Ce travail a été soutenu par des subventions du NIH AI071815 (CEH) et la Feuille de route nanomédecine Centre de développement attribution PN2EY016586 (MLD).

<ol>
	<li>Bastiani, L., Laal, S., Kim, M., Zolla-Pazner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+cell-dependent+alterations+in+envelope+components+of+human+immunodeficiency+virus+type+1+virions.">Host cell-dependent alterations in envelope components of human immunodeficiency virus type 1 virions.</a> J. Virol. 71, 3444-3450 (1997).</li><li>Dimitrov, D. S., Willey, R. L., Sato, H., Chang, L. J., Blumenthal, R., Martin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+human+immunodeficiency+virus+type+1+infection+kinetics.">Quantitation of human immunodeficiency virus type 1 infection kinetics.</a> J. Virol. 67, 2182-2190 (1993).</li><li>Dustin, M. 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F., Cantin, R., Lamontagne, G., Tremblay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+ICAM-1+glycoproteins+incorporated+on+human+immunodeficiency+virus+type+1+are+biologically+active+and+enhance+viral+infectivity.">Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity.</a> J. Virol. 71, 3588-3596 (1997).</li><li>Frank, I., Stoiber, H., Godar, S., Stockinger, H., Steindl, F., Katinger, H. W., Dierich, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+host+cell-surface-derived+molecules+by+HIV-1.">Acquisition of host cell-surface-derived molecules by HIV-1.</a> Aids. 10, 1611-1620 (1996).</li><li>Hioe, C. E., Bastiani, L., Hildreth, J. E., Zolla-Pazner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cellular+adhesion+molecules+in+HIV+type+1+infection+and+their+impact+on+virus+neutralization.">Role of cellular adhesion molecules in HIV type 1 infection and their impact on virus neutralization.</a> AIDS Res. Hum. Retroviruses. 14, S247-S254 (1998).</li><li>Hioe, C. E., Chien, P. C., Lu, C., Springer, T. A., Wang, X. H., Bandres, J., Tuen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LFA-1+expression+on+target+cells+promotes+human+immunodeficiency+virus+type+1+infection+and+transmission.">LFA-1 expression on target cells promotes human immunodeficiency virus type 1 infection and transmission.</a> J. Virol. 75, 1077-1082 (2001).</li><li>Jolly, C., Kashefi, K., Hollinshead, M., Sattentau, Q. J. <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+cell+to+cell+transfer+across+an+Env-induced,+actin-dependent+synapse.">HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse.</a> J. Exp. Med. 199, 283-293 (2004).</li><li>Jolly, C., Mitar, I., Sattentau, Q. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+an+intact+T-cell+actin+and+tubulin+cytoskeleton+for+efficient+assembly+and+spread+of+human+immunodeficiency+virus+type+1.">Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1.</a> J. Virol. 81, 5547-5560 (2007).</li><li>McDonald, D., Wu, L., Bohks, S. M., KewalRamani, V. N., Unutmaz, D., Hope, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+HIV+and+its+receptors+to+dendritic+cell-T+cell+junctions.">Recruitment of HIV and its receptors to dendritic cell-T cell junctions.</a> Science. 300, 1295-1297 (2003).</li><li>Pearce-Pratt, R., Malamud, D., Phillips, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+cytoskeleton+in+cell-to-cell+transmission+of+human+immunodeficiency+virus.">Role of the cytoskeleton in cell-to-cell transmission of human immunodeficiency virus.</a> J. Virol. , 682898-682905 (1994).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Rizzuto, C. D., Sodroski, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+virion+ICAM-1+to+human+immunodeficiency+virus+infectivity+and+sensitivity+to+neutralization.">Contribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivity to neutralization.</a> J. Virol. 71, 4847-4851 (1997).</li><li>Vardhana, S., Dustin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+Planar+Bilayers+for+the+Formation+of+Study+of+Immunological+Synapses+and+Kinapse.">Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse.</a> J. Vis. Exp. (19), e947-e947 (2008).</li><li>Vasiliver-Shamis, G., Cho, M. W., Hioe, C. E., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+envelope+gp120-induced+partial+T-cell+receptor+signaling+creates+an+F-actin-depleted+zone+in+the+virological+synapse.">Human immunodeficiency virus type 1 envelope gp120-induced partial T-cell receptor signaling creates an F-actin-depleted zone in the virological synapse.</a> J. Virol. 83, 11341-11355 (2009).</li><li>Vasiliver-Shamis, G., Tuen, M., Wu, T., Starr, T., Cameron, T., Thomson, R., Kaur, G., Liu, J., Visciano, M., Li, H., Kumar, R., Ansari, R., Han, D., Cho, M., Dustin, M. L., Hioe, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+envelope+gp120+induces+a+stop+signal+and+virological+synapse+formation+in+noninfected+CD4++T+cells.">Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells.</a> J. Virol. 82, 9445-9457 (2008).</li><li>Zhu, P., Liu, J., Bess, J., Chertova, E., Lifson, J., Grise, H., Ofek, G., Taylor, K., Roux, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+three-dimensional+structure+of+AIDS+virus+envelope+spikes.">Distribution and three-dimensional structure of AIDS virus envelope spikes.</a> Nature. 441, 847-852 (2006).</li></ol>

