Name: high throughput in vitro assessment of latency reversing agents on hiv transcription and splicing
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Description: Watch this Scientific Journal Video about High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing at JoVE.com

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>
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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 ...

Research

JoVE Journal

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

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In vitro Uncoating of HIV-1 Cores

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

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

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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 ...

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

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

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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

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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

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