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

To determine the exact steps in the life cycle of HSV-1 that were inhibited by Eval418, a time of addition experiment for Eval418 against HSV-1 was performed. HSV-1 or Vero cells were incubated with Eval418 at 10 μg/mL for different periods of time, and the inhibitory effects were measured by PRA ( Figure 4a ). After pre-incubation with HSV-1 or following addition to the cells during the attachment step of HSV-1, Eval418 exhibited potent inhibition of plaque formation, suggesting an initial inhibitory activity against HSV-1 infection. On the contrary, Eval418 scarcely reduced plaque quantity when pre-incubated with Vero cells or added to cells after infection and showed a weak inhibition against viral entry. These results suggested that Eval418 only exerted a viral inactivation effect and prevented initiation of HSV-1 infection but did not affect intracellular anti-HSV-1 activity. (c) Time-dependent inactivation activity of the Eval418 peptide. HSV-1 (60 PFU/well) was directly incubated with the Eval418 peptide over serial durations, and the mixture was applied to a plaque reduction assay on Vero cells. (d) Inhibitory activity of the Eval418 peptide against HSV-1 viral attachment. Eval418 was added to the cell culture medium at a series of concentrations simultaneously with HSV-1 at the viral attachment step and rinsed out before viral entry. Then, the inhibitory effect was measured by plaque reduction assay. (e) The inhibitory activity of the Eval418 peptide against HSV-1 at the viral entry step. After viral attachment, Eval418 was added and co-incubated with cells during the viral entry step. The inhibitory effect was then measured by plaque reduction assay. (f) Cellular localization of the Eval418 peptide in Vero cells. Eval418 was labeled by FITC, and the cellular localization of Eval418 was determined by confocal microscopy after incubation for 24 h with Vero cells. Scale bars: 10 µM. Figure 4b , HSV-1 was incubated with Eval418 over a series of concentrations, and the mixture was then used to infect Vero cells. The data from PRA experiments showed that plaque formation was inhibited by Eval418 in a dose-dependent manner and that the 50% inhibition concentration (IC50) was 2.48 μg/mL. The viral inactivation effect of Eval418 was also time-dependent at 10 μg/mL (Figure 4c ). When treated with Eval418 for 5 min, the PFU of HSV-1 decreased by 20%, suggesting a rapid-onset inactivation. After treatment with Eval418 for 15 min, the average reduction increased to 67.87%. The reduction was more than 90% after 30 min and almost 100% following Eval418 treatment for 1 h.