This work was supported by project grant APP1129320 and program grant APP1052979 from the NHMRC of Australia. We thank Dr. Adam Wheatley, Dr. Marina Alexander, Dr. Jenny L. Anderson and Michelle Y. Lee for providing essential constructs and advice for the successful completion of this work. We also thank the DMI Flow Facility staff for their advice and generous assistance in maintaining the flow cytometer used in this study.

NOTE: Procedures for cloning, transformation and sequencing are discussed elsewhere18,19. The protocols herein begin from the transfection of the mammalian expression vectors (Figure 3).
1. Transfection of HEK293T Cells with Dual Color Reporter Construct
<ol>
	<li>Cultivate HEK293T cells in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 &#956;g/mL) in a 5% CO2 incubator at 37 &#176;C. After thawing, passage HEK293T cells 2-3 times before using them in transfection experiments. This would give the cells time to recover from the thawing procedure. 
	CAUTION: Mycoplasma contamination of cell cultures remains a serious problem. Good laboratory practice and routine testing of cell cultures are essential to decrease the risk of mycoplasma contamination and avoid diffusion20.</li>
	<li>Split the cells 1:2 one day before seeding and transfer them into fresh DMEM medium. Cultivate the cells for 24 h in a 5% CO2 incubator at 37 &#176;C.</li>
	<li>The day before transfection, remove the medium from the flask by aspiration and wash the cells with Dulbecco&#8217;s phosphate buffered saline (DPBS) solution free of calcium (Ca2+) and magnesium (Mg2+) to remove traces of serum.</li>
	<li>Dispense 5 mL of trypsin-EDTA solution (0.05% - 10 mM in PBS) into the culture vessel and place it at 37 &#176;C in a 5% CO2 incubator for 2 to 5 min.</li>
	<li>When the cells are detached, add 5 mL of DMEM supplemented with 10% (v/v) FBS to further inhibit the trypsin activity which may damage the cells.</li>
	<li>Resuspend gently by pipetting the cell suspension up and down to break up the clumps.</li>
	<li>Dilute the cells 1:10 in trypan blue stain (0.4%).</li>
	<li>Count the live cells that do not take up trypan blue using a microscope (10x objective) and a haemocytometer.</li>
	<li>Calculate the number of viable cells per milliliter by taking the average of the cell count and multiplying it by 10,000 and by the dilution factor (1:10) from the trypan blue stain.</li>
	<li>Plate 2 x 104 cells in 100 &#956;L of DMEM medium supplemented with 10% FBS without antibiotics in a 96-well flat-bottom plate for 24 h.</li>
	<li>For each well, dilute 400 ng of pLTR.gp140/EGFP.RevD38/DsRed, 20 ng of pCMV-RevNL4.3 and 100 ng of pCMV-Tat101AD8-Flag DNA in 50 &#956;L of serum free medium. Mix gently. For experiments without Tat, use a matched empty Tat vector pcDNA3.1 (-). 
	NOTE: The use of endotoxin-free DNA is highly recommended. For that, prepare endotoxin-free DNA using a midi or maxiprep nucleic acid purification kit. Determine the DNA purity by measuring the 260/280 OD ratio, which should be between 1.7 and 1.9.</li>
	<li>Mix the lipid reagent gently before use, then dilute 0.65 &#956;L in 50 &#956;L of serum free medium. Mix gently and incubate for 5 min at room temperature.</li>
	<li>Combine the diluted DNA with the diluted lipid reagent.</li>
	<li>Mix gently and incubate for 20 min at room temperature. The solution may appear cloudy. 
	NOTE: Proceed to step 1.15. within 30 min.</li>
	<li>Dispense 100 &#956;L per well of the lipid reagent-DNA complexes drop-wise on top of the cells.</li>
	<li>Mix gently by rocking the plate back and forth.</li>
	<li>Incubate the plate for 5 h in a 5% CO2 humidified incubator at 37 &#176;C.
	<ol>
		<li>Each reaction mix is sufficient for a single well (96-well) transfection. Adjust the amount and volume of components according to the number of drug and combination that would be tested, and accounts for pipetting variations. All conditions are usually tested in triplicates.</li>
		<li>Transfect additional wells with 100 ng of CMV-driven EGFP and DsRed-Express DNA plasmid for compensation purposes.</li>
	</ol>
	</li>
</ol>
2. Treatment of Transfected HEK293T Cells with Latency Reversing Agents
NOTE: Prior to each assay, determine the physiological condition of each LRA by measuring the viability of the cells after exposure to high and low dose of the drug with cell proliferation assay.
