Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity

Abstract

Herpes simplex virus (HSV) membrane fusion represents an attractive target for anti-HSV therapy. To investigate the structural basis of HSV membrane fusion and identify new targets for inhibition, we have investigated the different membranotropic domains of HSV-1 gH envelope glycoprotein. We observed that fusion peptides when added exogenously are able to inhibit viral fusion likely by intercalating with viral fusion peptides upon adopting functional structure in membranes. Interestingly, peptides analogous to the predicted HSV-1 gH loop region inhibited viral plaque formation more significantly. Their inhibitory effect appears to be a consequence of their ability to partition into membranes and aggregate within them. Circular dichroism spectra showed that peptides self-associate in aqueous and lipidic solutions, therefore the inhibition of viral entry may occur via peptides association with their counterpart on wild-type gH. The antiviral activity of HSV-1 peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

Fig. 1

(A) HSV-1 gH protein diagram and secondary structure prevision and (B) hydrophobicity plot.

Fig. 2

gH peptides induced fusion of unilammellar vesicles. Peptide-promoted membrane fusion of PC/Chol (1:1) LUV as determined by lipid mixing; peptide aliquots were added to 0.1 mM LUV, containing 0.6% NBD and 0.6% Rho. The increase in the fluorescence was measured 15 min after the addition of peptide aliquots; reduced Triton-X-100 (0.05%, v/v) was referred to as 100% of fusion. The dose dependence of lipid mixing is reported.

Fig. 3

Inhibition of viral infectivity. (A) Vero cells were incubated with increasing concentrations of the peptides (10, 50, 100, 250, 500 μM) in the presence of the viral inoculum for 45 min at 37 °C. (B) Cells were exposed to active peptides at a concentration of 250 μM either prior to infection (cells pre-exposure), during attachment and entry (co-exposure), after virus penetration (post-exposure), or alternatively, the virus was pre-incubated with peptides for 1 h at 37 °C before addition to the cells (virus preincubation). (C) Cells were exposed to active peptides (gH493–512 and gH626–644) at a concentration of 250 and 500 μM and PIV-2 in the co-exposure mode. For all treatments, non-penetrated viruses were inactivated by low-pH citrate buffer after the 45 min incubation with cells at 37 °C. The cells were then, incubated for 48 h at 37 °C in DMEM supplemented with CMC and plaque numbers were scored. Experiments were performed in triplicate and the percentage of inhibition was calculated with respect to no-peptide control experiments. Error bars represent standard deviations.

Fig. 4

Secondary structure of gH peptides by CD spectroscopy. Circular dichroism spectra of gH626–644 (A) and gH493–512 (B) at different percentages of TFE.

Fig. 5

Circular dichroism spectra of gH. Circular dichroism spectra of gH626–644 (A) and gH493–512 (B) at different peptide concentrations.

Fig. 6

Native gel electrophoresis of gH peptides. Lane 1: molecular weight standard (188 kDa; 98 kDa; 62 kDa; 49 kDa; 38 kDa; 28 kDa; 15 kDa; 4 kDa); lane 2: gH626–644; lane 3: gH626–644 treated with 4 mM SDS; lane 4: gH626–644 treated with 5 mM SDS; lane 5: gH493–512; lane 6: gH493–512 treated with 4 mM SDS; lane 7: gH493–512 treated with 5 mM SDS.