This work was supported by NIH grants AI071815 (C.E.H.) and the Roadmap Nanomedicine Development Center award PN2EY016586 (M.L.D.).

1. Labeling GP120
Fluorescent dyes used in this protocol are effective for binding to proteins, are sufficiently photo-stabile for microscopy imaging, and match the excitation wavelengths of the lasers available with our microscope. These three criteria need to be met when selecting fluorescent molecules for tagging proteins.
<ol>
	<li>Exchange His6-tagged gp120 DH12 (a gift from Dr. Michael Cho, Iowa State University) protein buffer to sterile PBS using a centrifugal filter unit (30 kDa MWCO). Make sure gp120 concentration is not more than 1 mg/ml. Note: His6-tagged gp120 proteins from other X4 or R5 tropic strains have also been tested and are now available commercially from Immune Technologies.</li>
	<li>Add sodium bicarbonate pH 9.0 to your protein solution to obtain 50mM final concentration of sodium bicarbonate with ~pH 8.5.</li>
	<li>Add amine reactive Alexa Fluor 488 (AF488) fluorescent dye, which is dissolved in water at a 10-fold molar excess. Incubate this mixture for 1-2 hours at room temperature in the dark.</li>
	<li>To remove the excess dye, use the centrifugal filter unit as before and add fresh sterile PBS to the column. Repeat this step until all free dye is removed (3-4 washes with 4 ml PBS). Spin down until gp120 is concentrated to about 1 mg/ml. Note: Removing free dye by dialysis or centrifugal filter unit only works if the dye is water soluble, or else gel filtration can be used.</li>
	<li>To measure the fluorescence intensity per molecule of protein (F/P) of the labeled protein, determine the concentrations (in &mu;M) of the fluorescent dye at the appropriate wavelength (for example, at 488 nm for AlexaFluor 488) and protein (we use a NanoDrop spectrophotometer using the 'proteins and labels' setting) and then divide these two numbers giving you the F/P ratio. The same procedure is used for other proteins and dyes such as ICAM-1 and Cy5. Note: Other types of spectrophotometers can be used to obtain the protein concentration and fluorescent dye concentration to obtain the F/P ratio if a NanoDrop is not available.</li>
</ol>
2. Flow Cell Assembly and Bilayer Preparation
The general procedure for flow cell and bilayer preparation has been described in detail previously14. Here we outline specifically the protocol to prepare bilayers containing His6-tagged gp120 DH12 on a Bioptech flow cell. For studies involving infectious viruses or infected cells and when small volumes are necessary due to limited reagents, a disposable Ibidi flow chamber (sticky Slide I 0.2 Luer) may be used and followed with the same bi-layer preparation procedure in Steps 2.4-2.9. Information for Ibidi flow chambers can be obtained from the company website, <a href="http://www.ibidi.com/service/display_material/CA_8p_EN_150dpi.pdf">http://www.ibidi.com/service/display_material/CA_8p_EN_150dpi.pdf</a>.
<ol>
	<li>Prepare Pirhana solution (45 ml sulfuric acid (Trace Metal Grade 98%) and 15 ml 30% hydrogen peroxide) 14. With plastic clamps, immerse cover slips in the Pirhana solution for 15 minutes.</li>
	<li>Transfer cover slips to a beaker filled with pica-purified water and wash each one under running water for 2 min (one minute on each side). Place on rack to dry.</li>
	<li>Assemble Bioptechs FCSII chambers as previously described14.</li>
	<li>A liposome mixture containing 12.5% Ni2+ chelating lipids is used for capturing and presenting His6-gp120 on the bilayer surface16. Add 1 &mu;l drops of liposome mixture onto the microaqueduct slide without touching the tip to the glass. To prepare the maximum of 5 bilayers per flow cell, repeat up to 4 more times so that there are five 1&mu;l dots as shown previously14.</li>
	<li>Place cover slip on top of lipids. Cover the white retaining ring with a stainless steel clamp base unit, flip the whole apparatus over and seal. Mark location of bilayers with a marker. Incubate for 10 mins at room temperature.</li>
	<li>Attach tubing with two-way stopcock to left perfusion tube, as shown in video. Generate a positive meniscus at the end of the tubing with the 3-way stopcock on it (tubing has been previously filled with HEPES-buffered saline (HBS) containing human serum albumin (HSA) (HBS/HSA:50 ml 10x HBS [10x HEPES buffered saline: 200 mM HEPES, pH 7.2, 1.37 M NaCl, 50 mM KCl, 7 mM Na2HPO4, 60 mM D-glucose] 20 ml 25x HSA, 500 &mu;l 1M CaCl2, 1 ml 1 M MgCl2 and dH20 to bring a total volume to 500ml and filter with 0.22&mu;m filter) and is devoid of bubbles. Connect tubing to right perfusion tube and gently push buffer through flow cell.</li>
