Este trabajo fue apoyado por subvenciones del NIH AI071815 (CEH) y el Premio al Desarrollo de Nanomedicina Hoja de Ruta Centro 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+Bond+Dynamics+in+Contacts+between+T+Lymphocytes+and+Glass-supported+Planar+Bilayers+Reconstituted+with+the+Immunoglobulin-related+Adhesion+Molecule+CD58.">Adhesive Bond Dynamics in Contacts between T Lymphocytes and Glass-supported Planar Bilayers Reconstituted with the Immunoglobulin-related Adhesion Molecule CD58.</a> J. Biol. Chem. 272, 15782-15788 (1997).</li><li>Fortin, J. 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>

Los autores declaran no tener conflicto de intereses.

Estudios previos han VS visualizan en el sistema conjugado célula-célula, sin embargo estos estudios no proporcionan imágenes de alta resolución para visualizar las estructuras supramoleculares en la sinapsis. En nuestro laboratorio, se utilizó el vidrio apoyada sistema bicapa plana para representar la superficie de las células infectadas que expresan la gp120 envoltura del virus y la molécula de adhesión celular ICAM-1. En conjunción con microscopía TIRF, que detecta las señales de fluorescencia dentro de 100-200 nm desde la superficie del bicapa con una alta relación señal-ruido, hemos sido capaces de detectar la segregación supramolecular de la gp120 de la ICAM-1 en el VS. Además, el método estándar de inmunotinción puede aplicarse al sistema bicapa y se utiliza aquí para detectar y cuantificar la contratación específica de sesión Lck, pero no Fionia, a la zona de contacto en la gp120-SV 15. Por lo tanto, la bicapa plana ofrece un sistema experimental para imágenes de alta resolución de la interfaz de sinapsis en un plano 2D TIRF microscopia, así como los métodos de iluminación de campo amplio o confocal. Sin embargo, el sistema también tiene limitaciones como el ajuste de la movilidad ligando, fuera del plano de flexión, y las fluctuaciones de las membranas biológicas no se reproducen por bicapas planas. Además, este es un sistema in vitro y por lo tanto tiene otras limitaciones, tales como la falta de otras moléculas de la membrana que estarían presentes en una célula infectada y la maquinaria citoesqueleto que regula la movilidad molecular y la motilidad celular. Además, la dinámica y la distribución de las moléculas, tales como trímeros frente a monómeros de gp120, no podrán estar representados fisiológicamente en la bicapa. Sin embargo, aun con estas limitaciones, este sistema sigue siendo muy valiosa para el estudio del virus de las células o las interacciones célula-célula, y estos métodos pueden servir como una guía útil para los investigadores que están buscando imágenes de alta resolución para detectar la organización supramolecular que no es discernible en el sistema conjugado convencional célula-célula.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Su etiquetados gp120 del VIH </td><td> Regalo del Dr. Cho </td><td></td><td> DH12 </td></tr><tr><td> Su etiquetados gp120 del VIH </td><td> Tecnología Inmune </td><td></td><td> Varios X4 o R5 trópico </td></tr><tr><td> HSA </td><td> Williams Medical Company </td><td> 521302 </td><td> Albúmina sérica humana al 25% </td></tr><tr><td> Amicon centrífugas Filtros Ultra </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30K </td></tr><tr><td> Lck Conejo MAB </td><td> Señalización Celular </td><td> 2787 </td><td></td></tr><tr><td> p-Lck Rabbit pAb </td><td> Santa Cruz Biotechnology Inc. </td><td> Sc-101728 </td><td> Tyr 394 </td></tr><tr><td> Fionia Conejo 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 cabra anti-conejo IgG (H + L) </td></tr><tr><td> Alexa Fluor 488 </td><td> Molecular Probes </td><td> Una-20000 </td><td> carboxílico succinimidil éster </td></tr><tr><td> FCS2 Cámara </td><td> Bioptechs </td><td> 060319-2-03 </td><td></td></tr><tr><td> Microaqueduct Slide </td><td> Bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td> 30mm Redonda w / agujeros </td><td> Bioptechs </td><td> 1907-08-750 </td><td> 0,75 mm de espesor </td></tr><tr><td> Rectángulo Junta </td><td> Bioptechs </td><td> 1907-1422-250 </td><td> 0,25 mm de espesor 14x24 </td></tr><tr><td> Tubo Tygon </td><td> Bioptechs </td><td> 20202275 </td><td> 1/16 &quot;(25 pies) </td></tr><tr><td> Llave de tres vías </td><td> BioRad </td><td> 7328103 </td><td></td></tr><tr><td> Llave de paso de dos vías </td><td> BioRad </td><td> 7328102 </td><td></td></tr><tr><td> Slide Sticky E 0.2 Luer </td><td> Ibidi </td><td> 80168 </td><td></td></tr><tr><td> Cubreobjetos </td><td> Ibidi </td><td> 10812 </td><td></td></tr><tr><td> Jeringa 1ml </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> PERROS-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> Cubreobjetos </td><td> Bioptechs </td><td> 40-1313-0319 </td><td> 40mm </td></tr><tr><td> TIRF microscopio </td><td> Nikon </td><td></td><td></td></tr><tr><td> ImageJ </td><td> Los Institutos Nacionales de Salud </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

