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">
	<tbod...

<ol>
	<li>Chukkapalli, V., Inlora, J., Todd, G. C., 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=Evidence+in+support+of+RNA-mediated+inhibition+of+phosphatidylserine-dependent+HIV-1+Gag+membrane+binding+in+cells.">Evidence in support of RNA-mediated inhibition of phosphatidylserine-dependent HIV-1 Gag membrane binding in cells.</a> J Virol. 87 (12), 7155-7159 (2013).</li><li>Chukkapalli, V., Oh, S. J., 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=Opposing+mechanisms+involving+RNA+and+lipids+regulate+HIV-1+Gag+membrane+binding+through+the+highly+basic+region+of+the+matrix+domain.">Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain.</a> Proc Natl Acad Sci U S A. 107 (4), 1600-1605 (2010).</li><li>Chukkapalli, V., Hogue, I. B., Boyko, V., Hu, W. S., 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=Interaction+between+the+human+immunodeficiency+virus+type+1+Gag+matrix+domain+and+phosphatidylinositol-(4,5)-bisphosphate+is+essential+for+efficient+gag+membrane+binding.">Interaction between the human immunodeficiency virus type 1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient gag membrane binding.</a> J Virol. 82 (5), 2405-2417 (2008).</li><li>Zhou, W., Parent, L. J., Wills, J. W., Resh, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+membrane-binding+domain+within+the+amino-terminal+region+of+human+immunodeficiency+virus+type+1+Gag+protein+which+interacts+with+acidic+phospholipids.">Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids.</a> J Virol. 68 (4), 2556-2569 (1994).</li><li>Ono, A., Ablan, S. D., Lockett, S. J., Nagashima, K., Freed, E. O. <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+regulates+HIV-1+Gag+targeting+to+the+plasma+membrane.">Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane.</a> Proc Natl Acad Sci U S A. 101 (41), 14889-14894 (2004).</li><li>Saad, J. S., 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=Structural+basis+for+targeting+HIV-1+Gag+proteins+to+the+plasma+membrane+for+virus+assembly.">Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly.</a> Proc Natl Acad Sci U S A. 103 (30), 11364-11369 (2006).</li><li>Shkriabai, N., 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=Interactions+of+HIV-1+Gag+with+assembly+cofactors.">Interactions of HIV-1 Gag with assembly cofactors.</a> Biochemistry. 45 (13), 4077-4083 (2006).</li><li>Vlach, J., Saad, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trio+engagement+via+plasma+membrane+phospholipids+and+the+myristoyl+moiety+governs+HIV-1+matrix+binding+to+bilayers.">Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers.</a> Proc Natl Acad Sci U S A. 110 (9), 3525-3530 (2013).</li><li>Anraku, K., 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=Highly+sensitive+analysis+of+the+interaction+between+HIV-1+Gag+and+phosphoinositide+derivatives+based+on+surface+plasmon+resonance.">Highly sensitive analysis of the interaction between HIV-1 Gag and phosphoinositide derivatives based on surface plasmon resonance.</a> Biochemistry. 49 (25), 5109-5116 (2010).</li><li>Llewellyn, G. N., Grover, J. R., 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=HIV-1+Gag+associates+with+specific+uropod-directed+microdomains+in+a+manner+dependent+on+its+MA+highly+basic+region.">HIV-1 Gag associates with specific uropod-directed microdomains in a manner dependent on its MA highly basic region.</a> J Virol. 87 (11), 6441-6454 (2013).</li><li>Inlora, J., Chukkapalli, V., Derse, D., 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=Gag+localization+and+virus-like+particle+release+mediated+by+the+matrix+domain+of+human+T-lymphotropic+virus+type+1+Gag+are+less+dependent+on+phosphatidylinositol-(4,5)-bisphosphate+than+those+mediated+by+the+matrix+domain+of+HIV-1+Gag.">Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of HIV-1 Gag.</a> J Virol. 85 (8), 3802-3810 (2011).