We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.
The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an 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 "cell-like" 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.
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 'eukaryotic cytosol-like' 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.
Day 1: Expression of Gag Proteins Using the Wheat Germ Lysate-based In Vitro Transcription-translation System
1. Preparation of HIV-1 Gag
Mix-1 (Feeding mix) | |
Feeding solution | 900 µl |
Amino acids | 80 µl |
Methionine | 20 µl |
Total | 1,000 µl |
Mix-2 (Lysate mix) | |
Wheatgerm lysates | 15 µl |
RNase free water | 7 µl |
Amino acids | 4 µl |
Methionine | 1 µl |
Plasmid DNA in TE buffer (1 µg/µl)** | 4 µl |
Reaction buffer | 15 µl |
RNasin* | 4 µl |
Total | 50 µl |
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's instruction. However, plasmids prepared using conventional, but not endotoxin-free, plasmid isolation kits have been successfully used. The manufacturer'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 µg/µl have successfully been used.
Day 2: Prepare GUVs by Electroformation and Harvest Wheat Germ Lysates
2. Preparation of GUVs
Lipid mix | Molar ratio | Volume (in µl) | Stock concentration |
POPC+POPS+Chol | 46.6+23.3+30 | 19.74+10.18+6.57 | POPC, POPS, Chol:10 mg/ml |
POPC+POPS+Chol+Brain-PI(4,5)P2 | 40+20+30+10 | 16.11+8.3+6.25+58.19 | POPC, POPS, Chol:10 mg/ml |
Brain-PI(4,5)P2:1 mg/ml |
Table 2: Volumes of different lipids used to obtain GUVs.
Figure 1: The layout of the ITO-coated glass slide used for electroformation. Please click here to view a larger version of this figure.
Figure 2: Schematic representation of the electroformation chamber (side view). Please click here to view a larger version of this figure.
Figure 3: The positions at which the electrodes from the multimeter are placed for measuring frequency and current. Please click here to view a larger version of this figure.
3. Harvest In Vitro Translation Reactions
4. GUV Binding Assay
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 binding to GUVs composed of POPC+POPS+Chol+brain-PI(4,5)P2 is evident by the increase in fluorescence intensity on the membrane surface. This increase can be also seen in fluorescence intensity profiles (Figure 4, bottom) along the line crossing the GUV boundary (X-X' in Figure 4C and D).
Figure 4: Gag binds to brain-PI(4,5)P2-contianing GUVs. A confocal cross section of POPC+POPS+Chol (A and C) and POPC+POPS+Chol+PI(4,5)P2 GUVs (B and D). Distribution of Gag-YFP is shown in green. Enlarged images of the selected GUVs (yellow boxes) are shown in panels C and D. Fluorescence intensity profiles along randomly chosen lines drawn to cross the opposite sides of GUVs (X to X′) are shown below the images. Bar = 5 µm. Please click here to view a larger version of this figure.
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 complex mixture (e.g., in the presence of cytosolic components), which is not readily achieved in other real-time techniques such as surface plasmon resonance-based assays. Therefore, one can argue that the assay described here enables real-time assessment of Gag-acidic lipid interactions in a more native context than other assays used in the field.
However, there are limitations associated with the method as well. To ensure that GUVs in a given preparation have similar composition to each other and to the original mixture, electroformation must be performed well above the transition temperature of the lipids that have the highest transition temperature present in the mixture25,26. However, it is possible that exposure of lipids to higher temperatures for longer time may lead to lipid breakdown25,27. Therefore, to control the quality of GUVs, in addition to visual inspection enabled by inclusion of fluorescent lipid dyes (e.g., DiD), it is desirable to test the functionality of the lipid of interest [e.g., PI(4,5)P2] using a lipid-specific probe. Additionally, with GUV compositions that give rise to phase separation at a certain temperature, care must be taken to precisely maintain a desired temperature when observing the protein-GUV interaction. Another limitation is that solutions of higher ionic strength are incompatible with the electroformation protocol described here, and therefore GUVs are grown and maintained in 300 mM sucrose solution26. Notably, however, some studies have successfully used buffers at physiological ionic strengths in their modified electroformation protocols28.
