Hydrogen Peroxide Induce Human Cytomegalovirus Replication through the Activation of p38-MAPK Signaling Pathway

Human cytomegalovirus (HCMV) is a major risk factor in transplantation and AIDS patients, which induces high morbidity and mortality. These patients infected with HCMV experience an imbalance of redox homeostasis that cause accumulation of reactive oxygen species (ROS) at the cellular level. H2O2, the most common reactive oxygen species, is the main byproduct of oxidative metabolism. However, the function of H2O2 on HCMV infection is not yet fully understood and the effect and mechanism of N-acetylcysteine (NAC) on H2O2-stimulated HCMV replication is unclear. We, therefore, examined the effect of NAC on H2O2-induced HCMV production in human foreskin fibroblast cells. In the present study, we found that H2O2 enhanced HCMV lytic replication through promoting major immediate early (MIE) promoter activity and immediate early (IE) gene transcription. Conversely, NAC inhibited H2O2-upregulated viral IE gene expression and viral replication. The suppressive effect of NAC on CMV in an acute CMV-infected mouse model also showed a relationship between antioxidants and viral lytic replication. Intriguingly, the enhancement of HCMV replication via supplementation with H2O2 was accompanied with the activation of the p38 mitogen-activated protein kinase pathway. Similar to NAC, the p38 inhibitor SB203580 inhibited H2O2-induced p38 phosphorylation and HCMV upregulation, while upregulation of inducible ROS was unaffected. These results directly relate HCMV replication to H2O2, suggesting that treatment with antioxidants may be an attractive preventive and therapeutic strategy for HCMV.


Introduction
Human cytomegalovirus (HCMV), a β herpesvirus, is an enveloped, large and double-stranded DNA virus. Like most herpesviruses, HCMV is able to establish a latent state in its hosts after primary infection, which can result in serious health conditions when the virus reactivates and performs lytic replication [1]. HMCV infection can be asymptomatic among immunocompetent people, but it can become an important and common cause of morbidity and mortality in immunocompromised patients, such as those with AIDS, solid organ transplantation, and hematopoietic stem cell transplantation [1][2][3][4][5]. Previous studies have shown that HCMV infection is highly associated with atherosclerosis, cardiovascular diseases [6][7][8], and inflammatory bowel disease [9].
Several mechanisms of the regulation of HCMV and mouse cytomegalovirus (MCMV) latency, reactivation, and lytic replication, such as chromatin remodeling and mitogen-activated protein kinase (MAPK) pathways, have been reported by previous studies [10][11][12][13][14]. However, none of these proposed triggers can be the cause of all clinical cases of HCMV reactivation and replication.
Although the common physiological trigger that stimulates HCMV replication remains unclear, many clinical diseases are characterized by high levels of oxidative stress. Patients who undergo solid organ transplantation generally suffer from oxidative stress and inflammation associated with ischemia/reperfusion, organ rejection, and as a side effect of immunosuppressive therapy [15][16][17]. AIDS patients have high levels of oxidative stress and inflammation as a result of the defensive mechanism of the immune system in the response to HIV infection [18]. In cardiovascular diseases, oxidative stress and inflammation can also be found and are believed to contribute to the development of atherosclerosis [19,20]. Thus, one potential key to determining the trigger of HCMV replication may be oxidative stress.
As a byproduct of oxidative metabolism [21], H 2 O 2 was produced and released to impair redox homeostasis during oxidative stress. Mechanically, transcription of the major HCMV immediate early (IE) gene is driven by the complex major IE (MIE) promoter/enhancer. Within the region, there are several binding sites for known cellular transcription factors [22], such as NF-κB, CREB, ATF, and YY1. Studies have shown that high levels of oxidative stress result in activation of these transcription factors through mitogen-and stress-activated protein kinase (MSK), which is activated by the p38-MAPK pathway [23,24].
Thus, we hypothesize that the activation of p38-MAPK signaling plays a significant role in the HCMV replication caused by hydrogen peroxide, and this could be decreased by antioxidant treatment.

