The presence of a single N-terminal histidine residue enhances the fusogenic properties of a Membranotropic peptide derived from herpes simplex virus type 1 glycoprotein H

Abstract

Herpes simplex virus type 1 (HSV-1)-induced membrane fusion remains one of the most elusive mechanisms to be deciphered in viral entry. The structure resolution of glycoprotein gB has revealed the presence of fusogenic domains in this protein and pointed out the key role of gB in the entry mechanism of HSV-1. A second putative fusogenic glycoprotein is represented by the heterodimer comprising the membrane-anchored glycoprotein H (gH) and the small secreted glycoprotein L, which remains on the viral envelope in virtue of its non-covalent interaction with gH. Different domains scattered on the ectodomain of HSV-1 gH have been demonstrated to display membranotropic characteristics. The segment from amino acid 626 to 644 represents the most fusogenic region identified by studies with synthetic peptides and model membranes. Herein we have identified the minimal fusogenic sequence present on gH. An enlongation at the N terminus of a single histidine (His) has proved to profoundly increase the fusogenic activity of the original sequence. Nuclear magnetic resonance (NMR) studies have shown that the addition of the N-terminal His contributes to the formation and stabilization of an alpha-helical domain with high fusion propensity.

FIGURE 1.

Peptide-promoted membrane fusion of PC/Chol (1:1) LUVs as determined by lipid mixing. Peptide aliquots were added to 0.1 mm LUVs containing 0.6% NBD and 0.6% Rho. The increase in fluorescence was measured after the addition of peptide aliquots. Reduced Triton X-100 (0.05% v/v) was referred to as 100% of fusion. Dose dependence of lipid mixing is reported. Fusion experiments for longer and shorter peptides are reported in panel a, for N-terminal elongated peptides in panel b, and for C-terminal elongated peptides in panel c.

FIGURE 2.

Peptide-promoted membrane fusion of PC/Chol (1:1) LUVs as determined by lipid mixing. Peptide aliquots were added to 0. 1 mm LUVs containing 0.6% NBD and 0.6% Rho. The increase in fluorescence was measured after the addition of peptide aliquots. Reduced Triton X-100 (0.05% v/v) was referred to as 100% of fusion. Dose dependence of lipid mixing at 37 °C is reported.

FIGURE 3.

Tryptophan fluorescence spectra in buffer and in liposomes for gH-(626–644) and gH625–644.

FIGURE 4.

Binding isotherms obtained plotting X_b* versus C_f for gH-(626–644) and gH625–644.

FIGURE 5.

Stern-Volmer plots of acrylamide quenching of gH-(626-644), L627S, and L631S in buffer (open symbols) and in LUVs (closed symbols).

FIGURE 6.

Sensorgrams of the binding between various concentrations of gH-(626–644) and gH625–644 with the HPA chip (upper) and the L1 chip (lower).

FIGURE 7.

Chemical shift index secondary structure prediction and NOE effects of gH-(617–644) (upper panel) and gH-(626–639) (lower panel) in TFE/H₂O (80/20 v/v) at 300 K. The gray bars reported for gH-(617–644) indicate the Hα chemical shift variations with respect to gH-(626–644) (101).

FIGURE 8.

Minimized structure of gH-(617–644) (upper structures) and gH-(626–639) (lower structures). Panels a and b, superposition of the 20 lowest energy conformers aligned according to the minimal root mean square deviations of the backbone atoms is shown. Panels c and d, representative structures are shown. Side chains are shown in blue, and side chains assuming α-helical structure in gH-(626–639) are colored in green.

FIGURE 9.

α-Helical structure representation of gH-(617–644) (panel a). The helix is depicted in red, His-625 and Gly-626 side chains are reported in gold, and Trp-634 and Tyr-637 side chains are in green. Electrostatic surface potentials are shown in panels a and c. Polar and charged residues are shown in blue, and hydrophobic amino acids are in light gray.

FIGURE 10.

Cells were incubated with increasing concentrations of the peptides (10, 25, 50, and 100 μm) in the presence of the viral inoculum for 45 min at 37 °C. Nonpenetrated virus was inactivated, and cells were incubated for 48 h at 37 °C in Dulbecco's modified Eagle's medium supplemented with carboxymethylcellulose. Plaque numbers were scored, and the percentage of inhibition was calculated with respect to no-peptide control experiments. Data are reported in triplicate, and error bars represent S.D. Inhibition experiments for longer and shorter peptides are reported in panel a, for N-terminal elongated peptides in panel b, and for C-terminal elongated peptides in panel c.