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>

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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 latency in resting CD4+ T cells, dual fluorescent reporter systems ensued such as the RGH (Red-Green-HIV) reporter developed by Ivan Sadowski&#39;s group with an LTR-driven Gag-eGFP marker and a CMV-driven mCherry in place of Nef23. A similar dual color reporter virus, Duo-Fluo, was developed by E. Verdin laboratory with an LTR-driven EGFP expression and an mCherry fluorescent marker driven by an EF1&#945; promoter24. In this system, the EGFP was inserted at the 3&#39; end of the provirus in place of Nef. The deletion in the envelope in both these systems prevents multiple rounds of infections. Moreover, the combination of the two fluorescent proteins allows the detection of productively (eGFP+ mCherry+) and latently (eGFP- mCherry+) infected cells. However, several shortcomings of the dual color reporter viruses were noticeable in CD4+ T cells as the direct infection of latently infected cells is largely dependent on the cellular activation state of the T-cells23,24. In fact, very low rate of infection was observed using these two reporter viruses in resting CD4+ T cells: 0.1% for RGH23 and 0.2% for DuoFluo25. In addition, as the CMV promoter has been shown to be expressed at lower levels in non-activated cells26,27,28, an inactive CMV or EF1&#945;&#160;promoter would lead to underestimation of the latently infected populations.
We have generated and characterized a new HIV reporter vector to study the establishment of latency and virus reactivation in a high throughput assay. Several advantages to our screening method include i) cost effectiveness with the ability to assess transcription and splicing simultaneously, ii) the ability to test a large number of drugs or combinations in a single experiment, iii) quick assessment of the effect of LRAs by flow cytometry (30-45 min typical read time for 96 well plate independent of the number of fluorescent parameters), iv) high dynamic range, v) suitable for automated high throughput transfection and flow cytometry using robot arms and HTS platform, vi) fast curing of data using a flow cytometry workspace template, vii) optimal for suspension and adherent cells, viii) simplicity of the model and adaptability to luminescence and plate reader measurements (dual luciferase Firefly and Renilla), and ix) scalability (96- and 384-well pate formats).
The propensity of an LRA or a combination of LRAs to activate HIV LTR-driven transcription and splicing thus EGFP and DsRed fluorescent proteins expression could be examined by flow cytometry using our HIV splicing reporter. However, before testing the synergy of novel LRAs, it is highly recommended to determine the physiological conditions of each LRA by measuring the viability of the cells after exposure to a range of concentrations of the single drug. Therefore, including a marker of live and dead cells is essential to eliminate false positives and to obtain results of the highest quality. Another important aspect of flow cytometry analysis is the compensation controls or FMO. It is highly recommended to use singly stained samples ensuring minimal spillover of EGFP+ population into the DsRed and vice-versa. Moreover, it is essential to account for autofluorescence of the cells by including unstained cells. Finally, a regular control of the cytometer performance and sensitivity across the detectors is critical mostly when measuring samples for a study spanning a long period of time.
Although not discussed in the protocol section, there are several limitations of our splicing reporter system. For a more thorough discussion, please see our previous publication (Khoury et al., 2018). Export of the partially or unspliced variants of HIV mRNA is facilitated by the viral protein Rev and its association with the Rev Responsive Element (RRE) in these mRNA species29,30,31,32. The overexpression of Rev in our system allowed us to circumvent the major block of nuclear retention of multiply spliced and unspliced mRNAs observed in latently infected T cells17 and focus on understanding how the LRAs induce transcription and splicing thus virus reactivation. In addition, given that our system requires the transfection of 3 plasmids, we chose HEK293T cells because the high transfection efficiency of these cells. Owing the different temporal and spatial availability of cellular factors in T cells compared to a cancer cell line, it is important to validate the results of the splicing reporter acquired in HEK293T cells in T-cell lines and primary cells, which might provide great technical challenges due to their limited transfectability. Therefore, the use of alternative DNA delivery reagents for transfection such as DMRIE-C, which has been shown to be particularly effective for the transfection of suspension cells (e.g., Jurkat), or nucleofection methods such as Amaxa nucleofector or Neon transfection system that have been proven to facilitate efficient and reliable delivery of single or multiple plasmids to difficult-to-transfect cells including primary CD4+ T cells. Alternatively, it would be interesting to test this reporter system in the context of full-length virus in a primary cell model of latency using the aforementioned transfection methods. In fact, the differences in the availability of host transcription, elongation and splicing factors in a rCD4+ T cell may affect the capacity of an LRA to reactivate the latent provirus. Finally, our model does not address whether the LRA can affect transcription and splicing of bystander cells, and whether it can induce replication competent virus. However, we would suspect that by measuring an increase in cell-associated HIV US and MS RNA, this would lead to the production of replication competent virus in the culture supernatant. This could be confirmed by conducting the gold standard quantitative virus outgrowth assay (QVOA)33.
Due to the complex nature of latency and to ensure efficient virus reactivation, a multifaceted combinatorial strategy may be required34. Potential treatment needs to stimulate multiple pathways including transcription and splicing, which has been previously shown to play essential roles in the establishment of latency16,35,36,37. Our LTR-driven splicing reporter would allow a high throughput screening of drugs that can optimally target both steps, transcription and splicing, increasing the chance of finding molecules that can synergize efficiently, which would lead to a lower dose levels and thus toxicity of each drug.