Cell Culture, Chemical Reagents and Antibodies
Human foreskin fibroblast (HFF) cells of no more than 15 passages, HEK 293 cells, mouse embryonic fibroblast (MEF) cells, and MRC-5 cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 • C under a 5% CO 2 atmosphere. Confluent cell monolayers were starved from serum for 24 h before infection. Serum free DMEM was used during drug treatment, virus incubation, and infection until the cells were harvested.
The plasmid expression specific siRNA used to target human catalase (5 -GCCTGGGACCCAATTATCTTCATAT-3 ) and the scrambled control siRNA were purchased from Hanbio (Shanghai, China). The transfection of HEK 293 and HFF cells was performed using Lipofectamine 3000 reagent from Invitrogen (Carlsbad, CA, USA).

Virus Preparation, Titration and Infection
HCMV (AD169) and MCMV (Smith strain) stocks were prepared in MRC-5 cells and MEF cells, and aliquots were stored at −80 • C. Viral titers were determined using the 50% tissue culture infective dose (TCID 50 ) method, as previously described [26]. Briefly, HFF or MEF cells were incubated with mock, UV-inactivated HCMV (UV-HCMV) or HCMV for 1 h under serum free DMEM at a multiplicity of infection (MOI) of 0.5. Then, the medium was removed, the cells were washed with PBS and fresh serum free medium was added. All experiments were examined at least three times using Reed and Muench's method.

DCF Staining
The measurement of reactive oxygen species (ROS) production in response to H 2 O 2 was performed, as previously described [27]. After treatment with H 2 O 2 (0, 100, or 200 µM) for 24 h, cells were washed with PBS and incubated with 10 µM H 2 DCF-DA in warmed, serum free DMEM for 30 min in a CO 2 incubator at 37 • C. Cells were then washed three times with PBS and images were taken using a Leica microscope.

Detection of Cellular Catalase Activity and Intracellular H 2 O 2 Level
HFF cells cultured with H 2 O 2 (0, 100 or 200 µM) or ATA (0, 1.0, 2.0 or 4.0 mM) for 24 h were harvested by centrifugation. The cell pellets were sonicated in 100 µL cold PBS. After centrifugation at 10,000× g and 4 • C for 5 min, the supernatants were used for the detection of catalase activity and intracellular H 2 O 2 level. The assay kits were all purchased from Jiancheng Bioengineering Institute (Nanjing, China).

Real-Time PCR
Total DNA was isolated from the supernatants of infected cells using a cell and tissue genomic DNA extraction kit (BioTeke Corporation, Beijing, China). Changes in viral DNA loads were monitored using absolute quantitative real-time PCR. Viral DNA levels were detected using primers against the HCMV IE1 gene (forward primer, 5 -ATGTACGGGGGCATCTCTCT-3 and reverse primer, 5 -GGCTTGGTTATCAGAGGCCG-3 ) or the MCMV IE1 gene (forward primer, 5 -GTGGGCATGAAGTGTGGGTA-3 and reverse primer, 5 -CGCATCGAAAGACAACGCAA-3 ).

Western Blot Analysis
Cells pellets were lysed in lysis buffer (Promega) with a cocktail of protein inhibitors (Roche, Mannheim, Germany) and then centrifuged at 13,000× g and 4 • C for 10 min. In brief, 30 µg whole cell extract was heated for 5 min at 98 • C with Laemmli buffer, and then, samples were separated by 12% sodium dodecyl sulfate polymerase gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking in 5% (w/v) skim milk (Applygen, Beijing, China) or 5% (w/v) bovine serum albumin (BSA) (MP, Auckland, New Zealand), the blots were probed with primary antibodies overnight at 4 • C. Protein bands were detected using Western blotting luminol reagent (Santa Cruz Biotechnology). The membranes were incubated with Western Blot stripping buffer (CWBio) to re-probe for other proteins in the same membrane.

Animal Studies
BALB/c mice (male, 6-8 weeks old, 20-25 g body weight) were purchased from Vital River (Beijing, China). The animal study was performed according to the protocols approved by the Ethics Committee at the Beijing Institute of Transfusion Medicine and in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines.
Mice were treated intragastrically with 400 µL of 40 mM NAC in water every day, from 3 days before intraperitoneal inoculation with MCMV (Smith strain, 5 × 10 3 p.f.u). At the proper time (day 7, 14, 21 and 28 post infection), DNA was extracted from 100 µL whole blood and used to determine the viral DNA load. To detect infectious virions in mice organs, the salivary glands (50 mg) and the lung (50 mg) were collected and homogenized on day 14 and 28, and viral titer was calculated with TCID 50 assays in MEF monolayers.

