Name: detection of human immunodeficiency virus type 1 (hiv-1) antisense protein (asp) rna transcripts in patients by strand-specific rt-pcr
Uploaded: 2019-11-27T14:00:00
Description: Watch this Scientific Journal Video about Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR at JoVE.com

We thank Patrizia Amelio, Alessandra Noto, Craig Fenwick, and Matthieu Perreau for always being available to discuss our work and all the people in the Laboratory of AIDS Immunopathogenesis for their precious technical assistance. We also would like to thank John and Aaron Weddle from VSB Associated Inc. who contributed with excellent artwork. Finally, many special thanks to all the Patients, without whom this work would not have been possible. This work received no specific grant from any funding agency.

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-1HXB2 strain
<ol>
	<li>Day 1: PBMCs STIMULATION
	<ol>
		<li>Isolate PBMCs from a healthy donor buffy coat.</li>
		<li>Count and resuspend PBMCs at a concentration of 1x106/mL in complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FB...

<ol>
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S., Birch, K. E., Deacon, N. J., Mosse, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+control+of+human+immunodeficiency+virus+type+1+asp+expression+by+alternative+splicing+in+the+upstream+untranslated+region.">Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.</a> DNA Cell Biol. 31 (7), 1303-1313 (2012).</li><li>Laverdure, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Antisense+Transcription+Is+Preferentially+Activated+in+Primary+Monocyte-Derived+Cells.">HIV-1 Antisense Transcription Is Preferentially Activated in Primary Monocyte-Derived Cells.</a> Journal of Virology. 86 (24), 13785-13789 (2012).</li><li>Zapata, J. C., 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=The+Human+Immunodeficiency+Virus+1+ASP+RNA+promotes+viral+latency+by+recruiting+the+Polycomb+Repressor+Complex+2+and+promoting+nucleosome+assembly.">The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly.</a> Virology. 506, 34-44 (2017).</li><li>Haist, K., Ziegler, C., Botten, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strand-Specific+Quantitative+Reverse+Transcription-Polymerase+Chain+Reaction+Assay+for+Measurement+of+Arenavirus+Genomic+and+Antigenomic+RNAs.">Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs.</a> PLoS One. 10 (5), 0120043 (2015).</li><li>Lerat, H., 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=Specific+detection+of+hepatitis+C+virus+minus+strand+RNA+in+hematopoietic+cells.">Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.</a> The Journal of Clinical Investigation. 97 (3), 845-851 (1996).</li><li>Tuiskunen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-priming+of+reverse+transcriptase+impairs+strand-specific+detection+of+dengue+virus+RNA.">Self-priming of reverse transcriptase impairs strand-specific detection of dengue virus RNA.</a> J Gen Virol. 91, 1019-1027 (2010).</li><li>Mancarella, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+antisense+protein+(ASP)+RNA+transcripts+in+individuals+infected+with+human+immunodeficiency+virus+type+1+(HIV-1).">Detection of antisense protein (ASP) RNA transcripts in individuals infected with human immunodeficiency virus type 1 (HIV-1).</a> Journal of General Virology. , (2019).</li><li>Peyrefitte, C. N., Pastorino, B., Bessaud, M., Tolou, H. J., Couissinier-Paris, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+in+vitro+falsely-primed+cDNAs+that+prevent+specific+detection+of+virus+negative+strand+RNAs+in+dengue-infected+cells:+improvement+by+tagged+RT-PCR.">Evidence for in vitro falsely-primed cDNAs that prevent specific detection of virus negative strand RNAs in dengue-infected cells: improvement by tagged RT-PCR.</a> J Virol Methods. 113 (1), 19-28 (2003).</li><li>Boncristiani, H. F., Di Prisco, G., Pettis, J. S., Hamilton, M., Chen, Y. 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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&#39; end with sequences not related to HIV has pr...

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 translated2,3,4,5,6,7,8,9. 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-111,12. 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 &#176;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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD LSR II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BigDye Terminator v1.1 Cycle Sequencing Kit</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4337450</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set (100 mM)</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>10297018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 Streptavidin</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>11205D</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human CD4+ T Cell Isolation Kit</td>
			<td>Stemcell Technologies</td>
			<td>19052</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>S1010-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Solution Kit</td>
			<td>Becton Dickinson</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>HIV Gag p24 flow cytometry antibody - Kc57-FITC</td>
			<td>Beckman Coulter</td>
			<td>6604665</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-2</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-743</td>
			<td></td>
		</tr>
		<tr>
			<td>Lectin from Phaseolus vulgaris (PHA)</td>
			<td>Sigma-Aldrich</td>
			<td>61764-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Yellow Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>L34967</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human CD28</td>
			<td>Becton Dickinson</td>
			<td>55725</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human CD3</td>
			<td>Becton Dickinson</td>
			<td>55329</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and Probes</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>BioConcept</td>
			<td>4-01F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Taq DNA Polymerase High Fidelity</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 Medium</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR System</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4376600</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr>
			<td>TOPO TA Cloning Kit for Subcloning, with One Shot TOP10 chemically competent E. coli cells</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>K450001</td>
			<td></td>
		</tr>
		<tr>
			<td>TURBO DNase (2 U/&#181;L)</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>Veriti Thermal Cycler</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4375786</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of human immunodeficiency virus type 1 (hiv-1) antisense protein (asp) rna transcripts in patients by strand-specific rt-pcr

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&#39; 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.