Les auteurs déclarent aucun conflit d&#39;intérêts.

Des études antérieures ont VS visualisée dans le système conjugué de cellule à cellule, mais ces études n&#39;ont pas de fournir des images de résolution suffisamment élevée pour visualiser les structures supramoléculaires à la synapse. Dans notre laboratoire, nous avons utilisé le verre soutenu par système bicouche plane pour représenter la surface des cellules infectées exprimant le gp120 de l&#39;enveloppe du virus et la molécule d&#39;adhésion cellulaire ICAM-1. En collaboration avec la microscopie TIRF, qui détecte des signaux de fluorescence dans les 100-200 nm de la surface de la bicouche avec un fort rapport signal sur bruit, nous étions en mesure de détecter la ségrégation supramoléculaire de la gp120 de l&#39;ICAM-1 à la VS. En outre, la méthode immunomarquage norme peut être appliquée au système bicouche et a été utilisé ici afin de détecter et de quantifier le recrutement spécifique de Lck actif, mais pas Fionie, à la zone de gp120-contact à VS 15. Par conséquent, la bicouche plane offre un système expérimental pour l&#39;imagerie haute résolution de l&#39;interface synapse dans un plan 2D par la FRBR microscopie, ainsi que les méthodes d&#39;éclairage à grand champ ou confocale. Cependant, le système a aussi ses limites que l&#39;ajustement de mobilité ligand, out-of-plan de flexion, et les fluctuations des membranes biologiques ne sont pas reproduites par les bicouches planes. En outre, il s&#39;agit d&#39;un système in vitro et a donc d&#39;autres limitations, telles que le manque de molécules membranaires d&#39;autres qui seraient présents sur une cellule infectée et de la machinerie du cytosquelette qui réglemente la mobilité moléculaire et la motilité cellulaire. En outre, la dynamique et la distribution des molécules, telles que trimères contre monomères de la gp120, ne peuvent pas être représentés physiologiquement sur la bicouche. Néanmoins, même avec ces limitations, ce système est encore très précieux pour l&#39;étude du virus-cellule ou cellule-cellule interactions, et ces méthodes peuvent servir de guide utile aux chercheurs qui cherchent des images haute résolution pour détecter organisation supramoléculaire qui n&#39;est pas perceptible dans le système conjugué de cellule à cellule conventionnelle.