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 ser...

<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. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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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. 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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 ...

The overall goal of this procedure is to assess in vitro, the impact of latency reversing agents on HIV mRNA processing. The protocol provide a simple efficient and reliable method for assessing, simultaneously the effect of LRA on HIV transcription and splicing. This technique provided an insight on the ability of LRAs to induce virus reactivation and clearance of the latent reservoir.After cultivating cells, place 20, 000 of them in 100 microliters of DMEM medium, supplemented with 10%FBS, and without antibiotics in the wells of a 96-well flat bottom plate for 24 hours. The next day dilute the dual color reporter construct. Tat and Rev DNA in 50 microliters of serum free medium, and mix gently.Gently mix the lipid reagent, and then dilute 0.65 microliters of it per reaction in 50 microliters of serum free medium. Mix gently, and incubate at room temperature for five minutes. Combine the diluted lipid reagent with the diluted DNA and gently mix.Incubate at room temperature for 20 minutes. Then, dispense 100 microliters of this lipid reagent DNA complex drop wise, on top of the cells in each well. Rock the plate gently back and forth to mix.And incubate the plate in an humidified incubator, at 37 degrees celsius with 5%carbon dioxide for five hours. After this, transfect additional wells with 100 nanograms of CMV driven EGFP and DsRed express DNA plasmid for compensation purposes. To begin, dilute each latency reversing agent to the appropriate working condition with growth medium.Using a multi-channel pipe head, carefully aspirate the transfection medium, and replace it with 100 microliters of medium containing the appropriate latency reversing agent. Incubate in a humidified incubator at 37%celsius with 5%carbon dioxide for 48 hours. To begin staining the cells, use a multi-channel pipe head to add 100 microliters of PBS to each well.Gently pipe head up and down approximately five times. While making sure to avoid frothing to detach the cells into the media. Next, transfer the cells into the wells of a 96-well V-bottom plate.Centrifuge at 500 times gravity and at four degrees celsius for five minutes. Aspirate the medium and PBS without touching the cells. Wash the cells at least once with 200 microliters of protein and serum free PBS.Centrifuge at 500 times gravity and at four degrees celsius for five minutes. Tilt the plate to discard the supernatant and remove the wash buffer without touching the cells. After this dilute the fixed viability dye in protein and serum free PBS at a ratio of 1 to 1000, to prepare a working solution.Add 50 microliters of the diluted dye to each well, and pipe head up and down to mix. Incubate at four degrees celsius. While protected from light using foil for 10 to 15 minutes.Then wash the cells once or twice with 150 microliters of wash buffer. After this, centrifuge at 500 times gravity, and four degrees celsius for five minutes, and discard the supernatant. To fix the cells, add 100 microliters of freshly prepared 1%formaldehyde and wash buffer.And incubate at four degrees celsius while protected from light for 10 to 15 minutes. After this, wash the cells once or twice with 100 microliters of wash buffer. Centrifuge at 500 times gravity and at four degrees celsius for five minutes.Discard the supernatant, and resuspend the cell pellet in 70 microliters of wash buffer. After checking the cytometer settings performance and sensitivity by running calibration beads, adjust the forward and side scatter voltages with unstained sample, so that the main population is on screen and clearly discernible. Perform manual or automatic compensation by using the single stained samples, ensuring minimal spill over of EGFP positive population into the DsRed detector and vice versa.Using the fluorescence minus one controls, create plots and set the gates. Next, adjust the forward and side scatter voltages with unstained sample, so that the main population is on screen and clearly discernible. Acquire and record a minimum of 10, 000 viable cell events per sample.After this, use flow cytometry data analysis software to analyze the data as outlined in the text protocol. Representative results for the expression of HIV-1, unspliced and spliced products, after treatment with the bromodomain inhibitor JQ1, show that both JQ1 positive and Tat, significantly increase the percentage of cells expressing EGFP. Which is indicative of unspliced transcripts.JQ1 positive is also seen to significantly increase the percentage of cells expressing DsRed. And increase the proportion of spliced product to similar levels as Tat. This confirms the ability of JQ1 positive to turn on HIV transcription and splicing.However, treatment with a stereoisomer control JQ1 negative, abolishes the JQ1 positive effect on HIV transcription and splicing. Once mastered, this method can be carried out using drugs individually or in combination. Testing for their ability to synergize efficiently.Which would lead to a lower dose of the drug and toxicity. After each development, this technique paved the way for testing efficient drug combinations in primary models of latency in ex vivo patient samples.

high-throughput-vitro-assessment-latency-reversing-agents-on-hiv

Research

JoVE Journal

Immunology and Infection

5.7K Aufrufe.  University of Melbourne. Das übergeordnete Ziel dieses Verfahrens besteht darin, in vitro die Auswirkungen von Latenz-Umkehrmitteln auf die HIV-mRNA-Verarbeitung zu bewerten. Das Protokoll bietet eine einfache effiziente und zuverlässige Methode zur Bewertung, gleichzeitig die Wirkung von LRA auf HIV-Transkription und Spleißen. Diese Technik gab einen Einblick in die Fähigkeit von LRAs, Virusreaktivierung und Clearance des latenten Reservoirs zu induzieren.Nach dem Anbau von Zellen 20.000 davon in 100 Mikr...