Histidine-rich Modification of a Scorpion-derived Peptide Improves Bioavailability and Inhibitory Activity against HSV-1

Zeng, Zhengyang; Zhang, Runhong; Hong, Wei; Cheng, Yuting; Wang, Huijuan; Lang, Yange; Ji, Zhenglin; Wu, Yingliang; Li, Wenxin; Xie, Youli; Cao, Zhijian

The suppressive activities of Eval418 and its derivative peptides against HSV-1 proliferation in the post-entry assay were then determined (Figure 6g) . The extracellular and intracellular infectivity of HSV-1 were assessed by plaque forming assay, and the intracellular DNA quantity was measured by real-time PCR. The wild type peptide Eval418 exerted no effect, whereas the extracellular and intracellular infectivity of HSV-1 were significantly decreased by all four derivative peptides. Among these peptides, Eval418-FH5 exhibited the strongest inhibition and reduced the infectivity of extracellular and intracellular HSV-1 by 81.90% and 77.65%, respectively. Concentration-dependent inactivation activities of Eval418 derivative peptides. HSV-1 (60 PFU/well) was directly treated with Eval418-derived peptides. Vero cells were infected with the virus-peptide mixtures at a serial final concentration of the derivative peptides. (d) Time-dependent inactivation activities of Eval418 derivative peptides. Eval418-derived peptides were added to HSV-1 (60 PFU/well) and incubated over a series of indicated durations. The mixtures were then used to infect Vero cells, and the plaque reduction assay was completed. (e) Inhibitory activities of the Eval418-derived peptides when added to cells during the HSV-1 viral attachment step. Peptides were added to culture medium of Vero cells simultaneously with HSV-1 and incubated for 1 h at 4 ℃. The cells were then rinsed and replenished with a cover layer, and the plaque reduction assay was completed. (f) Inhibitory activities of the Eval418-derived peptides against viral entry of HSV-1. Cells were incubated with HSV-1 at 4 ℃ for 1 h. After HSV-1 was washed out, the derivative peptides were added and incubated at 37 ℃ for 1 h. The cells were then rinsed and replenished with a cover layer. The inhibitory rates were measured by plaque reduction assay. (g) Anti-HSV-1 activities of Eval418 and its derivative peptides during the post-entry step. Extracellular and intracellular HSV-1 infectivity levels were assessed by plaque forming assay, and the DNA content was assessed by real-time PCR. (h) Flow cytometry measurement of the cellular uptake of Eval418 and Eval418-FH5 peptides. Vero cells were incubated with FITC-labeled Eval418 or Eval418-FH5 for 24 h, and the average FITC intensity of each cell was measured by flow cytometry. ***P< 0.001. (i) Confocal microscopic examination of cellular localization of Eval418-FH5. Eval418-FH5 was labeled by FITC and used to treat Vero cells for 24 h. The cellular localization was determined using confocal microscopy. Scale bars: 10 µM.