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

Summary

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.

Abstract

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 "shock and kill" 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.

Introduction

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' 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 latency^2,3. 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 cells^4,5,6,7,8. 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'LTR and activation of HIV expression^{9,10,11,12,13}. 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 vivo^14,15. 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.RevΔ38/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 (DsRed) 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' 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, RevΔ38-DsRed. Briefly, DsRed was inserted into the 2^nd 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-Rev^NL4.3) 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.

Protocol

NOTE: Procedures for cloning, transformation and sequencing are discussed elsewhere^18,19. The protocols herein begin from the transfection of the mammalian expression vectors (Figure 3).

1. Transfection of HEK293T Cells with Dual Color Reporter Construct

1. Cultivate HEK293T cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL) in a 5% CO₂ incubator at 37 °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.
2. 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 °C.
3. The day before transfection, remove the medium from the flask by aspiration and wash the cells with Dulbecco’s phosphate buffered saline (DPBS) solution free of calcium (Ca²⁺) and magnesium (Mg²⁺) to remove traces of serum.
4. Dispense 5 mL of trypsin-EDTA solution (0.05% - 10 mM in PBS) into the culture vessel and place it at 37 °C in a 5% CO₂ incubator for 2 to 5 min.
5. 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.
6. Resuspend gently by pipetting the cell suspension up and down to break up the clumps.
7. Dilute the cells 1:10 in trypan blue stain (0.4%).
8. Count the live cells that do not take up trypan blue using a microscope (10x objective) and a haemocytometer.
9. 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.
10. Plate 2 x 10⁴ cells in 100 μL of DMEM medium supplemented with 10% FBS without antibiotics in a 96-well flat-bottom plate for 24 h.
11. For each well, dilute 400 ng of pLTR.gp140/EGFP.RevD38/DsRed, 20 ng of pCMV-Rev^NL4.3 and 100 ng of pCMV-Tat101^AD8-Flag DNA in 50 μL 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.
12. Mix the lipid reagent gently before use, then dilute 0.65 μL in 50 μL of serum free medium. Mix gently and incubate for 5 min at room temperature.
13. Combine the diluted DNA with the diluted lipid reagent.
14. Mix gently and incubate for 20 min at room temperature. The solution may appear cloudy.
    NOTE: Proceed to step 1.15. within 30 min.
15. Dispense 100 μL per well of the lipid reagent-DNA complexes drop-wise on top of the cells.
16. Mix gently by rocking the plate back and forth.
17. Incubate the plate for 5 h in a 5% CO₂ humidified incubator at 37 °C.
    1. 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.
    2. Transfect additional wells with 100 ng of CMV-driven EGFP and DsRed-Express DNA plasmid for compensation purposes.

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.

1. Dilute each LRA to the appropriate working concentration with growth medium (e.g., 1 μM for JQ1(+)).
   CAUTION: Reconstitute the lyophilized drugs with the appropriate solvent to a concentration of 10 mM. Store all stock LRAs at -80 °C in a single use aliquot (5 μL each) in order to avoid repeated freezing-thawing cycles.
2. Carefully aspirate transfection medium with a multichannel and replace with 100 μL 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.
3. Incubate the plate for 48 h in a 5% CO₂ humidified incubator at 37 °C.

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.