Statistical Analysis
All values are expressed as the means ± standard deviations. Statistical analyses were performed using SPSS statistical software V.17 (SPSS Inc., Chicago, IL, USA). Significant differences were evaluated by the two-tailed Student's t-test when two groups were compared, one-way analysis of variance (ANOVA) followed by the Dunnett's test when multiple groups were tested against a control group and the Bonferroni post hoc test when performing multiple comparisons between groups. A p-value lower than 0.05 was considered to indicate a statistically significant difference.

ROS Enhance HCMV Replication through Paracrine and Autocrine Mechanisms
After treatment with exogenous hydrogen peroxide for 24 h, HFF cells dose-dependently formed an increasing amount of ROS ( Figure 1A,B) and H 2 O 2 content ( Figure 1C).
To investigate the role of H 2 O 2 in HCMV lytic replication, we observed whether exogenous H 2 O 2 is sufficient to enhance HCMV replication at both the mRNA and protein level. HCMV MIE promoter activities were induced by H 2 O 2 (Figure 2A), and H 2 O 2 increased the expression of the IE gene in a dose-dependent manner ( Figure 2B). Furthermore, H 2 O 2 increased the levels of viral proteins, including pp72 and pp65 ( Figure 2C,D), as well as the production of HCMV DNA in the culture supernatant ( Figure 2E) and infectious virions ( Figure 2F).
Next, we verified whether an increase in intracellular H 2 O 2 is sufficient to induce HCMV replication. To confirm that the effect of ATA on HCMV replication was the result of an increase in the intracellular level of H 2 O 2 , we transiently expressed a catalase-specific siRNA in HFF cells. Compared to cells expressing a control siRNA, those transfected with a catalase-specific specific siRNA showed greatly lower catalase protein expression. Similar to ATA treatment, knockdown of catalase enhanced HCMV lytic pp72 and pp65 protein levels ( Figure 3G). Viruses 2015, 7 6 level ( Figure 3A right panel). ATA increased the MIE promoter activities, the IE1 gene transcripts, the expression of HCMV pp72 and pp65, and production of infectious virions ( Figure 3B-F).
To confirm that the effect of ATA on HCMV replication was the result of an increase in the intracellular level of H2O2, we transiently expressed a catalase-specific siRNA in HFF cells. Compared to cells expressing a control siRNA, those transfected with a catalase-specific specific siRNA showed greatly lower catalase protein expression. Similar to ATA treatment, knockdown of catalase enhanced HCMV lytic pp72 and pp65 protein levels ( Figure 3G).      level, as indicated by a reduction in the median fluorescent level, in H 2 -DCFH-treated HFF cells ( Figure 4A). As expected, NAC inhibited the upregulation of MIE promoter activities ( Figure 4B) and IE1 transcription ( Figure 4C) by supplementation with ATA and H 2 O 2 . The effects of H 2 O 2 on upregulation of HCMV MIE promoter activity and viral gene expression were also inhibited by another two scavengers, catalase and reduced glutathione ( Figure 4D). Furthermore, NAC impaired the H 2 O 2 -upregulated HCMV lytic protein and infectious virions ( Figure 4E-H) Viruses 2015, 7 9

H2O2 Scavengers Inhibit H2O2-Upregulated HCMV Lytic Replication
To detect whether H2O2 is required for HCMV lytic replication, we used a H2O2 scavenger to decrease the intracellular H2O2 level. Treatment with NAC, a common H2O2 scavenger, decreased the cellular H2O2 level, as indicated by a reduction in the median fluorescent level, in H2-DCFH-treated HFF cells ( Figure 4A). As expected, NAC inhibited the upregulation of MIE promoter activities ( Figure 4B) and IE1 transcription ( Figure 4C) by supplementation with ATA and H2O2. The effects of H2O2 on upregulation of HCMV MIE promoter activity and viral gene expression were also inhibited by another two scavengers, catalase and reduced glutathione ( Figure 4D). Furthermore, NAC impaired the H2O2-upregulated HCMV lytic protein and infectious virions ( Figure 4E-H). These results indicate that H2O2 scavengers, such as NAC and catalase, can suppress the stimulation of HCMV lytic replication by H2O2.

