Suppressive effects of pterostilbene on human cytomegalovirus (HCMV) infection and HCMV-induced cellular senescence

Foscarnet (FOS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). PTE was purchased from Shanghai Standard Technology Co., Ltd. (Shanghai, China) and dissolved in vehicle (DMSO) for a stock solution of 10 mM. Human diploid fibroblasts (WI-38 cells) and HCMV (Towne strain) were obtained from the American Tissue Culture Collection (ATCC, catalogue no. VR-977, Manassas, VA, USA). The QIAamp DNA mini kit was obtained from Qiagen (Hilden, Germany). Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), 100 × penicillin-streptomycin solution, and 0.25% trypsin-EDTA were purchased from Gibco-BRL Life Technologies (Grand Island, NY, USA). Mouse monoclonal antibodies against immediate-early (IE1/2, catalogue no. P1215) and early protein (UL44, catalogue no. CA006-100) of HCMV were obtained from Virusys Corporation (Randallstown, MD, USA). Anti-p16^INK4a (catalogue no. 92803S), anti-p21 (catalogue no. 2947S), and anti-p53 (catalogue no. 2527S) primary rabbit antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). The Senescence-associated β-galactosidase (SA-β-Gal) Staining Kit and polyvinylidene fluoride (PVDF) membrane were purchased from Cell Signaling Technology (Boston, MA, USA) and Bio-Rad Laboratories (Hercules, CA, USA), respectively. The 2 × Universal SYBR Green Fast qPCR Mix was obtained from Abclonal Technology (Wuhan, China). Cell lysis buffer for Western blotting, 5 × SDS‒PAGE protein sample loading buffer, Cell Counting Kit-8 and Reactive Oxygen Species (ROS) Assay Kit were all obtained from Beyotime Institute of Biotechnology (Shanghai, China). 10 × Tris-glycine SDS‒PAGE running buffer and 10 × Tris-glycine transfer buffer were obtained from Sangon Biotech (Shanghai, China). HRP-conjugated goat anti-mouse IgG, HRP-conjugated goat anti-rabbit IgG, and FITC-conjugated goat anti-mouse IgG were all purchased from Hangzhou Huaan Biotechnology (Hangzhou, China).

WI-38 cells were cultured in DMEM with 10% FBS and penicillin-streptomycin solution. WI-38 cells were considered young at a population doubling (PD) of 30 or less, whereas cells at PD 50 or higher were defined as replicative senescent. All experiments in this study were performed using young cells ranging from 25 to 30 PD.

Stocks of the Towne strain of HCMV were routinely prepared as previously mentioned [29‐31]. Before virus infection, the culture medium was replaced with 0.2% FBS for 48 h to synchronize cells in the G₀ phase. The cells were mock-infected or infected with HCMV (MOI = 0.5) for 2 h at 37 °C. Then, the inoculum was discarded, and the cells were washed and incubated with fresh culture medium with 0.2% FBS in the absence or in the presence of PTE. FOS (200 µg/mL) was used as a positive-drug control. The cell samples were harvested at the indicated times. In the PTE and FOS treatment groups, WI-38 cells were pretreated with PTE and FOS 2 h before virus infection.

The effect of PTE on cell viability was determined by CCK-8 assays. Briefly, WI-38 cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and incubated at 37 °C. Subsequently, culture medium containing different concentrations of PTE (1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) was added and incubated for 5 days. At the predetermined time, 10 μL of CCK-8 was added to each well. After incubation for 1–2 h at 37 °C, absorbance was measured using a Thermo Scientific microplate reader (Multiskan ™ FC) at 450 nm according to the standard protocol. The vehicle (DMSO) group was used as the control. Each group was assayed in triplicates (n = 3).

