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

<ol>
	<li>Fakan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+support+for+RNA+synthesis+in+the+cell+nucleus.">Structural support for RNA synthesis in the cell nucleus.</a> Methods Achiev. Exp. Pathol. 12, 105-140 (1986).</li><li>Jensen, P. O., Larsen, J., Christiansen, J., Larsen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+measurement+of+RNA+synthesis+using+bromouridine+labelling+and+bromodeoxyuridine+antibodies.">Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies.</a> Cytometry. 14, 455-458 (1993).</li><li>Jao, C. Y., Salic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+RNA+transcription+and+turnover+in+vivo+by+using+click+chemistry.">Exploring RNA transcription and turnover in vivo by using click chemistry.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 15779-15784 (2008).</li><li>Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+huisgen+cycloaddition+process:+copper(I)-catalyzed+regioselective+"ligation"+of+azides+and+terminal+alkynes.">A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes.</a> Angew Chem. Int. Ed. Engl. 41, 2596-2599 (2002).</li><li>Tornoe, C. W., Christensen, C., Meldal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidotriazoles+on+solid+phase:+[1,2,3]-triazoles+by+regiospecific+copper(i)-catalyzed+1,3-dipolar+cycloadditions+of+terminal+alkynes+to+azides.">Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.</a> J. Org. Chem. 67, 3057-3064 (2002).</li><li>Kalveram, B., Lihoradova, O., 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=NSs+protein+of+rift+valley+fever+virus+promotes+posttranslational+downregulation+of+the+TFIIH+subunit+p62.">NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.</a> J. Virol. 85, 6234-6243 (2011).</li><li>Kalveram, B., 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=Rift+Valley+fever+virus+NSs+inhibits+host+transcription+independently+of+the+degradation+of+dsRNA-dependent+protein+kinase+PKR.">Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.</a> Virology. , (2012).</li><li>Head, J. A., 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=Functional+analysis+of+Rift+Valley+fever+virus+NSs+encoding+a+partial+truncation.">Functional analysis of Rift Valley fever virus NSs encoding a partial truncation.</a> PLoS One. 7, e45730 (2012).</li><li>Lihoradova, O. 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. M., 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=.">.</a> In Fields Virology. , 1741-1789 (2007).</li><li>Le May, N., 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=TFIIH+transcription+factor,+a+target+for+the+Rift+Valley+hemorrhagic+fever+virus.">TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus.</a> Cell. 116, 541-550 (2004).</li><li>Caplen, H., Peters, C. J., Bishop, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagen-directed+attenuation+of+Rift+Valley+fever+virus+as+a+method+for+vaccine+development.">Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development.</a> J. Gen. Virol. 66, 2271-2277 (1985).</li><li>Ikegami, T., Won, S., Peters, C. J., Makino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+infectious+Rift+Valley+fever+virus+entirely+from+cDNA,+analysis+of+virus+lacking+the+NSs+gene,+and+expression+of+a+foreign+gene.">Rescue of infectious Rift Valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.</a> J. Virol. 80, 2933-2940 (2006).</li><li>Vyboh, K., Ajamian, L., Mouland, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+viral+RNA+by+fluorescence+in+situ+hybridization+(FISH).">Detection of viral RNA by fluorescence in situ hybridization (FISH).</a> J. Vis. Exp. , e4002 (2012).</li><li>Reich, E., Goldberg, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actinomycin+and+nucleic+acid+function.">Actinomycin and nucleic acid function.</a> Prog. Nucleic Acid Res. Mol. Biol. 3, 183-234 (1964).</li><li>Holmes, K. L., Lantz, L. M., Russ, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+of+fluorochromes+to+monoclonal+antibodies.">Conjugation of fluorochromes to monoclonal antibodies.</a> Curr. Protoc. Cytom. Chapter 4 (Unit 4 2), (2001).</li></ol>

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><tbody><tr><td>5-ethynyl-uridine</td><td>Berry 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></td></tr><tr><td>5 ml polystyrene tubes</td><td>BD Biosciences</td><td>352058</td><td></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></td></tr><tr><td>Bovine Serum Albumin (BSA) </td><td>Santa Cruz</td><td>sc-2323</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>C-8027</td><td>dissolve in H&lt;sub&gt;2&lt;/sub&gt;O for 100 mM stock</td></tr><tr><td>DAPI </td><td>Sigma</td><td>D-9542</td><td>dissolve in H&lt;sub&gt;2&lt;/sub&gt;O for 5 mg/ml stock</td></tr><tr><td>DMEM </td><td>Invitrogen</td><td>11965092</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000044</td><td></td></tr><tr><td>fluorescent azide (Alexa Fluor 594-coupled)</td><td>Invitrogen</td><td>A10270</td><td></td></tr><tr><td>fluorescent azide (Alexa Fluor 657-coupled)</td><td>Invitrogen</td><td>A10277</td><td></td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>A-5960</td><td>dissolve in H&lt;sub&gt;2&lt;/sub&gt;O for 500 mM </td></tr><tr><td>paper filter</td><td>VWRbrand</td><td>28333-087</td><td></td></tr><tr><td>paraformaldehyde </td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td>poly-L-lysine (MW &amp;#8805; 70000)</td><td>Sigma</td><td>P1274</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T-1503</td><td>dissolve in H&lt;sub&gt;2&lt;/sub&gt;O for 1.5 M stock, pH 8.5</td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25200056</td><td></td></tr><tr><td>Fluorescence Microscope</td><td></td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; Olympus IX71</td></tr><tr><td>Flow Cytometer</td><td></td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; 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

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 ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Biochemistry

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

Genetics

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...