We would like to thank Mohammad Saleem, Jing Wu and Krishnan Raghunathan for helpful discussions. We also thank Priya Begani for assistance during the filming. This work is supported by National Institutes of Health grants R01 AI071727 (to A.O.) and R01 GM110052 (to S.L.V).

Day 1: Expression of Gag Proteins Using the Wheat Germ Lysate-based In Vitro Transcription-translation System
1. Preparation of HIV-1 Gag
<ol>
	<li>Remove all the reagents of the commercial wheat germ CECF kit from freezers (wheat germ lysates are stored at -80 &#186;C; other reagents at -20 &#186;C). Thaw them on ice and mix the components as shown in Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mix-1 (Feeding mix)</td>
			<td></td>
		</tr>
		<tr>
			<td>Feeding solution</td>
			<td>900 &#181;l</td>
		</tr>
		<tr>
			<td>Amino acids</td>
			<td>80 &#181;l</td>
		</tr>
		<tr>
			<td>Methionine</td>
			<td>20 &#181;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>1,000 &#181;l</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mix-2 (Lysate mix)</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheatgerm lysates</td>
			<td>15 &#181;l</td>
		</tr>
		<tr>
			<td>RNase free water</td>
			<td>7 &#181;l</td>
		</tr>
		<tr>
			<td>Amino acids&#160;</td>
			<td>4 &#181;l</td>
		</tr>
		<tr>
			<td>Methionine</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>Plasmid DNA in TE buffer (1 &#181;g/&#181;l)**</td>
			<td>4 &#181;l</td>
		</tr>
		<tr>
			<td>Reaction buffer</td>
			<td>15 &#181;l</td>
		</tr>
		<tr>
			<td>RNasin*</td>
			<td>4 &#181;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>50 &#181;l</td>
		</tr>
	</tbody>
</table>
Table 1: Compositions of mixtures required for wheat germ reactions. 
Note: If the experiment is designed to examine the effect of RNA removal by RNase on Gag membrane binding, replace ribonuclease inhibitors with RNase-free water. Plasmid DNA should be free from impurities according to the manufacturer&#39;s instruction. However, plasmids prepared using conventional, but not endotoxin-free, plasmid isolation kits have been successfully used. The manufacturer&#39;s instruction also recommends using DNA dissolved in nuclease-free water. If using DNA suspended in TE [10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA], avoid using lower concentrations of DNA, since EDTA in TE may affect the yield. Plasmids dissolved in TE buffer at a concentration range of 0.8-1 &#181;g/&#181;l have successfully been used.
<ol start="2">
	<li>First add the Feeding mixture (Mix-1) into the reaction cassette (transparent chamber). Then add the lysate mix (Mix-2) to the other chamber (red). Do not introduce any bubbles while adding the mixtures into their respective chambers, since that may lead to inefficient exchange of solution components and therefore reduce the efficiency of reaction.</li>
	<li>Cover the chambers with the adhesive film provided with the kit.</li>
	<li>Insert the reaction cassette into the cassette holder and incubate the assembly for 24 hr at 24 &#186;C at a constant shaking speed of 900 rpm in an Eppendorf thermomixer R.</li>
</ol>
Day 2: Prepare GUVs by Electroformation and Harvest Wheat Germ Lysates
2. Preparation of GUVs
<ol>
	<li>Aliquot lipids dissolved in inorganic solvents such as chloroform or mixtures of chloroform and methanol in glass vials with screw caps lined with Teflon liners and wrapped with Parafilm. Handle lipids with Hamilton-type glass syringes, or if available, chloroform-resistant plastic tips. Take out glass vials containing the lipids to be used for GUV preparation from the -20 &#186;C freezer.
	<ol>
		<li>Once the vials are equilibrated to room temperature, make sure by visual inspection that the lipid suspensions are clear. The quality of lipids, particularly acidic lipids, such as palmitoyl-oleoyl phosphatidylserine (POPS) and PI(4,5)P2, is important for successful experiments. Do not use lipids that are in aliquots for longer than 2 months.</li>