H2O2 Scavenger NAC Inhibits MCMV Lytic Replication in Vivo
Because of the strict species specificity of CMV, it is difficult to establish an animal model of HCMV infection. MCMV, which is similar to HCMV biological characteristics [28,29], has been regularly used to mimic HCMV infection in vitro and in vivo. We sought to inhibit MCMV lytic replication in mice using H2O2 scavenger NAC. We found that oxidative stress production was induced during primary infection of MEF cells with MCMV ( Figure 5A,B). Conversely, supplement with NAC strongly inhibited MCMV infection of MEF ( Figure 5C).
To examine CMV lytic replication and verify the inhibitory effect of antioxidant NAC in vivo, we treated BALB/c mice intragastrically with 400 μL of 40 mM NAC. The viral DNA load in whole blood was measured on day 7, 14, 21, and 28 post-infection. We found that mice fed NAC had a lower viral load than those fed drinking water alone ( Figure 5D). To determine the production of infectious virions in mice organs, we used cell-free supernatants from ultrasonic homogenates of the salivary glands and the lung to infect MEF. We observed a high viral titer in cells infected with supernatants from the control group, while those infected with supernatants from NAC-treated mice had lower ones on days 14 and 28, respectively ( Figure 5E). Collectively, the results of these in vivo experiments indicate that the antioxidant NAC effectively decreased MCMV replication in these mice.

H 2 O 2 Scavenger NAC Inhibits MCMV Lytic Replication in Vivo
Because of the strict species specificity of CMV, it is difficult to establish an animal model of HCMV infection. MCMV, which is similar to HCMV biological characteristics [28,29], has been regularly used to mimic HCMV infection in vitro and in vivo. We sought to inhibit MCMV lytic replication in mice using H 2 O 2 scavenger NAC. We found that oxidative stress production was induced during primary infection of MEF cells with MCMV ( Figure 5A,B). Conversely, supplement with NAC strongly inhibited MCMV infection of MEF ( Figure 5C).
To examine CMV lytic replication and verify the inhibitory effect of antioxidant NAC in vivo, we treated BALB/c mice intragastrically with 400 µL of 40 mM NAC. The viral DNA load in whole blood was measured on day 7, 14, 21, and 28 post-infection. We found that mice fed NAC had a lower viral load than those fed drinking water alone ( Figure 5D). To determine the production of infectious virions in mice organs, we used cell-free supernatants from ultrasonic homogenates of the salivary glands and the lung to infect MEF. We observed a high viral titer in cells infected with supernatants from the control group, while those infected with supernatants from NAC-treated mice had lower ones on days 14 and 28, respectively ( Figure 5E). Collectively, the results of these in vivo experiments indicate that the antioxidant NAC effectively decreased MCMV replication in these mice.

H 2 O 2 Upregulates HCMV Replication by Activating the p38 MAPK Pathway
As previously presented (Figure 2), H 2 O 2 stimulates the upregulation of HCMV replication, but the mechanism is unclear. Here, the results revealed that p38-MAPK was rapidly and strongly activated by H 2 O 2 treatment, following a time-and dose-dependent pattern ( Figure 6A,B). In particular, p38-MAPK activation displayed a rapid onset within 1 h of treatment, followed by a progressive increase, returning to basal levels within 48 h, while a second peak was observed at 72 h after treatment ( Figure 6A). Increasing H 2 O 2 concentration led to an increase of p38-MAPK phosphorylation ( Figure 6B) and the minimal concentration of H 2 O 2 was 25 µM. Next, treatment with 10 µM SB203580 reduced both H 2 O 2 -and ATA-activated p38 phosphorylation ( Figure 6C). Since supplementation with NAC inhibited H 2 O 2 -and ATA-induced HCMV replication ( Figure 5), we suppose that NAC could inhibit H 2 O 2 -induced p38-MAPK activation. As expected, pretreatment with NAC (5 mM) also strongly decreased ATA-and H 2 O 2 -induced activation of p38-MAPK ( Figure 6D). This effect was consistent with a decline of H 2 O 2 -induced oxidative stress in cells ( Figure 6E,F), while SB203580 inhibited the H 2 O 2 -induced activation of p38 without affecting the redox status. At the same time, the upregulation of IE1 gene transcription, the expression of viral pp72 and pp65 proteins and the production of infectious virions were inhibited by treatment with SB203580 ( Figure 6G-I). These results indicated that H 2 O 2 upregulation of HCMV replication was mediated by the p38 MAPK pathway and that the inhibitory effect of NAC on H 2 O 2 -induced HCMV replication was also involved in this pathway.