<ol>
	<li>Dilute each LRA to the appropriate working concentration with growth medium (e.g., 1 &#956;M for JQ1(+)). 
	CAUTION: Reconstitute the lyophilized drugs with the appropriate solvent to a concentration of 10 mM. Store all stock LRAs at -80 &#176;C in a single use aliquot (5 &#956;L each) in order to avoid repeated freezing-thawing cycles.</li>
	<li>Carefully aspirate transfection medium with a multichannel and replace with 100 &#956;L medium containing appropriate LRA (Table 1). Add medium very gently alongside the wall of the well to avoid detaching the transfected HEK293T cells, which are known to easily detach from the culture plate surface.</li>
	<li>Incubate the plate for 48 h in a 5% CO2 humidified incubator at 37 &#176;C.</li>
</ol>
3. Staining of Transfected Cells with Fixable Viability Dye for Flow Cytometry Analysis
CAUTION: Removing dead cells and debris is essential to eliminate false positives and to obtain results of the highest quality.
<ol>
	<li>Detach the cells into the media using 100 &#956;L of phosphate-buffered saline (PBS) per well and by gently pipetting up-and-down (~5 times) with a multichannel, while avoiding frothing of the media. If needed, detach the cells from the plate using 35 &#181;L per well of trypsin-EDTA solution (0.05% - 10 mM in PBS) and incubating 5 min at 37 &#176;C, before neutralizing with medium containing serum.</li>
	<li>Transfer the cells to a 96-well V-bottom plate.</li>
	<li>Spin the cells for 5 min at 500 x g at 4 &#176;C, then carefully aspirate the medium/PBS without touching the cells.</li>
	<li>Wash the cells at least 1 time with 200 &#956;L of protein/serum free PBS.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C, then discard the supernatant by tilting the plate and removing the wash buffer without touching the cells.</li>
	<li>Prepare a working solution of fixable viability dye by diluting the viability dye 1:1000 in protein/serum free PBS. Prepare 50 &#181;L of the diluted stain per well. 
	NOTE: Viability dyes are available in a range of colors suitable for use with blue, red and violet lasers. Prepare a stock solution of fixable viability dye by resuspending one vial of the lyophilized dye (component A) with 50 &#956;L anhydrous DMSO (component B). Store at -20 &#176;C in a single use aliquot (1 &#956;L each), protected from the light.</li>
	<li>Add 50 &#956;L of the diluted viability dye to each well and mix the cells by pipetting up and down with a multichannel.</li>
	<li>Stain for 10-15 min at 4 &#176;C, protected from the light.</li>
	<li>Wash 1-2 times with 150 &#956;L of wash buffer (PBS with 1% bovine serum albumin and 2 mM EDTA).</li>
	<li>Centrifuge at 500 x&#160;g&#160;for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Fix the cells with 100 &#956;L of freshly prepared 1% formaldehyde in wash buffer for 10-15 min at 4 &#176;C in the dark. 
	CAUTION: Formaldehyde is very toxic. Prepare 1% formaldehyde solution in a fume hood to avoid inhalation while wearing gloves and safety glasses for protection.</li>
	<li>Wash the cells 1-2 times with 100 &#956;L of wash buffer.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in 70 &#956;L of wash buffer. 
	NOTE: The protocol can be paused here. Fixed cells could be stored at 4 &#176;C in the dark for analysis the next day on the flow cytometer.</li>
</ol>
4. EGFP and DsRed Measurements by Flow Cytometry and Data Analysis
NOTE: Analyze HIV transcription (% EGFP) and splicing (% DsRed) on a flow cytometer. Filter the sample before the run with a 70 &#956;m cell-strainer or 100 &#956;m nylon mesh to avoid clogging up the nozzle.
<ol>
	<li>Start up the cytometer and computer at least 10 min prior to use to ensure lasers warm up.</li>
	<li>Prior to running samples and commencing data acquisition, fill the sheath tank with 0.9% saline solution and ensure that a sodium hypochlorite tablet is added to the waste tank.</li>
	<li>Check the flow cell for air bubbles.</li>
	<li>Verify that the detectors and filters are appropriate for the experiment. 
	NOTE: For EGFP, use of blue laser (488 nm) and 530/30 bandpass, while DsRed Express is optimally detected using the yellow laser (561 nm) and 610/20 bandpass. A blue laser (488 nm) and 610/20 bandpass can also be used to detect DsRed expression.</li>
	<li>Check the cytometer performance and sensitivity across detectors by running calibration beads.</li>
	<li>Adjust the forward (FSC-A) and side (SSC-A) scatter voltages with unstained sample so that the main population is on-screen and clearly discernable. 