	<li>Block bilayer with ~300 &mu;l of casein containing 100 &mu;M NiCl2 by filling 1 ml syringe and attaching to the open portion of 3-way stopcock. Gently push buffer through and close both stopcocks before disengaging the syringe. Incubate for 30 min at room temperature.</li>
	<li>Add His6-tagged AF488-labeled gp120 (the concentration is determined as described previously in16). We use ~250 molecules of gp120/&mu;m2 to mimic the local density of Env clusters found on HIV-1 virion surface17. Similarly, we use ~250 molecules/&mu;m2 of ICAM-1 on the bilayer as previously reported 14. Load into flow cell as done with casein. Incubate for 30 min in dark (cover with foil) at room temperature. The same procedure is used to incorporate other proteins such as His12-tagged Cy5-labeled ICAM-1 onto the bilayer.</li>
	<li>Wash bilayers with 5 ml of buffer.</li>
</ol>
3. Quality Control of Bilayers via FRAP
Proteins in bilayers must be laterally diffusible. Before adding cells onto bilayers, it is important to assure the mobility of proteins in the prepared bilayer. Here we describe a Fluorescent Recovery After Photobleaching (FRAP) method3 that may be performed manually on most objective illuminated TIRF, wide field epifluorescence, or laser scanning confocal microscopes. The goal is to image fully uniform fields of fluorescence in the lipid bilayer, bleach a spot, and to monitor the return of unquenched molecules to the bleached area. A recovery of 50% in 2 minutes may be acceptable. All the major microscope manufacturers (Leica, Nikon, Olympus, Zeiss) sell objective illuminated TIRF systems that work well for this application. While each company provides variations of TIRF illumination and other operational features, the image quality is essentially the same.
<ol>
	<li>Use a low excitation intensity to minimize bleaching. Use a high numerical aperture objective such as a N.A. 1.3 to 1.45 40x, 60x or 100x. When using a standard fluorescent or TIRF microscope, to reduce the light intensity, put neutral density filters in the excitation light path. Most laser systems may be set for low illumination by setting an acusto optical tunable filter (AOTF) or acusto optical beam splitter (AOBS) with low voltage.</li>
	<li>Record a "pre-bleach" image. If using a cooled CCD camera, to compensate for low light conditions binning may be set to 2x2 or 4x4, the gain may be set high, or long exposures (for instance on the order of seconds) may be used. If using a confocal, the pinhole aperture may be opened; the gain set high, and slow scanning or averaging used.</li>
	<li>Bleach a spot. If using a standard fluorescence or TIRF microscope, remove all ND filters to allow for maximum intensity illumination, close the field diaphragm, and expose the sample for a few seconds. If using a laser scanning confocal, set the laser power maximum and zoom in approximately 4x to 8x to bleach a spot. A warning for confocal usage: do not zoom in too high because the bilayer may be damaged by excessively concentrated photons.</li>
	<li>At ten to fifteen second intervals, record images using the same illumination and recording settings as in 3.1 and 3.2. Within two to three minutes the spot should recover intensity approaching that of the "pre-bleach"image. Quick recovery is the sign of a successful mobile bilayer. If the spot remains dark, and especially if the edge retains high contrast, the fluorescent proteins are immobile in the bilayer and should not be used for the experiment.</li>
</ol>
Note: In our current microscope, we can deliver 0.9 mW of 641 nm light to a circular spot with an area of 240 &mu;m2 which bleaches fluorescent ICAM at typical concentrations in less than 4 seconds. Readers should find that critical alignment of an Hg or Xe lamp with bleaching times less than 30 seconds is more than sufficient to perform this assay in a repeatable manner. We would also refer you to reference3 for additional details.
4. Injection of Cells and Immunofluorescence Staining
<ol>
