This review was supported by National Institutes of Health grants, AI074420-02, Burroughs Wellcome Investigator Award, Hirschl Career Scientist Awards to B.K.C. and by the NSF Center for Biophotonics Science and Technology, Cooperative Agreement PHY012099, and a UCD Health System Research Award to T.H. UCD CTSC NCRR ULRR024146.

This method was used in the research reported in <a href="http://www.sciencemag.org/cgi/content/abstract/323/5922/1743">H&uuml;bner et al. Science 323: 1743-1747 (2009)</a>.
1. Overview
Cell-to-cell spread of HIV was originally described using HIV infected Jurkat cells as donor cells and primary CD4+ T lymphocytes as acceptor cells 1,2,3. In these studies, virus was detected in fixed cells using antibody staining. To track the transfer of virus from cell to cell in live cells, we exploit a recombinant, infectious molecular clone of HIV called HIV Gag-iGFP 4. It carries the green fluorescent protein (GFP) inserted internally into the Gag protein between the MA and CA domains. This molecular clone of HIV retains infectivity without the need for helper virus. Virus particles made from this clone are stoichiometrically loaded with green fluorescent protein, providing a strong fluorescence signal to monitor viral assembly and transfer between cells. When used in conjunction with inert cell-tracking fluorescent dyes, recipient cells can be discriminated from input donor cells, and allowing one to visualize the transfer of HIV from an infected T cell to an uninfected T cell 5. Virological synapse formation and transfer of virus from one cell to another can then be observed in living cells while they are cultured in a sealed, gas-permeable imaging chamber.
(Day 1)
2. Preparation of HIV Gag-iGFP Transfected Jurkat T Cells
<ol>
	<li>Prepare the human CD4+ T cell line Jurkat (ATCC, Manassas, VA) by carefully maintaining the cells at a concentration between 2 x 105 and 8 x 105 cells/mL in Jurkat culture media (RPMI 1640, 10% fetal bovine serum, 100 units/mL penicillin, and 100 &mu;g/mL streptomycin). Key to success: Culture of the Jurkat cells at concentrations in excess of 8 x 105 cells/mL will result in a reduction in transfection efficiency. 
Note: The transfection of HIV Gag-iGFP proviral DNA into T cells, as described below, results in the production of a replication-competent human immunodeficiency virus. As such, all procedures are to be undertaken only by trained laboratory personnel in certified biosafety level 2+ or biosafety level 3 (BL2+/BL3) tissue culture rooms. Work with infectious HIV in imaging facilities must be approved by your local institutional biosafety office.</li>
	<li>Transfect the Jurkats with the viral plasmid, HIV Gag-iGFP using the Amaxa nucleofection method (Lonza, Walkersville, MD). Pellet 5 x 106 cells by centrifugation for 10 min at 150 x g. Aspirate supernatant and wash the cells in sterile phosphate buffered saline (PBS). Centrifuge the cells and resuspend the pellet to 97 &mu;L of pre-warmed, nucleofector solution V containing the manufacturer's supplement. Add three &mu;L of endotoxin free HIV Gag-iGFP plasmid (1 &mu;g/&mu;L) to the cell suspension and mix gently. Transfer the cell suspension to a cuvette and nucleofect using program S-18. Immediately transfer the cells to 3 mL of prewarmed Jurkat culture media without antibiotics.</li>
	<li>To prepare CD4+ target cells for virological synapses, we obtain human peripheral blood mononuclear cells from buffy coats by using a standard Ficoll-Paque (GE Healthcare, Uppsala, Sweden) protocol. CD4+ T cells are then negatively selected using a magnetic bead isolation kit (Miltenyi Biotec, Auburn, CA). These may be cryogenically stored in liquid N2 in aliquots of 5 x 106 cells/500 &mu;L of freezing media (90% fetal bovine serum/10% DMSO). Thawed primary CD4+ T cells are cultured in RPMI 10% FBS, supplemented with 10 units/mL of IL-2.</li>
</ol>
(Day 2)
3. Recovery of Live Cells from Jurkats Transfected with HIV Gag-iGFP and Staining of Target Cells with Fluorescent Dyes
<ol>