</li><li>Inlora, J., Collins, D. R., Trubin, M. E., Chung, J. Y., 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=Membrane+binding+and+subcellular+localization+of+retroviral+Gag+proteins+are+differentially+regulated+by+MA+interactions+with+phosphatidylinositol-(4,5)-bisphosphate+and+RNA.">Membrane binding and subcellular localization of retroviral Gag proteins are differentially regulated by MA interactions with phosphatidylinositol-(4,5)-bisphosphate and RNA.</a> MBio. 5 (6), e02202 (2014).</li><li>Valentine, K. G., 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=Reverse+micelle+encapsulation+of+membrane-anchored+proteins+for+solution+NMR+studies.">Reverse micelle encapsulation of membrane-anchored proteins for solution NMR studies.</a> Structure. 18 (1), 9-16 (2010).</li><li>Dick, R. A., Goh, S. L., Feigenson, G. W., Vogt, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Gag+protein+can+sense+the+cholesterol+and+acyl+chain+environment+in+model+membranes.">HIV-1 Gag protein can sense the cholesterol and acyl chain environment in model membranes.</a> Proc Natl Acad Sci U S A. 109 (46), 18761-18766 (2012).</li><li>Dick, R. A., Kamynina, E., Vogt, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+multimerization+on+membrane+association+of+Rous+sarcoma+virus+and+HIV-1+MA+proteins.">Effect of multimerization on membrane association of Rous sarcoma virus and HIV-1 MA proteins.</a> J Virol. 87 (24), 13598-13608 (2013).</li><li>Alfadhli, A., Still, A., Barklis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+human+immunodeficiency+virus+type+1+matrix+binding+to+membranes+and+nucleic+acids.">Analysis of human immunodeficiency virus type 1 matrix binding to membranes and nucleic acids.</a> J Virol. 83 (23), 12196-12203 (2009).</li><li>Rodriguez, N., Pincet, F., Cribier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+vesicles+formed+by+gentle+hydration+and+electroformation:+a+comparison+by+fluorescence+microscopy.">Giant vesicles formed by gentle hydration and electroformation: a comparison by fluorescence microscopy.</a> Colloids Surf B Biointerfaces. 42 (2), 125-130 (2005).</li><li>Needham, D., McIntosh, T. J., Evans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermomechanical+and+transition+properties+of+dimyristoylphosphatidylcholine/cholesterol+bilayers.">Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers.</a> Biochemistry. 27 (13), 4668-4673 (1988).</li><li>Darszon, A., 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=Reassembly+of+protein-lipid+complexes+into+large+bilayer+vesicles:+perspectives+for+membrane+reconstitution.">Reassembly of protein-lipid complexes into large bilayer vesicles: perspectives for membrane reconstitution.</a> Proc Natl Acad Sci U S A. 77 (1), 239-243 (1980).</li><li>Angelova, M. I., Dimitrov, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+electroformation.">Liposome electroformation.</a> Faraday Discuss. Chem. Soc. (81), 303-311 (1986).</li><li>Angelova, M. I., Sol&#233;au, S., M&#233;l&#233;ard, P. h., Faucon, F., Bothorel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+giant+vesicles+by+external+AC+electric+fields.">Preparation of giant vesicles by external AC electric fields.</a> Progr Colloid Polymer Sci. 89, 127-131 (1992).</li><li>Stachowiak, J. 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=Unilamellar+vesicle+formation+and+encapsulation+by+microfluidic+jetting.">Unilamellar vesicle formation and encapsulation by microfluidic jetting.</a> Proc Natl Acad Sci U S A. 105 (12), 4697-4702 (2008).</li><li>Yamauchi, S., 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=The+consensus+motif+for+N-myristoylation+of+plant+proteins+in+a+wheat+germ+cell-free+translation+system.">The consensus motif for N-myristoylation of plant proteins in a wheat germ cell-free translation system.</a> FEBS J. 277 (17), 3596-3607 (2010).</li><li>Olety, B., Veatch, S. 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>

