Agradecemos al Dr. James Robinson por su generosa donación de la A32, 17b, 48d, y C11 mAbs. PGT 121 se obtuvo a través del Programa de reactivos de SIDA de los NIH, la División de SIDA, NIAID, NIH (Cat # 12343). Este trabajo fue apoyado por la Fundación Canadiense para la Innovación Líder del Programa # 29866, por un operativo CIHR # 257792, por un Establecimiento FRQS de Joven Científico Grant # 24639 de la FA y por un subsidio continuo CRCHUM, así como por un CIHR subvención catalizador # 126630 a AF y RM. AF es el destinatario de un FRSQ Chercheur Boursier Junior 1 beca # 24639. MV fue apoyado por un Premio de Investigación Doctoral CIHR # 291485.

1. Día 1 - Cultivo Celular <ol><li> Plate 2 x 10 4 células por pocillo en un 96 pocillos placa de cultivo celular opaco adecuado para la lectura de luminiscencia osteosarcoma humano (HOS). Utilice Medio Eagle Modificado de Dulbecco (DMEM) suplementado con 10% de suero bovino fetal (FBS) y 100 U / ml de penicilina-estreptomicina. Incubar hasta el día siguiente a 37 ° C, 5% de CO 2. </li></ol> 2. Día 2 - Polietilenimina (PEI) Transfección <ol><li> Preparar la mezcla de transfección de acuerdo con los pasos subsiguientes. Ajuste reactivos y cantidades de ADN de acuerdo con el número de pozos que van a ser transfectadas con el mismo Env. </li><li> Tubo A: Añadir 10 ng de plásmido Tat-codificación (tal como pTat-III 27) y 150 ng de plásmido que codifica la Env a 5 l de DMEM suplementado con 25 mM HEPES. </li><li> El plásmido Tat-codificación sólo es necesaria para utilizar plásmidos Tat-dependientes Env-codifican como pSVIII. </li><li> Tubo B: Añadir 450 ng PEI (desde un 1g / l solución) a 5 l de DMEM. </li><li> Añadir el contenido del tubo B al tubo A. Mezclar bien por agitación durante 10 segundos e incubar la mezcla de transfección de 10 minutos a temperatura ambiente (22 ° C). </li><li> Añadir 10 l de la mezcla de transfección por pocillo de la placa de 96 pocillos. Incubar durante 48 horas a 37 ° C, 5% de CO 2. </li></ol> 3. Día 4 - ELISA <ol><li> Lleve a cabo todos los experimentos a temperatura ambiente para reducir al mínimo posible la endocitosis de los complejos Env / anticuerpos. </li><li> Preparar 250 ml de tampón de lavado por placa que se utilizan al mismo tiempo. Tampón de Lavado es solución salina tamponada con Tris 1X (TBS) pH 7,5 (50 mM Tris-Cl, pH 7,5; NaCl 150 mM), suplementado con 1 mM de MgCl 2 y 1,8 mM CaCl 2. </li><li> Preparar 125 ml de tampón de bloqueo por plato añadiendo leche en polvo sin grasa 1% y 5 mM Tris pH 8,0 y tampón de lavado. </li><li> Retire el medio de cultivo de células y mezcla de transfección (sobrenadante) de la placa de 96 pocillos. </li><li> Añadir 100 μ; L de tampón de bloqueo por pocillo y se incuba 20 min a TA. </li><li> Eliminar el sobrenadante y añadir 50 l de anticuerpo (o suero) por pocillo, se diluyó a la concentración apropiada en tampón de bloqueo. Típicamente, usar una concentración de 1 g / ml. Incubar 1 hora a temperatura ambiente. </li><li> Lave 3 veces con 100 l tampón de bloqueo y luego repetir 3x proceso de lavado con 100 l tampón de lavado. </li><li> Aspirar el sobrenadante, añadir 100 l tampón de bloqueo, e incubar 5 minutos a temperatura ambiente. </li><li> Aspirar el sobrenadante y añadir 50 l de anticuerpo secundario, diluido 1/3000 en tampón de bloqueo. Variar dilución óptima de anticuerpo de acuerdo a las diferencias fabricante. Incubar 40 min a RT. </li><li> Lave 3 veces con 100 l tampón de bloqueo y luego repetir 3x proceso de lavado con 100 l tampón de lavado. </li></ol> 4. Adquisición de Datos <ol><li> Aspirar el sobrenadante de la placa y añadir 30 l 1x aumento de quimioluminiscencia (ECL) de sustrato por pocillo. </li><li> Adquirir chemiluminescence señal durante 1 seg / pocillo en un lector de placas adecuado de acuerdo con las instrucciones del fabricante. El tiempo de lectura puede variar de acuerdo a las diferencias de hardware. </li></ol>

<ol>
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Los autores declaran no tener conflictos de interés.

Este ensayo se optimiza para detectar la interacción de los mAb específicos con el VIH-1 trimérica Env expresada en la superficie celular. Una vez que el protocolo ha sido establecida, puede ser utilizado en medios a altos rendimientos con bajos costes de materiales globales y pequeñas cantidades de anticuerpos. Dado que este ensayo está basado en la transfección, puede ser fácilmente adaptado para la coexpresión de las proteínas celulares tales como CD4 con el fin de estudiar sus efectos sobre la conformación...