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>&lt;strong&gt;Cell culture&lt;/strong&gt;</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&#039;s Modified Eagle&#039;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&#039;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, CMV&lt;sub&gt;IE&lt;/sub&gt; promoter.</td></tr><tr><td>pDsRed-Express-N1</td><td>Clontech (TaKaRa)</td><td>632429</td><td>Expression of DsRed-Express in mammalian cells, CMV&lt;sub&gt;IE&lt;/sub&gt; promoter.</td></tr><tr><td>pLTR.gp140/EGFP.RevD38/DsRed</td><td>Addgene</td><td>115775</td><td></td></tr><tr><td>pCMV-Rev&lt;sup&gt;NL4.3&lt;/sup&gt;</td><td>Addgene</td><td>115776</td><td></td></tr><tr><td>pCMV-Tat101&lt;sup&gt;AD8&lt;/sup&gt;-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 &amp;mu;M</td></tr><tr><td>JQ1(-)</td><td>Cayman Chemical</td><td>11232</td><td>Stock at 10 mM in DMSO; working concentration 1 &amp;mu;M</td></tr><tr><td>Phorbol Myristate Acetate (PMA)</td><td>Sigma-Aldrich</td><td>16561-29-8</td><td>Stock at 100 &amp;mu;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 &amp;mu;g/&amp;mu;L in PBS; working concentration 10 &amp;mu;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 &amp;mu;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Flow cytometry reagents&lt;/strong&gt;</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&amp;amp;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tissue culture flasks (75 cm&lt;sup&gt;2&lt;/sup&gt;, 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 mm&lt;sup&gt;2&lt;/sup&gt;; Depth 0.100 mm; volume 0.1 mL; area minimum 0.0025 mm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Coverslips (Menzel-Gl&amp;auml;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FACS Diva</td><td>BD Biosciences</td><td></td><td>&lt;em&gt;Flow cytometer data acquisition and analysis program, version 8.0.1&lt;/em&gt;</td></tr><tr><td>FlowJo</td><td>FlowJo</td><td>FlowJo 10.4.2</td><td>&lt;em&gt;Flow cytometer data analysis program, FlowJo Engine v3.05481&lt;/em&gt;</td></tr><tr><td>Omega</td><td>BMG Labtech</td><td></td><td>&lt;em&gt;FLUOstar multi-user reader control, version 5.11&lt;/em&gt;</td></tr><tr><td>Omega - Data Analysis</td><td>BMG Labtech</td><td>MARS</td><td>&lt;em&gt;FLUOstar data analysis, version 3.20R2&lt;/em&gt;</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>Excel:mac 2011</td><td>&lt;em&gt;version 14.0.0&lt;/em&gt;</td></tr><tr><td>Prism</td><td>GraphPad</td><td>Prism 7</td><td>&lt;em&gt;version 7.0c&lt;/em&gt;</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 proportion of spliced product (7.37 FC over DMSO) to a similar level as Tat (59.6 and 5.83 FC over DMSO, respectively) confirming the ability of JQ1(+) to turn on HIV transcription and splicing. On the other hand, treatment with the stereoisomer control JQ1(-) abolishes JQ1(+) effect on HIV transcription and splicing confirming the success of the protocol.
In a recent publication, we have shown by RNA analysis an accumulation of HIV spliced transcripts following JQ1(+) treatment21. In fact, our data revealed consistent changes in the levels of unspliced and spliced transcripts that were mirrored by the EGFP and DsRed protein expression in this model following treatment with JQ1(+). These results indicate that EGFP and DsRed expressing cells reflect the ability of the bromodomain inhibitor JQ1(+) to induce HIV transcription and splicing.
In summary, we validated the use of the pLTR.gp140/EGFP.Rev&#916;38/DsRed reporter in a high-throughput set-up for the assessment of the effect of LRAs on HIV-1 transcription and splicing. Sub-optimal amount of LRA or increased toxicity could lead to scud results. Optimizing the amount of drug used while preserving cell viability can improve the accuracy of the results.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58753/58753fig1.jpg" /> 
Figure 1: Schematic of the model used to determine the effects of latency reversing agents on LTR-driven transcription and splicing. The LTR construct expresses either unspliced envelope (Env) protein fused to enhanced green fluorescent protein (EGFP) or spliced &#916;38rev protein with DsRed. This figure has been reprinted from Khoury et al.21. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58753/58753fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58753/58753fig2.jpg" /> 
Figure 2: Constructs design. (A)&#160;pLTR.gp140/EGFP.Rev&#916;38/DsRed splicing reporter allows the expression of envelope fused to EGFP (gp140-EGFP) and non-functional Rev protein truncated at amino acid 38 fused to DsRed fluorescent protein (Rev&#916;38-DsRed).&#160;(B)&#160;pCMV-RevNL4.3 allows the expression of Rev protein. (C)&#160;pCMV-Tat101AD8-Flag allows the expression of Tat101(AD8) protein with a C-terminal flag-tag. The expression plasmids are constructed using PCR overlaps and restriction digest. All vector maps were created using SnapGene software, version 4.1.9. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58753/58753fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58753/58753fig3.jpg" /> 
