The work was supported in part by Public Health Service grant 1R01AI081568 from NIAID to Y. W. and by the generous donations of the donors and riders of the 2009 NYCDC AIDS Ride organized by M. Rosen and Day2 Inc.

1. Transfection of Plasmids for Production of the Rev-dependent Lentiviral Particles
Set up: The Rev-dependent GFP lentiviral vector, pNL-GFP-RRE-SA, was described previously4,5,6. To assemble into a viral particle, the plasmid was contransfected into HEK293 T cells with an HIV-1 packaging construct, pCMV&Delta;8.2 (kindly provided by Dr. Dider Trono), and a plasmid carrying the VSV-G glycoprotein (pHCMV-G). The transfection was carried out using the calcium phosphate method.
Preparation of buffers: 10 X HBS (Hepes Buffered Saline) was prepared by dissolving 5 g of Hepes, 8 g of NaCl, 0.37 g of KCl, 1 g of Dextrose, 0.103 g of Na2HPO4 (anhydrous) in 100 mL of H2O. Aliquot the10 X HBS buffer into 1 mL aliquots and store at -20&deg;C. At the time of transfection, convert the 10 X HBS to 2 X HBS with H2O (1: 5 dilution), and adjust the pH to between 7.05 - 7.12. (for 10 mL 2 X HBS, it usually requires approximately 50 &mu;L of 1M NaOH). Filter sterilize the 2X HBS buffer by passing through a 0.22 &mu;M Millipore filter. CaCl2 buffer was made by dissolving CaCl2 in 10 mM Hepes to a final concentration 2M. Adjust pH to 5.8, filter sterilize the buffer and store at 4&deg;C.
Procedure:
<ol>
	<li>One day prior to transfection, seed 2 x 106 HEK293T cells in a 100 mm Petri-dish, and grow cells overnight in 10 mL DMEM + 10% FBS at 37&deg;C, 5% CO2.</li>
	<li>The next day, remove supernatant from cells and replace with 5 mL fresh DMEM + 10% FBS, continuously culture cells for 4 hours.</li>
	<li>In a 5 mL polystyrene tube, add 480 &mu;L 2X HBS buffer.</li>
	<li>In a second 5 mL polystyrene tube, add 60 &mu;L 2M CaCl2 buffer, the plasmid DNA, and transfection TE buffer (1 mM Tris, 25 mM EDTA, pH 8.0) to a total volume of 480 &mu;L. For each Petri-dish, plasmids should be added in the ratio of 10 &mu;g of pNL-GFP-RRE-SA, 7.5 &mu;g of pCMV&Delta;R8.3, and 2.5 mg of pHCMV-G.</li>
	<li>Add the plasmid DNA-CaCl2 mixture dropwise to the 2 X HBS tube, and incubate at room temperature for 30 minutes. A fine precipitate will form.</li>
	<li>Add the 960 &mu;L of the mixture dropwise by pipette to the cells. Incubate at 37&deg;C, 5% CO2 overnight.</li>
	<li>The next day, remove the supernatant and add 10 mL fresh DMEM + 10% FBS, and continuously incubate the cells at 37&deg;C, 5% CO2 overnight.</li>
	<li>At 48 hours post transfection, harvest the virus by removing the supernatant and transferring it into 50 mL sterile tubes. Add fresh 10 mL fresh DMEM + 10% FBS into each Petri-dish and continue to culture overnight. Store the virus supernatant at 4&deg;C.</li>
	<li>Continue to harvest at 72 hours post transfection by collecting the supernatant. Combine all the supernatants, and centrifuge the supernatants at 500 x g for 15 minutes to pellet and remove cell debris.</li>
	<li>The supernatant were collected and filtered through a 0.22 &mu;M Millipore filter. The virus was further concentrated through size-exclusion columns. </li>
</ol>
2. Concentrate Viral Particles and Determine Viral Titer
<ol>
	<li>Viral particles were concentrated by a size-exclusion column (100,000 MW cut off, Centricoin). Viral supernatant was loaded into the column, and centrifuged at 6,000 x g at 4&deg;C for 15 minutes.</li>
	<li>Concentrated viral supernatant was collected, aliquoted, and stored at -80&deg;C.</li>
	<li>The viral titer was determined by infection of a TNF-treated, HIV-1 positive Jacket cell line, J1.1 7. Cells were cultured in a 96 well plate at 2.5 x 105 cells per mL (100 mL total volume), and infected with 100 mL of serially diluted vNL-GFP-RRE-SA for a week. The titer (TCID50) of the particle was estimated by counting the GFP positive wells following the method of Reed and Muench 8.</li>
</ol>
3. Marking HIV-1 Positive Cells with the Lentiviral Particle, vNL-GFP-RRE-SA
Set up: a human CD4 T cell line, CEM-SS, was first infected with HIV-1. At 48 hours post infection, cells were superinfected with the lentiviral particles, vNL-GFP-RRE-SA. Following superinfection for another two days, GFP-positive cells were analyzed by flow cytometry. As a control, HIV-1 uninfected CEM-SS cells were identically infected with vNL-GFP-RRE-SA. Only the HIV-1-infected CEM-SS cells gave rise to GFP positive cells but not the HIV-1 uninfected CEM-SS cells.
Procedure:
<ol>
	<li>Two hours before infection, add polybrane to a final concentration 4 mg / mL. Count cells and take 2 x 105 cells for each infection. Infect cells with 200 ng (p24) of HIV-1NL4-3 for 2 hours.</li>
	<li>Washing cells by centrifuge at 300 x g for 10 minutes, resuspend cells into 2 x 105 cells / mL, and culture at 37&deg;C for 48 hours.</li>
	<li>Count cells and take 2 x 105 cells per infection with vNL-GFP-RRE-SA. Add 100 mL of vNL-GFP-RRE-SA and incubate at 37&deg;C overnight. Wash cells and resuspend into 2 x 105 cells / mL.</li>
	<li>Perform flow cytometry analysis at 48 hours post superinfection. Infected cells were pelleted and resuspended into 500 mL 1% paraformaldehede, incubated at room temperature for 20 minutes, and then analyzed on a FACScan analyzer (Becton Dickenson).</li>
</ol>
4. Representative Results
If the experiments are successfully performed, a sizable GFP population will be detected in HIV-1-infected CEM-SS cells by the flow cytometer, whereas, in the control, GFP positive cells will not be detected in HIV-1 uninfected CEM-SS cells.
If the experiments are successfully performed, the Rev-dependent lentiviral vector, vNL-GFP-RRE-SA, will permit the highly specific detection of replicating HIV in living cell populations, through measurement of green fluorescence protein (GFP) expression. In this example, harvested CEM-SS cells were stained with a PE-labeled rat monoclonal antibody against mouse CD24, HSA, and then analyzed on a flow cytometer for both HSA and GFP expression.
<img src="/files/ftp_upload/2198/2198fig1.jpg" alt="Figure 1" /> Figure 1. As shown here, a sizable GFP population was detected in HIV-1-infected cells superinfected with vNL-GFP-RRE-SA (Figure 1c). In contrast, GFP positive cells were not detected in either HIV-1 uninfected CEM-SS cells without lentiviral superinfection (Figure 1a) or HIV-1 uninfected cells with lentiviral superinfection (Figure 1b).