	NOTE: FSC (Forward-scattered light) is a measurement of light diffraction in a flat angle, which depends on the volume of the cell (cell surface area or size), while SSC (side-scattered light) is a measurement of light diffraction in a right angle, which is proportional to cell granularity and internal complexity.</li>
	<li>Perform manual or automatic compensation by using the single-stained samples ensuring minimal spillover of EGFP+ population into the DsRed detector and vice-versa. 
	CAUTION: For compensation purposes, use a sample of cells expressing each of the single color fluorescent protein as well as cells singly stained with the fixable viability dye. Do not use FITC and PE compensation beads for EGFP and DsRed fluorescent proteins compensations due to different spectral characteristics. Compensation controls should be bright enough to resolve positive and negative population.</li>
	<li>Create plots and set the gates using fluorescence minus one (FMO) controls.</li>
	<li>Acquire and record a minimum of 10,000 viable cell events per sample. Run samples at medium or low speed to avoid doublets passing through the laser intercept. Use pulse geometry gating such as SSC-A versus SSC-H to eliminate doublets. Check the stability of the run by plotting time versus SSC-A to see how even the flow is during the run.</li>
	<li>At the end of the run, clean the flow cytometry fluidics with concentrated cleaning agents then water for 5 min each.</li>
	<li>Shutdown the system, discard the waste and re-fill the sheath tank after acquisition.</li>
	<li>Analyze the data using a flow cytometry data analysis software. Exclude cell debris and clump (doublets) based on forward and side scatter, then eliminate the dead cells using the viability dye stain (negative population = live cells). Identify the cells expressing EGFP and DsRed (Figure 4), as well as the percentage of spliced product DsRed/(DsRed + EGFP) (Figure 5).</li>
	<li>Determine the effect of the LRA on HIV transcription and splicing by comparing untreated cells and the cells exposed to individual or a combination of LRAs (Figure 5).</li>
</ol>

<ol>
	<li>Siliciano, J. D., 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=Long-term+follow-up+studies+confirm+the+stability+of+the+latent+reservoir+for+HIV-1+in+resting+CD4++T+cells.">Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells.</a> Nature Medicine. 9 (6), 727-728 (2003).</li><li>Khoury, G., 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=Ch.+8.">Ch. 8.</a> HIV vaccine and cure - The Path Towards Finding an Effective Cure and Vaccine. 1075, (2018).</li><li>Van Lint, C., Bouchat, S., Marcello, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+transcription+and+latency:+an+update.">HIV-1 transcription and latency: an update.</a> Retrovirology. 10, 67 (2013).</li><li>Archin, N. 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=HIV-1+Expression+Within+Resting+CD4++T+Cells+After+Multiple+Doses+of+Vorinostat.">HIV-1 Expression Within Resting CD4+ T Cells After Multiple Doses of Vorinostat.</a> Journal of Infectious Diseases. 210, 728-735 (2014).</li><li>Elliott, J. H., 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=Activation+of+HIV+Transcription+with+Short-Course+Vorinostat+in+HIV-Infected+Patients+on+Suppressive+Antiretroviral+Therapy.">Activation of HIV Transcription with Short-Course Vorinostat in HIV-Infected Patients on Suppressive Antiretroviral Therapy.</a> PLoS Pathogens. 10, (2014).</li><li>Leth, S., 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=Combined+effect+of+Vacc-4x,+recombinant+human+granulocyte+macrophage+colony-stimulating+factor+vaccination,+and+romidepsin+on+the+HIV-1+reservoir+(REDUC):+a+single-arm,+phase+1B/2A+trial.">Combined effect of Vacc-4x, recombinant human granulocyte macrophage colony-stimulating factor vaccination, and romidepsin on the HIV-1 reservoir (REDUC): a single-arm, phase 1B/2A trial.</a> The Lancet HIV. 3, e463-e472 (2016).</li><li>Rasmussen, T. 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=Panobinostat,+a+histone+deacetylase+inhibitor,+for+latent-virus+reactivation+in+HIV-infected+patients+on+suppressive+antiretroviral+therapy:+a+phase+1/2,+single+group,+clinical+trial.">Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial.</a> The Lancet HIV. 1, e13-e21 (2014).</li><li>S&#248;gaard, O. S., 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=The+Depsipeptide+Romidepsin+Reverses+HIV-1+Latency+In+Vivo.">The Depsipeptide Romidepsin Reverses HIV-1 Latency In Vivo.</a> PLoS Pathogens. 11, (2015).