For the “Entry” assay, PTE or FOS was added to the cells for 2 h, followed by HCMV inoculation for 1 h. Then, we removed the culture medium containing HCMV and the drug and washed the cells three times with preheated PBS. Finally, DMEM with 0.2% FBS was added to each well and maintained until sample collection. For the “Post-Entry” assay, first, HCMV was inoculated into WI-38 cells for 1 h at 37 °C. Then, we discarded the virus and washed the cells thrice with preheated PBS to remove unbound virus. Finally, DMEM with 0.2% FBS containing PTE or FOS was added to each well and maintained until sample collection. For all the experimental groups, the concentrations of PTE and FOS were 10 µM and 200 µg/mL, respectively. WI-38 cells were infected with HCMV (MOI = 5) and harvested 3 dpi. The HCMV DNA copy numbe and viral proteins in the infected cells were quantified by qPCR and Western blotting.

The cell samples were prepared as described above and lysed with cell lysis buffer. Then, 50 μg of protein extract was subjected to SDS‒PAGE and transferred to PVDF membranes. Blocking was performed with 5% nonfat dry milk for 1 h at room temperature, and the membranes were incubated with the corresponding antibodies mentioned above (IE1/2-1:500 dilution, UL44-1:10000 dilution, p16-1:1000 dilution, p21-1:1000 dilution, p53-1:1000 dilution). HCMV-infected alone, and FOS-pretreated groups were used as controls.

The cell samples were prepared as described above and the cell culture supernatant was discarded. Then, the cells were washed with PBS and harvested with 0.25% trypsin-EDTA. HCMV DNA was extracted with a QIAamp DNA Mini Kit according to the manufacturers’ instructions. The copy number of HCMV DNA was evaluated using qPCR assay with 2 × Universal SYBR Green Fast qPCR Mix [30, 31]. The HCMV UL 123, pp150 and GAPDH primers used in this study were as follows: HCMV UL123-forward 5′-TCTGCCAGGACATCTTTCTC-3′ and reverse 5′-GTGACCAAGGCCAC.

GACGTT-3′; HCMV pp150-forward 5′- GGTTTCTGGCTCGTGGATGTCG-3′ and reverse 5′- CACACAACACCGTCGTCCGATTAC-3′; GAPDH-forward 5′-CTGTTG.

CTGTAGCCAAATTCGT-3′ and reverse5′-ACCCACTCCTCCACCTTTGAC-3′. The qPCR procedure was as follows: 3 min at 95 °C for initial denaturation, 40 cycles of 5 s at 95 °C and 30 s at 60 °C. Each group was assayed in triplicates (n = 3). The difference in HCMV DNA copy number between the PTE treatment group and the HCMV infection alone group was calculated using the 2^−△△Ct method. First, the housekeeping gene GAPDH was used to normalize the Ct values of all treatment and control samples, i.e., ΔCt = Ct (sample) − Ct (GAPDH). Second, the Ct values of the PTE treatment groups were compared to those of the control samples. ΔΔCt = ΔCt (sample) − ΔCt (control sample). The relative expression of the target gene is represented by 2^−△△Ct.

IFA was carried out in a 48-well plate, and PTE or FOS treatment and HCMV infection were the same as above. The cell samples were fixed for 30 min using precooled acetone/methanol (v/v, 1:1) at − 20 °C. Then, the fixed samples were incubated with 5% nonfat dry milk for blocking for 1 h at room temperature. After five washes with PBS, the cell samples were treated with HCMV IE1/2 mouse monoclonal antibody overnight at 4 °C, followed by incubation with secondary antibody for 1 h at room temperature. The fluorescence signal was observed using a Zeiss microscope (Axio Vert.A1).

TCID₅₀ assays were performed in 96-well plates. When WI-38 cells grew to 80% confluence, they formed a monolayer. HCMV samples were serially diluted tenfold. Subsequently, an aliquot of 100 μL from each dilution was inoculated into WI-38 cells and maintained at 37 °C in a 5% CO₂ incubator (Heraeus Instruments). The experiment was performed simultaneously in 8 replicates per dilution. The cytopathic effect (CPE) was monitored and recorded daily until the CPE wells did not change. Finally, the virus titre was calculated according to the classical Reed-Muench method [31, 32]. The falf-maximal inhibitory concentration (IC50) was determined based on the results of the TCID50 assay according to nonlinear trajectory analysis using GraphPad Prism.