	<li>Allow the Jurkat cells to recover from the transfection overnight. Then remove the cellular debris by centrifugation over a ficoll hypaque density gradient material. Dispense 1.5 mL of Ficoll-Paque to the bottom of a 15 mL conical tube. Gently overlay this gradient material with the transfected Jurkats in a 5 mL volume of RPMI. Centrifuge the cells at 400 x g for 20 min at room temperature with the brake off. Carefully remove the cells from the media/Ficoll interface using a pipette. Transfer them to a new 15 mL conical tube. Dilute the cells with RMPI to a total volume of 15 mL and centrifuge at 300 x g for 10 min. Resuspend the pellet in 3 mL of Jurkat culture media in a 2 cm well (6-well plate) and return the cells to the tissue culture incubator.</li>
	<li>To distinguish target cells from donor cells in cell-cell transfer experiments we prelabel the CD4+ target cells with fluorescent dyes. Primary CD4+ T cells are labeled the evening before use. We have had excellent success labeling T cells with both CellTracker and CellTrace dyes (Invitrogen, Carlsbad, CA). While the dye concentration and incubation times must be optimized depending on the particular dye and cell type used, a representative protocol follows. Pellet 4 x 106 primary CD4+ T cells by spinning at 400 x g for 10 min. Wash the cells with 5 mL of PBS and resuspend in 2 mL of PBS. Add CellTracker Orange (CMTMR, Invitrogen, Carlsbad, CA) to a final concentration of 1.5 &mu;M and incubate at 37&deg;C for 20 min. Add 8 mL of complete Jurkat media and centrifuge at 400 x g for 10 min. Resuspend the cells in 3 mL of complete media supplemented with 10 units/mL of IL-2 and place in the tissue culture incubator overnight.</li>
</ol>
(Day 3)
4. Monitoring Cell-to-cell Transfer of HIV Gag-iGFP using Live Cell Imaging
We routinely use HIV Gag-iGFP transfected Jurkat cells 48 hours post-transfection. However, we have successfully used cells as early as 24 hours, and as late as 72 hours post-transfection.
<ol>
	<li>Approximately 48 hours post-transfection, HIV Gag-iGFP expressing Jurkats are counted, washed with CO2-independent media (Invitrogen, Carlsbad, CA), and resuspended in live cell imaging media (CO2 independent media, 10% Fetal Bovine Serum, 100 U/mL penicillin, and 100 &mu;g/mL streptomycin, 10 units/mL IL-2) at a concentration of 1-3 x 107 cells/mL.</li>
	<li>In a similar fashion, wash and resuspend labeled primary CD4+ T cells at a concentration of 1-3 x 107 cells/mL in live cell imaging media.</li>
	<li>Mix HIV Gag-iGFP-transfected Jurkats with primary CD4+ T cells at a 1:2 or 1:3 ratio and load into a tissue culture treated, gas permeable, microchamber (Ibidi, Verona, WI). For example, mix 20 &mu;L of HIV Gag-iGFP transfected Jurkats with 40 &mu;L of labeled primary CD4+ T cells. Load 50 &mu;L of this mixture into an Ibidi chamber and seal the chamber with plugs. Secure the plugs by wrapping the plug/Ibidi chamber interface with parafilm.</li>
</ol>
5. Considerations for microscopy platforms: spinning disk confocal imaging
After loading an Ibidi imaging chamber with cells, we immediately mount the device onto an inverted optical microscope (iX71 Olympus, Center Valley, PA). The Ibidi chamber should be transferred to the microscope only by BL2+-trained laboratory personnel wearing appropriate safety garments. Although live-cell imaging commonly uses expensive incubation chambers to maintain a stable 37&deg;C environment, this was achieved using a simple and economical thermostatic heater (ASI 400, Nevtek, Williamsville, VA) along with a T-type thermocouple tip mounted next to the sample for proper temperature feedback. We have found using CO2-independent media allows us to maintain high cell viability while avoiding the use of a CO2 chamber. To buffer the platform against temperature fluctuations in the room and to block out background room light, a large black shroud is draped over the whole microscope/heater setup creating an insulated air pocket around the system. This greatly reduces temperature variation and associated temperature-related sample drift, but requires pre-heating for 30 minutes prior to mounting the sample.
6. Data Acquisition, Transfer and Image Processing
