Inhibition of human cytomegalovirus immediate-early gene expression and replication by the ethyl acetate (EtOAc) fraction of Elaeocarpus sylvestris in vitro

Maintenance and propagation of primary human foreskin fibroblasts (HFF), HEK293 cells and the Towne strain of HCMV (HCMV-Towne) have been described previously [12]. Plant materials (Elaeocarpus sylvestris) were collected from the Jeju Biodiversity Research Institute at Jeju island in Korea (specimen number JBR-083).

Elaeocarpus sylvestris leaves were collected from Jeju island in Korea through Jeju Biodiversity Research Institute (Specimen number JBR-083). Dried Elaeocarpus sylvestris was exhaustively extracted with 70% ethanol (EtOH) twice at room temperature for 24 h. The ESE was concentrated under reduced pressure at 40 °C using a rotary evaporator to yield a semisolid dark-yellow residue. The extract was re-suspended in distilled water and successively fractionated using a series of solvents, including n-Hexane, dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), n-butanol (n-BuOH) and ddH₂O (Fig. 1) [13].

Isolation, amplification and quantification of HCMV DNA were performed as described previously [11].

HFF cells were infected with serially diluted HCMV-Towne and treated with either DMSO or the EtOAc fraction at concentrations of 5, 10, 25 or 50 μg/ml. At 3 days after infection, cells were overlaid with 2 ml agar and 2 ml medium, and again at 10 days after infection. At 14 days after infection, cells were fixed with 10% formaldehyde at room temperature for 10 min, followed by staining with 0.03% methylene blue at room temperature for 5 min. The numbers of plaques were counted and expressed as plaque forming units per ml (pfu/ml).

Cells were harvested, fractionated and transferred onto nitrocellulose membranes as described previously [14]. Antibodies to HCMV IE (IE1-72 kDa and IE2-86 kDa), ICP 36 and UL83 were purchased from Virusys (Taneytown, MD, USA). The anti-tubulin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Enhanced chemiluminescence detection reagent (Pierce, Rockford, Il.) and secondary peroxidase-labeled anti-mouse or anti-rabbit immunoglobulin G antibodies (Amersham Biosciences, Piscataway, NJ) were used according to the manufacturer’s recommendations.

Cell viability was assessed using the CellTiter-Glo luminescent cell viability assay (Promega, WI), which determines the ATP levels in metabolically active cells, according to the manufacturer’s directions.

The reporter construct, pJHA324, containing HCMV MIE enhancer/promoter-driven firefly luciferase was generated from pCATwt760 and kindly provided by Dr. Jin-Hyun Ahn [15, 16]. NF-κB-dependent promoter-driven firefly luciferase was described previously [17]. OmisFect™ was used for transient transfection according to the manufacturer’s instructions (Omicsbio, Taipei City, Taiwan). Luciferase assays were performed as described previously [18].

Statistical analyses were performed using JMP software (SAS Institute, Cary, NC). At least three independent experiments were conducted, and data were expressed as the mean ± standard deviation.

ESE was fractionated via sequential solvent extraction, and the effects of individual fractions on HCMV replication was determined using qPCR with primers specific for UL123. At a low (0.1) multiplicity of infection (MOI), HCMV replication was reduced by each solvent fraction of ESE fractions at a concentration of 50 μg/ml (Fig. 2a). However, the EtOAC and CH₂Cl₂ fractions of ESE induced significant reduction of HCMV replication after infection at a high (1) MOI (Fig. 2b). The EtOAc fraction of ESE suppressed HCMV replication to a similar extent as ESE (Fig. 2a and b, compare lanes 6 and 3). Interestingly, the CH₂Cl₂ fraction of ESE exerted a stronger inhibitory effect against HCMV replication than ESE (Fig. 2a and b, compare lanes 5 and 3).

