Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

Summary

RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP.

Abstract

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3' end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

Introduction

The antisense protein (ASP) gene is an open reading frame (ORF) located on the negative strand of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene, spanning the junction gp120/gp41¹. Over the past 30 years, several reports have shown that the HIV ASP gene is indeed transcribed and translated^{2,3,4,5,6,7,8,9}. Although ASP antisense transcripts have been fully characterized in vitro, until recently information about the actual production of ASP RNA in patients was still missing.

The sequence of ASP is reverse and complementary to env. This represents a major obstacle when trying to detect transcripts for ASP. Standard reverse transcription-polymerase chain reaction (RT-PCR) methods use gene-specific antisense primers to synthesize complementary DNAs (cDNAs) of the right polarity. This approach, however, does not allow to determine the orientation (sense or antisense) of the initial RNA template, since RNA hairpins or loops can prime RT in both directions in absence of primers¹⁰, a phenomenon known as RT self-priming. Most ASP investigators sidestep the problem of RT self-priming using primers tagged with sequences that are not related to HIV-1^11,12. This strategy, however, does not eliminate the occurrence of the phenomenon, and may lead to potential carry-over of non-specific cDNAs into the PCR¹¹.

We have recently developed a novel strand-specific RT-PCR assay for the study of antisense RNA and we have used it for ASP RNA detection in a cohort of six HIV-infected patients, as shown in Table 1. The procedure described below has been previously published by Antonio Mancarella et al.¹³. In our protocol, we avoid the production of non-specific cDNAs by a dual approach. Firstly, we eliminate RNA secondary structures by denaturing RNA at high temperature (94 °C), and secondly, we reverse transcribe ASP RNA using a biotinylated ASP-specific primer and affinity-purify the resulting cDNA. By this approach, we are able to amplify only our target cDNA, since other non-specific RT products are either prevented from being generated (high temperature denaturation of RNA) or eliminated prior to PCR (affinity purification).

Protocol

This study was approved by the Institutional Review Board of the Centre Hospitalier Universitaire Vaudois (CHUV).

1. Infection of peripheral blood mononuclear cells (PBMCs) with HIV-1_HXB2 strain

1. Day 1: PBMCs STIMULATION
   1. Isolate PBMCs from a healthy donor buffy coat.
   2. Count and resuspend PBMCs at a concentration of 1x10⁶/mL in complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (R-10), with addition of 50 U/mL of interleukin-2 (IL-2) and 3 µg/mL phytohaemagglutinin (PHA).
   3. Pipet up and down and split the cells in two six-well plates (3 mL of cell suspension/well).
   4. Incubate for 3 days at 37 °C and 5% CO₂.
2. Day 3: PBMCs INFECTION
   NOTE: Use one of the two plates as in step 1.1.3 as negative control (do not infect these cells).
   CAUTION: Perform the entire procedure in a biosafety level III laboratory (P3).
   1. Pool PBMCs from one plate into a 50 mL falcon tube and centrifuge at 300 x g for 10 min.
   2. Discard the supernatant and resuspend the cells in 2.2 mL of R-10 containing 20 U/mL of IL-2 and 2 µg/mL polybrene.
   3. Thaw vials containing the HIV-1_HXB2 virus and add 1.8 mL of viral suspension (0.376 ng/µL) to the cells.
   4. Incubate the cells for 2 h at 37 °C, gently swirling the tube every 30 min.
   5. Centrifuge the cells at 300 x g for 10 min.
   6. Discard the supernatant and resuspend the cells at 1x10⁶/mL in R-10 containing IL-2 (50 U/mL).
   7. Transfer the cell suspension to a 24-well plate at a concentration of 1 mL/well and incubate for 5 days at 37 °C and 5% CO₂.
   8. Harvest 1 well each day and transfer the cells to 1.5 mL tube (labeled with the day of infection).
      NOTE: Before centrifugation, transfer 150 µL of cell suspension to a flow cytometry tube for PBMCs infection quality control (see step 1.3)
   9. Centrifuge the tubes at 400 x g for 10 min and discard the supernatant.
   10. Freeze the cell pellets at -80 °C until use.
3. PBMCs infection: quality control
   1. For each day of infection, collect 150 µL of infected PBMCs and transfer them into a flow cytometry tube.
   2. Add 1 mL of phosphate-buffered saline (PBS) and centrifuge at 400 x g for 5 min.
   3. Discard the supernatant and add 50 µL of PBS containing 1 µL of Aqua live/dead dye (previously diluted 1:10 with PBS).
   4. Incubate at 4 °C for 15 min.
   5. Add 1 mL of PBS, centrifuge at 400 x g for 5 min, and discard supernatant.
   6. Add 250 µL of Fixation/Permeabilization solution and incubate for 20 min at room temperature in the dark.
   7. Add 1 mL of 1x Perm/Wash Buffer (10x stock solution containing both FBS and saponin diluted 1:10) and centrifuge at 400 x g for 5 min.
   8. Discard the supernatant and add 50 µL of PBS containing HIV Gag p24 fluorescein isothiocyanate (FITC)-conjugated antibody (diluted 1:10).
   9. Incubate for 20 min at room temperature in the dark.
   10. Add 1 mL of PBS, centrifuge at 400 x g for 5 min, and discard the supernatant.
   11. Add 150 µL of x CellFIX (10x stock solution containing 10% formaldehyde, 3.55% methanol, 0.93% sodium azide diluted 1:10) and analyze cells by flow cytometry.

