Este trabalho foi financiado pelo NIH concede AI071815 (CEH) e do Roteiro Nanomedicina prêmio Centro de Desenvolvimento PN2EY016586 (MLD).

<ol>
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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>

Os autores declaram nenhum conflito de interesses.

Estudos anteriores têm VS visualizado no sistema conjugado célula-célula, porém esses estudos não fornecem imagens de resolução alta o suficiente para visualizar as estruturas supramoleculares na sinapse. No nosso laboratório, utilizamos o vidro suportada sistema bicamada planar para representar a superfície das células infectadas que expressam o gp120 envelope do vírus e da molécula de adesão celular ICAM-1. Em conjunção com TIRF microscopia, que detecta sinais de fluorescência dentro 100-200 nm a partir da superfície bicamada com uma relação sinal-ruído elevado, fomos capazes de detectar a segregação supramolecular da gp120 a partir de ICAM-1 no VS. Além disso, o método de imunocoloração padrão pode ser aplicada ao sistema de bicamada e foi utilizado aqui para detectar e quantificar o recrutamento específico de Lck activa, mas não Fyn, para a área de contacto gp120-at VS 15. Assim, a bicamada planar oferece um sistema experimental para geração de imagens de alta resolução de interface de sinapse num plano 2D por TIRF microescopia, bem como métodos de iluminação de campo ou gama-confocal. No entanto, o sistema também tem limitações como ajustar mobilidade ligante, fora do plano de flexão, e as flutuações de membranas biológicas não são reproduzidas por bicamadas planas. Além disso, este é um sistema in vitro e, portanto, tem outras limitações, tais como falta de moléculas da membrana outros que estariam presentes em uma célula infectada e as máquinas citoesqueleto, que regula a mobilidade molecular e da motilidade celular. Além disso, as dinâmicas e distribuição das moléculas, tais como trímeros, versus monómeros de gp120, não podem ser representados fisiologicamente sobre a bicamada. No entanto, mesmo com estas limitações, este sistema é ainda muito valiosa para o estudo do vírus na célula ou interacções célula-célula, e estes métodos podem servir como um guia útil para os investigadores que procuram imagens de alta resolução para detectar organização supramolecular que não é perceptível no sistema conjugado convencional célula-célula.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Sua HIV marcados gp120 </td><td> Presente do Dr. Cho </td><td></td><td> DH12 </td></tr><tr><td> Sua HIV marcados gp120 </td><td> Tecnologia imune </td><td></td><td> Vários X4 ou R5 trópico </td></tr><tr><td> HSA </td><td> Williams Medical Company </td><td> 521302 </td><td> Soro Humano 25% de albumina </td></tr><tr><td> Amicon Ultra Filtros Centrífugos </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30K </td></tr><tr><td> Lck Coelho mAb </td><td> Sinalização celular </td><td> 2787 </td><td></td></tr><tr><td> p-Lck coelho pAb </td><td> Santa Cruz Biotechnology Inc. </td><td> Sc-101728 </td><td> Tyr 394 </td></tr><tr><td> Fyn Coelho 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> Cabra anti-coelho IgG (H + L) </td></tr><tr><td> Alexa Fluor 488 </td><td> Molecular Probes </td><td> Um-20000 </td><td> carboxilico succinimidil éster </td></tr><tr><td> FCS2 Câmara </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> Rodada 30 milímetros w / furos </td><td> Bioptechs </td><td> 1907-08-750 </td><td> 0,75 milímetros de espessura </td></tr><tr><td> Retângulo Junta </td><td> Bioptechs </td><td> 1907-1422-250 </td><td> 14x24 0,25 milímetros de espessura </td></tr><tr><td> Tubulação de Tygon </td><td> Bioptechs </td><td> 20202275 </td><td> 1/16 &quot;(25 pés) </td></tr><tr><td> Torneira de três vias </td><td> BioRad </td><td> 7328103 </td><td></td></tr><tr><td> Two-way Torneira </td><td> BioRad </td><td> 7328102 </td><td></td></tr><tr><td> Deslize pegajoso eu 0,2 Luer </td><td> Ibidi </td><td> 80168 </td><td></td></tr><tr><td> Cubra óculos </td><td> Ibidi </td><td> 10812 </td><td></td></tr><tr><td> Seringa de 1ml </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> CÃES-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> Vidro lamela </td><td> Bioptechs </td><td> 40-1313-0319 </td><td> 40 milímetros </td></tr><tr><td> Microscópio 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

Vírus da imunodeficiência humana tipo 1 (HIV-1) a infecção ocorre de forma mais eficiente através de célula para célula de transmissão 2,10,11. Esta célula para a transferência de células entre células T CD4 + envolve a formação de uma sinapse virológica (VS), que é uma F-actina-dependente junção célula-célula formada a partir do envolvimento do HIV-1 envelope gp120 na célula infectada com CD4 e o receptor de quimiocina (CKR) CCR5 ou CXCR4 na célula alvo 8. Além disso a gp120 e seus receptores, outras proteínas da membrana, em particular a molécula de adesão LFA-1 e os seus ligandos, a família ICAM, desempenham um papel importante na formação VS e transmissão de vírus em que estejam presentes na superfície das células infectadas pelo vírus de doadores e células alvo, bem como no envelope de HIV-1 viriões 1,4,5,6,7,13. Formação VS é também acompanhada por eventos de sinalização intracelulares que são transduzidas como um resultado da gp120-engajamento dos seus receptores. Na verdade, temos recentemente mostrou que os CD4 &lt;sup&gt; + interacção das células T com gp120 induz o recrutamento e fosforilação de moléculas de sinalização associados com o signalosome TCR incluindo Lck, CD3ζ, ZAP70, LAT, SLP-76, ITK, e PLCγ 15. Neste artigo, apresentamos um método para visualizar arranjo supramolecular e eventos membrana proximais de sinalização que ocorrem durante a formação do VS. Nós tirar vantagem do vidro suportada sistema bi-camada plana como um modelo reducionista para representar a superfície de células infectadas pelo HIV munidos do envelope viral gp120 e da molécula de adesão celular ICAM-1. O protocolo descreve procedimentos gerais para a monitorização do HIV-1 gp120 induzida por VS montagem e os eventos de sinal de activação que incluem i) preparação bi-camada e de montagem de uma célula de fluxo, ii) A injecção de células e de coloração de imunofluorescência para detectar moléculas de sinalização intracelulares em células interagindo com HIV-1 de aquisição de imagem gp120 e ICAM-1 em bi-camadas, iii) por microscopia TIRF, umand iv) análise dos dados. Este sistema gera imagens de alta resolução de VS interface além que a obtida com o sistema de célula-célula convencional uma vez que permite a detecção de grupos distintos de componentes individuais moleculares de VS, juntamente com moléculas de sinalização específicas recrutados para essas sub-domínios.

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

Este artigo descreve um método para visualizar a formação de uma sinapse envelope de HIV-1 induzida por virológica em vidro suportados bicamadas planares por reflexão total interna de fluorescência microscopia (TIRF). O método pode também ser combinado com coloração de imunofluorescência para detectar a activação e redistribuição de moléculas de sinalização que ocorrem durante o HIV-1 a formação de sinapses envelope induzida virológica.

Imagem do HIV-1 Synapse Envelope induzida virológica e Sinalização em bicamadas lipídicas 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 visualizações.  New York University Langone School of Medicine. Este artigo descreve um método para visualizar a formação de uma sinapse envelope de HIV-1 induzida por virológica em vidro suportados bicamadas planares por reflexão total interna de fluorescência microscopia (TIRF). O método pode também ser combinado com coloração de imunofluorescência para detectar a activação e redistribuição de moléculas de sinalização que ocorrem durante o HIV-1 a formação de sinapses envelope induzida virológica.

Vídeo: 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.

Inferência genotípica do HIV-1 Tropismo Usando de base populacional de Seqüenciamento 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 ...

Imunologia e Infecção

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]).

Previsão do HIV-1 de uso co-receptor (tropismo) por análise de seqüência usando uma abordagem 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.

Em desencapsulamento vitro do 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 ...

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.

Novas ferramentas para expandir as células T reguladoras de indivíduos HIV-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.

Avaliação conformacional do HIV-1 trimérico glicoproteínas do envelope Usando um ELISA Ensaio Celular

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-Resolution Imaging da Célula Natural Killer imunológica Synapse em um vidro com suporte 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 ...

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.

Isolamento de exossomas a partir do plasma de indivíduos HIV-1 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.

Visualização de HIV-1 Gag vinculação a gigante Unilamelares vesículas (GUV) Membranas

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.

Medição de<em&gt; In Vitro</em&gt; Actividade integração do HIV-1 de pré-integração Complexos

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.