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>
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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...

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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 ...

The overall goal of this procedure is to visualize HIV-1 Gag binding on giant unilamellar vesicle membranes. This method can help answer key questions in the Bio Assembly field. Such as the roles created by lipids with different osteo chains in membrane binding or virostructural proteins, such as vitro viral Gag proteins.The main advantage of this technique is that the membrane binding of Gag can be detected visually on giant unilamellar vesicle membranes. directly after mixing result potentially deleterious, lengthy Post Mixing steps. Preparation of HIV-1 Gag is covered in the text protocol, and must be done one day in advance of preparing the GUVs.Take out lipid stocks from the freezer and equilibrate to room temperature. Meanwhile, set the temperature of the heat block at least five degrees celsius higher than the highest melting temperature of the lipids in use. In this demonstration, the temperature is set to 65 degrees celsius, the highest temperature we have used.The lipids must be dissolved in inorganic solvents, stored in glass vials with Teflon-lined screw caps, and wrapped in Parafilm. Once thawed, make sure the suspensions are cleared. Calculate the required lipid volumes according to the desired moldar/lipid ratios.Then, aliquot the calculated volumes into a clean, screw-capped, glass vial using a chloroform-resistant device, such as a Hamilton-type glass syringe. Adjust the total volume with an appropriate organic solvent, such as chloroform. Next, prepare two new ITO coated slides for each electroformation chamber.Verify that the conductive side of the slide is facing up by checking the resistance using a multimeter, which should be about 200 ohms. Then, gently clean their surfaces using 70%ethanol and lint free wipes. Next, clean the outer surface of the syringe needle by rinsing it with chloroform, and place the syringe on the heat block.Also place the cleaned ITO-coated slides on the heat block, conductive side up. In a few minutes, the slides will reach temperature. Then, add 40 to 50 microliters of the lipid mixture to one of the slides.The lipid should be spread out quickly, but as uniformly as possible. Next, add another half volume of the lipid mixture to another glass slide, and immediately spread it out to make a uniform lipid film. Be quick and gentle to avoid damaging the thin ITO coating.If the opacity of the film appears excessively nonuniform, redo the procedure using a new slide. Next, make a second pair of slides for a different lipid mixture as the first. A uniform layer of lipids is needed for optimal GUV yields, and compositional uniformity of GUVs.Store the slides in a vacuum dessication chamber for 60 to 90 minutes to remove trace levels of organic solvent. For the next step, set an incubator to the temperature of the heat block used in the previous steps, and warm a stock of 300 millimolar sucrose solution to that temperature. Also, prepare a PDMS gasket by gently wiping it with 70%ethanol, and letting it dry completely.Once the slides have dried, place them on a clean surface and gently but firmly press the gasket onto one slide so that the gasket forms a watertight seal. Confirm the absence of gas between the PDMS gasket and the slide. Then, completely fill the gasket with the prewarmed sucrose solution.Do not leave any bubbles. Now, press the other coated slide on top of the gasket aligned with the other slide. Ensure that there are no bubbles, and fasten the chamber using binder clips.Next, remove the excess sucrose solution by aspiration, and clean the slides using lint free wipes. To proceed, fasten the slide assembly to a copper bar such that the conductive side of each slide is in uniform contact with the bar. Then, connect the copper bar to the function generator output, and place the assembly inside the incubator with the function generator remaining outside.Once at temperature, program the function generator to apply a 10 Hz sine wave and a potential difference of one volt for 90 minutes. Make certain that the voltage is one volt using a multimeter. After 90 minutes, slowly decrease the frequency to two Hz and continue the electroformation for another 10 minutes.During this time, harvest the HIV-1 Gag from the in vitro translation reaction set up the day before. Remove unwanted aggregates by spinning down the lysates and carefully transfer the supernatant into another tube. After 10 minutes at two Hz, switch off the function generator, turn off the incubator, and leave the incubator door open until the assembly returns to room temperature.Once cooled, carefully bring the slide assembly to the bench and gently disassemble the chamber by removing the top slide with forceps. Transfer the GUVs with a clipped 1000 microliter pipette tip to a new tube. Handle this tube gently and use the GUVs within 90 minutes.Next, prepare an imaging chamber. Attach a PDMS sheet containing a small hole onto a clean cover slip. Press it down gently and firmly for a tight seal.Then, mix five microliters of the in vitro translation reaction with five microliters of GUVs. After a few minutes, transfer the mixture to the imaging chamber and proceed with imaging. To prevent evaporation, the imaging chamber can be covered with a cover slip.Using the described protocol, GUVs were prepared composed of POPC, POPS, and cholesterol in a 4.7 to 2.3 to 3 molar ratio. Florescence measurement of Gag YFP signals on a GUV shows that they are not detectable above the background at the GUV surface. However, Gag-YFP protein bound efficiently to the GUVs when brain-PIP2 was included into the lipid mixture.Gag binding is evident by the increase in florescence intensity on the membrane surface, which, when scanned, appears as a sharp peak. Once mastered, provided that the protein is available in a florescently labeled form, this technique can be done in four to five powers. While attempting this procedure, it's important to remember to be gentle when handling the GUVs, and always use fresh lipids when possible.This technique has allowed researchers in the field of retrovirus assembly to examine membrane interactions of retroviral Gag proteins in vitro without lengthy Post Mixing procedures, which may alter the latent state of the interactions.

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Research

JoVE Journal

Immunology and Infection

8.7K visualizações.  University of Michigan Medical School. The overall goal of this procedure is to visualize HIV-1 Gag binding on giant unilamellar vesicle membranes. This method can help answer key questions in the Bio Assembly field. Such as the roles created by lipids with different osteo chains in membrane binding or virostructural proteins, such as vitro viral Gag proteins.The main advantage of this technique is that the membrane binding of Gag can be detected visually on giant unilamellar vesicle membranes. directly after mixing result ...