To ascertain whether the inhibitory effects of solvent fractions against HCMV replication are not related to cytotoxicity, HFF cells were treated with ESE solvent fractions at a concentration of 50 μg/ml, and cell viability was determined at 0, 24, 48 or 72 h after treatment using the CellTiter-Glo® Luminescent cell viability assay. Compared to 0 h, cellular ATP levels were elevated at 24, 48 and 72 h possibly due to active cellular metabolism and/or proliferation (Fig. 3). Surprisingly, the CH₂Cl₂ fraction of ESE induced a significant decrease in the viability of HFF cells (Fig. 3), suggesting that reduction of HCMV replication by the CH₂Cl₂ fraction of ESE is potentially mediated by its cytotoxicity. On the other hand, the EtOAc fraction of ESE did not exert significant cytotoxic effects against HFF cells until 48 h after treatment (Fig. 3). Compared to 0 h, the EtOAc fraction of ESE slightly reduced cellular ATP levels by 13% at 72 h after treatment (Fig. 3). Nevertheless, replenishment of the EtOAc fraction of ESE at 72 h exhibited no further effect on cellular ATP levels, and levels of cellular proteins including the p38 mitogen-activated protein kinase (p38 MAPK) and c-jun N-terminal kinase (JNK) were not attenuated by the treatment of the EtOAc fraction of ESE for 72 h (data not shown). Therefore, further experiments were conducted to elucidate the anti-HCMV activity and mechanism(s) of action of the EtOAc fraction.

To determine the inhibitory concentration of the EtOAc fraction of ESE that reduces the number of HCMV plaques by 50% (IC₅₀), HFF cells were infected with serially diluted HCMV-Towne and treated with either DMSO or the EtOAc fraction of ESE at concentrations of 5, 10, 25 or 50 μg/ml. Cells were re-treated with DMSO or the EtOAc fraction every 3 days following infection, and the plaque reduction assay was performed to determine pfu/ml (Fig. 4). The IC₅₀ value of the EtOAc fraction of ESE was estimated as 39 ± 2.65 μg/ml.

To determine the effects of the EtOAc fraction on HCMV lytic gene expression, HFF cells were infected with HCMV-Towne at an MOI of 1 and treated with either DMSO or the EtOAC fraction of ESE (50 μg/ml). At 0, 24, 48 or 72 h after infection, the levels of HCMV IE (IE1-72 kDa and IE2-86 kDa), E (infected cell protein 36, ICP 36) and L (UL83) proteins were determined via western blot (Fig. 5). In DMSO-treated cells, HCMV IE1-72 kDa protein was detected at 24 h after infection, and other lytic genes were strongly induced at 48 and 72 h after infection (Fig. 5, compare lane 1 with lanes 2 to 4). Notably, the EtOAc fraction induced a significant reduction in the levels of IE proteins and, in turn, E and L proteins (Fig. 5, compare lane 5 with lanes 6 to 8). The data collectively indicate that the EtOAc fraction of ESE contains an active constituent(s) that inhibits HCMV lytic gene expression and replication.

In view of the finding that the EtOAc fraction of ESE disrupts HCMV IE protein expression, its effects on HCMV MIE enhancer/promoter activation were investigated. HEK293 cells were transfected with HCMV MIE enhancer/promoter-driven firefly luciferase and control Renilla luciferase plasmids and treated with either DMSO, ESE or the EtOAC fraction of ESE at concentrations of 5, 10, 25 or 50 μg/ml. At 5 and 10 μg/ml, both ESE and its EtOAc fraction reduced HCMV MIE enhancer/promoter activity by 40 and 48%, respectively (Fig. 6a). Interestingly, the EtOAc fraction of ESE exerted a stronger inhibitory effect on HCMV MIE enhancer/promoter activity than ESE and reduced activity in a dose-dependent manner (Fig. 6a). On the other hand, the EtOAc fraction of ESE did not exhibit an inhibitory effect on NF-κB-dependent promoter activity (Fig. 6b). Based on the results, we proposed that the EtOAc fraction reduces HCMV IE gene expression by down-regulating MIE enhancer/promoter activity.