Saikosaponin B2, Punicalin, and Punicalagin in Vitro Block Cellular Entry of Feline Herpesvirus-1

Abstract

In the realm of clinical practice, nucleoside analogs are the prevailing antiviral drugs employed to combat feline herpesvirus-1 (FHV-1) infections. However, these drugs, initially formulated for herpes simplex virus (HSV) infections, operate through a singular mechanism and are susceptible to the emergence of drug resistance. These challenges underscore the imperative to innovate and develop alternative antiviral medications featuring unique mechanisms of action, such as viral entry inhibitors. This research endeavors to address this pressing need. Utilizing Bio-layer interferometry (BLI), we meticulously screened drugs to identify natural compounds exhibiting high binding affinity for the herpesvirus functional protein envelope glycoprotein B (gB). The selected drugs underwent a rigorous assessment to gauge their antiviral activity against feline herpesvirus-1 (FHV-1) and to elucidate their mode of action. Our findings unequivocally demonstrated that Saikosaponin B2, Punicalin, and Punicalagin displayed robust antiviral efficacy against FHV-1 at concentrations devoid of cytotoxicity. Specifically, these compounds, Saikosaponin B2, Punicalin, and Punicalagin, are effective in exerting their antiviral effects in the early stages of viral infection without compromising the integrity of the viral particle. Considering the potency and efficacy exhibited by Saikosaponin B2, Punicalin, and Punicalagin in impeding the early entry of FHV-1, it is foreseeable that their chemical structures will be further explored and developed as promising antiviral agents against FHV-1 infection.

Keywords: Bio-layer interferometry; envelope glycoprotein B; feline herpesvirus-1; natural compounds; virus entry inhibitor.

Figure 3

Identified natural compounds directly interact with gB through BLI binding kinetics assay. (A) Immobilization of gB to Ni-NTA probe and Reference Ni-NTA probe. (B) BLI sensorgram structures of 16 compounds showing their binding to gB. For each compound, a 1:1 fitting curve was constructed. The chemical structure and binding dissociation coefficients of the compounds are shown. The KD (M) value represents the affinity constant, Kon (1/Ms) represents the rate of association, and Kdis (1/s) represents the rate of dissociation.

Figure 6

Saikosaponin B2, Punicalin, and Punicalagin target the early stages of FHV-1 entry. (A–C) Effect of Saikosaponin B2, Punicalin, and Punicalagin on free virus particles, virus entry, and viral fusion. (D,E) Saikosaponin B2, Punicalin, and Punicalagin were used to pretreat CRFK cells or applied to CRFK cells infected with FHV-1. Data are representative of three independent experiments. Data were analyzed using Student’s t-test (*, p < 0.05; **, p < 0.01; ****, p < 0.0001).

Figure 7

Saikosaponin B2, Punicalin, and Punicalagin target the early stages of FHV-1 entry/fusion. (A) Saikosaponin B2, Punicalin, and Punicalagin inhibit the adsorption of FHV-1. FHV-1 was labeled with DIOC using 10 MOI and whether the drug was added or not and incubated in CRFK cells at 4 °C for 2 h, formaldehyde-fixed and DAPI-stained, and DIOC fluorescence was detected through laser confocal microscopy. (B) Saikosaponin B2 inhibited the entry of FHV-1. FHV-1 was labeled with R18 using 10 MOI and whether the drug was added or not. The cells were incubated in CRFK cells at 4 °C for 2 h and then continued to be incubated at 37 °C for 2 h. The cells were formaldehyde-fixed and stained with DAPI, and the R18 fluorescence was detected through laser confocal microscopy. (C,D) Quantification of fluorescence intensity was carried out using ImageJ. Data are representative of three independent experiments. Data were analyzed using Student’s t-test (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 1

Schematic diagram of the experimental program.

Figure 2

Image of FHV-1 gB Western Blot.

Figure 4

Antiviral activity of Saikosaponin B2, Punicalin, and Punicalagin against FHV-1 in CRFK cells. (A) Dilute the drug in cell maintenance medium containing 50 TCID₅₀ FHV-1. Add the diluted drug to a 96-well plate with cells that have grown into a monolayer, with each well receiving 100 μL. In the cell culture incubator, observe cytopathic effects (CPE) in 80% of cells in the positive control group. Examine the condition of each well under an inverted optical microscope and calculate and plot the inhibition curve using GraphPad Prism. (B) Dilute the drug in serum-free cell culture medium containing 50 TCID₅₀ FHV-1. Add the diluted drug to a 24-well plate with cells that have grown into a monolayer, with each well receiving 500 μL. After 2 h of incubation in the cell culture incubator, remove the supernatant, and overlay with 1% agarose containing 2% FBS. Continue incubation in the cell culture incubator for 48 h. After fixation with 4% paraformaldehyde and staining with crystal violet, capture images using a CTL fluorescence spot reader.

Figure 5

Saikosaponin B2 (50 μg/mL), Punicalin (0.75 μg/mL), and Punicalagin (1.5 μg/mL) inhibition of CPE produced by FHV-1 on CRFK cells.

Figure 8

Saikosaponin B2, Punicalin, and Punicalagin altered the thermal stability of gB. Changes in Tm (Tm, the temperature at which 50% of the protein remains soluble) values of gB proteins in the presence or absence of drugs. The results showed a rightward shift in the measured Tm in the drug group compared to no drug. Each set of trials was repeated four times.

Figure 9

Electron microscopy of drug-treated virus particles. Saikosaponin B2, Punicalin, and Punicalagin do not disrupt the structural integrity of virus particles.