</li><li>Bartholomeeusen, K., Xiang, Y., Fujinaga, K., Peterlin, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodomain+and+extra-terminal+(BET)+bromodomain+inhibition+activate+transcription+via+transient+release+of+Positive+Transcription+Elongation+Factor+b+(P-TEFb)+from+7SK+small+nuclear+ribonucleoprotein.">Bromodomain and extra-terminal (BET) bromodomain inhibition activate transcription via transient release of Positive Transcription Elongation Factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein.</a> Journal of Biological Chemistry. 287, 36609-36616 (2012).</li><li>Boehm, D., 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=BET+bromodomain-targeting+compounds+reactivate+HIV+from+latency+via+a+Tat-independent+mechanism.">BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism.</a> Cell Cycle. 12, 452-462 (2013).</li><li>Contreras, X., 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=Suberoylanilide+hydroxamic+acid+reactivates+HIV+from+latently+infected+cells.">Suberoylanilide hydroxamic acid reactivates HIV from latently infected cells.</a> Journal of Biological Chemistry. 284 (11), 6782-6789 (2009).</li><li>Rasmussen, T. 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=Comparison+of+HDAC+inhibitors+in+clinical+development:+effect+on+HIV+production+in+latently+infected+cells+and+T-cell+activation.">Comparison of HDAC inhibitors in clinical development: effect on HIV production in latently infected cells and T-cell activation.</a> Human Vaccines &amp; Immunotherapeutics. 9 (5), 993-1001 (2013).</li><li>Wei, D. G., 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=Histone+deacetylase+inhibitor+romidepsin+induces+HIV+expression+in+CD4+T+cells+from+patients+on+suppressive+antiretroviral+therapy+at+concentrations+achieved+by+clinical+dosing.">Histone deacetylase inhibitor romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing.</a> PLoS Pathogens. 10 (4), e1004071 (2014).</li><li>Blazkova, J., 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=Effect+of+histone+deacetylase+inhibitors+on+HIV+production+in+latently+infected,+resting+CD4(+)+T+cells+from+infected+individuals+receiving+effective+antiretroviral+therapy.">Effect of histone deacetylase inhibitors on HIV production in latently infected, resting CD4(+) T cells from infected individuals receiving effective antiretroviral therapy.</a> Journal of Infectious Diseases. 206 (5), 765-769 (2012).</li><li>Bullen, C. K., Laird, G. M., Durand, C. M., Siliciano, J. D., Siliciano, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+ex+vivo+approaches+distinguish+effective+and+ineffective+single+agents+for+reversing+HIV-1+latency+in+vivo.">New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo.</a> Nature Medicine. 20 (4), 425-429 (2014).</li><li>Yukl, S. 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=HIV+latency+in+isolated+patient+CD4+T+cells+may+be+due+to+blocks+in+HIV+transcriptional+elongation,+completion,+and+splicing.">HIV latency in isolated patient CD4+T cells may be due to blocks in HIV transcriptional elongation, completion, and splicing.</a> Science Translational Medicine. 10, (2018).</li><li>Lassen, K. G., Ramyar, K. X., Bailey, J. R., Zhou, Y., Siliciano, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+retention+of+multiply+spliced+HIV-1+RNA+in+resting+CD4+T+cells.">Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+T cells.</a> PLoS Pathogens. 2, 0650-0661 (2006).</li><li>Alexander, M. R., Wheatley, A. K., Center, R. J., Purcell, D. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transcription+through+an+intron+requires+the+binding+of+an+Sm-type+U1+snRNP+with+intact+stem+loop+II+to+the+splice+donor.">Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor.</a> Nucleic Acids Research. 38, 3041-3053 (2010).</li><li>Anderson, J. L., Johnson, A. T., Howard, J. L., Purcell, D. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+Linear+and+Discontinuous+Ribosome+Scanning+Are+Used+for+Translation+Initiation+from+Bicistronic+Human+Immunodeficiency+Virus+Type+1+env+mRNAs.">Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs.</a> Journal of Virology. 81, 4664-4676 (2007).</li><li>Nikfarjam, L., Farzaneh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+detection+of+Mycoplasma+contamination+in+cell+culture.">Prevention and detection of Mycoplasma contamination in cell culture.</a> Cell J. 13 (4), 203-212 (2012).</li><li>Khoury, G., 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=HIV+latency+reversing+agents+act+through+Tat+post+translational+modifications.">HIV latency reversing agents act through Tat post translational modifications.</a> Retrovirology. 15 (1), 36 (2018).</li><li>Hakre, S., Chavez, L., Shirakawa, K., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+latency:+experimental+systems+and+molecular+models.">HIV latency: experimental systems and molecular models.</a> FEMS Microbiology Reviews. 36 (3), 706-716 (2012).