for 12 hr.</li>
</ol>
Note: If adapting this protocol for use with another virus, it is advisable to perform a time course experiment to determine the optimal time point for labeling and harvesting. Set up this time course so that the infection times are staggered and all samples can be labeled, harvested, and stained at the same time.
<ol start="4">
	<li>At 12 h.p.i., replace growth medium with medium containing 0.5 mM EU. To one of the mock infected wells, also add 5 &mu;g/ml ActD. This will serve as the control for transcriptional suppression, as ActD inhibits DNA-dependent RNA synthesis. Return cells to the incubator.</li>
	<li>At 13 h.p.i., wash cells once with PBS and harvest by trypsinization of cells. Wash harvested cells three times with PBS containing 1 mM EDTA (PBS-EDTA).</li>
</ol>
Note: During this and subsequent steps, do not centrifuge cells faster than 500 - 1,000 x g to prevent cell breakage. Centrifuge cells for 1 - 2 min to sediment.
Note: If a surface immunostaining is planned following the click reaction, harvest using a rubber policeman or disposable cell scraper instead.
<ol start="6">
	<li>Fix cells by resuspending them in 1 ml of 4% PFA and incubate for 30 min at RT. Refer to step 1.6 for instructions on how to prepare 4% PFA.</li>
	<li>Wash once with 1 ml PBS-EDTA per well. Proceed immediately to step 6.</li>
</ol>
Note: It is important to immediately proceed with the click reaction, as the RNA degenerates if kept at this stage for prolonged periods of time. Similarly, if performing a time course experiment, be sure to label, fix and stain all samples at the same time.
6. Click Reaction to Detect Labeled RNA
<ol>
	<li>Permeabilize cells by resuspending in 0.5 ml of 0.2% Triton X-100 in PBS. Incubate for 10 min at RT.</li>
</ol>
Note: All solutions used in steps 6.1 to 6.3 need to be nuclease free.
Note: If cells do not sediment properly during this step, increase the centrifugation time to 5 min. Sedimentation behavior will improve once cells are suspended in FACS buffer (step 6.4).
<ol start="2">
	<li>Wash once with 1 ml PBS.</li>
</ol>
Note: Do not use EDTA in this step as this will chelate the Cu2+ ions necessary for the click reaction.
<ol start="3">
	<li>Resuspend cells in 200 &mu;l click staining solution (100 mM Tris pH 8.5, 1 mM CuSO4, 200nM azide labeled with fluorescent dye [excitation/emission maximum: 650/665 nm], 100 mM ascorbic acid). Incubate for 1 hr at RT in the dark.</li>
</ol>
Note: Prepare the click staining solution fresh immediately prior to this step. Stock solutions of 1.5 M Tris pH 8.5, 100 mM CuSO4, and 500 mM ascorbic acid may be stored at 4 &deg;C. However, discard any ascorbic acid stock solution that has turned yellow.
Note: If cells clump together during this step, resuspend by pipetting up and down several times.
Note: Be sure to select a fluorophore that can be detected by your flow cytometer.
Note: Also prepare one sample of infected cells which are not subjected to the click staining procedure; these will be needed for calibration of the flow cytometer. Either use half of the infected cells or include an additional infected well at step 5.2. These control cells will be immunostained at step 7.1.
<ol start="4">
	<li>Wash twice with FACS buffer (PBS containing 1 mM EDTA and 0.5% (w/v) BSA).</li>
</ol>
Note: If no immunostaining of viral proteins is desired, cells can be analyzed immediately (proceed to step 8).
7. Immunofluorescence to Detect Expression of Viral Proteins
<ol>
	<li>Resuspend cells in 200 &mu;l of primary antibody (anti-RVFV) diluted in FACS buffer (1:10,000) and incubate for 1 hr at RT in the dark.</li>
</ol>
Note: Alternatively, this step can be performed O/N at 4 &deg;C in the dark.
Note: Also prepare one sample of uninfected cells which were subjected to the click staining procedure but will not be immunostained, these will be needed for calibration of the flow cytometer. Either use half of the mock infected cells or include an additional uninfected well at step 5.2.
Note: When adapting this protocol for use with a different virus, determine the optimal dilution for your primary antibody first.
<ol start="2">
	<li>Wash cells twice in FACS buffer.</li>
	<li>Resuspend cells in 200 &mu;l of secondary antibody (anti-mouse IgG conjugated to fluorescent dye [excitation/emission maximum: 495/519 nm]) diluted in FACS buffer (1:1,000) and incubate for 1 hr at RT in the dark.</li>
	<li>Wash cells once in FACS buffer.</li>
</ol>
Note: At this point, cells can be stored O/N at 4 &deg;C in the dark.
8. Acquisition of Data
<ol>
	<li>Resuspend cells in 500 &mu;l FACS buffer, transfer into 5 ml polystyrene tubes and analyze using a flow cytometer. Use MP-12 infected cells (no click reaction, anti-RVFV staining only) and mock infected cells (click reaction only) to calibrate the instrument.</li>
</ol>
Note: Be sure to select appropriate fluorophores which can be detected by your instrument. For reference, the following are the laser lines and filter sets used to acquire the data shown in this publication.
Fluorophore with an excitation/emission spectrum of 495/519: 
Laser: 488 nm 
Filter: 530/30
Fluorophore with an excitation/emission spectrum of 650/665: 
Laser: 640 nm 
Filter: 670/20

