Samarangenin B from Limonium sinense suppresses herpes simplex virus type 1 replication in Vero cells by regulation of viral macromolecular synthesis

Abstract

Inhibitory effects of ethanolic extracts from 10 Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. By a bioassay-guided fractionation procedure, samarangenin B (Sam B) was isolated from Limonium sinense; Sam B significantly suppressed HSV-1 multiplication in Vero cells without apparent cytotoxicity. Time-of-addition experiments suggested that the inhibitory action of Sam B on HSV-1 replication was not due to the blocking of virus adsorption. In an attempt to further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to viral multiplication was examined, including viral immediate-early (alpha), early (beta), and late (gamma) gene expression and DNA replication. Results indicated that levels of glycoprotein B (gB), gC, gD, gG, and infected-cell protein 5 (ICP5) expression and gB mRNA expression in Vero cells were impeded by Sam B. Data from PCR showed that replication of HSV-1 DNA in Vero cells was arrested by Sam B. Furthermore, Sam B decreased DNA polymerase, ICP0, and ICP4 gene expression in Vero cells. Results of an electrophoretic mobility shift assay demonstrated that Sam B interrupted the formation of an alpha-trans-induction factor/C1/Oct-1/GARAT multiprotein complex. The mechanisms of antiviral action of Sam B seem to be mediated, at least in part, by inhibiting HSV-1 alpha gene expression, including expression of the ICP0 and ICP4 genes, by blocking beta transcripts such as DNA polymerase mRNA, and by arresting HSV-1 DNA synthesis and structural protein expression in Vero cells. These results show that Sam B is an antiviral agent against HSV-1 replication.

FIG. 1.

Effects of ethanolic extracts from 10 Chinese herbs on HSV-1 replication in Vero cells determined by plaque reduction assay. Vero cells (3.5 × 10⁵/dish) were incubated with 100 PFU of HSV-1 and 10 μM acyclovir, 100 μg of each extract/ml (A), or the indicated concentrations of L. sinense extracts (B). After adsorption for 1 h, 1% methylcellulose was added to each well. On the fifth day p.i., the virus plaques formed in Vero cells were counted by crystal violet staining. Each bar represents the mean of three independent experiments. VL, Ventilago leiocarpa, ER, Ecdysanthera rosea, EU, Ecdysanthera utilis, HL, Hippobroma longiflora, AB, Ardrisia brevicaulis, SD, Selaginella delicatula, LS, L. sinense, AJ, Ardrisia japonica, AV, Ardrisia violacea, AE, Andendron benthamianum.

FIG. 2.

The structure of Sam B purified from L. sinense.

FIG. 3.

Cytopathic effect of HSV-1 on Vero cells treated with or without Sam B. HSV-1-infected (MOI, 3) and uninfected Vero cells were treated with or without Sam B (25 μM). At 22 h p.i., cells were examined by microscopy (magnification, ×180). Bar, 50 μm.

FIG. 4.

Effects of Sam B on HSV-1 replication and Vero cell viability and growth. (A) Inhibitory effects of 10 μM acyclovir and indicated concentration of Sam B on HSV-1 replication were determined by plaque reduction assay. Each bar represents the mean of three independent experiments. (B) Vero cells (3.5 × 10⁵ in 25-cm² flasks) were treated with medium, 0.1% DMSO, or 25, 50, or 100 μM Sam B for 5 days. Then total, viable, and nonviable cells were counted after staining with trypan blue. Each bar represents the mean of three independent experiments. (C) Vero cells (5 × 10³/well of a 96-well plate) were treated with Sam B at the indicated concentrations or were treated without Sam B for 5 days. Then tritiated thymidine, tritiated uridine, or tritiated leucine was added to each well (1 μCi/well) to detect cell growth. After a 16-h incubation, the cells were harvested by an automatic harvester and then radioactivity was measured by scintillation counting. Each bar represents the mean of three independent experiments.

FIG. 5.

Kinetics of inhibition of HSV-1 replication by Sam B. (A) Vero cells (5 × 10⁶) were infected with HSV-1 at an MOI of 3 in the presence (▾) or absence of acyclovir (10 μM) or Sam B (25 μM). Treatments were as follows: Sam B was added at the same time as HSV-1 (▿), Sam B was present after the adsorption period (□), and Sam B was present for 2.5 h and removed before the adsorption phase by washing the cells with medium three times (▪). Then cell supernatants were collected at 0, 2, 4, 6, 8, 16, 24, and 36 h p.i. and the viral titers were determined by a plaque forming assay. Each point represents the mean of three independent experiments. (B) Vero cells (5 × 10⁶) were infected with HSV-1 (MOI, 3), and Sam B (25 μM) was added at the indicated times. Cell supernatants were collected at 25 h p.i., and HSV-1 titers were determined as described in Materials and Methods. Each bar represents the mean of three independent experiments.