</li><li>Dahabieh, M. S., 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=Direct+non-productive+HIV-1+infection+in+a+T-cell+line+is+driven+by+cellular+activation+state+and+NFkappaB.">Direct non-productive HIV-1 infection in a T-cell line is driven by cellular activation state and NFkappaB.</a> Retrovirology. 11, 17 (2014).</li><li>Calvanese, V., Chavez, L., Laurent, T., Ding, S., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-color+HIV+reporters+trace+a+population+of+latently+infected+cells+and+enable+their+purification.">Dual-color HIV reporters trace a population of latently infected cells and enable their purification.</a> Virology. 446 (1-2), 283-292 (2013).</li><li>Chavez, L., Calvanese, V., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+Latency+Is+Established+Directly+and+Early+in+Both+Resting+and+Activated+Primary+CD4+T+Cells.">HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells.</a> PLoS Pathogens. 11 (6), e1004955 (2015).</li><li>Hunninghake, G. W., Monick, M. M., Liu, B., Stinski, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoter-regulatory+region+of+the+major+immediate-early+gene+of+human+cytomegalovirus+responds+to+T-lymphocyte+stimulation+and+contains+functional+cyclic+AMP-response+elements.">The promoter-regulatory region of the major immediate-early gene of human cytomegalovirus responds to T-lymphocyte stimulation and contains functional cyclic AMP-response elements.</a> Journal of Virology. 63 (7), 3026-3033 (1989).</li><li>Reeves, M., Sinclair, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspects+of+human+cytomegalovirus+latency+and+reactivation.">Aspects of human cytomegalovirus latency and reactivation.</a> Current Topics in Microbiology and Immunology. 325, 297-313 (2008).</li><li>Sambucetti, L. C., Cherrington, J. M., Wilkinson, G. W., Mocarski, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappa+B+activation+of+the+cytomegalovirus+enhancer+is+mediated+by+a+viral+transactivator+and+by+T+cell+stimulation.">NF-kappa B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation.</a> EMBO Journal. 8 (13), 4251-4258 (1989).</li><li>Kula, 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+the+HIV-1+RNA+associated+proteome+identifies+Matrin+3+as+a+nuclear+cofactor+of+Rev+function.">Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function.</a> Retrovirology. 8, 60 (2011).</li><li>Kula, A., Marcello, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Post-Transcriptional+Regulation+of+HIV-1+Gene+Expression.">Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression.</a> Biology (Basel). 1 (2), 116-133 (2012).</li><li>Yedavalli, V. S., Jeang, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rev-ing+up+post-transcriptional+HIV-1+RNA+expression.">Rev-ing up post-transcriptional HIV-1 RNA expression.</a> RNA Biology. 8 (2), 195-199 (2011).</li><li>Zolotukhin, A. S., 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=PSF+acts+through+the+human+immunodeficiency+virus+type+1+mRNA+instability+elements+to+regulate+virus+expression.">PSF acts through the human immunodeficiency virus type 1 mRNA instability elements to regulate virus expression.</a> Molecular and Cellular Biology. 23 (18), 6618-6630 (2003).</li><li>Laird, G. M., Rosenbloom, D. I., Lai, J., Siliciano, R. F., Siliciano, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+Frequency+of+Latent+HIV-1+in+Resting+CD4(+)+T+Cells+Using+a+Limiting+Dilution+Coculture+Assay.">Measuring the Frequency of Latent HIV-1 in Resting CD4(+) T Cells Using a Limiting Dilution Coculture Assay.</a> Methods in Molecular Biology. 1354, 239-253 (2016).</li><li>Cary, D. C., Peterlin, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+latent+reservoir+to+achieve+functional+HIV+cure.">Targeting the latent reservoir to achieve functional HIV cure.</a> F1000Res. 5, (2016).</li><li>Han, Y., 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=Resting+CD4++T+cells+from+human+immunodeficiency+virus+type+1+(HIV-1)-infected+individuals+carry+integrated+HIV-1+genomes+within+actively+transcribed+host+genes.">Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes.</a> Journal of Virology. 78 (12), 6122-6133 (2004).</li><li>Laird, G. 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=Ex+vivo+analysis+identifies+effective+HIV-1+latency+-+reversing+drug+combinations.">Ex vivo analysis identifies effective HIV-1 latency - reversing drug combinations.</a> Journal of Clinical Investigation. 125, 1901-1912 (2015).</li><li>Lenasi, T., Contreras, X., Peterlin, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+interference+antagonizes+proviral+gene+expression+to+promote+HIV+latency.">Transcriptional interference antagonizes proviral gene expression to promote HIV latency.</a> Cell Host Microbe. 4 (2), 123-133 (2008).</li></ol>

The authors have nothing to disclose.