H2O2 Upregulates HCMV Replication by Activating the p38 MAPK Pathway
As previously presented (Figure 2), H2O2 stimulates the upregulation of HCMV replication, but the mechanism is unclear. Here, the results revealed that p38-MAPK was rapidly and strongly activated by H2O2 treatment, following a time-and dose-dependent pattern ( Figure 6A,B). In particular, p38-MAPK activation displayed a rapid onset within 1 h of treatment, followed by a progressive increase, returning to basal levels within 48 h, while a second peak was observed at 72 h after treatment ( Figure 6A). Increasing H2O2 concentration led to an increase of p38-MAPK phosphorylation ( Figure 6B) and the minimal concentration of H2O2 was 25 μM. Next, treatment with 10 μM SB203580 reduced both H2O2and ATA-activated p38 phosphorylation ( Figure 6C). Since supplementation with NAC inhibited H2O2-and ATA-induced HCMV replication ( Figure 5), we suppose that NAC could inhibit H2O2-induced p38-MAPK activation. As expected, pretreatment with NAC (5 mM) also strongly decreased ATA-and H2O2-induced activation of p38-MAPK ( Figure 6D). This effect was consistent with a decline of H2O2-induced oxidative stress in cells ( Figure 6E,F), while SB203580 inhibited the H2O2-induced activation of p38 without affecting the redox status. At the same time, the upregulation of IE1 gene transcription, the expression of viral pp72 and pp65 proteins and the production of infectious virions were inhibited by treatment with SB203580 ( Figure 6G-I). These results indicated that H2O2 upregulation of HCMV replication was mediated by the p38 MAPK pathway and that the inhibitory effect of NAC on H2O2-induced HCMV replication was also involved in this pathway.

Discussion
In this study, we investigated the role of H2O2 in the regulation of viral lytic replication in HFF. We demonstrate that hydrogen peroxide upregulates HCMV lytic replication through extracellular and intracellular mechanisms in fibroblasts. In addition, pretreatment with antioxidants inhibits HCMV replication in vitro and in vivo. Mechanistically, the p38-MAPK pathway contributes to the stimulation of HCMV replication by H2O2.
Since AIDS, organ transplantation, and atherosclerosis are characterized by oxidative stress, and HCMV infection is of high morbidity and mortality among these patients, oxidative stress might be a crucial physiological factor that upregulates HCMV lytic replication in these cases. Regularly, p < 0.05;ˆˆp < 0.01 orˆˆˆp < 0.001 for p38 inhibitor SB203580-treated cells versus H 2 O 2 or ATA-treated cells.