	</ol>
	</li>
	<li>Pre-warm a heat block to the desired temperature. This temperature must be at least 5 &#186;C higher than the melting temperature (Tm) of the lipid(s) with the highest Tm.</li>
	<li>Calculate the lipid volumes needed according to the desired molar lipid ratios. For studying HIV-1 Gag binding here, use the following molar ratios as shown in Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lipid mix</td>
			<td>Molar ratio</td>
			<td>Volume (in &#181;l)</td>
			<td>Stock concentration&#160;</td>
		</tr>
		<tr>
			<td>POPC+POPS+Chol&#160;</td>
			<td>46.6+23.3+30</td>
			<td>19.74+10.18+6.57</td>
			<td>POPC, POPS, Chol:10 mg/ml</td>
		</tr>
		<tr>
			<td rowspan="2">POPC+POPS+Chol+Brain-PI(4,5)P2&#160;&#160;&#160;</td>
			<td rowspan="2">40+20+30+10</td>
			<td rowspan="2">16.11+8.3+6.25+58.19</td>
			<td>POPC, POPS, Chol:10 mg/ml</td>
		</tr>
		<tr>
			<td>Brain-PI(4,5)P2:1 mg/ml&#160;</td>
		</tr>
	</tbody>
</table>
Table 2: Volumes of different lipids used to obtain GUVs.
<ol start="4">
	<li>Add the desired volumes of lipids into a clean screw-capped glass vial.</li>
	<li>Use ITO-coated slides of dimensions 25 x 50 x 1.1 mm3 and resistance 70-100 &#937; (as described in the catalog). Place two ITO-coated slides on a clean bench. Each electroformation chamber requires two indium-tin-oxide (ITO)-coated glass slides. Verify that the conductive side of the glass slide is facing up by checking the resistance using a multimeter. It should show a value of approximately 200 &#937;. Make sure the surface of the slides are clean by gently wiping with 70% ethanol using lint-free wipes.</li>
	<li>Clean the outer surface of a needle of a syringe by rinsing it with chloroform and place the syringe on the heat block.</li>
	<li>Place a new ITO-coated glass slide on the heat block with conductive side facing up and allow it to equilibrate to the desired temperature usually about 2-3 min. Do not reuse the ITO-coated glass slides for a potential risk that the ITO-coating on the glass slides might be damaged during cleaning procedures.</li>
	<li>Use a volume of 40-50 &#181;l of the lipid mixture for each slide for uniform and yet quick spreading of the lipids on the glass slide. Adjust the total volume with an appropriate organic solvent such as chloroform.
	<ol>
		<li>Place one half of the total volume of the lipid mixture on the glass slide and immediately spread the lipid using the cleaned syringe (step 2.6) to leave a uniform lipid film (moving the needle 2-3 times across the slide). Spread the lipid film gently (but quickly) to avoid any damage to the thin ITO coating. Non-uniform spreading or rough spreading may lead to sub-optimal yields or computational heterogeneity of GUVs.</li>
		<li>Therefore, ensure uniform spread by inspecting the lipid film after spread before proceeding to the next step. If the opacity of the film appears excessively non-uniform, redo the procedure using a new slide.</li>
	</ol>
	</li>
	<li>Place the coated slide inside a Petri dish with the coated side facing up.</li>
	<li>Repeat Steps 2.6-2.9 for the rest of the mixture using another ITO-coated slide.</li>
	<li>Place the petri dish containing both the slides in a conventional vacuum desiccation chamber for 60-90 min to remove any trace of organic solvents.</li>
	<li>During this process, set the incubator to the same temperature as that of the heat block (65 &#186;C in most cases) and pre-warm 300 mM sucrose solution in the incubator.</li>
	<li>After drying, bring the petri dish to the bench and place the glass slide on a clean surface.</li>
	<li>Gently clean a PDMS gasket (Figure 1) using 70% ethanol and dry it completely.</li>