	<li>Warm up flow cell to 37 &deg;C (this can be done in a 37 &deg;C incubator with no CO2 for about 30 min).</li>
	<li>Add activated human CD4+ T cells (2-5x106/flow cell) in HBS/HSA ~400 &mu;l by 1 ml syringe. Let cells attach to bilayer for ~45 min in the 37 &deg;C incubator. If the Ibidi chambers are used, supply additional buffer every 15-20 min.</li>
	<li>Fix the cells by injecting 2% paraformaldehyde and incubate for 10 mins at 37&deg;C.</li>
	<li>Wash 3x with 1ml room temperature PBS buffer. Permeabilize cells by injecting 0.1% Triton X-100 for 5 min at room temperature.</li>
	<li>Wash 3x with 1ml of room temperature PBS buffer (when probing for phosphorylated proteins, you can add sodium vanadate at 1mM final concentration or another phosphatase inhibitor in PBS to prevent phosphate removal during processing). Block using casein with 5% goat serum for 25 mins at room temperature. The type of animal serum depends on the secondary antibody.</li>
	<li>Wash 3x with 1 ml of room temperature PBS buffer. Add primary antibody in ~300 &mu;l buffer/flow cell for 30 min - 1 hr at room temperature. To detect the initial membrane-proximal activation signal, we use antibodies specific for phosphorylated Lck (pLck), total Lck or Fyn.</li>
	<li>Wash 3x with 1 ml of room temperature PBS buffer. Add appropriate fluorescently labeled secondary antibody in buffer as done with primary antibody. For our experiments, we use Alexa Fluor 568-labeled goat anti-rabbit secondary. Incubate for 20 min at room temperature. Wash 3x with 1 ml of PBS buffer.</li>
	<li>Note: It is essential to have a control bilayer that is treated with only secondary antibody (no primary antibody). Follow steps 4.1-4.5 and skip 4.6, then proceed with step 4.7. This will determine the level of non-specific binding of your secondary antibody. Alternatively, one can use directly labeled primary antibodies.</li>
</ol>
5. Image acquisition by TIRF microscopy
TIRF microscopy allows excitation of fluorescent signals limited to a 250 nm or thinner plane at the glass substrate and cell interface. This guarantees image acquisition of only signals from the lipid bilayer and from the immediately apposed cell membranes. Therefore, only molecules at the synapse are imaged. Because TIRF images only the membrane-proximal area at the bottom of the cell, proteins that are internalized or redistributed into the dorsal membrane will not be detected. It may then be important to additionally acquire images by standard widefield or confocal microscopy to determine accumulation or distribution of the proteins at the synaptic area as compared to the other volumes of the cell.
All the major microscope manufacturers (Leica, Nikon, Olympus, Zeiss) sell objective illuminated TIRF systems that work well for this application. While each company provides variations of TIRF illumination and other operational features, the image quality is essentially the same. Each TIRF microscope has its own details for operation, but the following general rules apply to obtain high-resolution imaging.
<ul>
	<li>Illumination should be low to avoid bleaching.</li>
	<li>The camera should have a linear response.</li>
	<li>The camera should be capable of imaging a wide dynamic range with no saturation.</li>
	<li>All settings should be set constant for quantitative analyses.</li>
</ul>
6. Data Analysis
To study the intracellular signals activated as a result of CD4+ T cell interaction with gp120 at the VS, quantitative analyses of TIRF images are done to determine whether or not intracellular signaling molecules such as Lck and Fyn are activated and recruited to the synapse 15. Here we describe a simple method applicable for most image analysis application packages such as ImageJ and Metamorph. We have used ImageJ, which runs on Windows, Macintosh, and Linux, for our method here 12.
In the Analyze &gt; Set Measurements tab, check the boxes for Area and Mean gray value. When you make a region of interest on the image that is a polygon or a freehand traced closed shape and do Analyze &gt; Measure, the software will put the data from this measurement in a results table. The Area will be in number of pixels or in &mu;m2. The Mean is the average intensity in arbitrary units. It must have background intensity subtracted from it. Multiplying the mean minus background by area returns the integrated intensity of the protein in arbitrary units.
 