Figure 3: Assay design for rapid assessment of HIV latency reversing agents. The current method for the assessment of the effect of LRAs on HIV-1 transcription and splicing includes i) seeding of HEK293T cells one day prior to ii) transfection with expressions vectors followed by iii) LRA treatment. iv) Cells are analyzed by flow cytometry 48 h post-treatment. Using this protocol, 32 (in triplicate) to 96 conditions could be tested per plate. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58753/58753fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Representative example of the plate set-up and controls needed. Columns 1 to 3 correspond to negative (-) controls with no drug and solvent only (DMSO 1:5000) as well as positive (+) controls such as Tat (100 ng), PMA/PHA = phorbol myristate acetate/phytohaemagglutinin (10 nM PMA, 10 &#956;g/mL PHA), JQ1 (+/-) (1 &#956;M), VOR = vorinostat (0.5 &#956;M), and PAN = panobinostat (30 nM). Unknown LRAs are tested in triplicate over a range of concentrations (15.625 to 1000 nM). For compensation purposes, cells expressing each of the single-color fluorescent protein (EGFP+ and DsRed+) as well as cells stained with the viability dye and unstained cells are included in each run. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58753/Table1.xls">Please click here to download this file.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58753/58753fig4.jpg" /> 
Figure 4: Gating strategy used in the flow cytometry analysis. The first step in the gating strategy is based on the forward and side scatter, which allows the distinction of the cells of interest based on the size and granularity properties of these cells. It is recommended that this gating be as generous as possible to include all events, while removing cellular debris and dead cells, which are found at the bottom left corner of the density plot. Then remove doublets from the dataset using the pulse geometry gating, SSC-A versus SSC-H, and further narrow on the live cells using the viability dye. Finally, a dump channel (violet) and EGFP or DsRed are used to define to cells of interest. The use of fluorescence minus one (FMO) controls are critical in defining the population of interest and addressing the issues of spillover from another fluorochrome in the channel of interest. After acquisition, data are analyzed using flow cytometry data analysis software. This figure has been reprinted in adapted form Khoury et al.21. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58753/58753fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58753/58753fig5.jpg" /> 
Figure 5: Representative results of the effect of bromodomain inhibitors on HIV transcription and splicing. (A)&#160;Examples of two-parameter dual color fluorescence EGFP versus DsRed density plots from untransfected cells (- Tat), cells transfected with 100 ng of pCMV-Tat101AD8-Flag (+ Tat) and cells treated with DMSO, JQ1 (+) or JQ1 (-). (B)&#160;The mean percentage (%) of cells expressing EGFP, DsRed or spliced product (DsRed/DsRed+EGFP) from 3 independent experiments is shown. Comparisons of each condition to DMSO were made using the 2-way ANOVA test. Only statistically significant comparisons are shown. *p&#160;&#60; 0.05; **p&#160;&#60; 0.01; ***p&#160;&#60; 0.001. The black lines represent the mean&#177;SEM. DMSO (1:5000), JQ1 (+) and JQ1(-) (1 &#956;M). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58753/58753fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Universidad San Sebastian

Research

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

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Genetics

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

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 ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

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

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

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

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

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

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

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 ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Studying ER Stress and UPR Activation During HIV-1 Infection in T-Cells

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. 
Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2&#945;, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

Author Spotlight: Exploring the Role of Unfolded Protein Response in HIV-1 Replication and Infectivity

Here, we describe some established methods to determine endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, with particular emphasis on HIV-1 infection. This article also describes a set of protocols to investigate the effect of ER stress/UPR on HIV-1 replication and virion infectivity.

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses ...

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

Summary

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Chapters in this video

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

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

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

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

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

Humanized NOD/SCID/IL2rγ^null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication