עבודה זו נתמכה על ידי NIH מענקים AI071815 (CEH) ו Nanomedicine מפת הדרכים לפיתוח מרכז פרס PN2EY016586 (MLD).

<ol>
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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>

החוקרים מצהירים שום ניגוד עניינים.

מחקרים קודמים VS דמיינו במערכת תאים תאים הצמוד, אולם מחקרים אלה לא סיפקו תמונות ברזולוציה גבוהה מספיק כדי להמחיש את מבנים מולקולריים בסינפסה. במעבדה שלנו, השתמשנו זכוכית נתמך במערכת bilayer מישוריים לייצג את פני השטח של תאים נגועים המבטאים את המעטפה הנגיף gp120 ו מולקולה הידבקות הסלולר ICAM-1. יחד עם מיקרוסקופיה TIRF, אשר מזהה אותות הקרינה בתוך 100-200 ננומטר מפני השטח bilayer עם יחס אות לרעש גבוה, הצלחנו לזהות הפרדה מולקולריים של gp120 מ ICAM-1 ב VS. יתר על כן, שיטת immunostaining רגיל יכול להיות מיושם על מערכת bilayer ושימש כאן כדי לזהות ולכמת את הגיוס הספציפי של פעיל Lck, אבל לא פין, לאזור gp120 מגע ב VS 15. לפיכך, bilayer מישוריים מציעה מערכת ניסיונית עבור הדמיה ברזולוציה גבוהה של ממשק סינפסה במטוס 2D ידי TIRF מיקרוscopy, כמו גם שיטות תאורה שדה רחב או confocal. עם זאת, המערכת כוללת גם מגבלות כמו התאמת ניידות ליגנד, מחוץ למטוס כיפוף, וכן תנודות של ממברנות ביולוגיות לא לשכפל ידי bilayers מישוריים. יתר על כן, זו מערכת in-vitro, ולכן יש מגבלות אחרות, כמו חוסר מולקולות בממברנה אחרים שיהיו נוכחים על תא נגוע מכונות cytoskeleton המווסת את הניידות מולקולרית תנועתיות הסלולר. כמו כן, הדינמיקה והפצה של מולקולות, כמו trimers לעומת מונומרים של gp120, לא יכול להיות מיוצג על bilayer פיזיולוגית. עם זאת, גם במגבלות אלה, המערכת הזאת עדיין בעל ערך רב לחקר וירוסים תא או תאים תאים אינטראקציות, ושיטות אלה יכולים לשמש כמדריך שימושי חוקרים שמחפשים תמונות ברזולוציה גבוהה לאתר הארגון סופראמולקולרית שאינו ניכר במערכת תאים תאים הקונבנציונלית המצומד.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> HIV מתויג שלו gp120 </td><td> מתנת ד&quot;ר צ </td><td></td><td> DH12 </td></tr><tr><td> HIV מתויג שלו gp120 </td><td> החיסון טכנולוגיה </td><td></td><td> X4 שונים או R5 חוג </td></tr><tr><td> HSA </td><td> וויליאמס חברת מדיקל </td><td> 521302 </td><td> האדם סרום אלבומין 25% </td></tr><tr><td> Amicon מסננים צנטריפוגליות אולטרה </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30K </td></tr><tr><td> Lck הארנב MAB </td><td> התא איתות </td><td> 2787 </td><td></td></tr><tr><td> P-Lck הארנב PAB </td><td> סנטה קרוז ביוטכנולוגיה בע&quot;מ </td><td> SC-101728 </td><td> Tyr 394 </td></tr><tr><td> Fyn הארנב MAB </td><td> Millipore </td><td> 04-353 </td><td></td></tr><tr><td> Alexa פלואוריד 568 ° 2; Ab </td><td> בדיקות מולקולריות </td><td> A11034 </td><td> נגד ארנב עיזים IgG (H + L) </td></tr><tr><td> Alexa פלואוריד 488 </td><td> בדיקות מולקולריות </td><td> A-20000 </td><td> carboxylic חומצה succinimidyl אסתר </td></tr><tr><td> FCS2 הקאמרית </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> 30 מ&quot;מ עגול w / חורים </td><td> Bioptechs </td><td> 1907-08-750 </td><td> 0.75mm עבה </td></tr><tr><td> מלבן אטם </td><td> Bioptechs </td><td> 1907-1422-250 </td><td> 14x24 0.25mm עבה </td></tr><tr><td> Tygon Tubing </td><td> Bioptechs </td><td> 20202275 </td><td> 1/16 &quot;(25ft) </td></tr><tr><td> שלוש דרך שסתום </td><td> BioRad </td><td> 7328103 </td><td></td></tr><tr><td> דו דרך שסתום </td><td> BioRad </td><td> 7328102 </td><td></td></tr><tr><td> Slide דביק אני 0.2 Luer </td><td> Ibidi </td><td> 80168 </td><td></td></tr><tr><td> כיסוי משקפיים </td><td> Ibidi </td><td> 10812 </td><td></td></tr><tr><td> 1ml המזרק </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> כלבים, נ.ת. ע </td><td> AVANTI ליפידים פולאר </td><td> 790404C </td><td></td></tr><tr><td> DOPC </td><td> AVANTI ליפידים פולאר </td><td> 850375C </td><td></td></tr><tr><td> זכוכית coverslip </td><td> Bioptechs </td><td> 40-1313-0319 </td><td> 40mm </td></tr><tr><td> TIRF מיקרוסקופ </td><td> ניקון </td><td></td><td></td></tr><tr><td> ImageJ </td><td> המכונים הלאומיים לבריאות </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

וירוס הכשל החיסוני האנושי סוג 1 (HIV-1) ההדבקה מתרחשת בצורה היעילה ביותר דרך תא שידור סלולרי 2,10,11. זה התא העברת התא בין CD4 + T תאים כרוך היווצרות הסינפסה virological (VS), שהוא ה-F-אקטין תלויה תאים תאים צומת הוקמה על האירוסין של מעטפת ה-HIV 1-gp120 על התא הנגוע עם CD4 ו הקולטן chemokine (CKR) CCR5 או CXCR4 על תא המטרה 8. בנוסף gp120 ואת הקולטנים שלו, חלבוני קרום אחרים, בעיקר מולקולות הדבקה LFA-1 ו ligands שלה, משפחת ICAM, לשחק תפקיד מרכזי ביצירת VS והעברת נגיף כפי שהם נמצאים על פני השטח של התאים הנגועים בנגיף התורמות ואת היעד תאים, כמו גם על המעטפה של HIV-1 virions 1,4,5,6,7,13. היווצרות VS מלווה גם אירועים איתות תאיים, כי הם transduced כתוצאה מעורבות-gp120 של הקולטנים שלו. אכן, יש לנו לאחרונה הראה כי CD4 &lt;sup&gt; + אינטראקציה עם תא T gp120 גורם גיוס זרחון של מולקולות איתות הקשורים signalosome TCR כולל Lck, CD3ζ, ZAP70, LAT, SLP-76, Itk, ו PLCγ 15. במאמר זה, אנו מציגים שיטה לחזות הסדר סופראמולקולרית ו קרום הפרוקסימלי איתות אירועים המתרחשים במהלך היווצרות VS. אנו מנצלים את מערכת הזכוכית נתמך שכבה דו מישורי כמודל הרדוקציוניסטית לייצג את פני השטח של תאים נגועים ב-HIV הנושאים את מעטפת הנגיף gp120 ו מולקולה הידבקות הסלולר ICAM-1. הפרוטוקול מתאר צעדים כלליים ניטור HIV-1 gp120-Induced VS הרכבה והפעלה האות אירועים הכוללים i) שכבת דו הכנה והרכבה בתא זרימה, II) הזרקה של תאים צביעה immunofluorescence לזהות מולקולות איתות תאיים על תאים אינטראקציה עם רכישת ה-HIV-1 תמונה gp120 ו ICAM-1 על דו שכבות, III) על ידי מיקרוסקופ TIRF,ND ד) ניתוח הנתונים. מערכת זו מפיקה תמונות ברזולוציה גבוהה של ממשק VS מעבר לזה שהושג עם מערכת תאים תאים קונבנציונלי שכן היא מאפשרת זיהוי של קבוצות שונות של רכיבים מולקולריים בודדים של VS יחד עם מולקולות איתות מסוימות שגויסו אלה תת תחומים.

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

מאמר זה מתאר שיטה לחזות היווצרות הסינפסה virological HIV-1 המעטפה הנגרמת על bilayers זכוכית הנתמכים מישוריים על ידי מיקרוסקופ פלואורסצנטי סך פנימית (TIRF) השתקפות. השיטה ניתן גם לשלב עם כתמים immunofluorescence לזהות הפעלה חלוקה מחדש של מולקולות איתות המתרחשים במהלך היווצרות ה-HIV-1 המעטפה הנגרמת סינפסה virological.

הדמיה של HIV-1 מעטפה הנגרמת סינפסה Virological והאיתות על bilayers שומנים סינתטי

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

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JoVE Journal

Immunology and Infection

12.1K Views.  New York University Langone School of Medicine. מאמר זה מתאר שיטה לחזות היווצרות הסינפסה virological HIV-1 המעטפה הנגרמת על bilayers זכוכית הנתמכים מישוריים על ידי מיקרוסקופ פלואורסצנטי סך פנימית (TIRF) השתקפות. השיטה ניתן גם לשלב עם כתמים immunofluorescence לזהות הפעלה חלוקה מחדש של מולקולות איתות המתרחשים במהלך היווצרות ה-HIV-1 המ?...

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.

הסקה Genotypic של כמיהות HIV-1 שימוש אוכלוסייה מבוססת רצף של 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 ...

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]).

ניבוי של שימוש בנגיף HIV-1 Coreceptor (זיקה) על ידי ניתוח רצף באמצעות גישת genotypic

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.

ב Uncoating במבחנה של HIV-1 ליבות

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.

כלים חדשים כדי להרחיב תאי T רגולטוריים מאנשים HIV-1 נגוע-

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.

הערכת קונפורמציה של HIV-1 trimeric מעטפת גליקופרוטאינים שימוש ELISA Assay מבוסס Cell

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.

הדמיה ברזולוציה סופר של תא הרוצח הטבעי החיסונית סינפסה על מישורי שומנים bilayer נתמך-זכוכית

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.

בידוד של Exosomes מהפלזמה של HIV-1 אנשים חיוביים

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.

ויזואליזציה של HIV-1 Gag מחייב את הענק Unilamellar שלפוחיות (בוס) ממברנות

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.

מדידה<em&gt; במבחנה</em&gt; פעילות האינטגרציה של HIV-1 Preintegration מכלולים

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.