2. Stimulation of human CD4+ T cells with anti-CD3/CD28 antibodies

1. Isolate CD4+ T cells from PBMCs of both HIV-1 infected patients and healthy donors.
2. Prepare human anti-CD3/CD28 antibody mix by diluting anti-CD3 (1:100) and anti-CD28 (1:50) in PBS.
3. Coat a 48-well plate by adding 200 µL of antibody mix per well to an appropriate number of wells and incubate at 37 °C for 2 h.
4. Aspirate the antibody solution and add 1 mL of CD4+T cells at 1x10⁶/mL to the anti-CD3/CD28 antibody-coated wells.
   NOTE: Stimulate CD4+ T cells up to 5 days.

3. Reverse transcription

NOTE: To obtain patient-specific primers, proviral DNA isolated from CD4+T cells from each patient was amplified using HIV-1_HXB2 Pan ASP primers. Patient specific primers were designed using the proviral sequence internal to the Pan ASP primers. All primers and probes used in this study are listed in Table 2.

1. Isolating total RNA from cells
   1. Quantify RNA and eliminate DNA contamination by treating samples with DNase.
   2. Perform RT reactions in a volume of 50 µL. Transfer 0.1-1 µg of total RNA into an appropriate number of wells of a 96-well PCR plate. Prepare the RNA mixture by adding the following components to the RNA:
      5 µL of biotinylated ASP reverse primer (20 µM)
      2.5 µL of deoxynucleotide triphosphates (dNTPs) (10 mM)
      25 µL of diethylpyrocarbonate (DEPC)-treated distilled water (dH₂O).
      Prepare the endogenous RT controls by adding 5 µL of nuclease-free water in place of the biotinylated ASP reverse primer.
   3. Place the 96 well PCR plate into a thermocycler and heat the RNA mixture to 94 °C for 5 min.
   4. Immediately cool down the RNA mixture in iced water for at least 1 min.
   5. Prepare the reaction mixture by adding the following components to a 1.5 mL tube (calculate the total number of samples plus 1):
      10 µL of 5x RT buffer
      2.5 µL of 0.1 M of 1,4-dithiothreitol (DTT)
      2.5 µL of RNase out (40 units/µL)
      2.5 µL of RT enzyme (200 units/µL)
   6. Transfer 17.5 µL of this mixture to each of the RNA-containing wells. Prepare the RT-minus controls replacing reverse transcriptase with 2.5 µL of nuclease-free water.
   7. Mix gently and incubate the plate at 55 °C for 60 min using a thermocycler.
   8. Inactivate the reactions by heating at 70 °C for 15 min.

4. Affinity purification of ASP biotinylated cDNA

NOTE: Do not purify RT-minus reactions.

1. Prepare 1 L of 2x washing/binding buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA) and 2 M NaCl. Filter the solution.
   1. Prepare 50 mL of 1x washing/binding buffer using PCR grade water.
   2. For each reaction, use 10 µL of streptavidin-conjugated magnetic beads. Based on the number of reactions, transfer an appropriate volume of beads to a 1.5 mL tube.
   3. Wash the beads by adding an equal volume of 2x washing/binding buffer and vortex for 15 s.
   4. Place the tube into a magnetic separation rack and incubate for 3 min at room temperature.
   5. Carefully remove the supernatant without disturbing the beads and add the same volume of washing/binding 2x buffer as the initial volume of beads.
   6. Vortex for 30 s and transfer 10 µL of the beads suspension to an appropriate number of 1.5 mL tubes.
   7. Place the tubes in a magnetic separation rack and incubate for 3 min at room temperature.
   8. Discard the supernatant and add 50 µL of washing/binding 2x buffer.
   9. Add 50 µL of biotinylated cDNA to the corresponding tubes containing the beads.
   10. Incubate for 30 min at room temperature rotating gently.
   11. Separate the beads from the supernatant by a magnet separation rack for 3 min at room temperature.
   12. Wash the beads by adding 200 µL of 1x washing/binding buffer. Place the tubes in a magnet separation rack for 3 min at room temperature and discard the supernatant. Repeat twice.
   13. Resuspend the beads in 10 µL of PCR grade water.

5. Standard PCR

NOTE: The aim of the standard PCR is to amplify the entire ORF of the ASP gene. The amplification products are then cloned into pCR2.1 plasmid to develop standard curves for ASP RNA quantification by real-time PCR (see paragraph real time PCR).

1. Perform standard PCRs in a total volume of 50 µL. Prepare an appropriate volume of PCR master mix (number of samples plus 1). For each sample, add the following components to a 1.5 mL tube:
   1 µL of 10 µM ASP primers (forward and reverse)
   1 µL of 10 mM dNTPs
   Magnesium chloride (MgCl₂) (concentration depends on the primers used)
   dH₂O to a final volume of 49 µL
   1. Mix well, quickly spin down contents and transfer 49 µL/well of the PCR master mix to a 96-well PCR plate.
   2. Carefully vortex the tubes containing the beads used for ASP cDNA affinity purification for 15 s.
   3. Add 1 µL of beads into the corresponding wells and run the PCR using the following program: 95 °C for 2 min, 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 40 s, then 68 °C for 7 min.
   4. Separate the PCR products onto 1 % agarose gel, cut the bands and clone the amplified products into pCR2.1 plasmid.
   5. Sequence and analyze the sequences of each clone.

6. Real time quantitative PCR (qPCR)

NOTE: Develop patient-specific primers and probes using the approach described in step 3 "Reverse Transcription". For qPCR include ASP plasmid dilutions for standard curves. Plasmids containing patient-specific inserts are developed as mentioned in the note at the beginning of step 5 "Standard PCR".

1. Prepare the standard curve using 1:10 ASP plasmid serial dilutions starting from 3 x 10⁶ copies/µL down to 3 copies/µL.
   1. First round PCR (pre-amp). Perform reactions in a total volume of 25 µL. Prepare an appropriate volume of PCR master mix (number of samples plus 1). For each sample, add the following components to a 1.5 mL tube:
      0.5 µL of ASP or env primer mix (final concentration 0.2 µM each) (forward and reverse)
      0.5 µL of dNTPs mix (final concentration 0.2 mM each)
      MgCl₂ (concentration depends of the primer pairs)
      dH₂O to a final volume of 24 µL
   2. Transfer 24 µL of PCR master mix to an appropriate number of wells of a 96-well PCR plate.
   3. Carefully vortex the tubes containing the beads with the cDNA for 15 s.
   4. Add 1 µL of beads to the corresponding wells. Do the same for plasmid dilutions.
   5. Run the PCR using the following algorithm: denaturation for 2 min at 95 °C ; 30 s at 95 °C, 30 s at 55 °C, 40 s at 68 °C for 18 cycles; extension at 68 °C for 7 min.
   6. Dilute the first round PCRs reactions 1:5 with PCR grade water.
   7. Second round qPCR. Perform reactions in a total volume of 20 µL. Prepare an appropriate volume of PCR master mix (number of samples plus 1). For each sample, add the following components to a 1.5 mL tube:
      1.8 µL 10 µM primers (forward and reverse)
      1 µL of 5 µM probe
      6.2 µL of dH₂O
   8. Transfer 19 µL of qPCR master mix into an appropriate number of wells of a 96-well qPCR plate and add 1 µL of the diluted first round PCR reactions to the corresponding wells. Do the same for the plasmid dilutions.
   9. Run the qPCR with the following algorithm: denaturation for 10 min at 95 °C; 15 s at 95 °C, 1 min at 60 °C for 40 cycles.