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Son étiqueté gp120 du VIH </td><td> Don de M. Cho </td><td></td><td> DH12 </td></tr><tr><td> Son étiqueté gp120 du VIH </td><td> Technologie immunitaire </td><td></td><td> Divers X4 ou tropisme R5 </td></tr><tr><td> HSA </td><td> Williams Medical Company </td><td> 521302 </td><td> L&#39;albumine sérique humaine 25% </td></tr><tr><td> Amicon Ultra Filtres centrifuges </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30K </td></tr><tr><td> Lck lapin mAb </td><td> Signalisation cellulaire </td><td> 2787 </td><td></td></tr><tr><td> p-Lck lapin pAb </td><td> Santa Cruz Biotechnology Inc </td><td> Sc-101728 </td><td> Tyr 394 </td></tr><tr><td> Fyn lapin mAb </td><td> Millipore </td><td> 04-353 </td><td></td></tr><tr><td> Alexa Fluor 568 2 °; Ab </td><td> Molecular Probes </td><td> A11034 </td><td> De chèvre anti-IgG de lapin (H + L) </td></tr><tr><td> Alexa Fluor 488 </td><td> Molecular Probes </td><td> Un-20000 </td><td> carboxylique ester succinimidylique </td></tr><tr><td> FCS2 Chambre </td><td> Bioptechs </td><td> 060319-2-03 </td><td></td></tr><tr><td> Microaqueduct diapositives </td><td> Bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td> 30mm ronde w / trous </td><td> Bioptechs </td><td> 1907-08-750 </td><td> 0.75mm d&#39;épaisseur </td></tr><tr><td> Rectangle Joint </td><td> Bioptechs </td><td> 1907-1422-250 </td><td> 14x24 0,25 d&#39;épaisseur </td></tr><tr><td> Tubes Tygon </td><td> Bioptechs </td><td> 20202275 </td><td> 1/16 &quot;(25ft) </td></tr><tr><td> Robinet à trois voies </td><td> BioRad </td><td> 7328103 </td><td></td></tr><tr><td> Deux voies Robinet </td><td> BioRad </td><td> 7328102 </td><td></td></tr><tr><td> Diapositive it I 0,2 Luer </td><td> Ibidi </td><td> 80168 </td><td></td></tr><tr><td> Couvrir verres </td><td> Ibidi </td><td> 10812 </td><td></td></tr><tr><td> Seringue de 1 ml </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> CHIENS-NTA </td><td> Avanti Polar Lipids </td><td> 790404C </td><td></td></tr><tr><td> DOPC </td><td> Avanti Polar Lipids </td><td> 850375C </td><td></td></tr><tr><td> Une lamelle en verre </td><td> Bioptechs </td><td> 40-1313-0319 </td><td> 40mm </td></tr><tr><td> Microscope TIRF </td><td> Nikon </td><td></td><td></td></tr><tr><td> ImageJ </td><td> National Institutes of Health </td><td></td><td> <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/~~V</a> </td></tr></tbody></table>

imaging of hiv-1 envelope-induced virological synapse and signaling on synthetic lipid bilayers