1. Detach the cells into the media using 100 μL 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 µL per well of trypsin-EDTA solution (0.05% - 10 mM in PBS) and incubating 5 min at 37 °C, before neutralizing with medium containing serum.
2. Transfer the cells to a 96-well V-bottom plate.
3. Spin the cells for 5 min at 500 x g at 4 °C, then carefully aspirate the medium/PBS without touching the cells.
4. Wash the cells at least 1 time with 200 μL of protein/serum free PBS.
5. Centrifuge at 500 x g for 5 min at 4 °C, then discard the supernatant by tilting the plate and removing the wash buffer without touching the cells.
6. Prepare a working solution of fixable viability dye by diluting the viability dye 1:1000 in protein/serum free PBS. Prepare 50 µL 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 μL anhydrous DMSO (component B). Store at -20 °C in a single use aliquot (1 μL each), protected from the light.
7. Add 50 μL of the diluted viability dye to each well and mix the cells by pipetting up and down with a multichannel.
8. Stain for 10-15 min at 4 °C, protected from the light.
9. Wash 1-2 times with 150 μL of wash buffer (PBS with 1% bovine serum albumin and 2 mM EDTA).
10. Centrifuge at 500 x g for 5 min at 4 °C. Discard the supernatant.
11. Fix the cells with 100 μL of freshly prepared 1% formaldehyde in wash buffer for 10-15 min at 4 °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.
12. Wash the cells 1-2 times with 100 μL of wash buffer.
13. Centrifuge at 500 x g for 5 min at 4 °C. Discard the supernatant.
14. Resuspend the cell pellet in 70 μL of wash buffer.
    NOTE: The protocol can be paused here. Fixed cells could be stored at 4 °C in the dark for analysis the next day on the flow cytometer.

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 μm cell-strainer or 100 μm nylon mesh to avoid clogging up the nozzle.

1. Start up the cytometer and computer at least 10 min prior to use to ensure lasers warm up.
2. 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.
3. Check the flow cell for air bubbles.
4. 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.
5. Check the cytometer performance and sensitivity across detectors by running calibration beads.
6. 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.
7. 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.
8. Create plots and set the gates using fluorescence minus one (FMO) controls.
9. 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.
10. At the end of the run, clean the flow cytometry fluidics with concentrated cleaning agents then water for 5 min each.
11. Shutdown the system, discard the waste and re-fill the sheath tank after acquisition.
12. 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).
13. 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).

Results

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...

Discussion

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'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...