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 RNAs10,14,15,16,17. In order to prevent this phen...

Watch this Scientific Journal Video about Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR at JoVE.com

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

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.

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

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...

Conventional RT-PCRs do not allow for discrimination between the sense and anti-sense sequences when used to detect RNAs encoded by overlapping genes. This protocol makes it possible to detect specifically anti-sense RNAs. The use of a biotynylated primers along with high RNA denaturation temperatures allows for amplification of anti-sense cDNAs, preventing the formation of non-specific RT products.We have used this method to demonstrate that ASP either as an RNA anti-sense or a protein precursor, it's a new HIV antigen, does it potentially a novel HIV target for therapy. This method can be applied to any system, in which anti-sense RNAs from overlapping genes can be detected. Start by designing patient specific primers using the pro-viral DNA sequence internal to the PanASP primers.Next, quantify the RNA and eliminate DNA contamination by treating samples with DNase. Transfer between 0.1 and one microgram of total RNA into the appropriate wells on a 96 well PCR plate. Then set up the reverse transcription, or RT reaction, according to manuscript directions.Prepare the endogenous RT controls by adding five microliters of nuclease free water instead of the ASP reverse primer. Put the plate into the thermocycler and heat the RNA to 94 degrees Celsius for five minutes. Then immediately cool it in iced water for at least one minute.Prepare the reaction mixture in a 1.5 milliliter tube as described in the text manuscript, then transfer 17.5 microliters to each of the RNA containing wells. RNA denaturation must be performed at 94 degrees Centigrade. Without total denaturation, syntheses of non-specific products from sense RNA may occur, leading to amplification of non-specific cDNAs.Mix the contents of the wells gently and incubate the plate at 55 degrees Celsius for 60 minutes. Then, inactivate the reactions by heating the plate to 70 degrees Celsius for 15 minutes. Prepare one liter of 2X washing and binding buffer according to manuscript directions and filter the solution.Next, prepare 50 milliliters of 1X washing and binding buffer using PCR grade water. Transfer the appropriate number of streptavidin-conjugated magnetic beads to a 1.5 milliliter tube then wash them by adding an equal volume of 2X washing and binding buffer and vortexing the tube for 15 seconds. Place the tube into a magnetic separation rack and incubate it for three minutes at room temperature.Then, carefully remove the supernatant without disturbing the beads and add the same volume of the 2X washing and binding buffer as the initial volume of the beads. Vortex the beads for 30 seconds and transfer 10 microliters of the suspension to an appropriate number of 1.5 milliliter tubes. Place the tubes in the magnetic separation rack and incubate them for three minutes at room temperature.Then, discard the supernatant and add 50 microliters of 2X washing and binding buffer. Add 50 microliters of biotynylated cDNA to the corresponding tubes with the beads and incubate them for 30 minutes while gently rotating. After the incubation, place the tubes on the magnetic separation rack for three minutes then wash the beads with 200 microliters of 1X washing and binding buffer, followed by a three minute incubation on the separation rack.Discard the supernatant and repeat the wash twice. Then, resuspend the beads in 10 microliters of PCR grade water. Prepare an appropriate volume of PCR Master Mix according to the manuscript directions.Mix it well and quickly spin it down, then transfer 49 microliters per well to a 96 well PCR plate. Carefully vortex the tubes with the purified cDNA for 15 seconds and add one microliter of the suspension to the corresponding wells. Run the PCR and separate the products on a 1%agarose gel.Cut the bands from the gel and clone the amplified products into a PCR 2.1 plasmid. Then sequence the clones and analyze each sequence. The initial RT reactions were performed with the regular non-biotynylated anti-sense primer and resulted in successful amplification of ASP.However, a band of the same molecular weight and primer minus controls was also amplified. To bypass this problem, the specific anti-sense primer was labeled with biotyn and the resulting anti-sense cDNA was purified prior to PCR. This made it possible to amplify the ASP sequence with greatly reduced contamination from the non-specific cDNA in the primer minus controls.Further optimization of this method was achieved by complete denaturation of RNA at 94 degrees Celsius prior to RT, followed by immediate cooling on ice water, which resulted in effective amplification of the ASP band and no non-specific products. The kinetics of ASP RNA were measured and CD4 cells isolated from three HIV positive subjects with detectable viremia and an absence of therapy following stimulation with anti-CD3/CD28. Quantification of ASP RNA was also possible in patients undergoing ART, with non-detectable viremia.In two out of three patients, ASP RNA was detected in low levels at three to five days post stimulation. Two patients were analyzed for ASP and env RNAs, one untreated and one treated. For the untreated patient, both RNAs could be detected.For the treated patient, ASP and env were barely detectable after several days of stimulation. When attempting this procedure, it is important to remember that denaturation linearizes RNA so that secondary structures able to prime RT are destroyed. Whereas, washing of the beads flushes away non-biotynylated cDNAs.After purification of the cDNAs with the correct polarity, qPCR can be performed to analyze gene expression. Moreover, cDNAs can also be cloned and used for sequence analysis. This method allows studying genes overlapping in opposite orientation and may be useful in elucidating regulation and pathogenesis of a bacteria and a virus-related diseases including some types of cancer.

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