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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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). ...

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