HIV-1 uncoating studies in the Aiken laboratory were supported by NIH grants AI40364, AI50423 and AI076121. Several key materials, including reagents used in the p24 ELISA, were provided by the NIH AIDS Research and Reference Program, Division of AIDS, NIAID, NIH.

1. Production of HIV-1 particles
Before proceeding, you must obtain permission and follow the guidelines from the Biosafety office of your institution to work in a biosafety facility approved for infectious HIV. You must ensure that proper care and safety precautions are followed during working in the biosafety laboratory.
Production of HIV-1 particles is typically performed by transient transfection of 293T cells with HIV-1 proviral DNA using a calcium phosphate transfection method7, 8. High titer virus stocks grown by culture of infected T cells are also suitable.
<ol>
	<li>Culture 293T cells in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and supplemented with antibiotics [Penicillin (100 IU/ml) and Streptomycin (100 &mu;g/ml)] at 37&deg;C, 5% CO2.</li>
	<li>Detach cells from nearly confluent dishes using 0.25% Trypsin-EDTA and seed 2x106 cells in 9 ml per 100 mm culture dish a day before transfection. Seed six 100 mm culture dishes for production of virions. If concentrated preparation of cores is required, more dishes could be seeded for transfection.</li>
	<li>On the next day, the cell monolayers should be about 25% confluent and ready for transfection. For six dishes of cells to be transfected, bring 120 &mu;g of proviral DNA up to a volume of 2.7 ml with sterile water. To this mixture add 0.3 ml of 2.5 M CaCl2 and 3 ml of 2XBBS, and mix properly by pipetting up and down several times. Allow the mixture to stand at room temperature for 10-20 minutes.</li>
	<li>Add 1 ml of the resulting mixture dropwise to the center of each dish while swirling gently to mix. Place the dishes overnight (~16 hours) in an incubator set at 35&deg;C and 3% CO2.</li>
	<li>Aspirate the culture medium and gently add 5 ml PBS.</li>
	<li>Aspirate PBS and add 5 ml of fresh medium. Culture the cells in incubator at 37&deg;C and 5% CO2 for additional 24-48 hours.</li>
	<li>Collect the culture supernatant containing viral particles, transfer it to a 50 ml conical centrifuge tube. Combine the supernatants from all dishes and centrifuge at 1,500 x g for 5 min to pellet cells and debris. Filter the supernatant using 0.45 &mu;m pore-size syringe filters. Precautions should be taken to minimize aerosol formation while working with the live virus. This includes use of conical tubes with O-rings or rotor bucket covers during the centrifugation step. If this is not possible, the screw caps of the tubes should be wrapped with parafilm.</li>
</ol>
2. Concentration of HIV-1 virions by ultracentrifugation
<ol>
	<li>Place the culture supernatant (30 ml) in a 38.5 ml polyallomer centrifuge tube (for Beckman SW32Ti rotor or equivalent). Gently underlay the virus containing supernatant with 5 ml solution of 20% sucrose in PBS using a 5 ml pipet.</li>
	<li>Centrifuge for 3 hours at 32,000 rpm at 4&deg;C (175,000 x g at rmax) to pellet virus particles.</li>
	<li>Remove supernatant by aspiration, taking care not to disturb the pellet at the bottom of the tube. Add 0.5 ml of 1XSTE buffer (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 mM EDTA) to the tube and place at 4&deg;C for 1 hour to loosen the pellet. Using a wide-bore 1 ml pipet tip gently pipet up and down a few times to detach the pellet from the bottom of the tube. Next, transfer the loosened pellet to a 1.5 ml microcentrifuge tube, taking care to avoid foaming. Incubate at 4&deg;C for another 1-3 hours to allow the small clumps of virus to disperse. During this period, prepare the sucrose gradient as described in step 3.1.</li>
	<li>Gently pipet the virus suspension up and down several times and centrifuge at 8,000 rpm (6,000 x g) at 4&deg;C in a refrigerated microfuge for 1 min to remove residual clumps. Keep the sample cold during this process.</li>
</ol>
3. Sucrose gradient centrifugation to isolate cores
HIV-1 cores are isolated using a "spin-thru" method9 which is a modification of the previously described method for the purification of HIV-2 cores10.
<ol>
	<li>Prepare a 12 ml linear 30% to 70% sucrose gradient in 1XSTE buffer in a 14.5 ml polyallomer centrifuge tube for SW32.1Ti rotor. To prepare gradient, use a 20 ml gradient former; place 6 ml of 70% sucrose solution on the near side (close to the outlet port) and 6 ml of 30% sucrose solution on the far side. Make sure that air bubbles are not trapped in the channel connecting the two chambers. Use the Auto Densi-Flow gradient former to pump the gradient from bottom to the top of the tube containing 1 ml of 85% sucrose solution at the bottom. Gently place the gradient at 4&deg;C until cooled (2-4 hours).</li>
	<li>Using a wide-bore 1 ml pipet tip gently overlay the gradient with 0.25 ml of 15% sucrose solution in STE containing 1% Triton X-100.</li>
	<li>Gently overlay with 0.25 ml of 7.5% sucrose solution in STE using a wide-bore 1 ml pipet tip. Be careful not to mix the layers.</li>
	<li>Gently overlay with 0.5 ml virus suspension from step 2.4. This step should be performed carefully to avoid any disturbance in the gradient and mixing of the underlaid sucrose layers.</li>
	<li>Place the tubes in the pre-cooled SW32.1Ti bucket and centrifuge at 32,000 rpm (187,000 x g at rmax) overnight (16-20 hours) at 4&deg;C. To avoid pausing of the centrifuge, do not start centrifugation until the vacuum in the centrifuge reaches 250 &mu;m</li>
	<li>Collect 1 ml fractions from top to bottom of the gradient using the Auto-Densi-Flow gradient fractionator. Place the tubes on ice immediately upon collection. Mix the contents of the tubes by inverting few times, and withdraw 50 &mu;l of each fraction for quantification by ELISA or reverse transcriptase activity assay.</li>
</ol>
4. Localization of HIV-1 cores and storage of cores
<ol>
	<li>Before performing uncoating assays, it is important to determine which fractions contain intact HIV-1 cores. This can be determined by p24 ELISA or reverse transcriptase activity assay using the 50 &mu;l aliquots from step 3.6.</li>
