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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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><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 10 Hz and 1 V 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,&lt;br/&gt; Berlin, Germany</td><td>BR1401001</td><td></td></tr><tr><td>DiD</td><td>Life Technologies, Carlsbad, CA&amp;nbsp;</td><td>D7757</td><td></td></tr><tr><td>POPC</td><td>Avanti Polar Lipids, Alabaster, AL&amp;nbsp;</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)P&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</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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Universidad de Cartagena UDEC

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JoVE Journal

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

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.

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 ...

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

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 ...

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 ...

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 ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Biology

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

Bioengineering

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...