Antiviral Activity of a Turbot (Scophthalmus maximus) NK-Lysin Peptide by Inhibition of Low-pH Virus-Induced Membrane Fusion

Abstract

Global health is under attack by increasingly-frequent pandemics of viral origin. Antimicrobial peptides are a valuable tool to combat pathogenic microorganisms. Previous studies from our group have shown that the membrane-lytic region of turbot (Scophthalmus maximus) NK-lysine short peptide (Nkl_71⁻100) exerts an anti-protozoal activity, probably due to membrane rupture. In addition, NK-lysine protein is highly expressed in zebrafish in response to viral infections. In this work several biophysical methods, such as vesicle aggregation, leakage and fluorescence anisotropy, are employed to investigate the interaction of Nkl_71⁻100 with different glycerophospholipid vesicles. At acidic pH, Nkl_71⁻100 preferably interacts with phosphatidylserine (PS), disrupts PS membranes, and allows the content leakage from vesicles. Furthermore, Nkl_71⁻100 exerts strong antiviral activity against spring viremia of carp virus (SVCV) by inhibiting not only the binding of viral particles to host cells, but also the fusion of virus and cell membranes, which requires a low pH context. Such antiviral activity seems to be related to the important role that PS plays in these steps of the replication cycle of SVCV, a feature that is shared by other families of virus-comprising members with health and veterinary relevance. Consequently, Nkl_71⁻100 is shown as a promising broad-spectrum antiviral candidate.

Keywords: NK-lysin; Nkl71–100; SVCV; aggregation; antiviral; leakage; phosphatidylserine; phospholipid vesicles; viral fusion.

Figure 1

Vesicle aggregation induced by the interaction of the Nkl_71–100 with phospholipid vesicles. To 1 ml of vesicles (0.14 mM) of PC (A), PG (A) or PS (B) in medium buffer at either pH 3 (full circles and continuous lines) or pH 7 (empty circles and dash lines), different amounts of Nkl_71–100 were added to reach a final concentration of peptide ranging 0–25 µM. The absorbance was measured after incubating the sample for 1 h at 37 °C and presented as its variation respect to solvent control values. Results are shown as mean ± s. d. from three different experiments.

Figure 2

Carboxifluorescein leakage from phospholipid vesicles induced by Nkl_71–100. To 100 µL of carboxifluorescein loaded PS vesicles (0.14 mM) in medium buffer at either pH 3 (orange) or pH 7 (gray), different amounts of Nkl_71–100 were added to reach a final concentration of peptide ranging 0–24 µM. The mixtures were incubated at 37 °C for 30 min and the fluorescence intensity was recorded with excitation and emission wavelengths set at 492 nm and 520 nm, respectively. Results are shown in percentage (mean ± s. d., n = 3), considering maximal leakage that obtained upon addition of 0.5% Triton X-100.

Figure 3

Effect of Nkl_71–100 on the thermotropic behavior of DMPS vesicles. DPH-labeled DMPS LUVs vesicles, with or without added Nkl_71–100 peptide, were submitted to a temperature ramp, and the anisotropy of the fluorescent probe was measured. The phospholipid concentration was 0.14 mM, the peptide 18.4 µM, and the DPH probe to lipid molar ratio 1 to 500. The lipid phase transition (T_m) for each condition is indicated.

Figure 4

Viability of EPC cells after treatment with Nkl_71–100. EPC monolayers were treated with increasing concentrations of Nkl_71–100 for 24 h at 22 °C before performing the MTT assay. Cell viability is shown as the percentage (mean ± s.d.) relative to untreated cells from three independent experiments performed in tetraplicate.

Figure 5

Infectivity of SVCV after preincubation with Nkl_71–100. EPC monolayers were infected with SVCV (MOI 10⁻³) co-incubated with increasing concentrations of Nkl_71–100 (0–32 μM) for 24 h. Same mixes performed right before the inoculation to cell monolayers were also included (t = 0). After 20 h of infection, cells were fixed, SVCV-infected foci detected by fluorescent immune-labelling and counted. (A) Results are presented as percentage of foci in comparison to those found in corresponding untreated SVCV-infected monolayers, shown as the mean (±s.d.) from two independent experiments performed in triplicate. Solid lines correspond to the best fit to a dose-response curve, from which the IC₅₀ values that are included in the graph were calculated. Multiple t test analysis did not give any significant difference between the two datasets. (B) Representative images (merging both bright and fluorescent fields) of SVCV-infected cell monolayers with different focus occurrence patterns are shown.

Figure 6

Effect of the timing of the Nkl_71–100 treatment on the infectivity of SVCV. EPC monolayers were treated 2 h before the inoculation of SVCV (pre-adsorption) and just after the adsorption period for either 2 or 20 h (post-adsorption) with increasing concentrations of Nkl_71–100 (0–32 μM). Cells were infected with SVCV at MOI 10⁻³. After 20 h of infection, cells were fixed, SVCV-infected foci detected by fluorescent immune-labelling and counted. Results are presented as percentage of foci relative to those found in corresponding untreated SVCV-infected monolayers, shown as the mean (±s.d.) from three different experiments performed in triplicate. Solid lines correspond to the best fit to a dose-response curve, from which the IC₅₀ values were calculated, if possible, and included in the graph.

Figure 7

Effect of Nkl_71–100 on the SVCV attachment to host cell surface. The amount of SVCV particles just adsorbed to host cell in the presence (8 and 24 μM) and absence of Nkl_71–100 was determined by quantifying their gene copies by RT-qPCR. SVCV was used at MOI 1. Results are shown as percentage of amplified copies (normalized to cellular ef1a ones) relative to the value corresponding to untreated monolayers. Each mean (±s.d.) is calculated from three different experiments performed in duplicate. The significance of the changes between treated and untreated groups were indicated as: * p < 0.05 and ** p < 0.01.

Figure 8

Effect of Nkl_71–100 on the virus-cell interaction through the SVCV-gpG-mediated fusion activity. The evaluation of the ability of Nkl_71–100 to inhibit the membrane fusogenic activity of SVCV gpG was determined as previously described by [56]. Treatments included Nkl_71–100 at 8 and 24 μM which were either incubated with cells for 2 h and then removed prior to the fusion triggering or just added in that moment. A. Results are shown as percentage of syncytia relative to that present in corresponding untreated monolayers. Each mean (±s.d.) is calculated from two different experiments performed in triplicate. The significance of the changes between treated and untreated groups were indicated as: ** p < 0.01 and *** p < 0.001. B. Representative bright field images of SVCV-gpG-mediated syncytia taken at two different magnifications.