Discussion
In this study, we investigated the role of H 2 O 2 in the regulation of viral lytic replication in HFF. We demonstrate that hydrogen peroxide upregulates HCMV lytic replication through extracellular and intracellular mechanisms in fibroblasts. In addition, pretreatment with antioxidants inhibits HCMV replication in vitro and in vivo. Mechanistically, the p38-MAPK pathway contributes to the stimulation of HCMV replication by H 2 O 2 .
Since AIDS, organ transplantation, and atherosclerosis are characterized by oxidative stress, and HCMV infection is of high morbidity and mortality among these patients, oxidative stress might be a crucial physiological factor that upregulates HCMV lytic replication in these cases. Regularly, the reactive oxygen species H 2 O 2 is often used to induce intracellular oxidative stress in vitro [30] and our results demonstrated that treatment with exogenous H 2 O 2 for 24 h impairs cellular redox homeostasis and acted as an active ROS in cells (Figure 1). ATA, a small molecule irreversible inhibitor of the H 2 O 2 -scavenging enzyme catalase, was utilized to induce intracellular H 2 O 2 in cells ( Figure 3A) and thereby performed the same role as exogenous H 2 O 2 ( Figure 3B-F).
HCMV lytic replication is initiated by IE1 transcription, which is activated by MIE promoter/enhancer activity. Studies have shown that ROS can enhance IE transcription products in human endothelial cells and smooth muscle cells [31,32], but this has rarely been declared in fibroblasts. Interestingly, it has been shown that oxidative stress can lead to the reactivation and replication of KSHV, another member of the herpesvirus family, in PEL cells and endothelial cells [33,34]. The luciferase reporter assay showed that treatment with H 2 O 2 enhances the activity of HCMV MIE promoter in a dose-dependent manner. Consistent with this result, we detected an increasing expression of viral IE1 gene and production of virions under the treatment of both H 2 O 2 -and ATA-induced oxidative stress. These results indicated that viral gene transcription and viral replication in the permissible cells, HFF, was initiated by H 2 O 2 -upregulated HCMV major immediately promoter activity.
Cellular antioxidants, such as superoxide dismutase (SOD) and catalase (CAT), protect cells from oxidative stress. SOD catalyzes the transition of superoxide into H 2 O 2 , which can be further converted into H 2 O and O 2 by catalase. Antioxidants used to inhibit oxidative stress have been shown to block the replication of RNA viruses, including influenza virus, EV71, and HIV-1 [35,36]. However, it remains unclear whether H 2 O 2 scavengers can inhibit H 2 O 2 -induced HCMV replication. In this study, NAC treatment resulted in inhibition of H 2 O 2 -induced oxidative stress and H 2 O 2 -upregulated HCMV replication. Similar to NAC, catalase and reduced glutathione also inhibited H 2 O 2 induced MIE promoter transcription and IE gene expression. In addition, we illustrated that ROS were required for upregulation of HCMV replication induced by CAT inhibition and depletion. These results support the hypothesis that the reactive oxygen species hydrogen peroxide is a key factor in the enhancement of HCMV gene expression and replication and that this effect could be inhibited by treatment with H 2 O 2 scavengers in vitro. Therefore, our results exhibited a critical role for H 2 O 2 and cellular antioxidants in regulating HCMV replication.
Accumulating evidence has suggested an essential role for oxidative stress during viral infection [37][38][39][40][41]. Oxidative stress can be considered a protective means of the cell, which can contribute to apoptosis [42], and thus prevent the virus from replicating and infecting other cells. Interestingly, CMV appears to utilize virus-specific mechanisms to protect the cell from the effects of ROS and maintain a redox homeostasis [43]. However, the results showed that the levels of ROS increased remarkably upon MCMV infection, with the increase first appearing at approximately 2 h after infection ( Figure 5A) and sustained until 72 h post-infection. ( Figure 5B). Thus, it seems antioxidant therapy could be a potential treatment method for primary MCMV infection. In support of the results of previous studies we conducted, NAC was shown to prevent MCMV replication and production in vitro ( Figure 5C). Significantly, NAC strongly reduced MCMV DNA load in whole blood and the production of infectious virions in the salivary gland and the lung.
It is widely accepted that the HCMV major immediate early promoter contains several types of transcription factor binding sites [44], such as NF-κB, that can be induced by H 2 O 2 [45]. Furthermore, previous studies have shown that H 2 O 2 can induce NF-κB transcription through multiple signaling pathways [23,46], including the JNK and p38 MAPK pathways. Furthermore, studies have shown that p38 MAPK, which is mediated by MSK1, is involved in NF-κB transactivation by H 2 O 2 stimulus [23,24], but very little is known regarding the possible linkage between this pathway under H 2 O 2 -upregulated HCMV replication in fibroblast cells. Here, consistent with the results of previous studies, we showed that p38 was rapidly and strongly activated by H 2 O 2 treatment. Additionally, co-culturing fibroblasts with HCMV and the p38 specific inhibitor SB203580 decreased the phosphorylation of p38 and HCMV transcription and production. Similar to SB203580, NAC also hampered the activation of p38 by H 2 O 2 and inhibited the viral replication. However, NAC inhibited the H 2 O 2 -induced phosphorylation of p38 through inhibition of H 2 O 2 -induced oxidative stress, while SB203580 directly inhibited the p38 activation without affecting the production of ROS. Thus, we first demonstrated that H 2 O 2 induced HCMV replication through the ROS/P38 MAPK signaling pathway.
Conclusively, our findings suggest that further studies of the antiviral and immune-modulatory effects of antioxidants are warranted. Furthermore, targeting of hydrogen peroxide and H 2 O 2 -mediated signaling is a potential therapeutic or preventive approach in HCMV infection.