	<li>Place it on the lipid-coated glass slide as shown in Figure 1 and gently and firmly press so that the gasket forms a water tight seal. Confirm the absence of gaps between the PDMS gasket and the slide. This results in the formation of a shallow chamber that holds the sucrose solution.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54293/54293fig1.jpg" /> 
Figure 1: The layout of the ITO-coated glass slide used for electroformation. <a href="https://www.jove.com/files/ftp_upload/54293/54293fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="16">
	<li>Fill the chamber with pre-warmed sucrose solution without any bubbles.</li>
	<li>Place the other coated slide on top to make a tight seal. Ensure that there are no bubbles. The final assembly should appear as shown in Figure 2. Fasten the chamber using binder clips.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54293/54293fig2.jpg" /> 
Figure 2: Schematic representation of the electroformation chamber (side view). <a href="https://www.jove.com/files/ftp_upload/54293/54293fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="18">
	<li>Remove excess sucrose by aspiration and clean the slides using lint-free wipes. Fasten the slides to the copper bar so that the conductive sides are in uniform contact with the bar. Ensure that only one conductive side of the glass slide is in contact with one copper bar.</li>
	<li>Connect the copper bar to the function generator output using the alligator clips. Place the assembly inside the incubator to allow the assembly to equilibrate to the desired temperature.</li>
	<li>Apply a sine wave frequency of 10 Hz and a potential difference of 1 V for 90 min. Ensure that the voltage is 1 V by a multimeter as shown in Figure 3. Continue the process for 90 min.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/54293/54293fig3.jpg" /> 
Figure 3: The positions at which the electrodes from the multimeter are placed for measuring frequency and current. <a href="https://www.jove.com/files/ftp_upload/54293/54293fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="21">
	<li>After 90 min, slowly decrease the frequency to 2 Hz and continue electroformation for another 10 min. During this time, harvest in vitro translation reactions (steps 3.1-3.2).</li>
	<li>Switch off the function generator and allow the assembly to cool down to room temperature slowly (by turning off the incubator and leaving the incubator door slightly opened).</li>
	<li>After cooling, disconnect the cables to the generator from the slide assemblies. GUVs may be very fragile and therefore need to be handled very gently. Carefully bring the assembly to the bench.</li>
	<li>Disassemble the chamber by gently removing the top slide using forceps. Harvest the sucrose suspension containing GUVs using a cut 1,000 &#181;l tip and transfer to a clean tube. Handle the tube gently to prevent disruption of GUVs.</li>
	<li>Use the GUVs immediately. GUVs are stable for at least 90 min after harvesting when stored at room temperature.</li>
</ol>
3. Harvest In Vitro Translation Reactions
<ol>
	<li>Harvest the in vitro translation reactions containing desired proteins from the red chamber using a micropipette into a tube.</li>
	<li>To remove aggregated proteins, spin the lysates at 16,200 x g for 15 min on a tabletop centrifuge and carefully transfer the supernatant into another tube.</li>
</ol>
4. GUV Binding Assay
<ol>
	<li>Using a cut tip, mix 5 &#181;l of in vitro translation reactions containing translated proteins and 5 &#181;l of GUVs in a tube and incubate at room temperature for 2-3 min.</li>
	<li>Attach a PDMS sheet with a small hole (3-4 mm diameter) on a clean cover slip and ensure tight seal by gently and firmly pressing it against the coverslip.</li>
	<li>Place the 10 &#181;l mixture from step 4.1 into the small hole.</li>
	<li>Image the mixture under an inverted epi- or confocal fluorescence microscope at room temperature. 
	Note: To prevent evaporation, the imaging chamber can be covered by a coverslip.</li>
</ol>

