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 viv....

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....

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

List of materials used in this article

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