The level of cellular senescence was assessed by an SA-β-gal staining kit essentially as described in a previously published paper [30]. Briefly, WI-38 cells were cultured in 6-well plates with different drug-treatments until the indicated time. Then, the cells were washed three times with PBS and fixed for 5 min in 0.25% glutaraldehyde. After this, the cells were incubated with SA-β-gal staining solution at 37 °C for 3–16 h. Finally, the fields of SA-β-gal-positive cells (blue-green cells) were randomly selected and then counted, and the percentage of senescent cells was evaluated from three independent experiments.

The ROS assay was performed in 6-well plates, and the cell samples with different treatments were the same as those shown above. The intracellular ROS level was calculated with an ROS Assay Kit according to the manufacturer’s instructions. First, the ROS probe 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) was diluted with FBS-free DMEM to a final concentration of 10 μM. Then, the cells were washed twice with PBS and treated with H₂DCFDA for 20–30 min at 37 °C. Then, the cells were washed three times with FBS-free DMEM. Finally, the cells were digested with trypsin and collected to measure the intracellular fluorescence intensity by flow cytometry.

To explore the effects of PTE on the viability of WI-38 cells, we conducted a CCK-8 assay to analyse the potential cytotoxicity of PTE. The result was shown in Fig. 1B, C, no obvious cytotoxicity was detected after 5 days of treatment in the concentration range of 1 to 30 μM (P > 0.05); however, when the concentration reached 40 μM or higher, compared with the control, PTE showed obvious cytotoxicity, and the difference was extremely significant (P < 0.001). Thus, the half-maximal cytotoxicity concentration (CC50) of PTE treatment was 35.15 μM in WI-38 cells (Fig. 1C). Thus, we chose 10 μM as the concentration for the next study unless specifically indicated. Moreover, the cells treated with 5 μM, 10 μM or 20 μM PTE showed normal fibrous morphological features (Fig. 1D, upper panel).

Previously, we observed obvious morphological changes and cytopathic effects (CPE) in HCMV-infected cells [30, 31]. As shown in Fig. 1D, WI-38 cells infected with HCMV alone at an MOI of 0.5 displayed typical CPE characterized by cell rounding and enlargement with inclusion bodies at 5 dpi, while pretreatment of the cells with 5 μM PTE significantly reduced the degree of CPE, and the morphology of WI-38 cells treated with 10 μM and 20 μM was similar to that of the mock-infected group. These results indicated that PTE could protect the morphological features of the WI-38 cells infected with HCMV and further suggested that PTE might have anti-HCMV activity.

To verify the anti-HCMV activity of PTE, we performed Western blotting, qPCR, IFA and TCID50 assays to assess viral protein expression, viral gene copy number and virus titre. First, we evaluated the protein expression levels of HCMV IE1/2 and UL44. Compared with those of the vehicle control group, the expression levels of the IE1/2 and UL44 proteins were reduced rapidly after treatment with PTE at the indicated concentrtions (Fig. 2A, B). The results were consistent with the UL123 and PP150 copy numbers shown in Fig. 2C, D. The UL123 and PP150 copy numbers decreased rapidly at 5 μM or higher (P < 0.01) compared with those of the HCMV infection group. The results indicated that PTE inhibited HCMV DNA replication.

Second, we carried out IFA to further assess the anti-HCMV activity of PTE on WI-38 cells (Fig. 2E, F). Similar to the results of published papers [30, 31], the IE1/2 positive signal was concentrated in the nuclei of the HCMV-infected cells. Compared with that of the HCMV-infected alone group, the number of IE1/2-positive cells decreased dramatically when the cells were treated with PTE (10 μM).

