Scorpion Venom Antimicrobial Peptide Derivative BmKn2-T5 Inhibits Enterovirus 71 in the Early Stages of the Viral Life Cycle In Vitro

Abstract

Enterovirus 71 (EV71), a typical representative of unenveloped RNA viruses, is the main pathogenic factor responsible for hand, foot, and mouth disease (HFMD) in infants. This disease seriously threatens the health and lives of humans worldwide, especially in the Asia-Pacific region. Numerous animal antimicrobial peptides have been found with protective functions against viruses, bacteria, fungi, parasites, and other pathogens, but there are few studies on the use of scorpion-derived antimicrobial peptides against unenveloped viruses. Here, we investigated the antiviral activities of scorpion venom antimicrobial peptide BmKn2 and five derivatives, finding that BmKn2 and its derivative BmKn2-T5 exhibit a significant inhibitory effect on EV71. Although both peptides exhibit characteristics typical of amphiphilic α-helices in terms of their secondary structure, BmKn2-T5 displayed lower cellular cytotoxicity than BmKn2. BmKn2-T5 was further found to inhibit EV71 in a dose-dependent manner in vitro. Moreover, time-of-drug-addition experiments showed that BmKn2-T5 mainly restricts EV71, but not its virion or replication, at the early stages of the viral cycle. Interestingly, BmKn2-T5 was also found to suppress the replication of the enveloped viruses DENV, ZIKV, and HSV-1 in the early stages of the viral cycle, which suggests they may share a common early infection step with EV71. Together, the results of our study identified that the scorpion-derived antimicrobial peptide BmKn2-T5 showed valuable antiviral properties against EV71 in vitro, but also against other enveloped viruses, making it a potential new candidate therapeutic molecule.

Keywords: BmKn2-T5; EV71; antimicrobial peptides; antiviral activity; scorpion venom peptides.

Figure 1

Screening of scorpion venom antimicrobial peptide BmKn2 and its derivatives against EV71. (A) Multiple alignment of BmKn2 and five derivatives. Conserved amino acids are highlighted in yellow. (B) Inhibitory activity of BmKn2 and its five derivatives against EV71-induced CPE in RD cells. RD cells infected with EV71 at an MOI of 1 were treated with 10 μg/mL BmKn2 or its derivatives, and the morphology of RD cells was recorded at a magnification of 100× after 24 h post-infection. (C) Inhibitory activity of BmKn2 against EV71 assessed by qRT-PCR. (D) Inhibitory activity of BmKn2-T5 against EV71 assessed by qRT-PCR. Data are presented as the means ± SD from three independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 2

Analysis of BmKn2 and BmKn2-T5 structural characteristics. (A,B) Three-dimensional structures of BmKn2 and BmKn2-T5 obtained using I-TASSER. (C,D) Helical wheel plots of BmKn2 and BmKn2-T5 obtained using the HeliQuest server. In the helical wheel plots, arrows represent the direction of summed vectors of hydrophobicity, and residues marked in blue and yellow represent positively charged alkaline and hydrophobic amino acids, respectively. Meanwhile, the residue marked in purple represents Ser, and residues marked in gray represent Gly and Ala. (E,F) CD spectrum analysis of BmKn2 and BmKn2-T5. The CD spectra were recorded in water, 30% TFE, and 70% TFE solutions, respectively. N-ter, N-terminus; C-ter, C-terminus.

Figure 3

Dose-dependent antiviral effect of BmKn2-T5 on EV71 in RD cells. Cytotoxicity of (A) BmKn2 and (B) BmKn2-T5 in RD cells. The cell viability of RD cells treated with different concentrations of BmKn2 and BmKn2-T5 was measured using an MTS assay. (C) Inhibitory activity of BmKn2-T5 with different concentrations to EV71-induced CPE in RD cells. RD cells infected with EV71 at an MOI of 1 were treated with different concentrations of BmKn2-T5, and the morphology of RD cells was recorded at a magnification of 100× at 24 h post-infection. Dose-dependent inhibitory effect of BmKn2-T5 on EV71 analyzed using (D) qRT-PCR and (E) Western blotting. Data are presented as the means ± SD from three independent experiments (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Original images of (E) can be found in Figure S1.

Figure 4

Effect of BmKn2-T5 on different stages of EV71 life cycle. (A) Schematic diagram for studying the action stage of BmKn2-T5 on EV71. (B) Effect of BmKn2-T5 on EV71 free virion in RD cells. EV71 attachment, entry, and replication in RD cells detected using (C) qRT-PCR and (D) Western blotting. (E) Schematic diagram of BmKn2-T5 treatment for different time points on EV71 infection. (F) Effect of BmKn2-T5 added for different time points on EV71 infection. RD cells infected with EV71 were incubated with 10 μg/mL BmKn2-T5 for the indicated times, and intracellular EV71 RNA was analyzed using qRT-PCR at 12 h post-infection, respectively. Data are presented as the means ± SD from three independent experiments (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Original images of (D) can be found in Figure S2.

Figure 5

Antiviral activities of BmKn2-T5 against DENV, ZIKV, and HSV-1 at noncytotoxic concentrations. Inhibitory effect of BmKn2-T5 on (A) DENV2 and (B) ZIKV. Vero cells infected with ZIKV at MOI of 0.1 were treated with different concentrations of BmKn2-T5 for 24 h, and the level of intracellular DENV2 or ZIKV RNA was analyzed using qRT-PCR. (C,D) Inhibitory effect of BmKn2-T5 on HSV-1 infection in vitro. Vero cells infected with HSV-1 were treated with different concentrations of BmKn2-T5 for 1 h, and intracellular HSV-1 was then detected using plaque assays. Meanwhile, plaques were quantified by counting, and the results are described in percentages for the statistical analysis. (E,F) Effect of BmKn2-T5 on DENV or ZIKV attachment, entry, and replication in RD cells detected using qRT-PCR. Data are presented as the means ± SD from three independent experiments (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001).