Given the difficulty in measuring virus reactivation ex vivo, a wide range of in vitro models were developed over the time in order to study HIV latency including latently infected T cell-lines (J-Lats, ACH2, U1), primary models of latent infection of resting (O&#39;Doherty, Lewin, Greene and Spina models) or pre-activated CD4+ T cells (Sahu, Marini, Planelles, Siliciano, Karn models) with single round or replication competent reporter viruses22. To model the physiological conditions of HIV latenc...

Despite effective long-term antiretroviral therapy, HIV persists in a latent state as an integrated provirus in memory CD4+ T cells1. The chromatin structure of the HIV-1 5&#39; long terminal repeat (LTR) promoter and epigenetic modifications such as histone methylation and deacetylation by DNA methyltransferases (DNMT) and histone deacetylases (HDAC) are important mechanisms leading to transcriptional repression and thus post-integration latency2,3. A large variety of latency reversing agents (LRAs) has been investigated for their efficacy to induce virus production in vitro and in vivo from latently infected resting CD4+ T cells4,5,6,7,8. Among the LRAs tested, HDACi (HDAC inhibitors) and BET bromodomain inhibitors (BETis) induce chromatin decondensation and release of the positive transcription elongation factor b (P-TEFb) respectively, leading to subsequent relieve of the transcriptional repression at the 5&#39;LTR and activation of HIV expression9,10,11,12,13. However, the magnitude of reactivation achieved by these LRAs was limited as only a modest increase in cell-associated unspliced HIV mRNA (US RNA), indicative of viral transcription, was observed ex vivo14,15. Importantly, these LRAs also failed to induce a reduction in the frequency of latently infected cells.
HIV expression may be further restricted by inefficient splicing16 as well as defects in nuclear export of multiply spliced HIV RNA (MS RNA)17. Thus, identifying new classes of LRAs that are more potent and can affect distinct aspects of virus production post-integration are needed. In addition, the development of novel assays that help defining the optimal compounds to efficiently reverse latency is required.
Here, a protocol is presented, which utilizes a high-throughput approach for functional assessment of the impact of interventions on HIV LTR-driven transcription and splicing. In brief, a new LTR-driven dual color reporter system pLTR.gp140/EGFP.Rev&#916;38/DsRed (Figure 1) is used to assess HIV reactivation by flow cytometry. In this fluorescent reporter, the expression of unspliced HIV mRNA (4 kb) leads to enhanced green fluorescent protein (EGFP) expression, while the expression of spliced mRNA (2 kb) would lead to Discosoma sp. red&#160;(DsRed)&#160;fluorescent protein expression. Briefly, we used a fluorescent Env-EGFP fusion protein, gp140unc.EGFP, where the coding sequence of EGFP was placed in frame with an un-cleaved and truncated form of the envelope (Env). Changes were introduced to ablate the cleavage site preventing the dissociation of Env into gp120 and gp41-EGFP, and to truncate the gp160 protein prior to the transmembrane domain creating a soluble Env analogue, which facilitates the correct folding and expression of EGFP. Upon the expression within a cell, Rev localizes to the nucleus where it mediates the nuclear-cytoplasmic export of the 4 kb env mRNA via the interaction with the rev responsive element (RRE). The truncation of Env does not compromise the RRE, which lies between gp120 and gp41, and the A7 3&#39; splice site. In this system, splicing at HIV-1 splice donor 4 (SD4) and splice acceptor 7 (SA7) results in the production of a 2 kb mRNA encoding a non-functional Rev protein truncated at amino acid 38 fused to DsRed fluorescent protein, Rev&#916;38-DsRed. Briefly, DsRed was inserted into the 2nd exon of Rev at amino acid 38 by overlap extension18. To facilitate the nuclear export of unspliced mRNA, a mammalian expression vector encoding Rev (pCMV-RevNL4.3) was co-transfected with the fluorescent reporter construct (Figure 2). This unique reporter construct described here is useful in high-throughput assessment of HIV transcription and splicing, without the need to use viral vectors.