Finally, a TCID50 assay was conducted to further investigate the changes in viral titres induced by PTE treatment. The result was showed in Fig. 2G. PTE inhibited the production of HCMV progeny virions in a dose-dependent manner at the indicated concentrations (0.01 μM, 0.1 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM), and the half-maximal inhibitory concentration (IC50) was determined to be 1.315 μM. The selectivity index (SI) of PTE was calculated as 26.73. In conclusion, PTE could suppress HCMV infection in host WI-38 cells with respect to protein expression, UL123 and PP150 gene expression and viral titres.

To determine at what point during viral infection PTE mediated its inhibitory effect, we examined the expression level of viral protein and the UL123 gene using Entry (Fig. 3A) and Post-Entry assays (Fig. 3B) at 3 dpi. In the Entry assay group, WI-38 cells were pretreated with PTE before HCMV attachment and entry. There was no significant change in the expression levels of IE1/2 and UL44 compared to those of the control group (P > 0.05). However, we observed that the viral protein decreased dramatically when HCMV was first allowed to enter the cell and then subjected to PTE treatment. The inhibitory effect was similar to that in the FOS-treated sample, as FOS mainly targets DNA polymerase, which affects the HCMV cycle of the replication period [8]. The results suggested that PTE predominantly acts after virus entry and membrane fusion (Fig. 3C, D). Moreover, the copy number of UL123 in all the above groups was calculated to verify this result. Similar to viral protein level results, the copy number of UL123 decreased significantly only in Post-Entry assay (Fig. 3E) (P < 0.001).

WI-38 cells are also widely used as a model of cell senescence, and HCMV infection could induce cellular senescence in previous studies [29, 30, 33, 34]. The accumulation of a lysosomal enzyme termed SA-β-Gal was the first and is still the most widely used biomarker to detect senescent cells in cultured cells; hence, we performed classical SA-β-Gal staining to determine whether HCMV infection could promote the progression of cell senescence in young WI-38 cells (PD30). As shown in Fig. 4, HCMV infection (HCMV infection alone or with vehicle treatment) at an MOI of 0.5 could induce 60–80% cell senescence at 5 dpi, and this result was similar to previous studies [29, 30]. While the percentage of SA-β-Gal-positive cells decreased to 20% and 10% after treatment with 5 μM and 10 μM PTE, respectively, the effect of 10 μM PTE was the same as that of the control group (P > 0.05). Compared to that of the HCMV-alone group, the number of SA-β-Gal-positive cells in the two treatment groups with different concentrations of PTE (5 μM, 10 μM) showed significant differences (P < 0.01). These results indicated that PTE significantly suppressed the senescence phenotype in WI-38 cells infected with HCMV.

In addition to the increasing number of SA-β-Gal-positive cells, the accumulation of p16 and p21 is another characteristic of senescent cells and was also used as a biomarker of cellular senescence. p16 and p21 are two cyclin-dependent kinase inhibitors that are components of tumour suppressor pathways governed by p53, and their expression levels play critical roles in the establishment and maintenance of senescence-associated growth arrest [35]. We then evaluated the expression levels of p16, p21 and p53 in the HCMV-infected cells of different MOIs with or without PTE treatment. We found that HCMV infection increased the expression levels of all three proteins in an MOI-dependent manner, and this result was in accordance with previous studies [29, 30]. However, PTE treatment rapidly alleviated this effect (Fig. 5). These results suggested that PTE had a significant inhibitory effect on HCMV-induced expression of p16, p21 and p53.

Oxidative stress is closely related to cell senescence and might function as a common trigger for activation of the senescence programme [36, 37]. Oxidative stress is also involved in the process of viral infection [38]. Our previous studies found that HCMV infection induced a sharp rise in ROS production in young WI-38 cells [29, 30]. Hence, we also analysed the effects of PTE on HCMV-induced ROS production. As shown in Fig. 6, HCMV infection elevated intercellular ROS production, while PTE treatment significantly alleviate this effect (P < 0.001). This result suggested that PTE could suppress HCMV-induced ROS production.