Development of this assay was supported by Viiv Healthcare and the Canadian Institutes of Health Research (CIHR) and through a GlaxoSmithKline/CIHR Chair in Clinical Virology for Dr. Harrigan.
Support provided by Pfizer and ViiV Healthcare.

<ol>
	<li>Feng, Y., Broder, C. C., Kennedy, P. E. <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+entry+cofactor:+functional+cDNA+cloning+of+a+seven+transmembrane,+G+protein-coupled+receptor.">HIV-1 entry cofactor: functional cDNA cloning of a seven transmembrane, G protein-coupled receptor.</a> Science. 272, 872-877 (1996).</li><li>Dragic, T., Litwin, V., Allaway, G. P. <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+entry+into+CD4++cells+is+mediated+by+the+chemokine+receptor+CC-CKR-5.">HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.</a> Nature. 381, 667-673 (1996).</li><li>Dorr, P., Westby, M., Dobbs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+(UK-427,857),+a+potent,+orally+bioavailable,+and+selective+small-molecule+inhibitor+of+chemokine+receptor+CCR5+with+broad-spectrum+anti-human+immunodeficiency+virus+type+1+activity.">Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.</a> Antimicrob. Agents Chemother. 49, 4721-4732 (2005).</li><li>Gulick, R. M., Lalezari, J., Goodrich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+for+previously+treated+patients+with+R5+HIV-1+infection.">Maraviroc for previously treated patients with R5 HIV-1 infection.</a> N. Engl. J. Med. 359, 1429-1441 (2008).</li><li>Cooper, D. A., Heera, J., Goodrich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+versus+efavirenz,+both+in+combination+with+zidovudine/lamivudine,+for+the+treatment+of+antiretroviral-na&#237;ve+subjects+with+CCR5-tropic+HIV-1.">Maraviroc versus efavirenz, both in combination with zidovudine/lamivudine, for the treatment of antiretroviral-na&#237;ve subjects with CCR5-tropic HIV-1.</a> J. Infect. Dis. 201, 803-813 (2010).</li><li>Low, A. J., McGovern, R. A., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trofile+HIV+co-receptor+usage+assay.">Trofile HIV co-receptor usage assay.</a> Expert Opin. Med. Diagnostics. 3, 181-191 (2000).</li><li>Sing, T., Low, A. J., Beerenwinkel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+HIV+co-receptor+usage+based+on+genetic+and+clinical+covariates.">Predicting HIV co-receptor usage based on genetic and clinical covariates.</a> Antiviral Ther. 12, 1097-1106 (2007).</li><li>Harrigan, P. R., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+HIV+tropism+using+population-based+V3+genotypic+analysis:+a+retrospective+virological+outcome+analysis+using+stored+plasma+screening+samples+from+MOTIVATE-1.">Screening for HIV tropism using population-based V3 genotypic analysis: a retrospective virological outcome analysis using stored plasma screening samples from MOTIVATE-1.</a> , (2009).</li><li>Harrigan, P. R., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+clinically+relevant+cut-points+for+the+determination+of+HIV+co-receptor+usage+to+predict+maraviroc+responses+in+treatment+experienced+(TE)+patients+using+population+V3+genotyping.">Optimization of clinically relevant cut-points for the determination of HIV co-receptor usage to predict maraviroc responses in treatment experienced (TE) patients using population V3 genotyping.</a> , (2009).</li><li>Swenson, L. C., McGovern, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+clinically+relevant+cutoffs+for+determining+HIV+co-receptor+use+by+population+and+"deep"+sequencing+methods.">Optimization of clinically relevant cutoffs for determining HIV co-receptor use by population and "deep" sequencing methods.</a> , (2009).</li><li>Swenson, L., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+screening+for+HIV+tropism+versus+both+population-based+and+"deep"+sequencing.">Phenotypic screening for HIV tropism versus both population-based and "deep" sequencing.</a> , (2009).</li><li>Swenson, L. C., Moores, A., Low, A. J., Thielen, A., Dong, W., Woods, C., Jensen, M. A., Wynhoven, B., Chan, D., Glascock, C., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Detection+of+CXCR4-Using+HIV+by+V3+Genotyping:+Application+of+Population-based+and+'Deep'+Sequencing+to+Plasma+RNA+and+Proviral+DNA.">Improved Detection of CXCR4-Using HIV by V3 Genotyping: Application of Population-based and 'Deep' Sequencing to Plasma RNA and Proviral DNA.</a> Journal of Acquired Immune Deficiency Syndromes. 54, 506-510 (2010).</li><li>Swenson, L. C., Boehme, R., Thielen, A., McGovern, R. A., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+determination+of+HIV-1+tropism+in+the+clinical+setting.">Genotypic determination of HIV-1 tropism in the clinical setting.</a> HIV Therapy. 4, 293-303 (2010).</li></ol>

P. Richard Harrigan has conflicts of interest with ViiV Healthcare, Virco, Merck, Abbott, and Quest. This video-article is sponsored by ViiV Healthcare.