FIG. 6.

Effects of Sam B on HSV-1 γ gene expression in Vero cells detected by Western blot analysis and RT-PCR. Vero cells (5 × 10⁶) were infected with HSV-1 (MOI, 3) or were not infected in the presence or absence of Sam B (25 μM). (A) Lysates (20 μg of protein) were collected at 16 h p.i. and run on an SDS-10% PAGE gel and analyzed by immunoblotting with an anti-gB, -gC, -gD, -gG, or -ICP5 antibody. (B) The total cellular RNA was isolated from Vero cells at 16 h p.i. RT-PCR was done as described in Materials and Methods. Following the reaction, the amplified product was run on a 2% agarose gel. Lane 1, uninfected Vero cells; lane 2, HSV-1-infected cells; lane 3, cells treated with HSV-1 and Sam B at the same time; lane 4, infected cells treated with Sam B at 12 h to 16 h p.i. The graph indicates the ratio of γ protein or gB mRNA to β-actin mRNA. Each bar represents the mean of three independent experiments.

FIG. 7.

Effect of Sam B on HSV-1 DNA synthesis in Vero cells detected by PCR. Cells (5 × 10⁶) were infected with HSV-1 (MOI, 3) or were not infected in the presence or absence of Sam B (25 μM). The cells were harvested at 2, 4, 8, and 16 h p.i., and total DNA was extracted with phenol-chloroform. The PCR was done as described in Materials and Methods. Following the reaction, the amplified product was run on a 2% agarose gel. (A) DNA was extracted from uninfected Vero cells at 16 h p.i. (lane 1) or from HSV-1-infected cells at 2, 4, 8, and 16 h p.i. (lanes 2 to 5). (B) Sam B and HSV-1 were added to the cells at the same time, and the total cellular DNA was extracted at 2 (lane1), 4 (lane 2), 8 (lane 3), and 16 h (lane 4) p.i. Lane 5, total cellular DNA extracted from infected cells treated with Sam B at 12 to 16 h p.i. Each band was quantitated by densitometer, and the ratio of HSV-1 DNA to β-actin DNA was calculated. Each bar represents the mean of three independent experiments.

FIG. 8.

Effects of Sam B on DNA polymerase gene transcription in Vero cells detected by Northern blot analysis. Vero cells (5 × 10⁶) were infected with HSV-1 (MOI, 3) or were not infected in the presence or absence of Sam B (25 μM). (A) Total cellular RNA was isolated from the cells at 6 h p.i. and analyzed in 6.66% formaldehyde-agarose gel and hybridized with digoxigenin-labeled DNA polymerase cDNA or β-actin cDNA. Lanes 1 and 4, HSV-1-infected cells treated with or without DMSO; lane 2, HSV-1-infected cells treated with Sam B; lane 3, uninfected cells. (B) Each exposed band was quantitated by densitometer, and the ratio of DNA polymerase mRNA to β-actin mRNA was calculated. Each bar is the mean of three independent experiments.

FIG. 9.

Effects of Sam B on HSV-1 ICP0 and ICP4 gene expression and formation of IEC in Vero cells detected by Western blotting, RT-PCR, and EMSA, respectively. Vero cells (5 × 10⁶) were infected with HSV-1 (MOI, 3) or were not infected in the presence or absence of Sam B (25 μM). (A) Lysates (20 μg of protein) were collected at 4 h p.i. and run on an SDS-10% PAGE gel and analyzed by immunoblotting with an anti-ICP0 or -ICP4 antibody. (C) Total cellular RNA was isolated from Vero cells at 4 h p.i. and analyzed by RT-PCR. Lane 1, uninfected Vero cells; lanes 2 and 4, HSV-1-infected cells treated with or without DMSO; lane 3, infected cells treated with Sam B. (B and D) Bar graphs indicating the ratio of ICP0 or ICP4 to β-actin proteins or mRNAs. Each bar represents the mean of three independent experiments. (E) EMSA was performed as described in Materials and Methods. Nuclear extracts from HSV-1-infected (lanes 1 and 4) or uninfected Vero cells (lane 3) were incubated with a ³²P-end-labeled GARAT probe. The effects of Sam B on the formation of IEC in virus-infected nuclear extracts pretreated with 25 μM Sam B for 5 min and then mixed with the probes (lane 2) were detected. Lane 5, results of adding a 50-fold excess of unlabeled probe to the reaction mixture.