	<li>The recovery of CA associated with the cores is often related to the stability of the cores. Measure the core associated CA by p24 ELISA9 using a sample from each fraction. Alternatively, measure the reverse transcriptase activity of each fraction. Using both methods the peak should be around fraction 10.</li>
	<li>Pool the fractions containing cores. If the cores are not to be used for uncoating assay immediately, aliquot the pooled cores into 0.2 to 0.3 ml aliquots, flash-freeze in liquid N2 and store at -80&deg;C. Cores frozen in this manner are suitable for the uncoating assay. The yield of the core-associated CA is typically 1-2 &mu;g of p24 per milliliter or about 15% of the total virion-associated p24.</li>
</ol>
5. Kinetic assay of HIV-1 uncoating
<ol>
	<li>The amount of HIV-1 cores required per uncoating reaction is approximately 50 ng. To perform the in vitro uncoating assay, predilute the aliquot of cores with an equal volume of cold 1XSTE buffer to reduce the viscosity of the suspension and to minimize pipetting errors.</li>
	<li>Further dilute 100 &mu;l of the cores into 0.15 ml of cold 1XSTE buffer containing 10 &mu;g/ml of BSA in a 1.5 ml microcentrifuge tube. We supplement the STE buffer with BSA (10 &mu;g/ml) to minimize adsorption of CA to the walls of the tube. Mix by inverting the tube several times. Do not vortex the samples. Incubate the tubes in a water bath at 37&deg;C for timed intervals (15, 30, 60 and 120 min). The tubes should be immersed in the water bath at least up to the level of the internal sample in order to ensure even warming.</li>
	<li>During incubation, gently mix the contents of the tube periodically (usually 5-10 min) by flicking the tube. For a zero minute control, dilute 100 &mu;l of cores into 0.15 ml of cold 1XSTE buffer and incubate on ice for the entire length of the experiment (120 min). The zero minute control gives the basal uncoating value and is used to determine increase in uncoating of cores incubated at 37&deg;C for different timed intervals.</li>
	<li>At the end of incubation period, stop the uncoating process by placing the tubes in ice for 10 minutes.</li>
	<li>Centrifuge the tubes at 45,000 rpm (TLA-55 rotor; 125,000 x g at rmax) for 20 minutes at 4&deg;C. This will pellet the intact cores and the free CA released as a result of uncoating of cores will remain in the supernatant. The rotor should be precooled at 4&deg;C prior to loading the samples.</li>
	<li>Transfer the supernatant to new precooled microfuge tubes preferably using gel-loading tips stored at room temperature. Please note that the pellet will not be visible to the naked eye, so take extreme care while pipetting out the supernatant. Resuspend the pellets in 250 &mu;l of ELISA sample diluent (0.5% Triton X-100 and 5% Donor calf serum in PBS). To ensure efficient dissolution of the pellet, vortex after adding the ELISA sample diluent. Quantify the CA content of the pellet and supernatant using p24 ELISA as described in the following section.</li>
	<li>The extent of uncoating is obtained by calculating the fraction of CA present in the supernatant.</li>
</ol>
6. Assay for CA by p24 ELISA
<ol>
	<li>Many commercial ELISA kits are available and can be used to quantify CA. Use any commercial or "in-house" ELISA and include p24 standards to determine the linearity of the assay. Dilutions of the samples should be assayed to ensure accuracy.</li>
	<li>We have developed a homemade ELISA which we routinely use to assay for CA. The procedure is adapted from a previous report11 and is performed using capture and detector antibodies available from NIH AIDS Research and Reference Reagent Program.</li>
</ol>
7. Representative Results:
An example of a result from ELISA for determining fractions containing core is shown in Figure 1. After collecting fractions (1 ml each), 50 &mu;l of sample from each fraction was used to make serial dilutions and analyzed by ELISA. The total p24 obtained by adding the values for twelve fractions was 14.5 &mu;g. As shown in the figure fractions 8 to 10 contained cores. The p24 value for the core-containing fractions was 2.17 &mu;g. The percentage of core-associated p24 (14.97%) was determined by taking the ratio of p24 value for the core fractions (2.17 &mu;g) to the total p24 value (14.5 &mu;g). Fraction 1 or 2 will always contain highest amount of p24; this represents free CA which is not associated with the cores. This is followed by a gradual reduction in p24 values with each subsequent fraction, then a sharp increase and finally a sharp drop in the p24 values. If proper care is taken while loading the gradient then the peak of core-associated CA is found around fraction 10.
A typical result obtained by assaying the kinetics of uncoating of HIV-1 cores is shown in Figure 2. The graph shows a time-dependent increase in uncoating of wild-type HIV-1 cores. The percent uncoating value was obtained by calculating the fraction of CA present in the supernatant. With practice, the assay is highly reproducible and is useful for determining the stability of viral cores in vitro. Please note that the reproducibility of the assay is highly dependent on the proper handling of the samples during the procedure. Since the cores are heat labile, the samples should be maintained at 4&deg;C throughout the assay except during the incubation period.
<img alt="Figure 1" src="/files/ftp_upload/3384/3384fig1.jpg" /> 
Figure 1 Equilibrium density gradient centrifugation of HIV-1 cores. A concentrated virus suspension was applied to the top of a 30 to 70% linear sucrose gradient overlaid with 1% Triton-X-100. Following overnight centrifugation at 187,000 x g and 4&deg;C, 1 ml fractions were collected from top (Fraction 1) to bottom (Fraction 12) and p24 was quantified by ELISA.
<img alt="Figure 2" src="/files/ftp_upload/3384/3384fig2.jpg" /> 
Figure 2 Time course of HIV-1 uncoating in vitro. Purified HIV-1 cores were diluted and incubated at 37&deg;C for the indicated time intervals. The zero min sample was incubated on ice for the entire length of the experiment. Following incubation, samples were ultracentrifuged at 125,000 x g for 20 minutes at 4&deg;C. Supernatant and pellets were separated and analyzed by p24 ELISA. The extent of uncoating was determined as described in the protocol text. Each uncoating reaction was performed in duplicate; error bars span the range of the two values.

