Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

Summary

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.

Abstract

The structural protein of HIV-1, Pr55^Gag (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.

Introduction

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 Pr55^Gag (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 th....

Protocol

Day 1: Expression of Gag Proteins Using the Wheat Germ Lysate-based In Vitro Transcription-translation System

1. Preparation of HIV-1 Gag

1. Remove all the reagents of the commercial wheat germ CECF kit from freezers (wheat germ lysates are stored at -80 ºC; other reagents at -20 ºC). Thaw them on ice and mix the components as shown in Table 1.

Mix-1 (Feeding mix)
Feeding solution	900 µl
Ami....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 10 Hz and 1 V 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