The method presented here is a standard sequencing method applied to tropism testing. The clinical application of HIV envelope V3 loop sequencing to predict viral tropism has until late been limited. This method has been shown, in retrospective analyses of the maraviroc (ViiV Healthcare) clinical trials, to be at minimum equally able to predict viral tropism in comparison to other validated assays used clinically.
There are many benefits to performing genotypic analysis of the V3 loop for tropism prediction. First and foremost, this procedure can be performed at any facility with operational sequencing equipment, greatly increasing the accessibility and reducing the turnaround time of tropism testing. In comparison, the phenotypic Enhanced Sensitivity Trofile Assay (ESTA) (Monogram Biosciences), which has served as the gold standard for tropism testing, is performed at one centre in Southern California. Additional benefits include the requirement of less starting material and a minimum required plasma viral load of 500copies/mL. As well, the operational costs of running the genotypic assay are relatively low in comparison to those of a phenotypic assay. The use of the ReCall software in particular eliminates the requirement for manual sequence review, which tends to be a labour intensive part of genotype analyses.
The method presented here is fairly straightforward, with no particular step outweighing another in importance. We do suggest running PCR and sequencing in triplicate to increase the odds of capturing minority species within the viral population of a sample. Replicates greater than three can be performed, however we have found triplicates to be both efficient and reliable. We also suggest using the One-Step Reverse Transcriptase PCR Kit (Qiagen) which performs both RT-PCR and first round PCR in the same reaction while maintaining high sensitivity and specificity.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RNA extraction using NucliSens easyMAG version 1.0</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NucliSens easyMAG Magnetic Silica</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280133</td>
<td>(48 x 0.6 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG Lysis Buffer</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280134</td>
<td>(4 x 1000 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG Extraction Buffer 1</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280130</td>
<td>(4 x 1000 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG Extraction Buffer 2</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280131</td>
<td>(4 x 1000 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG Extraction Buffer 3</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280132</td>
<td>(4 x 1000 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG version 1.0 </td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Class II A2 biological safety cabinet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>One-Step Reverse Transcriptase PCR and Second Round PCR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DEPC-treated water</td>
<td>&nbsp;</td>
<td>Ambion Corp.</td>
<td>cat. # 9922</td>
<td>&nbsp;</td>
<tr>
<td>SuperScrip III One-Step RT-PCR System with Platinum Taq High Fidelty</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>cat#12574-035</td>
<td>Kit components used: 
- SuperScript III RT/ Platinum Taq High Fidelty Enzyme Mix 
- 2X Reaction Mix (a buffer containing 0.4mM of each dNTP, 2.4mM MgSO4)</td>
</tr>
<tr>
<td>Expand High Fidelity PCR System</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>cat. # 1 759 078</td>
<td>Kit components used: 
- Expand High Fidelity Buffer 10X conc. with 15 mM MgCl2 (lids labelled 2) 
- Expand HF PCR Enzyme Mix (3.5U/&mu;l) 
- MgCl2 Stock Solution (25mM) (lids labelled 4)</td>
</tr>
<tr>
<td>dNTPs: dATP, dGTP, dCTP, dTTP</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>cat. # 11969064001</td>
<td>(100 mM)</td>
</tr>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat. # S0389-500G</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat.# B5525</td>
<td>&nbsp;</td>
<tr>
<td>Cresol Red</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat.# 114472-5G</td>
<td>indicator grade</td>
</tr>
<tr>
<td>Thermocycler </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gel Electrophoresis</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50X TAE buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>cat. # 24710030</td>
<td>(1000 ml) </td>
</tr>
<tr>
<td>SYBR Safe DNA gel stain</td>
<td>&nbsp;</td>
<td>Invitrogen Life Technologies</td>
<td>cat. # S33102</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (ultra pure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>cat. # 15510-027</td>
<td>&nbsp;</td>
<tr>
<td>E-Gel Low Range Quantitative DNA Ladder</td>
<td>&nbsp;</td>
<td>Invitrogen Life Technologies</td>
<td>cat. # 12373-031</td>
<td>&nbsp;</td>
<tr>
<td>Reagent Grade Type II water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gel apparatus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sequencing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ABI PRISM BigDye Terminator Cycle Sequencing Kit</td>
<td>&nbsp;</td>
<td>Applied Biosciences</td>
<td>cat. # 4337458</td>
<td>(25000 reactions)</td>
</tr>
<tr>
<td>Hi-Di Formamide</td>
<td>&nbsp;</td>
<td>Applied Biosciences</td>
<td>cat. # 4311320</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Acetate (NaOAc)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat. # S-2889</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Cat.# 03690-100ML</td>
<td>0.5M, pH=8.0</td>
</tr>
<tr>
<td>TRIZMA Hydrochloride Buffer Solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat. # T-2819</td>
<td>1 M, pH 9.0</td>
</tr>
<tr>
<td>Magnesium Chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat. # M-1028</td>
<td>1 M</td>
</tr>
<tr>
<td>95% Ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Running Buffer (10X) with EDTA</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>cat. # 4335613</td>
<td>500 mL</td>
</tr>
<tr>
<td>Polymer Pop-7 (25mL) Or Polymer Pop-7 (10mL)</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>cat. # 44363929 or cat. # 4352759</td>
<td>&nbsp;</td>
<tr>
<td>3700/3730 BigDye Terminator v3.1 Sequencing Standard Kit</td>
<td>&nbsp;</td>
<td>Applied Biosciences</td>
<td>Cat# 4336943</td>
<td>&nbsp;</td>
<tr>
<td>Thermocycler </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge with plate holders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3730xl DNA Analyzer</td>
<td>&nbsp;</td>
<td>Applied Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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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.

Watch this Scientific Journal Video about Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3 at JoVE.com

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

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 ...

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Research

JoVE Journal

Immunology and Infection

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

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant &beta;-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2 used the ab initio algorithm 3 accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

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.

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, ...

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. ...

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 ...

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 ...

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.