<ol>
	<li>Garcia, J. A., Wu, F. K., Mitsuyasu, R., Gaynor, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+cellular+proteins+involved+in+the+transcriptional+regulation+of+the+human+immunodeficiency+virus.">Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.</a> EMBO Journal. 6, 3761-3770 (1987).</li><li>Merzouki, A. <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+gp120/160+expressing+cells+upregulate+HIV-1+LTR+directed+gene+expression+in+a+cell+line+transfected+with+HIV-1+LTR-reporter+gene+constructs.">HIV-1 gp120/160 expressing cells upregulate HIV-1 LTR directed gene expression in a cell line transfected with HIV-1 LTR-reporter gene constructs.</a> Cell Mol Biol (Noisy-le-grand. 41, 445-452 (1995).</li><li>Aguilar-Cordova, E., Chinen, J., Donehower, L., Lewis, D. E., Belmont, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+reporter+cell+line+for+HIV-1+tat+activity,+HIV-1+inhibitors,+and+T+cell+activation+effects.">A sensitive reporter cell line for HIV-1 tat activity, HIV-1 inhibitors, and T cell activation effects.</a> AIDS Res Hum Retroviruses. 10, 295-301 (1994).</li><li>Wu, Y., Beddall, M. H., Marsh, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rev-dependent+lentiviral+expression+vector.">Rev-dependent lentiviral expression vector.</a> Retrovirology. 4, 12-12 (2007).</li><li>Wu, Y., Beddall, M. H., Marsh, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rev-dependent+indicator+T+cell+line.">Rev-dependent indicator T cell line.</a> Current HIV Research. 5, 395-403 (2007).</li><li>Young, J., Tang, Z., Yu, Q., Yu, D., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+killing+of+HIV-1-positive+macrophages+and+T+Cells+by+the+Rev-dependent+lentivirus+carrying+anthrolysin+O+from+Bacillus+anthracis.">Selective killing of HIV-1-positive macrophages and T Cells by the Rev-dependent lentivirus carrying anthrolysin O from Bacillus anthracis.</a> Retrovirology. 5, 36-36 (2008).</li><li>Perez, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+HIV-1-infected+T+cell+clone+defective+in+IL-2+production+and+Ca2++mobilization+after+CD3+stimulation.">An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization after CD3 stimulation.</a> Journal of Immunology. 147, 3145-3148 (1991).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27, 493-497 (1938).</li></ol>