Images are taken using a 60x 1.42 NA oil immersion objective (UPlanApo N, Olympus) as the high numerical aperture (NA) greatly improves resolution. To create 3D confocal images, scanning is performed by a spinning disk confocal unit (CSU-10, Yokagawa, Japan) that simultaneously uses &gt;800 confocal spots to dramatically increase the scan rate. To compliment its speed, the CSU-10 is paired with a highly sensitive electron-multiplying charged-coupled device (EMCCD, iXon+ 897, Andor Technologies, Ireland) camera to allow fast, low light imaging which reduces both photobleaching and phototoxicity. The fluorescence light source is a multi-wavelength ArKr ion gas laser (Innova 70C, Coherent, Santa Clara) where the desired wavelength (488 nm) and laser power measured right before the microscope objective (&lt;1 mW) are selected by an acousto-optic tunable filter (AOTF), (Andor Technologies). To further reduce photobleaching and extend total imaging time, the AOTF rapidly (microseconds) shuts off the laser beam every time the camera is inactive (10-20 ms) while the newly acquired image is delivered from the CCD (15-30 ms exposure) to the computer. The laser light is coupled into the spinning disk system using a 405/488/568/647 quad band dichroic mirror (Semrock, Rochester, NY). Fluorescence emission is filtered using a 525/50 bandpass filter (Semrock) inside a filter wheel (Ludl Electronic Products, Hawthorne, NY) mounted between the EMCCD camera and the confocal spinning disk unit. All hardware and acquisition, including a z-stage (Mad City Labs, Madison, WI), are controlled by the Andor iQ v1.8 software. To maximize the speed of acquisition at around 1.2-1.9 second 3D image stacks, we crop the recording region of the EMCCD's camera down to the cell-pair's immediate area. Furthermore, at the expense of some information in z, a large z-step between 0.45-0.75 &mu;m can be used to speed up acquisition. Imaging under these conditions can last up to 6 hours - using continuous 20-60 minute segments - with minimal photobleaching. Altogether, each data segment typically occupies 5-20 Gb of space as tens of thousands of images are taken. To facilitate the transport and analysis of the large data files, each acquisition set needs to be broken down and exported as 1 Gb TIFF image file segments using the Andor iQ v1.8 software. From here, many excellent software packages are available for image analysis, such as Metamorph, Volocity and ImageJ (we recommend its variation Fiji).
When two fluorescent markers are to be tracked, we simultaneously record both of them without slowing down acquisition by using a "split screen mode." Both markers are excited by two different ArKr laser lines at once (often 488 and 568 nm). Inserted directly before the EMCCD camera is an image splitter (OptoSplit II, Cairn, Kent, UK) that separates the two different images. Each image is then independently filtered using fluorescence filters (Semrock) and projected side-by-side on the camera at once creating the "split-screen" effect. This would then require manual cropping and aligning of images later in image processing.
7. Considerations for Image Processing and Data Analysis
We typically perform image analysis with Volocity (PerkinElmer, Waltham, MA) on Macintosh computers (Apple, Inc). Spinning disk laser confocal microscopy images are first adjusted in their intensity to correct for photobleaching, then deconvolved with Volocity. Intensity measurements and tracking of Gag puncta is performed with the Volocity Quantification module. For image sets where the movement relative to a pre-formed synapse needs to be calculated, an automated tracking algorithm is employed which tracks the synaptic button throughout the entire sequence. Manual inspection of the regions of interest defined by the autotracking software must be performed to confirm that the software has correctly tracked the desired object. For objects where the contrast with the surrounding objects is too weak, manual tracking may be performed on a frame-by-frame basis. The Volocity software package allows the export of XYZ location, volume and integrated signal within the desired objects. The distance from the synapse and velocity of the tracked object are calculated by normalizing movements to the center of the synaptic button.
8. Representative Results
Within 10 to 15 minutes of mixing one should be able to visualize HIV Gag-iGFP expressing Jurkat cells adhering to primary CD4+ T cells. Imaging these conjugates, one can assess the movements of Gag puncta in infected cells as well as in target cells after synaspe. One may observe the movement of Gag-iGFP puncta into target cells soon after synapses are formed.