<table>
	<tbody>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells (Human Embryonic Kidney cells)</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>Replicates vectors carrying the SV40 region of replication.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM 1x + GlutaMAX-I)</td>
			<td>Gibco</td>
			<td>10569-010</td>
			<td>+ 4.5 g/L D-Glucose + 110 mg/L Sodium Pyruvate</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td>Origin Australia</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4458</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS), no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain, 0.4%</td>
			<td>Gibco</td>
			<td>15250</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Lipid transfection reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM I (1x) reduced serum medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td>Serum free medium</td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi</td>
			<td>Marcherey-Nagel</td>
			<td>740414.50</td>
			<td></td>
		</tr>
		<tr>
			<td>pEGFP-N1 plasmid</td>
			<td>Clontech (TaKaRa)</td>
			<td>6085-1</td>
			<td>Expression of EGFP in mammalian cells, CMVIE promoter.</td>
		</tr>
		<tr>
			<td>pDsRed-Express-N1</td>
			<td>Clontech (TaKaRa)</td>
			<td>632429</td>
			<td>Expression of DsRed-Express in mammalian cells, CMVIE promoter.</td>
		</tr>
		<tr>
			<td>pLTR.gp140/EGFP.RevD38/DsRed</td>
			<td>Addgene</td>
			<td>115775</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-RevNL4.3</td>
			<td>Addgene</td>
			<td>115776</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-Tat101AD8-Flag</td>
			<td>Addgene</td>
			<td>115777</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Millipore</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>JQ1(+)</td>
			<td>Cayman Chemical</td>
			<td>11187</td>
			<td>Stock at 10 mM in DMSO; working concentration 1 &#956;M</td>
		</tr>
		<tr>
			<td>JQ1(-)</td>
			<td>Cayman Chemical</td>
			<td>11232</td>
			<td>Stock at 10 mM in DMSO; working concentration 1 &#956;M</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Sigma-Aldrich</td>
			<td>16561-29-8</td>
			<td>Stock at 100 &#956;g/mL in DMSO; working concentration 10 nM</td>
		</tr>
		<tr>
			<td>Phytohaemagglutinin (PHA)</td>
			<td>Remel</td>
			<td>HA15/R30852701</td>
			<td>Stock at 1 &#956;g/&#956;L in PBS; working concentration 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Vorinostat (VOR)</td>
			<td>Cayman Chemical</td>
			<td>10009929</td>
			<td>Stock at 10 mM in DMSO; working concentration 0.5 &#956;M</td>
		</tr>
		<tr>
			<td>Panobinostat (PAN)</td>
			<td>TRC</td>
			<td>P180500</td>
			<td>Stock at 10 mM in DMSO; working concentration 30 nM</td>
		</tr>
		<tr>
			<td>CellTiter 96 AQueous One Solution Cell Proliferation Assay</td>
			<td>Promega</td>
			<td>63581</td>
			<td></td>
		</tr>
		<tr>
			<td>Venor GeM Classic</td>
			<td>Minerva Biolabs</td>
			<td>11-1100</td>
			<td>Mycoplasma Detection Kit, PCR-based</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Flow cytometry reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSR Fortessa</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer (4 lasers-blue, red, violet and yellow)</td>
		</tr>
		<tr>
			<td>LSR II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer (3 lasers-blue, red and violet)</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td>Life Technologies</td>
			<td>L34976</td>
			<td>Viability dye: for 633 or 635 nm excitation, 400 assays. Component A and B are both provided in the kit.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5M pH8</td>
			<td>Gibco</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde Solution 37/10 (37%)</td>
			<td>Chem-Supply</td>
			<td>FA010</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Diva CS&#38;T Research Beads</td>
			<td>BD Biosciences</td>
			<td>655050</td>
			<td>Calibration beads</td>
		</tr>
		<tr>
			<td>Sphero Rainbow Calibration Particles (8 peaks)</td>
			<td>BD Biosciences</td>
			<td>559123</td>
			<td>3.0 - 3.4 mm</td>
		</tr>
		<tr>
			<td>Sheath solution</td>
			<td>Chem-Supply</td>
			<td>SA046</td>
			<td>90 g NaCl in 10 L water</td>
		</tr>
		<tr>
			<td>HAZ-Tabs</td>
			<td>Guest Medical</td>
			<td>H8801</td>
			<td>Chlorine release tablets for disinfection</td>
		</tr>
		<tr>
			<td>Decon 90</td>
			<td>Decon Laboratories Limited</td>
			<td>N/A</td>
			<td>Concentrated cleaning agents of flow cytometer. Working solution Decon 90 5%.</td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite (12-13% Solution)</td>
			<td>Labco</td>
			<td>SODHYPO-5L</td>