<ol>
	<li>Ganser, B. K., Li, S., Klishko, V. Y., Finch, J. T., Sundquist, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+analysis+of+conical+models+for+the+HIV-1+core.">Assembly and analysis of conical models for the HIV-1 core.</a> Science. 283, 80-83 (1999).</li><li>Li, S., Hill, C. P., Sundquist, W. I., Finch, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+reconstructions+of+helical+assemblies+of+the+HIV-1+CA+protein.">Image reconstructions of helical assemblies of the HIV-1 CA protein.</a> Nature. 407, 409-413 (2000).</li><li>Aiken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+and+cellular+factors+that+regulate+HIV-1+uncoating.">Viral and cellular factors that regulate HIV-1 uncoating.</a> Curr. Opin. HIV. AIDS. 1, 194-199 (2006).</li><li>Forshey, B. M., Schwedler, U. v. o. n., Sundquist, W. I., Aiken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+a+human+immunodeficiency+virus+type+1+core+of+optimal+stability+is+crucial+for+viral+replication.">Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication.</a> J. Virol. 76, 5667-5677 (2002).</li><li>Forshey, B. M., Aiken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disassembly+of+human+immunodeficiency+virus+type+1+cores+in+vitro+reveals+association+of+Nef+with+the+subviral+ribonucleoprotein+complex.">Disassembly of human immunodeficiency virus type 1 cores in vitro reveals association of Nef with the subviral ribonucleoprotein complex.</a> J. Virol. 77, 4409-4414 (2003).</li><li>Wacharapornin, P., Lauhakirti, D., Auewarakul, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+capsid+mutations+on+HIV-1+uncoating.">The effect of capsid mutations on HIV-1 uncoating.</a> Virology. 358, 48-54 (2007).</li><li>Chen, C., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transformation+of+mammalian+cells+by+plasmid+DNA.">High-efficiency transformation of mammalian cells by plasmid DNA.</a> Mol Cell Biol. 7, 2745-2752 (1987).</li><li>Aiken, C., Prasad, V. R., Ganjam, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology. 485, 41-53 (2009).</li><li>Kotov, A., Zhou, J., Flicker, P., Aiken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Nef+with+the+human+immunodeficiency+virus+type+1+core.">Association of Nef with the human immunodeficiency virus type 1 core.</a> J. Virol. 73, 8824-8830 (1999).</li><li>Kewalramani, V. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+structural+analysis+of+isolated+mature+cores+of+human+immunodeficiency+virus+type+1.">Biochemical and structural analysis of isolated mature cores of human immunodeficiency virus type 1.</a> J. Virol. 74, 1168-1177 (2000).</li><li>Briggs, J. A., Wilk, T., Welker, R., Kr&#228;usslich, H. G., Fuller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+organization+of+authentic,+mature+HIV-1+virions+and+cores.">Structural organization of authentic, mature HIV-1 virions and cores.</a> EMBO J. 22, 1707-1715 (2003).</li><li>Auewarakul, P., Wacharapornin, P., Srichatrapimuk, S., Chutipongtanate, S., Puthavathana, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoating+of+HIV-1+requires+cellular+activation.">Uncoating of HIV-1 requires cellular activation.</a> Virology. 337, 93-101 (2005).</li><li>Accola, M. A., Ohagen, A., G&#246;ttlinger, H. 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No conflicts of interest declared.