<ol>
	<li>Set the measurements to Mean and Area.</li>
	<li>Open images for one experimental condition.</li>
	<li>For each cell, trace and measure the region of interest (mean) and trace and measure a region outside the region of interest (background).</li>
	<li>When done with one experimental condition, either copy measurements and paste into Excel or other spreadsheet program or save the measurements.</li>
	<li>Return to step 6.2 for each experimental condition.</li>
	<li>When measurements are completed, calculate the results. The formulas are: 
	Average intensity = mean - background 
	Integrated intensity = Average intensity * area</li>
</ol>
7. Representative Results
To measure activation and recruitment of the initial membrane-proximal signaling molecules Lck and Fyn as a result of interaction with gp120 at the VS, primary human CD4+ T cells were introduced onto bilayers bearing gp120 and ICAM-1 15. Cells introduced to bilayers with only ICAM-1 served as a control to define the basal levels of signaling. Specific fluorescence intensity was measured within the gp120 contact area for cells on gp120 and ICAM-1 containing bilayers and within the whole contact area for cells interacting with the ICAM-1 bilayer. An increase of average fluorescence intensity is a sign of augmented recruitment and activation of the signaling molecule to the VS. This will be further demonstrated by a comparable increase in the integrated fluorescence intensity. If however, there is no change in the integrated fluorescence intensity, this indicates a redistribution of the signaling molecule.
After the cells interacted with bilayers containing gp120 and ICAM-1, total Lck and pLck(Y394) were recruited to the VS interface and colocalized with gp120 (Figs. 1 &amp; 2). The average intensity of total Lck (Fig. 1) was higher on the bilayer containing both gp120 and ICAM-1 than with ICAM-1 alone, but the integrated intensity levels (Fig. 1) were similar, suggesting that Lck is redistributed into a central cluster upon CD4+ T cell binding to gp120. However, quantification of pLck(Y394) (Fig. 2) showed that the average intensity on gp120 and ICAM-1 containing bilayers was higher than that on ICAM-1 alone bilayers, and the integrated intensity was higher on bilayers with both gp120 and ICAM-1 than on ICAM-1 alone bilayers. This indicates that while the levels of total Lck at the interface are similar in cells adhering onto gp120 and ICAM-1 and ICAM-1 alone bilayers, gp120 binding increased phosphorylation of residue Y394 at the Lck activation loop. In contrast, Fyn was not recruited to the VS (Fig. 3), as more Fyn was present in the contact area of cells on ICAM-1 alone bilayers than on bilayers containing both gp120 and ICAM-1. Consequently, we conclude that Lck, not Fyn, is the active kinase in the HIV-1 gp120-induced VS.
Figures: Membrane-proximal signaling at HIV-1 gp120-induced VS. Images of representative cells on the gp120+ICAM-1 bilayer (top panels) and the ICAM-1 bilayer (bottom panels) are shown. Fluorescence intensities of the individual cells were quantified within manually traced areas of the cell footprints as illustrated by the region marked with the yellow line in Figure 1. Quantification of average and integrated intensities detected by TIRF microscopy are presented in the left and right graphs, respectively. A total of 30 to 350 cells were quantified for each condition. Bars = 5 &mu;m. Data from one of three repeat experiments are shown.
<img alt="Figure 1" src="/files/ftp_upload/3757/3757fig1.jpg" /> 
Figure 1. CD4+ T cells were introduced onto bilayers containing gp120 and ICAM-1 or ICAM-1 alone for 45 min and then fixed and stained for total Lck.
<img alt="Figure 2" src="/files/ftp_upload/3757/3757fig2.jpg" /> 
Figure 2. CD4+ T cells were introduced onto bilayers containing gp120 and ICAM-1 or ICAM-1 alone for 45 min and then fixed and stained for pLck(Y394).
<img alt="Figure 3" src="/files/ftp_upload/3757/3757fig3.jpg" /> 
Figure 3. CD4+ T cells were introduced onto bilayers containing gp120 and ICAM-1 or ICAM-1 alone for 45 min and then fixed and stained for total Fyn.