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L., Ono, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-(4,5)-Bisphosphate+Acyl+Chains+Differentiate+Membrane+Binding+of+HIV-1+Gag+from+That+of+the+Phospholipase+Cdelta1+Pleckstrin+Homology+Domain.">Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cdelta1 Pleckstrin Homology Domain.</a> J Virol. 89 (15), 7861-7873 (2015).</li><li>Morales-Penningston, N. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUV+preparation+and+imaging:+minimizing+artifacts.">GUV preparation and imaging: minimizing artifacts.</a> Biochim Biophys Acta. 1798 (7), 1324-1332 (2010).</li><li>Veatch, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-formation+and+fluorescence+microscopy+of+giant+vesicles+with+coexisting+liquid+phases.">Electro-formation and fluorescence microscopy of giant vesicles with coexisting liquid phases.</a> Methods Mol Biol. 398, 59-72 (2007).</li><li>Ayuyan, A. G., Cohen, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+peroxides+promote+large+rafts:+effects+of+excitation+of+probes+in+fluorescence+microscopy+and+electrochemical+reactions+during+vesicle+formation.">Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation.</a> Biophys J. 91 (6), 2172-2183 (2006).</li><li>Montes, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroformation+of+giant+unilamellar+vesicles+from+native+membranes+and+organic+lipid+mixtures+for+the+study+of+lipid+domains+under+physiological+ionic-strength+conditions.">Electroformation of giant unilamellar vesicles from native membranes and organic lipid mixtures for the study of lipid domains under physiological ionic-strength conditions.</a> Methods Mol Biol. 606, 105-114 (2010).</li><li>Olety, B., Ono, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+played+by+acidic+lipids+in+HIV-1+Gag+membrane+binding.">Roles played by acidic lipids in HIV-1 Gag membrane binding.</a> Virus Res. 193, 108-115 (2014).</li><li>Keller, H., Krausslich, H. G., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimerizable+HIV+Gag+derivative+binds+to+the+liquid-disordered+phase+in+model+membranes.">Multimerizable HIV Gag derivative binds to the liquid-disordered phase in model membranes.</a> Cell Microbiol. 15 (2), 237-247 (2013).</li><li>Carlson, L. A., Hurley, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reconstitution+of+the+ordered+assembly+of+the+endosomal+sorting+complex+required+for+transport+at+membrane-bound+HIV-1+Gag+clusters.">In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters.</a> Proc Natl Acad Sci U S A. 109 (42), 16928-16933 (2012).</li><li>Gui, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+minimal+in+vitro+system+for+analyzing+HIV-1+Gag-mediated+budding.">A novel minimal in vitro system for analyzing HIV-1 Gag-mediated budding.</a> J Biol Phys. 41 (2), 135-149 (2015).</li><li>Prevost, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRSp53+senses+negative+membrane+curvature+and+phase+separates+along+membrane+tubules.">IRSp53 senses negative membrane curvature and phase separates along membrane tubules.</a> Nat Commun. 6, 8529 (2015).</li></ol>

We declare that we have no conflicting interests.

The GUV binding assay as described above provides a good alternative in scenarios where other protein-lipid interaction assays have their limitations. This assay allows us to examine interactions between myristoylated full-length Gag and acidic lipids with native-length acyl chains in the lipid bilayer context and do so without lengthy flotation centrifugation through high-density sucrose gradients or other post-binding processing of Gag-lipid complexes. Another advantage is that the behavior of Gag can be examined in a ...

Human immunodeficiency virus type 1 (HIV-1) is an enveloped virus that assembles at and buds from the plasma membrane (PM) in most cell types. The assembly of HIV-1 virus particles is driven by the 55 kDa viral core protein called Pr55Gag (Gag). Gag is synthesized as a precursor polyprotein composed of four major structural domains, namely, matrix, capsid, nucleocapsid, and p6, as well as two spacer peptides SP1 and SP2. During assembly, the matrix (MA) domain is responsible for targeting of Gag to the assembly site, the capsid (CA) domain mediates Gag-Gag interactions, the nucleocapsid (NC) domain recruits viral genomic RNA, and p6 recruits host factors that aid virus particle scission from the plasma membrane. Gag also undergoes a co-translational modification by the addition of a 14-carbon fatty acid or myristate moiety at its N-terminus.
Membrane binding of Gag is an essential requirement for the viral assembly, since mutants that are defective in membrane binding fail to produce virus particles. We and others have shown that membrane binding of Gag is mediated by bipartite signals within the MA domain: the N-terminal myristate moiety that mediates hydrophobic interactions with the lipid bilayer and a cluster of basic residues within the MA domain termed as highly basic region (HBR) that interacts with acidic lipids on the PM1-4. Studies of Gag-membrane interactions using ectopic expression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), an enzyme that catalyzes the hydrolysis of PM-specific acidic phospholipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] to phosphatidylinositol-4-phosphate, in cells suggested that Gag-PM localization is mediated by PI(4,5)P23,5 . However, in vitro studies aiming at understanding more mechanistic details of Gag-PI(4,5)P2 interactions have proven to be challenging for a number of reasons. For example, purification of full-length myristoylated HIV-1 Gag for biochemical experiments has been technically difficult at least in part due to the tendency of Gag to aggregate during purification. Hence, truncated forms of HIV-1 Gag, such as Myr-MA or Myr-MA-CA, or the non-myristoylated form have been frequently used in studies that necessitate Gag purification (e.g., nuclear magnetic resonance, protein footprinting, and surface plasmon resonance6-9). Alternatively, coupled in vitro transcription-translation reactions have been used to produce full-length myristoylated HIV-1 Gag in other biochemical studies1,2. Typically in this system, a Gag-encoding plasmid is transcribed and translated with eukaryotic cell lysates (e.g., rabbit reticulocyte lysates) that are devoid of any cellular membranes and messenger RNAs but contain the machinery for transcription and translation. After the reaction, cell lysates containing Gag are mixed with membranes for analysis of Gag interactions with lipids. In addition to the ease of preparing full-length myristoylated Gag, methods using the in vitro transcription translation system have an advantage that Gag synthesis and subsequent membrane binding reactions occur in an &#39;eukaryotic cytosol-like&#39; milieu that may better represent physiological conditions. This property contributed to the studies that showed that RNA molecules bound to the MA domain regulate Gag binding to acidic lipids in a competitive manner1,2,10-12. However, since the total amount of Gag proteins obtained in these cell lysates are not high, metabolic labeling of proteins with radiolabeled amino acids is necessary for their detection.
Depending on the method to measure Gag-lipid interactions, a variety of membrane preparations have been used. Each of these methods has its strengths and limitations. Most NMR-based assays require the use of lipids with short acyl chains that are water-soluble (e.g., C4- and C8-PI(4,5)P2)6,8. While NMR methods to test binding of Gag to the lipids that have long acyl chains found in cells are being developed, they have been used only with myristoylated or nonmyristoylated MA thus far8,13. Alternatively, liposomes prepared from lipids that have native length acyl chains have been used in biochemical methods such as liposome flotation or fluorescent liposome bead binding assays2,3,10,14-16. However, liposomes used in these assays have small diameters, and thus their membranes have steep and positive curvatures. In contrast, during the early phase of particle assembly in HIV-1-infected cells, Gag binds to the PM, which is nearly planar on the scale of Gag, and subsequently induces negative curvature during budding. Therefore, liposome membranes with steep and positive curvature might not be ideal lipid bilayers to study Gag-lipid interactions. As for liposome flotation assays, another potential caveat is that exposure of Gag-lipid complexes to hypertonic sucrose gradient during centrifugation may affect the experimental outcome. To alleviate these limitations and provide a complementary experimental system, assays for Gag binding to giant unilamellar vesicles (GUVs) have been developed in recent years. GUVs are single lipid bilayer vesicles whose diameters extend to several tens of micrometers. Thus, the curvature of these membranes resembles the PM on the scale of Gag. Furthermore, due to its large size, which enables visual inspection under optical microscopes, membrane binding of fluorescently tagged or labeled Gag proteins to these vesicles upon mixing can be easily determined without subsequent processing of Gag-lipid complexes.