Virus de inmunodeficiencia humana tipo 1 (VIH-1) la infección se produce más eficientemente a través de célula a célula de transmisión 2,10,11. Esta célula a célula de transferencia entre células T CD4 + implica la formación de una sinapsis virológica (VS), que es un F-actina dependiente de célula-célula unión formada sobre el compromiso de VIH-1 sobre la gp120 en la célula infectada con CD4 y el receptor de quimioquinas (CKR) CCR5 o CXCR4 en la célula diana 8. Además de la gp120 y sus receptores, otras proteínas de membrana, en particular la molécula de adhesión de LFA-1 y sus ligandos, la familia ICAM, juegan un papel importante en la formación de VS y la transmisión del virus, ya que están presentes en la superficie de las células del donante infectadas por virus y las células diana, así como sobre la envoltura de VIH-1 viriones 1,4,5,6,7,13. VS formación también es acompañada por eventos de señalización intracelulares que se transducen como resultado de la gp120-compromiso de sus receptores. De hecho, recientemente hemos mostraron que las células CD4 &lt;sup&gt; + la interacción de las células T con gp120 induce el reclutamiento y la fosforilación de moléculas de señalización asociadas a la signalosome TCR como Lck, CD3, ZAP70, LAT, SLP-76, ITK, y 15 PLCγ. En este artículo se presenta un método para visualizar acuerdo supramolecular y proximal a la membrana señalización de eventos que tienen lugar durante la formación de VS. Tomamos ventaja del vidrio-apoyado plana bi-capa del sistema como un modelo reduccionista para representar la superficie de células infectadas por VIH que lleven la envoltura viral gp120 y la molécula de adhesión celular ICAM-1. El protocolo describe los procedimientos generales para el seguimiento de la gp120 del VIH-1 inducida por VS montaje y eventos de la señal de activación que incluyen i) bi-capa de preparación y montaje en una celda de flujo, ii) la inyección de las células y la tinción de inmunofluorescencia para la detección de moléculas de señalización intracelular en las células que interactúan con el VIH-1 de adquisición de imágenes gp120 y la ICAM-1 en bi-capas, iii) por microscopía TIRF, unnd iv) el análisis de datos. Este sistema genera imágenes de alta resolución de la interfaz de VS más allá de lo logrado con el convencional de células-células del sistema ya que permite la detección de distintos grupos de los distintos componentes moleculares de VS, junto con las moléculas de señalización específicos de contratación de estos sub-dominios.

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

En este artículo se describe un método para visualizar la formación de una sinapsis virológica de VIH-1 sobre de cristal inducido por el apoyo bicapas planas de reflexión total interna de fluorescencia (TIRF) microscopía. El método también se puede combinar con la tinción de inmunofluorescencia para detectar la activación y la redistribución de las moléculas de señalización que se producen durante el VIH-1 sobre inducida por la formación de sinapsis virológicas.

Imagen del VIH-1 sobre inducida sinapsis virológica y señalización en la bicapa lipídica sintéticas

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 vistas.  New York University Langone School of Medicine. En este artículo se describe un método para visualizar la formación de una sinapsis virológica de VIH-1 sobre de cristal inducido por el apoyo bicapas planas de reflexión total interna de fluorescencia (TIRF) microscopía. El método también se puede combinar con la tinción de inmunofluorescencia para detectar la activación y la redistribución de las moléculas de señalización que se producen durante el VIH-1 sobre inducida por la formación de sinapsis viroló...

Video: 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.

Inferencia genotípica del VIH-1 tropismo utilizando la población basada en la secuencia de 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 ...

Investigación

Inmunología e Infección

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]).

Predicción del VIH-1 usos correceptor (tropismo) por análisis de secuencia utilizando un enfoque genotípica

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 uncoating in vitro del 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.

Nuevas herramientas para expandir células T reguladoras de las personas 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.

Evaluación conformacional del VIH-1 glicoproteínas de la envoltura trimérica El uso de un ELISA ensayo basado en células

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-resolución de imagen de la célula Natural Killer inmunológica Synapse en un apoyada-Glass planar bicapa lipídica

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.

Aislamiento de exosomas del plasma de VIH-1 en individuos positivos

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.

Visualización de VIH-1 Gag unión a Giant unilamelares vesículas membranas (GUV)

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.

Medida de<em&gt; In Vitro</em&gt; Integración Actividad del VIH-1 preintegration Complejos

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.