One critical aspect is the amount of Gag. A rabbit reticulocyte lysate-based system is often used for preparation of full-length HIV-1 Gag in liposome flotation assays. However, the amount of YFP-tagged full-length Gag obtained in this system was insufficient for visualization of Gag binding to GUVs. To address this problem, the wheat germ lysate-based cell-free transcription-translation system, which yields approximately 8-10 fold higher amount of Gag, was used in the current assay. However, if GUV binding experiments need to be performed in the absence of eukaryotic cell lysate components, one can use purified proteins labeled with a fluorophore as has been done recently (described below).
It has been shown that specific and efficient binding of Gag to membranes is a cooperative process regulated by various factors such as myristate, RNA, lipid headgroups and acyl chains, and Gag-Gag interactions (reviewed in reference29). The in vitro system describe here can be extended to address the roles of factors individually or in combination to understand the order of events in the cooperative process of Gag membrane binding. Thus far, the described assay system elucidated an unexpected role of unsaturated PI(4,5)P2 acyl chains in binding of Gag but not that of a canonical mammalian-cell-origin PI(4,5)P2-binding domain (the pleckstrin homology domain of phospholipase C delta 1)24, a finding that had not been revealed by liposome flotation assays. GUV-based assay systems have also been used to understand other aspects of Gag biology. Using a Gag derivative containing an artificial inducible multimerization motif and GUVs with raft-like and non-raft like domains, Keller et al. examined the relationship between Gag partitioning to 'raft-like' domains and multimerization30. A study by Carlson et al. has used the GUV system and purified and fluorescently labeled Gag to understand the sequential order of recruitment of various endosomal sorting complexes required for transport (ESCRT) proteins by membrane-bound Gag31. Recently, another study reported reconstitution of the Gag-induced vesicle budding process on the GUV using Gag conjugated with a fluorophore32. HIV-1 Gag multimerization leads to formation of submembrane Gag lattice that is thought to curve the membranes during assembly. Therefore, with further improvement in Gag-GUV assay systems, the relationship between membrane curvature changes and Gag membrane binding, which is arguably the least well understood aspect of HIV assembly, can be analyzed as is being done for other cellular curvature-sensitive proteins33.
In summary, using this protocol, one can obtain sufficient amounts of myristoylated full-length Gag and visualize Gag binding to GUV membranes. This protocol can be further developed to examine various stages of HIV-1 assembly and budding processes.
The authors have nothing to disclose.
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).
Digital Multimeter | Meterman | 30XR | A generic multimeter that can measure resistance and volts will serve the purpose. |
Function Generator | Instek | GFG-8216A | A generic function generator that is capable of generating a sine wave at 10hz and 1V is sufficient. |
ITO coated glass slides | Delta Technologies, Loveland, CO | CG-90IN | |
Incubator | Hoefer | Any incubator that can accurately maintain temperature will be sufficient | |
Vacuum chamber | Nalgene | ||
Thermomixer R | Eppendorf | 21516-166 | |
Syringe | Hamilton | 80400 | Gauge 22S, Syringe number 702 |
PDMS | Sylgard elastomer base kit, Dow-Corning | Sylgard, 184 | |
RTS 100 Wheat Germ CECF Kit | BiotechRabbit, Berlin, Germany |
BR1401001 | |
DiD | Life Technologies, Carlsbad, CA | D7757 | |
POPC | Avanti Polar Lipids, Alabaster, AL | 850457C | |
POPS | Avanti Polar Lipids | 840034C | |
Cholesterol | Avanti Polar Lipids | 700041P | |
Brain-PI(4,5)P2 | Avanti Polar Lipids | 840046X |