Humana tipo 1 del virus de inmunodeficiencia (VIH-1) de entrada, mediada por las glicoproteínas de la envoltura viral triméricos (ENV) es el primer paso del ciclo infeccioso. Siendo el único antígeno viral expuesto presentado en la superficie de los viriones, la Env trímero provoca anticuerpos neutralizantes y no neutralizantes. Como tal, representa un candidato interesante para el diseño inmunógeno de la vacuna. Sin embargo, los ensayos de vacunación con Env en formas solubles o recombinantes suscitó respuestas con sólo una eficacia mínima contra más primaria del VIH-1 aislados de 1-3. No obstante, la eficacia parcial observada en el ensayo de la vacuna RV144 4 renovado interés en el VIH-1 Env como candidato inmunógeno. Esto fue corroborado por un reciente estudio describiendo que los anticuerpos anti-Env vacuna-suscitó fueron suficientes para generar un cierto grado de protección contra el VIS y el VIH desafía 5. Después de ser sintetizada en el retículo endoplásmico, el glycoprote Enven precursora, gp160, experimenta varias modificaciones post-traduccionales que son críticos para su capacidad para alimentar el proceso de fusión viral. El precursor de Env debe doblar correctamente y asociado en trímeros antes de ser escindida en sus gp120 y gp41 transmembrana extra-citoplasmáticos subunidades 6-10, con interacciones no covalentes que mantienen el enlace con gp120-gp41. La maquinaria de la célula infectada también es en gran medida responsable de glicosilar Env, que comprende aproximadamente 50% de su masa total 11,12. La compleja estructura resultante permite Env para ser conformacionalmente flexible de 13,14, mientras que proporciona una metaestabilidad que se piensa para permitir Env para adaptarse y ocultar ciertos epítopos altamente inmunogénicos que de otro modo estar expuestos 15-19, subrayando la importancia de comprender mejor las diferentes conformaciones muestreada por el trimer Env nativa. Hasta la fecha, varias técnicas se han desarrollado y utilizado con éxito para estudiar Env ch conformacionalanges. Sin embargo, varían en sus limitaciones, está a menudo restringido a contextos específicos Env. Por ejemplo, la resonancia de plasmón de superficie o ensayos de inmunoprecipitación utilizando anticuerpos monoclonales específicos de conformación (mAbs), se basan ya sea en moléculas Env solubles o solubilizados monoméricas que se sabe que son inmunogenéticamente diferentes de sus formas triméricas 20,21. Estudios recientes también sugieren que la escisión afecta conformaciones resultantes Env en la exposición de los epítopos reconocidos principalmente por anticuerpos no neutralizantes 14,22,23. A continuación se describe en detalle un método que permite la determinación rápida y fácil de la conformación de celularmente-expresado Env trímeros 18,24-26. Después de la transfección transitoria de Env en una línea de células adherentes humana la unión de los anticuerpos de Env-específicos se detecta utilizando una reacción de quimioluminiscencia simple. Esta técnica también se puede utilizar para caracterizar la preferencia conformacional de conformación depen-anticuerpos abolladura. Por lo tanto, este ensayo proporciona un método robusto y altamente flexible de detección.

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			<td height="21" style="height:21px;width:216px;">HOS cells</td>
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			<td height="42" style="height:42px;width:216px;">White Opaque Tissue Culture Plate, 96 well, Flat Bottom</td>
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			<td height="42" style="height:42px;width:216px;">Polyethylenimine, linear, 25000 MW</td>
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			<td>Prepared in 1mg/ml solution</td>
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			<td height="42" style="height:42px;width:216px;">Goat Anti-Human IgG, Peroxidase Conjugated</td>
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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.

Usando el procedimiento general descrito anteriormente, se adaptó el protocolo para ensayar el impacto de CD4 soluble (sCD4) y CD4 celular coexpressed en la exposición de los epítopos CD4i en cualquiera de tipo salvaje (wt) o mutado Env, como se describió previamente 18,24, 25,28. La figura 1 representa esquemáticamente el procedimiento general y la exposición de epítopos CD4i después del tratamiento con sCD4 o por coexpresión de CD4 celular 18. En la Figura 2,</st...

Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

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

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 ...

conformational-evaluation-hiv-1-trimeric-envelope-glycoproteins-using

Research

JoVE Journal

Immunology and Infection

14.2K vistas.  Université de Montréal. 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.

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

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.

Marcado específico del VIH-1 células positivas utilizando un vector lentiviral Ap-dependientes que expresan la proteína verde fluorescente

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, ...

Investigación

Inmunología e Infección

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 ...

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Determinación de estructuras moleculares de glicoproteínas de la envoltura del VIH con Cryo-tomografía electrónica y automatizada promedio Sub-tomografía

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

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 ...

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

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

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

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 ...

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.

La evaluación de la innata de detección de VIH-1 CD4 infectados<sup&gt; +</sup&gt; Células T por las células dendríticas plasmocitoides Usando un<em&gt; Ex vivo</em&gt; Co-cultura del sistema.

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

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Un promedio de glicoproteína de la envoltura viral Espigas de Electron cryotomography reconstrucciones utilizando Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

El recocido mediada nucleocápside electroforesis (NOMBRE) ensayo permite la identificación rápida del VIH-1 inhibidores de la nucleocápside

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

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. ...