Methods
Chemicals and reagents
The cyclic pentapeptide, Aspergillipeptide D, was isolated from the fungal strain Aspergillus SCSIO 41501 [14]. ACV (acyclovir) and 2-(2,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from SigmaAldrich (St. Louis, MO, USA). Trizol Reagent was purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were bought from Gibco-BRL (Gland Island, NY, USA). Aspergillipeptide D and ACV were dissolved in dimethylsulfoxide (DMSO), and the final concentrations of DMSO were less than 0.1%. Restriction enzymes were purchased from Takara Bio (Shiga, Japan).
Cells and viruses
African green monkey kidney cells (Vero; ATCC CCL81) were cultured in DMEM supplemented with 10% heat-inactivated FBS. The maintenance medium used for virus dilutions was DMEM supplemented with 2% heat-inactivated FBS. HSV-1/F (ATCC VR-733) was preserved in our lab. HSV-1/Blue, a TK mutant derived from HSV-1 (KOS) [15], two ACV-resistant clinical HSV-1 strains (HSV-1/106 and HSV-1/153) were a kind gift from Tao Peng (Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences). All viruses were propagated in Vero cells and stored at − 80 °C until further use.
MTT assay
The MTT assay was performed according to the standard protocol. Briefly, Vero cells were cultured in 96-well plates. After the cell confluence reached 90%, various concentrations of compound were added to the plate, with each concentration having three replicates. After 48 h of incubation, 10 μl MTT solution (5 mg/mL) was added to each well, and the plate was incubated for 4 h in the dark. Then, the MTT solution was discarded, and 100 μl DMSO was added to each well. Plates were incubated for 15 min at room temperature with gently shaking. The optical density (OD) at 570 and 630 nm was measured for each well with an enzyme immunoassay reader (Bio-Rad, Hercules, CA, USA). The 50% cytotoxicity concentration (CC50) was defined as the concentration to reduce 50% cell viability.
Viral titer determination using plaque assay
Vero cells were cultured in 96-well plates. And the next day ten-fold serial dilutions of with and without treatment of extracts of HSV-1 were prepared prior to infection. Vero cell monolayers were then infected with different dilutions of 100 μl HSV-1 and allowed to adsorb for 2 h at 37 °C and 5% CO2. Unabsorbed viruses were aspirated, and plates were then overlaid with a nutrient medium-containing agar and incubated at 37 °C and 5% CO2 for 3 days. Plaques were visualized by staining cells with crystal violet and counting within 50 h. The plaque assay was carried out in triplicate. Virus was quantified by serial dilution and titration assay. The TCID50 (50% tissue culture infectious dose) was calculated using the formula of Reed and Muench method [16]:
Log10 50% end point dilution = log10 of dilution showing a mortality next above 50% - (difference of logarithms × logarithm of dilution factor).
Difference of logarithms = [(mortality at dilution next above 50%)-50%] / [(mortality next above 50%) - (mortality next below 50%)].
Plaque reduction assay
Experimental wells of 24-well plates containing confluent monolayers of Vero cells were infected with virus suspensions to produce 50 plaques per well. After 2 h incubation at 37 °C and 5% CO2, unabsorbed virions were aspirated. Aspergillipeptide D solution (25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, and 0.78125 μM, respectively) was then added to the appropriate wells, followed by nutrient medium containing agar; the plates were incubated at 37 °C and 5% CO2 for 3 days. Plaques were counted as described above. The antiviral activity was calculated by the following formula:
$$ \mathrm{Antiviral}\ \mathrm{activity}\ \left(\%\right)=\frac{\mathrm{plaque}\ \mathrm{number}\left(\mathrm{control}\right)-\mathrm{plaque}\ \mathrm{number}\left(\mathrm{assay}\right)}{\mathrm{plaque}\ \mathrm{number}\left(\mathrm{control}\right)}\times 100\% $$
Virus inactivation assay
Culture Vero cells into 24-well plates (1.5*105 cells/well), and the next day 100 μl of virus inoculum (50 PFUs per well) and 100 μl of Aspergillipeptide D solution (different concentrations) were mixed and incubated for 2 h at 37 °C. Then the mixture was added into cell wells and incubated at 37 °C for 2 h. The inoculated were removed. Cells were replenished with cover layer and 3 days later were fixed, stained as described above.
Virus attachment assay
Culture Vero cells into 24-well plates (1.5*105 cells/well), and the next day cells were pre-cooled at 4 °C for 1 h and washed by cold PBS. Virus inoculum (50 PFUs per well) and Aspergillipeptide D at indicated concentrations were added into cell wells, and the mixture was incubated at 4 °C for another 2 h to allow virus attaching to the cells. The virus inoculum was removed. Cells were replenished with cover layer and 3 days later were fixed, stained as described above.
