Analysis of a membrane interacting region of herpes simplex virus type 1 glycoprotein H

Abstract

Glycoprotein H (gH) of herpes simplex virus type I (HSV-1) is involved in the complex mechanism of membrane fusion of the viral envelope with the host cell. Membrane interacting regions and potential fusion peptides have been identified in HSV-1 gH as well as glycoprotein B (gB). Because of the complex fusion mechanism of HSV-1, which requires four viral glycoproteins, and because there are only structural data for gB and glycoprotein D, many questions regarding the mechanism by which HSV-1 fuses its envelope with the host cell membrane remain unresolved. Previous studies have shown that peptides derived from certain regions of gH have the potential to interact with membranes, and based on these findings we have generated a set of peptides containing mutations in one of these domains, gH-(626-644), to investigate further the functional role of this region. Using a combination of biochemical, spectroscopic, and nuclear magnetic resonance techniques, we showed that the alpha-helical nature of this stretch of amino acids in gH is important for membrane interaction and that the aromatic residues, tryptophan and tyrosine, are critical for induction of fusion.

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% rhodamine. 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. In figure is reported the dose dependence of lipid mixing.

FIGURE 2.

A, fluorescence spectra in buffer and in liposomes for gH-(626–644) and Y637S labeled with NBD. B, tryptophan fluorescence spectra in buffer and in liposomes for gH-(626–644) and Y637S.

FIGURE 3.

Proteolytic digestion of membrane-bound NBD-labeled peptides. The fluorescence emission spectra of the NBD-labeled peptide was monitored at 530 nm with excitation set at 467 nm. A, fresh preparation of the peptide gH-(626–644); B, peptide gH-(626–644) after 24 h from solubilization in buffer; C, results obtained for all the labeled peptides. AU, absorbance unit.

FIGURE 4.

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

FIGURE 5.

Binding isotherms obtained plotting X_b^*versus C_f for NBD-gH-(626–644), NBD-R642S, and NBD-Y637S (A, C, and E); binding isotherms obtained plotting X_b^*versus C_f for gH-(626–644), R642S, and Y637S (B, D, and F).

FIGURE 6.

Circular dichroism spectra of peptides at different percentages of TFE.

FIGURE 7.

Circular dichroism spectra of peptides in 10 mm SDS.

FIGURE 8.

NOE effects and CSI of gH-(626–644) (upper panel), R642S (central panel), and L627S (lower panel) in TFE/H₂O (80:20, v/v) at 300 K.

FIGURE 9.

Minimized structures of gH-(626–644) (upper structure), R642S (central structure), and L627S (lower structure) in TFE/H₂O (80:20). A, superposition of the 20 best energy conformers aligned according to the minimal root mean square deviation of the backbone atoms Only the polypeptide backbone is shown. B, representative structures. Side chains are shown in magenta.

FIGURE 10.

Side-chain orientations of gH-(626–644) (upper structure), R642S (central structure), and L627S (lower structure) peptides. The van der Waals surface of the representative conformer in TFE/H₂O (80:20) is shown. Polar and charged residues are shown in red; hydrophobic amino acids are colored in light gray; mutated residues in R642S and L627S peptides are colored in yellow.

FIGURE 11.

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 DMEM 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.