Materials

Name	Company	Catalog Number	Comments
Cell culture
HEK293T cells (Human Embryonic Kidney cells)	ATCC	CRL-3216	Replicates vectors carrying the SV40 region of replication.
Dulbecco's Modified Eagle's Medium (DMEM 1x + GlutaMAX-I)	Gibco	10569-010	+ 4.5 g/L D-Glucose + 110 mg/L Sodium Pyruvate
Fetal Bovine serum	Gibco	10099-141	Origin Australia
Penicillin-Streptomycin	Sigma	P4458
Dulbecco's phosphate buffered saline (DPBS), no calcium, no magnesium	Gibco	14190-136
Trypan blue Stain, 0.4%	Gibco	15250
Trypsin-EDTA (0.05%), phenol red	Gibco	25300054
Lipofectamine 2000	Invitrogen	11668-019	Lipid transfection reagent
Opti-MEM I (1x) reduced serum medium	Gibco	31985-070	Serum free medium
NucleoBond Xtra Maxi	Marcherey-Nagel	740414.50
pEGFP-N1 plasmid	Clontech (TaKaRa)	6085-1	Expression of EGFP in mammalian cells, CMVIE promoter.
pDsRed-Express-N1	Clontech (TaKaRa)	632429	Expression of DsRed-Express in mammalian cells, CMVIE promoter.
pLTR.gp140/EGFP.RevD38/DsRed	Addgene	115775
pCMV-RevNL4.3	Addgene	115776
pCMV-Tat101AD8-Flag	Addgene	115777
Dimethyl sulfoxide (DMSO)	Millipore	67-68-5
JQ1(+)	Cayman Chemical	11187	Stock at 10 mM in DMSO; working concentration 1 μM
JQ1(-)	Cayman Chemical	11232	Stock at 10 mM in DMSO; working concentration 1 μM
Phorbol Myristate Acetate (PMA)	Sigma-Aldrich	16561-29-8	Stock at 100 μg/mL in DMSO; working concentration 10 nM
Phytohaemagglutinin (PHA)	Remel	HA15/R30852701	Stock at 1 μg/μL in PBS; working concentration 10 μg/mL
Vorinostat (VOR)	Cayman Chemical	10009929	Stock at 10 mM in DMSO; working concentration 0.5 μM
Panobinostat (PAN)	TRC	P180500	Stock at 10 mM in DMSO; working concentration 30 nM
CellTiter 96 AQueous One Solution Cell Proliferation Assay	Promega	63581
Venor GeM Classic	Minerva Biolabs	11-1100	Mycoplasma Detection Kit, PCR-based
Name	Company	Catalog Number	Comments
Flow cytometry reagents
LSR Fortessa	BD Biosciences		Flow cytometer (4 lasers-blue, red, violet and yellow)
LSR II	BD Biosciences		Flow cytometer (3 lasers-blue, red and violet)
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit	Life Technologies	L34976	Viability dye: for 633 or 635 nm excitation, 400 assays. Component A and B are both provided in the kit.
Bovine Serum Albumin	Sigma	A2153
EDTA 0.5M pH8	Gibco	15575-038
Formaldehyde Solution 37/10 (37%)	Chem-Supply	FA010
BD FACS Diva CS&T Research Beads	BD Biosciences	655050	Calibration beads
Sphero Rainbow Calibration Particles (8 peaks)	BD Biosciences	559123	3.0 - 3.4 mm
Sheath solution	Chem-Supply	SA046	90 g NaCl in 10 L water
HAZ-Tabs	Guest Medical	H8801	Chlorine release tablets for disinfection
Decon 90	Decon Laboratories Limited	N/A	Concentrated cleaning agents of flow cytometer. Working solution Decon 90 5%.
Sodium Hypochlorite (12-13% Solution)	Labco	SODHYPO-5L	Concentrated cleaning agents of flow cytometer. Working solution bleach 1%.
7x	MPBio	IM76670	Concentrated cleaning agents of flow cytometer. Working solution 7x 1%.
Name	Company	Catalog Number	Comments
Materials
Tissue culture flasks (75 cm2, canted neck, cap vented)	Corning	430641U
Tissue culture plates (96 well flat bottom with lid)	Costar	3599
Tissue culture plates (96 well V-bottom without lid)	Costar	3896
Centrifuge tubes (10 mL)	SARSTEDT	62.9924.284	100x16 mm
Centrifuge tubes (50 mL)	CellStar	227261	30x115 mm
Microcentrifuge tubes (1.5 mL)	Corning Axygen	MCT-150-C
Serological Pipette (25 mL), sterile	Corning	CLS4489-200EA
Serological Pipette (10 mL), sterile	Corning	CLS4488-200EA
Serological Pipette (5 mL), sterile	Corning	CLS4487-200EA
Reagent reservoirs (50 mL), sterile	Corning	CLS4470-200EA
5 mL Round-Bottom polystyrene test tube, with cell-strainer cap	Corning	352235	12 x 75 mm style, 70 mm
Nylon Mesh	SEFAR	03-100/32	100 mm
Titertube Micro test tubes, bulk	BIO-RAD	2239391	microfacs tubes
5 mL Round-Bottom polystyrene test tube, without cap	Corning	352008	12x75 mm style
Snap Caps for 12x75 mm Test Tubes	Corning	352032
Counting chamber, Neubauer improved double net ruling, bright-line (Haemocytometer, LO-Laboroptik)	ProSciTech	SVZ4NIOU	3x3 large squares of 1 mm2; Depth 0.100 mm; volume 0.1 mL; area minimum 0.0025 mm2
Coverslips (Menzel-Gläser)	Grale Scientific	HCS2026	20 x 26 mm
Microscope	Nikon TMS	310528
Centrifuge 5810R refrigerated	Eppendorf	5811000487	with rotor A-4-81 including adapters for 15/50 mL conical tubes
FLUOstar Omega microplate reader	BMG Labtech	N/A	Plate reader for cell proliferation assay. Filter 490 nm.
Name	Company	Catalog Number	Comments
Softwares
FACS Diva	BD Biosciences		Flow cytometer data acquisition and analysis program, version 8.0.1
FlowJo	FlowJo	FlowJo 10.4.2	Flow cytometer data analysis program, FlowJo Engine v3.05481
Omega	BMG Labtech		FLUOstar multi-user reader control, version 5.11
Omega - Data Analysis	BMG Labtech	MARS	FLUOstar data analysis, version 3.20R2
Microsoft Excel	Microsoft	Excel:mac 2011	version 14.0.0
Prism	GraphPad	Prism 7	version 7.0c