The method described here to purify HIV-1 cores to study uncoating in vitro is useful for studying this phase of the HIV-1 life cycle, especially due to unavailability of a validated method to analyze HIV-1 uncoating in target cells. Although this method uses equilibrium ultracentrifugation to purify cores, others have used direct pelleting after brief lysis with 1% Triton-X-10012, 13 or pelleting through a sucrose cushion overlaid with 0.1% Triton-X-10014, 15.
T...

<table border="1">
	<tbody>
		<tr>
			<td width="190">Name of the reagent</td>
			<td width="197">Company</td>
			<td width="114">Catalog number</td>
			<td width="161">Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cellgro</td>
			<td>21-031-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Mallinckrodt Baker Inc</td>
			<td>9002-93-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Acros</td>
			<td>9005-64-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Cellgro</td>
			<td>25-053-CI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2XBBS</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Composition: 50mM BES [pH 6.95], 280 mM NaCl and 1.5 mM Na2HPO4. Adjust pH to 6.95 at room temperature. Filter sterilize &amp; store in aliquots at -20&deg;C.</td>
		</tr>
		<tr>
			<td>Coating antibody: Monoclonal antibody to p24 (183-H12-5C)</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>3537</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Primary antibody: HIV-Ig (hyperimmune human patient serum)</td>
			<td>
			NIH AIDS Researchand Reference Reagent Program</td>
			<td>3957</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>
			Secondary antibody:Goat anti-human IgG, peroxidase conjugated</td>
			<td>Pierce</td>
			<td>31130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HRP substrate</td>
			<td>KPL Inc</td>
			<td>50-76-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Immulon 2HB 96 well plates</td>
			<td>Thermo Scientific</td>
			<td>3455</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SW32Ti and SW32.1 Ti rotors and compatible ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TLA-55 rotor and tabletop ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>High speed microfuge tubes</td>
			<td>Beckman Coulter</td>
			<td>326823, 358123, 357448</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Auto-Densi Flow density gradient fractionator</td>
			<td>Labconco Corp.</td>
			<td>4517000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gradient Former</td>
			<td>CBS Scientific Co. Inc.</td>
			<td>GM-20</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about In vitro Uncoating of HIV-1 Cores at JoVE.com

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.

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 ...

Research

JoVE Journal

Immunology and Infection

15.6K Views.  Vanderbilt University School of Medicine. 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.

Video: In vitro Uncoating of HIV-1 Cores

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 ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

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. ...

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...