No conflicts of interest declared.

As with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel new approach to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Biosafety Cabinet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&deg;C 5%CO2 incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>FACSCalibur</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>Allega&trade;6KR</td>
<td>&nbsp;</td>
<tr>
<td>4&deg;C regrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>-20&deg;C refrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> -80&deg;C refrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell counter</td>
<td>&nbsp;</td>
<td>Nexcelom</td>
<td>Cellometer&trade; AutoT4</td>
<td>&nbsp;</td>
<tr>
<td>HEK293T cell line</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CEM-SS cell line</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Jurkat cell line J1.1</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-118</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21870</td>
<td>&nbsp;</td>
<tr>
<td>HI FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438-018</td>
<td>&nbsp;</td>
<tr>
<td>HIV-1NL4-3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>prepared by tranfsection of HeLa cells with cloned proviral DNA</td>
</tr>
<tr>
<td>pNL-GFP-RRE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>copmlete deletion of all HIV ORFs of pNL4-3</td>
</tr>
<tr>
<td>pNL-GFP-RRE-SA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Insert HIV-1 A5 and D4 into pNL-GFP-RRE</td>
</tr>
<tr>
<td>pCMV&Delta;8.2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>provided by Dr. Dider Trono</td>
</tr>
<tr>
<td>pHCMV-G</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 x HBS (Hepes Buffered Saline)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11344-041</td>
<td>&nbsp;</td>
<tr>
<td>2M CaCl2 buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-013-154-10</td>
<td>&nbsp;</td>
<tr>
<td>1% paraformaldehy</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>158127</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50ml Falcon tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>358206</td>
<td>&nbsp;</td>
<tr>
<td>5ml round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352063</td>
<td>&nbsp;</td>
<tr>
<td>0.45&mu;M Millipore filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SLHA033SS</td>
<td>&nbsp;</td>
<tr>
<td>0.20&mu;M Millipore filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SLFG85000</td>
<td>&nbsp;</td>
<tr>
<td>100mm Petri-dish</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>CLS430588</td>
<td>&nbsp;</td>
<tr>
<td>Size-exclusion column (100,000MW cut off)</td>
<td>&nbsp;</td>
<td>SartoriuStedim biotech</td>
<td>VS2041</td>
<td>&nbsp;</td>
<tr>
<td>2ml frozen tube</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>SCT-200</td>
<td>&nbsp;</td>
<tr>
<td>96-well plate</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>353227</td>
<td>&nbsp;</td>
<tr>
<td>1.7mL Microcentrifuge Tube</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT- 175-C</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

specific marking of hiv-1 positive cells using a rev-dependent lentiviral vector expressing the green fluorescent protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

Watch this Scientific Journal Video about Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein at JoVE.com

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

specific-marking-hiv-1-positive-cells-using-rev-dependent-lentiviral

Research

JoVE Journal

Immunology and Infection

12.8K Views.  George Mason University. We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP. 

Video: Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...