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Rift+Valley+Fever+Virus+MP-12+Strain+Encoding+NSs+of+Punta+Toro+Virus+or+Sandfly+Fever+Sicilian+Virus.">Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus.</a> PLoS Negl Trop Dis. 7, e2181 (2013).</li><li>Kalveram, B., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toscana+Virus+NSs+Protein+Promotes+Degradation+of+Double-Stranded+RNA-Dependent+Protein+Kinase.">Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase.</a> J. Virol. , (2013).</li><li>Schmaljohn, C., Nichol, S., Knipe, D. 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No conflicts of interest declared.

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...

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 laboratories. BrU can be detected through immunostaining with anti-BrU antibodies, which may require harsh permeabilization to ensure access of the antibody to the nucleus or partial denaturation of RNA, as RNA secondary structure might be a steric hindrance for antibody binding.
The protocol described here utilizes the recently developed click chemistry3 to measure transcriptional activity in cells infected with the RVFV strain MP-12. Click chemistry relies on a highly selective and efficient copper(I)-catalyzed cycloaddition reaction between an alkyne and an azide moiety4,5. In this protocol, nascent RNA in infected cells is first labeled through incorporation of the uridine analog EU. In a second step, the incorporated EU is detected through reaction with a fluorescent azide. The main advantages of this method are (1) that it does not require the use of radioactive isotopes and (2) that it does not require harsh permeabilization or denaturation of RNA. With a molecular weight of less than 50 Da, access of the fluorescent azide is not impeded by insufficient permeabilization or RNA secondary structure. Furthermore, researchers are not limited in their choice of primary antibodies when combining the detection of RNA with a classical immunostaining for viral proteins.
Two different methods are described in this protocol: one for visualizing transcription via fluorescence microscopy (steps 1-4), and one for quantifying transcription through flow cytometry (steps 5-8). Both of these methods combine labeling of nascent RNA with an intracellular immunostaining for viral proteins, which allows for a correlation between transcriptional activity and viral infection on a cell-by-cell basis.
The authors have successfully used the protocol described here to determine transcriptional activity in cells infected with the RVFV strain MP-126, cells infected with different MP-12 mutants7,8, 9, and cells infected with Toscana virus (TOSV)10. The protocol described here can be easily adapted for use with different viruses and has the potential to be modified to include immunofluorescence staining of specific viral or cellular proteins.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>5-ethynyl-uridine</td>
			<td>Berry &amp; Associates</td>
			<td>PY 7563</td>
			<td>dissolve in DMSO for 100 mM stock</td>
		</tr>
		<tr>
			<td>12-mm round coverslips</td>
			<td>Fisherbrand</td>
			<td>12-545-82</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 ml polystyrene tubes</td>
			<td>BD Biosciences</td>
			<td>352058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Actinomycin D (ActD)</td>
			<td>Sigma</td>
			<td>9415</td>
			<td>dissolve in DMSO for 10 mM stock</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Santa Cruz</td>
			<td>sc-2323</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>Sigma</td>
			<td>C-8027</td>
			<td>dissolve in H2O for 100 mM stock</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D-9542</td>
			<td>dissolve in H2O for 5 mg/ml stock</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>16000044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fluorescent azide (Alexa Fluor 594-coupled)</td>
			<td>Invitrogen</td>
			<td>A10270</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fluorescent azide (Alexa Fluor 657-coupled)</td>
			<td>Invitrogen</td>
			<td>A10277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A-5960</td>
			<td>dissolve in H2O for 500 mM</td>
		</tr>
		<tr>
			<td>paper filter</td>
			<td>VWRbrand</td>
			<td>28333-087</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>poly-L-lysine (MW &ge; 70000)</td>
			<td>Sigma</td>
			<td>P1274</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T-1503</td>
			<td>dissolve in H2O for 1.5 M stock, pH 8.5</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>e.g. Olympus IX71</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>e.g. BD Biosciences LSRII Fortessa</td>
		</tr>
	</tbody>
</table>

using click chemistry to measure the effect of viral infection on host-cell rna synthesis

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.

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...

Watch this Scientific Journal Video about Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis at JoVE.com

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

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.

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, ...

using-click-chemistry-to-measure-effect-viral-infection-on-host-cell

Research

JoVE Journal

Immunology and Infection

11.6K Views.  University of Texas Medical Branch. 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.

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

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...