Humaine de type virus de l&#39;immunodéficience 1 (VIH-1) infection survient le plus efficacement par l&#39;intermédiaire de cellules à 2,10,11 transmission de cellules. Cette cellule à cellule de transfert entre cellules CD4 + T comprend la formation d&#39;une synapse virologique (VS), qui est un F-actine cellulaire dépendante des cellules jonction formée lors de l&#39;engagement du VIH-1 enveloppe gp120 de la cellule infectée avec CD4 et le récepteur de chimiokine (CKR) CCR5 ou CXCR4 sur la cellule cible 8. En plus de la gp120 et ses récepteurs, des protéines membranaires autres, en particulier la molécule d&#39;adhésion LFA-1 et ses ligands, la famille ICAM, jouent un rôle dans la formation et la transmission du virus SV qu&#39;ils sont présents sur la surface des cellules infectées par un virus donneurs des cellules cibles et, ainsi que sur l&#39;enveloppe de VIH-1 virions 1,4,5,6,7,13. La formation VS s&#39;accompagne aussi d&#39;événements de signalisation intracellulaire qui sont transduits à la suite de la gp120-engrènement de ses récepteurs. En effet, nous avons récemment montré que les CD4 &lt;sup&gt; + d&#39;interaction des cellules T avec la gp120 induit le recrutement et la phosphorylation de molécules de signalisation associées à la signalosome TCR, y compris Lck, CD3ζ, ZAP70, LAT, SLP-76, ITK, et PLCγ 15. Dans cet article, nous présentons une méthode pour visualiser arrangement supramoléculaire et les événements de signalisation membrane proximal qui se déroulent lors de la formation VS. Nous profitons du verre soutenu par plane bi-couche du système comme un modèle réductionniste pour représenter la surface de cellules infectées portant l&#39;enveloppe virale gp120 et la molécule d&#39;adhésion cellulaire ICAM-1. Le protocole décrit les procédures générales pour la surveillance du VIH-1 gp120 induit VS assemblage et les événements d&#39;activation de signaux qui incluent i) bi-couche de préparation et d&#39;assemblage dans une cellule d&#39;écoulement, ii) l&#39;injection de cellules et immunofluorescence pour détecter des molécules de signalisation intracellulaires sur cellules en interaction par le VIH-1 gp120 d&#39;acquisition d&#39;image et ICAM-1 sur bi-couches, iii) par microscopie TIRF, unee iv) l&#39;analyse des données. Ce système génère des images à haute résolution de l&#39;interface VS-delà de celle obtenue avec le classique système de cellule-cellule, car elle permet la détection de groupes distincts de chaque composants moléculaires de VS long avec des molécules de signalisation spécifiques recrutés pour ces sous-domaines.

Watch this Scientific Journal Video about Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers at JoVE.com

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

Cet article décrit une méthode pour visualiser la formation d&#39;une synapse enveloppe du VIH-1 induite par le verre virologique soutenu bicouches planaires par fluorescence à réflexion interne totale (TIRF) microscopie. La méthode peut également être combiné avec immunofluorescence pour détecter l&#39;activation et la redistribution des molécules de signalisation qui se produisent pendant le VIH-1 induite par l&#39;enveloppe formation de la synapse virologique.

Imagerie de VIH-1 Enveloppe induite Synapse virologique et de signalisation sur bicouches lipidiques synthétiques

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

imaging-hiv-1-envelope-induced-virological-synapse-signaling-on

Research

JoVE Journal

Immunology and Infection

12.1K vues.  New York University Langone School of Medicine. Cet article décrit une méthode pour visualiser la formation d'une synapse enveloppe du VIH-1 induite par le verre virologique soutenu bicouches planaires par fluorescence à réflexion interne totale (TIRF) microscopie. La méthode peut également être combiné avec immunofluorescence pour détecter l'activation et la redistribution des molécules de signalisation qui se produisent pendant le VIH-1 induite par l'enveloppe formation de la synapse virologique...

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

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.

Inférence génotypique de tropisme du VIH-1 en utilisant la population basée séquençage du 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 ...

Recherche

Immunologie et infection

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]).

Prédiction du VIH-1 Utilisation corécepteur (tropisme) par analyse de séquence en utilisant une approche génotypique

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.

En décapsidation in vitro du VIH-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 ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

De nouveaux outils pour multiplier des cellules T régulatrices de personnes VIH-1-infected

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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.

Évaluation de conformation du VIH-1 trimère glycoprotéines d&#39;enveloppe l&#39;aide d&#39;un base de cellules test ELISA

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

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-résolution de l&#39;imagerie cellulaire Synapse immunologique Natural Killer sur un verre soutenu par Planar lipidique en deux couches

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

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.

Isolement des exosomes dans le plasma du VIH-1 individus positifs

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.

Visualisation du VIH-1 Gag Reliure Giant unilamellaires vésiculaires (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 ...

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.

Mesure de<em&gt; In vitro</em&gt; Activité d&#39;intégration du VIH-1 préintégration 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 ...

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.