Results

High temperature RNA denaturation coupled to affinity purification of biotinylated cDNA prevents amplification of non-specific ASP products during PCR in PBMCs infected in vitro and in CD4+ T cells isolated from patients. RT self-priming has been shown to occur during reverse transcription of antisense RNAs^{10,14,15,16,17}. In order to prevent this phen...

Discussion

In this report we describe a strand-specific RT assay applied to the detection of ASP RNA in CD4+ T cells isolated from individuals infected with HIV-1. The occurrence of non-specific priming during RT hampers the detection of RNA transcripts with the right polarity, leading to misinterpretation of the results. Previous groups have developed several strategies aimed at preventing primer-independent cDNA synthesis during the RT reaction. Tagging the reverse primer at the 3' end with sequences not related to HIV has pr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD LSR II	Becton Dickinson
BigDye Terminator v1.1 Cycle Sequencing Kit	Applied Biosystem, Thermo Fisher Scientific	4337450
dNTP Set (100 mM)	Invitrogen, Thermo Fisher Scientific	10297018
Dynabeads M-280 Streptavidin	Invitrogen, Thermo Fisher Scientific	11205D
EasySep Human CD4+ T Cell Isolation Kit	Stemcell Technologies	19052
Fetal Bovine Serum	Biowest	S1010-500
Fixation/Permeabilization Solution Kit	Becton Dickinson	554714
HIV Gag p24 flow cytometry antibody - Kc57-FITC	Beckman Coulter	6604665
Human IL-2	Miltenyi Biotec	130-097-743
Lectin from Phaseolus vulgaris (PHA)	Sigma-Aldrich	61764-1MG
LIVE/DEAD Fixable Yellow Dead Cell Stain Kit, for 405 nm excitation	Invitrogen, Thermo Fisher Scientific	L34967
Mouse Anti-Human CD28	Becton Dickinson	55725
Mouse Anti-Human CD3	Becton Dickinson	55329
Primers and Probes	Integrated DNA Technologies (IDT)
Penicillin-Streptomycin	BioConcept	4-01F00-H
Platinum Taq DNA Polymerase High Fidelity	Invitrogen, Thermo Fisher Scientific	11304011
Polybrene Infection / Transfection Reagent	Sigma-Aldrich	TR-1003-G
RNeasy Mini Kit	Qiagen	74104
Roswell Park Memorial Institute (RPMI) 1640 Medium	Gibco, Thermo Fisher Scientific	11875093
StepOnePlus Real-Time PCR System	Applied Biosystem, Thermo Fisher Scientific	4376600
SuperScript III Reverse Transcriptase	Invitrogen, Thermo Fisher Scientific	18080044
TaqMan Gene Expression Master Mix	Applied Biosystem, Thermo Fisher Scientific	4369016
TOPO TA Cloning Kit for Subcloning, with One Shot TOP10 chemically competent E. coli cells	Invitrogen, Thermo Fisher Scientific	K450001
TURBO DNase (2 U/µL)	Invitrogen, Thermo Fisher Scientific	AM2238
Veriti Thermal Cycler	Applied Biosystem, Thermo Fisher Scientific	4375786