<ol>
	<li>Blanco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+level+of+coreceptor-independent+HIV+transfer+induced+by+contacts+between+primary+CD4+T+cells.">High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells.</a> J Biol Chem. 279, 51305-51314 (2004).</li><li>Sourisseau, M., Sol-Foulon, N., Porrot, F., Blanchet, F., Schwartz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+human+immunodeficiency+virus+replication+in+mobile+lymphocytes.">Inefficient human immunodeficiency virus replication in mobile lymphocytes.</a> J Virol. 81, 1000-1012 (2007).</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>Hubner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+of+human+immunodeficiency+virus+type+1+(HIV-1)+Gag+localization+and+oligomerization+monitored+with+live+confocal+imaging+of+a+replication-competent,+fluorescently+tagged+HIV-1.">Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1.</a> J Virol. 81, 12596-12607 (2007).</li><li>Hubner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+video+microscopy+of+HIV+transfer+across+T+cell+virological+synapses.">Quantitative 3D video microscopy of HIV transfer across T cell virological synapses.</a> Science. 323, 1743-1747 (2009).</li></ol>

No conflicts of interest declared.

We describe here a simple method for visualizing the transfer of HIV from cell-cell. Recent research from our laboratory and others' suggests that this may be the predominant mode of HIV spread between T cells. The method described here employs a recombinant form of HIV, called HIV Gag-iGFP, which carries a genetically encoded green fluorescent protein tag in the core structural protein, Gag. Because of the high levels of GFP produced in each virus particle, this replication-competent clone of HIV enables researchers to track individual virus particles in real time. This virus should be especially useful in studies concerning the assembly and cell-cell transfer of HIV.
The transfer of HIV by cell-cell transfer is highly efficient. During a three hour co-culture, we routine see virus transferred to 20-30% of primary T cells as assessed by flow cytometry. Similar efficiencies are qualitatively seen during live cell imaging. Virally infected T cells begin forming synapses with uninfected primary T cells almost immediately after the initiation of co-culture. Interesting, not all primary T cells are capable of interacting with HIV-infected Jurkat cells despite forced interactions with laser tweezers (unpublished observations, GM). HIV Gag-iGFP will undoubtedly be useful for determining T cell subsets that are most susceptible to cell-cell transfer, both in vitro and in vivo.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Jurkat T Cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Leukocyte buffy coats from whole blood</td>
<td>&nbsp;</td>
<td>New York Blood Center</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nucleofector System and Solution V</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EndoFree Maxi Prep Kits</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HIV Gag-iGFP plasmid</td>
<td>&nbsp;</td>
<td>Chen Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CD4+ T Cell Isolation Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ficoll-Paque</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Celltracker/CellTrace Dyes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CO2-Independent Media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Imaging Chambers</td>
<td>&nbsp;</td>
<td>Ibidi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus IX-71 Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PlanApo N 1.42NA 60X oil objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Innova 70C ArKr ion gas laser</td>
<td>&nbsp;</td>
<td>Coherent </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CSU-10 Nipkow-type spinning disk confocal unit</td>
<td>&nbsp;</td>
<td>Yokogawa</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Di01-T405/488/568/647-13x15x0.5 dichroic beamsplitter</td>
<td>&nbsp;</td>
<td>Semrock</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>525/50 bandpass filter</td>
<td>&nbsp;</td>
<td>Semrock</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>iXon+ 897 electron-multiplied charged coupled devise camera</td>
<td>&nbsp;</td>
<td>Andor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ASI 400 Thermostatic Heater</td>
<td>&nbsp;</td>
<td>Nevtek</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

visualizing cell-to-cell transfer of hiv using fluorescent clones of hiv and live confocal microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

Watch this Scientific Journal Video about Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy at JoVE.com

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

visualizing-cell-to-cell-transfer-hiv-using-fluorescent-clones-hiv

Research

JoVE Journal

Immunology and Infection

16.4K vues.  Mount Sinai School of Medicine. Cette expérience visualisé est un guide pour utiliser un clone moléculaire fluorescent du VIH pour vivre des expériences d'imagerie confocale.

Vidéo: Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

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 ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

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 ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

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 ...

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. ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

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 ...