			<td>Concentrated cleaning agents of flow cytometer. Working solution bleach 1%.</td>
		</tr>
		<tr>
			<td>7x</td>
			<td>MPBio</td>
			<td>IM76670</td>
			<td>Concentrated cleaning agents of flow cytometer. Working solution 7x 1%.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks (75 cm2, canted neck, cap vented)</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plates (96 well flat bottom with lid)</td>
			<td>Costar</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plates (96 well V-bottom without lid)</td>
			<td>Costar</td>
			<td>3896</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (10 mL)</td>
			<td>SARSTEDT</td>
			<td>62.9924.284</td>
			<td>100x16 mm</td>
		</tr>
		<tr>
			<td>Centrifuge tubes (50 mL)</td>
			<td>CellStar</td>
			<td>227261</td>
			<td>30x115 mm</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5 mL)</td>
			<td>Corning Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette (25 mL), sterile</td>
			<td>Corning</td>
			<td>CLS4489-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette (10 mL), sterile</td>
			<td>Corning</td>
			<td>CLS4488-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette (5 mL), sterile</td>
			<td>Corning</td>
			<td>CLS4487-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoirs (50 mL), sterile</td>
			<td>Corning</td>
			<td>CLS4470-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round-Bottom polystyrene test tube, with cell-strainer cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>12 x 75 mm style, 70 mm</td>
		</tr>
		<tr>
			<td>Nylon Mesh</td>
			<td>SEFAR</td>
			<td>03-100/32</td>
			<td>100 mm</td>
		</tr>
		<tr>
			<td>Titertube Micro test tubes, bulk</td>
			<td>BIO-RAD</td>
			<td>2239391</td>
			<td>microfacs tubes</td>
		</tr>
		<tr>
			<td>5 mL Round-Bottom polystyrene test tube, without cap</td>
			<td>Corning</td>
			<td>352008</td>
			<td>12x75 mm style</td>
		</tr>
		<tr>
			<td>Snap Caps for 12x75 mm Test Tubes</td>
			<td>Corning</td>
			<td>352032</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting chamber, Neubauer improved double net ruling, bright-line (Haemocytometer, LO-Laboroptik)</td>
			<td>ProSciTech</td>
			<td>SVZ4NIOU</td>
			<td>3x3 large squares of 1 mm2; Depth 0.100 mm; volume 0.1 mL; area minimum 0.0025 mm2</td>
		</tr>
		<tr>
			<td>Coverslips (Menzel-Gl&#228;ser)</td>
			<td>Grale Scientific</td>
			<td>HCS2026</td>
			<td>20 x 26 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon TMS</td>
			<td>310528</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810R refrigerated</td>
			<td>Eppendorf</td>
			<td>5811000487</td>
			<td>with rotor A-4-81 including adapters for 15/50 mL conical tubes</td>
		</tr>
		<tr>
			<td>FLUOstar Omega microplate reader</td>
			<td>BMG Labtech</td>
			<td>N/A</td>
			<td>Plate reader for cell proliferation assay. Filter 490 nm.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Diva</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer data acquisition and analysis program, version 8.0.1</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo</td>
			<td>FlowJo 10.4.2</td>
			<td>Flow cytometer data analysis program, FlowJo Engine v3.05481</td>
		</tr>
		<tr>
			<td>Omega</td>
			<td>BMG Labtech</td>
			<td></td>
			<td>FLUOstar multi-user reader control, version 5.11</td>
		</tr>
		<tr>
			<td>Omega - Data Analysis</td>
			<td>BMG Labtech</td>
			<td>MARS</td>
			<td>FLUOstar data analysis, version 3.20R2</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>Excel:mac 2011</td>
			<td>version 14.0.0</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td>Prism 7</td>
			<td>version 7.0c</td>
		</tr>
	</tbody>
</table>

high throughput in vitro assessment of latency reversing agents on hiv transcription and splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

Representative results are shown in Figure 5 for the expression of HIV-1 unspliced (EGFP) and spliced (DsRed) products following treatment with bromodomain inhibitor JQ1. Both JQ1(+) and Tat significantly increased the percentage of cells expressing EGFP (2.18 and 4.13 FC over DMSO respectively; n = 3) indicative of unspliced transcripts. Moreover, JQ1(+) significantly increased the percentage of cells expressing DsRed (46.6 FC over DMSO) as well as the propo...

Watch this Scientific Journal Video about High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing at JoVE.com

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

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JoVE Journal

Immunology and Infection

5.7K Views.  University of Melbourne. A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

Video: High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...