Virus penetration assay
Culture Vero cells into 24-well plates (1.5*105 cells/well), and the next day cells were pre-cool at 4 °C for 1 h and washed by cold PBS then infected by virus (50 PFUs per well) for another 2 h at 4 °C to allow virus attaching to the cells. After that, the virus inoculum was removed, and cells were washed by cold PBS. Then different concentrations of Aspergillipeptide D were added and incubated at 37 °C for 10 min to maximize virus penetration. After incubation, PBS (pH = 3) was added into every well for 1 min to inactive the virus which failed to penetrate the cells. After that, the solution was neutralized and the neutral PBS was removed. Cells were replenished with cover layer and 3 days later were fixed, stained as described above.
Treatment effects after virus infection
Vero cells were cultured in 24-well plates. And next day cells were infected with HSV-1 (50 PFUs per well) for 2 h at 37 °C. After infection, the virus inoculum was removed, and cells were washed by PBS, and overlaid with Aspergillipeptide D at the indicated concentrations. After 3 days, cells were fixed, stained as described above.
The analysis of HSV-1 DNA synthesis
Vero cells were cultured in 24-well plates. And next day cells infected with HSV-1 (MOI = 3) were incubated with or without Aspergillipeptide D (25 μM) for 15 h. Viral DNA was extracted using GeneJET Viral DNA and RNA Purification Kit (Thermo). RT-PCR assay was used to quantify the viral DNA. Then the HSV-1 genome copy numbers were expressed relative to the virus control groups. The primer pairs are as follow: UL47 (F: 5′-GACGTA CGCGAT GAG ATC AA -3′, R: 5′-GTT ACC GGA TTA CGG GGA CT-3′).
Real-time PCR
Vero cells were cultured in 6-well plates. And next day cells infected with HSV-1 (MOI = 3) were incubated with Aspergillipeptide D (25 μM) for 3, 6, 9 h, respectively. Total RNA was isolated using Trizol (Invitrogen) and subjected to cDNA synthesis using a PrimeScript RT reagent kit (Takara). Real-time PCR (RT-PCR) was conducted to determine the expression levels of immediate early (IE) gene UL54, early (E) gene UL52 and late (L) gene UL27 of HSV-1/F and HSV-1/106 at 3, 6 and 9 h pi., respectively. The primer pairs were the same as described before [17].
Immunofluorescence assay
Vero cells were cultured in confocal dish, next day cells infected with HSV-1 (MOI = 3) at 37 °C for 2 h for viral adsorption. Cells were transferred into main medium with or without 25 μM Aspergillipeptides D and incubated for 9 h.p.i. Cells were fixed for 15 min with 4% paraformaldehyde (PFA) and permeabilized with 0.02% Triton X-100, both in PBS, and subsequently incubated with anti-gB antibody (Abcam) for 60 min and Alexa Fluor 488(1:1000) secondary antibody (Invitrogen) for 60 min. Then, the cells were stained with Golgi-Tracker Red or ER-Tracker Red (Beyotime, China). After each step the slides were washed repeatedly with PBS, and finally they were preserved with PBS. The additional nuclear staining with 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes) was per-formed for 20 min. Fluorescence was recorded in a confocal laser scan microscope (LSM 510 meta; Zeiss) [17].
Western blotting
Vero cells were seeded in 60 mm cell culture dish with the density of 1.5 × 106 cells/ dish. After 24 h, cells were infected with HSV-1 (MOI = 3) at 37 °C for 2 h. DMEM maintenance medium containing Aspergillipeptides D (25 μM) was added. At 6 and 9 h post-infection, the cells were washed three times with PBS, and were lysed using RIPA buffer (Beyotime). The equal amount (40 μg/sample) proteins were subjected to Western Blot analysis. A primary antibody against HSV-1 ICP0 (abcam1:1000), ICP8 (abcam 1:8000), VP5 (santa1:1000), gB (abcam1:1000) and gD (abcam1:1000) was used to detect the content changes of immediate early, early and late protein [18].
Co-immunoprecipitation (co-IP) and LC-MS analysis
Vero cells were seeded in 100 mm cell culture dish with the density of 3 × 106 cells/ dish. After 24 h, cells were treated with Aspergillipeptide D (25 μM) and infected with HSV-1 (MOI = 3) for 9 h. The cells were then lysed and the protein concentrations were measured and adjusted to 1 mg/ml. The lysate was precleared by adding 1.0 μg of the appropriate control IgG (normal mouse or rabbit IgG, corresponding to the host species of the primary antibody), together with 20 μl of resuspended volume of Protein A/G PLUSA agarose. Afterwards, the mixture was incubated at 4 °C for 30 min. The optimal dilution of primary antibody was added to the cell lysates (supernatant), incubated for 1 h at 4 °C, and then incubated at 4 °C overnight with 30 μl of resuspended volume of Protein A/G PLUS-Agarose. Next, the immunoprecipitation was collected, washed with PBS, and resuspended in 20 μl 1 × SDS PAGE buffer (Beyotime, China). The LC-MS Analysis were provided by the BGI (China).
Statistical analysis
Results were calculated as the mean ± SD, and statistical significance were determined by the Student’s t test. P values (P)<0.05 were considered statistically significant.