We here describe a protocol to study HIV-1 Gag membrane binding using GUVs obtained from an electroformation method. Various methods such as gentle hydration, gel-assisted hydration, microfluidic jetting, and electroformation17-22 have been used to obtain GUVs. For the protocol described here, the electroformation method is used primarily because of its efficiency in forming GUVs with acidic lipids and its relative ease of use without the need of expensive setups. Since visualization of Gag necessitates a fluorescent reporter, yellow fluorescent protein (YFP) is genetically added to the C-terminus of Gag (Gag-YFP). Gag-YFP proteins are obtained by in vitro transcription and translation reactions in wheat germ lysates based on the continuous exchange-continuous flow (CECF) technology. In this technology, both removal of inhibitory byproducts of the reactions and supply of reaction substrates and energy components are achieved in a dialysis-based mechanism. For these reactions, a plasmid encoding Gag-YFP under the control of a T7 promoter is used. Of note, as shown earlier, wheat germ lysates support myristoylation without additional components23,24. Using this method, it has been possible to obtain sufficient quantities of full-length myristoylated Gag-YFP for visualization of Gag on GUV membranes24. Here we describe the protocol with which HIV-1 Gag binding to PI(4,5)P2-containing GUV membranes can be examined without lengthy subsequent processing following binding reactions and propose that this method complements preexisting Gag-membrane binding assays and can be extended to further understand HIV-1 Gag-membrane binding.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digital Multimeter</td>
			<td>Meterman</td>
			<td>30XR</td>
			<td>A generic multimeter that can measure resistance and volts will serve the purpose.</td>
		</tr>
		<tr>
			<td>Function Generator</td>
			<td>Instek</td>
			<td>GFG-8216A</td>
			<td>A generic function generator that is capable of generating a sine wave at 10hz and 1V is sufficient.</td>
		</tr>
		<tr>
			<td>ITO coated glass slides</td>
			<td>Delta Technologies, Loveland, CO</td>
			<td>CG-90IN</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Hoefer</td>
			<td></td>
			<td>Any incubator that can accurately maintain temperature will be sufficient</td>
		</tr>
		<tr>
			<td>Vacuum chamber</td>
			<td>Nalgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer R</td>
			<td>Eppendorf</td>
			<td>21516-166</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>80400</td>
			<td>Gauge 22S, Syringe number 702</td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Sylgard elastomer base kit, Dow-Corning</td>
			<td>Sylgard, 184</td>
			<td></td>
		</tr>
		<tr>
			<td>RTS 100 Wheat Germ CECF Kit</td>
			<td>BiotechRabbit, 
			Berlin, Germany</td>
			<td>BR1401001</td>
			<td></td>
		</tr>
		<tr>
			<td>DiD</td>
			<td>Life Technologies, Carlsbad, CA&#160;</td>
			<td>D7757</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC</td>
			<td>Avanti Polar Lipids, Alabaster, AL&#160;</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>POPS</td>
			<td>Avanti Polar Lipids</td>
			<td>840034C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Avanti Polar Lipids</td>
			<td>700041P</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain-PI(4,5)P2&#160;</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the above protocol, we prepared GUVs composed of POPC+POPS+Chol (molar ratio: 4.66:2.33:3). This composition was chosen to approximately reflect the PS and cholesterol concentrations of the PM. Robust and efficient binding of Gag was observed only when brain-PI(4,5)P2 was included into the POPC+POPS+Chol mixture (POPC+POPS+Chol+brain-PI(4,5)P2 [molar ratio: 4:2:3:1] in this example) (Figure 4, compare panels B and D with A and C). Gag-YFP bindi...

Watch this Scientific Journal Video about Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes at JoVE.com

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.

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 ...

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Immunology and Infection

8.8K Views.  University of Michigan Medical School. 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. 

Video: Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

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 ...

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

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

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

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

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 ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

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. ...

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.