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The authors declare no conflict of interests.

Previous studies have visualized VS in the cell-cell conjugate system; however these studies did not provide images of high enough resolution to visualize the supramolecular structures at the synapse. In our lab, we utilized the glass-supported planar bilayer system to represent the surface of infected cells expressing the virus envelope gp120 and the cellular adhesion molecule ICAM-1. In conjunction with TIRF microscopy, which detects fluorescence signals within 100-200 nm from the bilayer surface with a high signal-to-noise ratio, we were able to detect supramolecular segregation of gp120 from ICAM-1 at the VS. Moreover, the standard immunostaining method can be applied to the bilayer system and was used here to detect and quantify the specific recruitment of active Lck, but not Fyn, to the gp120-contact area at VS 15. Hence, the planar bilayer offers an experimental system for high-resolution imaging of synapse interface in a 2D plane by TIRF microscopy, as well as wide-field or confocal illumination methods. However, the system also has limitations as adjusting ligand mobility, out-of-plane bending, and fluctuations of biological membranes are not reproduced by planar bilayers. Furthermore, this is an in-vitro system and therefore has other limitations, such as lack of other membrane molecules that would be present on an infected cell and the cytoskeleton machinery that regulates molecular mobility and cellular motility. Also, the dynamics and distribution of the molecules, such as trimers versus monomers of gp120, may not be represented physiologically on the bilayer. Nevertheless, even with these limitations, this system is still very valuable for studying virus-cell or cell-cell interactions, and these methods can serve as a useful guide to researchers who are seeking high-resolution images to detect supramolecular organization that is not discernible in the conventional cell-cell conjugate system.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>His tagged HIV gp120</td>
 <td>Gift from Dr. Cho</td>
 <td>&nbsp;</td>
 <td>DH12</td>
 </tr>
 <tr>
 <td>His tagged HIV gp120</td>
 <td>Immune Technology</td>
 <td>&nbsp;</td>
 <td>Various X4 or R5 tropic</td>
 </tr>
 <tr>
 <td>HSA</td>
 <td>Williams Medical Company</td>
 <td>521302</td>
 <td>Human Serum Albumin 25%</td>
 </tr>
 <tr>
 <td>Amicon Ultra Centrifugal Filters</td>
 <td>Millipore</td>
 <td>UFC803024</td>
 <td>Ultracel-30K</td>
 </tr>
 <tr>
 <td>Lck Rabbit mAb</td>
 <td>Cell Signaling</td>
 <td>2787</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>p-Lck Rabbit pAb</td>
 <td>Santa Cruz Biotechnology Inc.</td>
 <td>Sc-101728</td>
 <td>Tyr 394</td>
 </tr>
 <tr>
 <td>Fyn Rabbit mAb</td>
 <td>Millipore</td>
 <td>04-353</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Alexa Fluor 568 2&deg; Ab</td>
 <td>Molecular Probes</td>
 <td>A11034</td>
 <td>Goat anti-Rabbit IgG (H+L)</td>
 </tr>
 <tr>
 <td>Alexa Fluor 488</td>
 <td>Molecular Probes</td>
 <td>A-20000</td>
 <td>carboxylic acid succinimidyl ester </td>
 </tr>
 <tr>
 <td>FCS2 Chamber</td>
 <td>Bioptechs</td>
 <td>060319-2-03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microaqueduct Slide</td>
 <td>Bioptechs</td>
 <td>130119-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>30mm Round w/holes</td>
 <td>Bioptechs</td>
 <td>1907-08-750</td>
 <td>0.75mm thick</td>
 </tr>
 <tr>
 <td> Rectangle Gasket</td>
 <td>Bioptechs</td>
 <td>1907-1422-250</td>
 <td>14x24 0.25mm thick</td>
 </tr>
 <tr>
 <td>Tygon Tubing</td>
 <td>Bioptechs</td>
 <td>20202275</td>
 <td>1/16" (25ft)</td>
 </tr>
 <tr>
 <td>Three-way Stopcock</td>
 <td>BioRad</td>
 <td>7328103</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Two-way Stopcock</td>
 <td>BioRad</td>
 <td>7328102</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sticky Slide I 0.2 Luer</td>
 <td>Ibidi</td>
 <td>80168</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cover glasses</td>
 <td>Ibidi</td>
 <td>10812</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1ml syringe</td>
 <td>BD</td>
 <td>309659</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DOGS-NTA</td>
 <td>Avanti Polar Lipids</td>
 <td>790404C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DOPC</td>
 <td>Avanti Polar Lipids </td>
 <td>850375C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass coverslip</td>
 <td>Bioptechs</td>
 <td>40-1313-0319</td>
 <td>40mm</td>
 </tr>
 <tr>
 <td>TIRF microscope</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ImageJ</td>
 <td>National Institutes of Health</td>
 <td>&nbsp;</td>
 <td><a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a></td>
 </tr>
 </tbody>
</table>

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.

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

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.

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 Views.  New York University Langone School of Medicine. 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.

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.

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]).

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.

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.

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.

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.

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.

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.

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.

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.