Alkyl anchor-modified artificial viral capsid budding outside-to-inside and inside-to-outside giant vesicles

Abstract

The budding of human immunodeficiency virus from an infected host cell is induced by the modification of structural proteins bearing long-chain fatty acids, followed by their anchoring to the cell membrane. Although many model budding systems using giant unilamellar vesicles (GUVs) induced by various stimuli have been developed, constructing an artificial viral budding system of GUVs using only synthesized molecules remains challenging. Herein, we report the construction of an artificial viral capsid budding system from a lipid bilayer of GUV. The C-terminus of the β-annulus peptide was modified using an octyl chain as an alkyl anchor via a disulfide bond. The self-assembly of the β-annulus peptide with an octyl chain formed an artificial viral capsid aggregate. The fluorescence imaging and transmission electron microscopy observations revealed that the addition of the tetramethylrhodamine (TMR)-labeled octyl chain-bearing β-annulus peptide to the outer aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle outside-to-inside the mother GUV. Conversely, the encapsulation of the TMR-labeled octyl chain-bearing β-annulus peptide in the inner aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle inside-to-outside the mother GUV. Contrarily, the addition of the TMR-labeled β-annulus peptide to GUV barely induced budding. It was demonstrated that the higher the membrane fluidity of GUV, the more likely budding would be induced by the addition of the alkyl anchor-modified artificial viral capsid. The simple virus-mimicking material developed in this study, which buds off through membrane anchoring, can provide physicochemical insights into the mechanisms of natural viral budding from cells.

Keywords: Artificial viral capsid; alkyl anchor; budding; giant vesicle; self-assembly; β-annulus peptide.

Graphical abstract

Figure 1.

Schematic illustration of the alkyl anchor-modified artificial viral capsid budding outside-to-inside and inside-to-outside giant unilamellar vesicles (GUVs).

Figure 2.

(a) Schematic illustration of the construction of the enveloped artificial viral capsid via hydrophobic interaction. (b–j) size distributions obtained from DLS (b, e, h), TEM images (c, f, i), and ζ-potential (d, g, j) of the alkyl anchor-modified artificial viral capsid (b–d; 50 μM β-annulus–SS-octyl peptide), enveloped artificial viral capsid (e-g; 50 μM β-annulus–SS-octyl peptide, 5 mM POPC), and liposome (h–j; 5 mM POPC) in a 10 mM Tris-HCl buffer (pH 7.4, 5% DMSO) at 25°C. The TEM samples were stained with an EM stainer.

Figure 3.

Concentration dependence of the β-annulus–SS-octyl peptide on the scattering intensity determined by DLS of alkyl anchor-modified artificial viral capsid (a) and enveloped artificial viral capsid (b) in a 10 mM Tris-HCl buffer (pH 7.4, 5% DMSO) at 25°C.

Figure 4.

(a) Schematic illustration of the construction of the TMR/NBD-labeled enveloped artificial viral capsid. (b) Fluorescence emission spectra of the TMR/NBD-labeled enveloped artificial viral capsid comprising 10 μM NBD-PE, 4.99 mM POPC, 25 (blue) or 23.1 µM (red) β-annulus–SS-octyl peptide, and 0 (blue) or 1.9 μM (red) TMR–β-annulus-SS-octyl peptide excited at 460 nm in the 10 mM HEPES buffer (pH 7.4, 5% DMSO) at 25°C. Schematic illustration of FRET between the TMR–β-annulus-SS-octyl peptide and NBD-PE on the enveloped artificial viral capsid.

Figure 5.

(a,b) CLSM images of the NBD-labeled POPC GUVs (1 mM POPC, 100 μM cholesterol, 10 μM NBD-PE) before (a) and after (b) adding 2 μM TMR–β-annulus–SS-octyl outside GUV. (c) Time-series CLSM images of NBD-labeled POPC GUV budding outside-to-inside. The elapsed time from the start of observation is shown. Channels for NBD-PE (green) and TMR (magenta), as well as bright fields of the CLSM images. The inner aqueous phase of GUV comprised 150 mM sucrose and 350 mM glucose in a 10 mM HEPES buffer (pH 7.4), and the outer phase comprised 2 μM TMR–β-annulus–SS-octyl and 500 mM glucose in the 10 mM HEPES buffer containing 5% DMSO (pH 7.4).

Figure 6.

TEM images of the budding of POPC GUVs ([POPC] = 1 mM, [cholesterol] = 100 μM, [NBD-PE] = 10 μM), following the addition of 2 μM TMR–β-annulus–SS-octyl outside (a–c) and inside (d–f) GUV. The TEM samples were stained with an EM stainer.

Figure 7.

Ratio of the number of deformed POPC GUVs to the total number of POPC GUVs following the addition of 2 μM TMR–β-annulus–SS-octyl, TMR–β-annulus, or TMR–octyl outside (N > 100).

Figure 8.

CLSM images of the NBD-labeled GUVs (DOPC, POPC, DPPC) with the addition of 2 μM TMR–β-annulus–SS-octyl, TMR–β-annulus, or TMR–octyl outside. Channels for NBD-PE (green) and TMR (magenta) of the CLSM images.

Figure 9.

(a,b) CLSM images of NBD-labeled POPC GUVs (1 mM POPC, 100 μM cholesterol, 10 μM NBD-PE) before (a) and after (b) adding 2 μM TMR–β-annulus–SS-octyl inside GUV. (c) Time-series CLSM images of NBD-labeled POPC GUV budding inside-to-outside. The elapsed time from the start of observation is shown. Channels for NBD-PE (green) and TMR (magenta), as well as the bright field for the CLSM images. The inner aqueous phase of GUV comprises 2 μM TMR–β-annulus–SS-octyl, 150 mM sucrose, and 350 mM glucose in a 10 mM HEPES buffer containing 5% DMSO (pH 7.4), and the outer phase comprises 500 mM glucose in the 10 mM HEPES buffer (pH 7.4).

Figure 10.

Ratio of the number of deformed POPC GUVs to the total number of POPC GUVs following the addition of 2 μM TMR–β-annulus–SS-octyl, TMR–β-annulus, or TMR–octyl inside (N > 100).

Figure 11.

CLSM images of NBD-labeled GUVs (DOPC, POPC, DPPC) with the addition of 2 μM TMR–β-annulus–SS-octyl, TMR–β-annulus, or TMR–octyl inside. Channels for NBD-PE (green) and TMR (magenta) of the CLSM images.