Gallic acid inhibits Kaposi's Sarcoma‐associated herpesvirus lytic reactivation by suppressing RTA transcriptional activities

Abstract Kaposi's sarcoma‐associated herpesvirus (KSHV), an oncogenic virus, has two life cycle modes: the latent and lytic phases. KSHV lytic reactivation is known to be important both for viral propagation and for KSHV‐induced tumorigenesis. The KSHV replication and transcription activator (RTA) protein is essential for lytic reactivation. Gallic acid (GA), one of the most abundant phenolic acids in the plant kingdom, has been shown potential chemotherapeutic efficacy against microbial and cancer. However, the effects of GA on KSHV replication and KSHV‐induced tumorigenesis have not yet been reported. Here, we report that GA induces apoptotic cell death in BCBL‐1 cells in a dose‐dependent manner. GA inhibits KSHV reactivation and reduces the production of progeny virus from KSHV‐harboring cells. GA inhibits RTA transcriptional activities by suppressing its binding to target gene promoters. These results suggest that GA may represent a novel strategy for the treatment of KSHV infection and KSHV‐associated lymphomas.

Similar to other herpesviruses, the replicative cycle of KSHV exists as latency and lytic replication (Jenner & Boshoff, 2002). KSHV mostly persists in the latent state during which it has a restricted latent gene expression program but can be reactivated and transitioned to the lytic state when triggered by stress conditions such as hypoxia or HIV coinfection, or stimulated by other chemical signals such as 12-O-tetradecanoylphorbol-13-acetate (TPA), sodium butyrate (NaB), and valproate (VPA) (Cai et al., 2006;Davis et al., 2001;Varthakavi et al., 1999).
During the lytic phase, the full spectrum of lytic viral genes (immediate-early (IE), early (E), and late (L) genes), are sequentially expressed (Sun et al., 1999). The KSHV replication and transcription activator (RTA) is an IE gene that functions as a transcription factor to drive the temporally ordered expression of KSHV lytic genes leading to production of infectious viral particles; it activates many viral promoters, including those for PAN RNA, Ori-Lyt-associated RNA, ORF57, and K8, as well as its own promoter, by binding to RTA-responsive elements (RREs) (Guito & Lukac, 2012;Lukac et al., 1999;Sun et al., 1998). KSHV lytic reactivation is known to tion. Gallic acid (GA), one of the most abundant phenolic acids in the plant kingdom, has been shown potential chemotherapeutic efficacy against microbial and cancer.
However, the effects of GA on KSHV replication and KSHV-induced tumorigenesis have not yet been reported. Here, we report that GA induces apoptotic cell death in BCBL-1 cells in a dose-dependent manner. GA inhibits KSHV reactivation and reduces the production of progeny virus from KSHV-harboring cells. GA inhibits RTA transcriptional activities by suppressing its binding to target gene promoters. These results suggest that GA may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.

K E Y W O R D S
apoptosis, gallic acid, KSHV, lytic activation, RTA be important both for viral propagation and for KSHV-induced tumorigenesis (Purushothaman et al., 2015), so any interference that disrupts KSHV lytic switch could contribute to the treatment of KSHV-related malignancies.
Gallic acid (GA) is one of the most abundant phenolic acids in the plant kingdom, with extensive application in the food and pharmaceutical industries (Fernandes & Salgado, 2016). Besides the edible uses of GA in the food industry, there are ample evidences that show the potential chemotherapeutic efficacy of GA against microbial, cancer, inflammatory, cardiovascular diseases, and neuropsychological diseases (Choubey et al., 2015). However, the effects of GA on KSHV replication and KSHV-induced tumorigenesis have not yet been reported.
In this study, we investigated the antitumor and antiviral activity of gallic acid against human primary effusion lymphoma (PEL) cells. Our data demonstrate that GA induces apoptotic cell death in PEL cells. We also examined the effects of GA on KSHV replication and reactivation. GA inhibits RTA transcriptional activities via a mechanism involving modulating its binding to target gene promoters. These results suggest that GA may represent a novel strategy for the treatment of KSHV infection and KSHVassociated lymphomas.

| Reagents
GA, VPA, NaB, TPA, and tetracycline were purchased from Sigma-Aldrich Chemical Co. GA was dissolved in DMSO at 1 M as a stock solution. VPA and NaB were dissolved in sterile ddH 2 O at 1 M as a stock solution. TPA was dissolved at 200 μg/ml concentration with sterile ddH 2 O. Tetracycline was dissolved in DMSO at 1 mg/ml as a stock solution.
HRP-conjugated goat anti-mouse IgG and anti-rabbit IgG were purchased from Santa Cruz Biotechnology. Mouse monoclonal antibody against Flag and rabbit monoclonal antibodies against GAPDH and tubulin were obtained from Cell Signaling Technology.

| Cell viability assays
The effect of GA on PEL cell viability was determined by CCK-8 assay. BCBL-1 cells (1 × 10 4 cells/well) were seeded onto 96-well plates. After incubation for 2 hr, cells were treated with or without various concentrations of GA for 48 hr. Cell viability was determined by CCK-8 assay. The untreated cells were utilized as control, and the cell viability was compared with the control. Each treatment was performed in triplicate, and three independent experiments were performed. Error bars represent the standard errors.

| Two-layered soft agar colony formation assay
For soft agar assay, experiments were carried out in 12-well plates coated with a base layer of RPMI1640 containing 0.5% agar, 20% fetal bovine serum, and GA or DMSO. 5 × 10 3 BCBL-1 cells were seeded per well in RPMI1640 containing 0.35% agar, 20% fetal bovine serum, and GA or DMSO for 7 days. Colonies were visualized using a stereomicroscope (Olympus).

| Quantitative reverse transcription-PCR (qRT-PCR)
Total RNA was extracted from cells using TRIzol (Life Technologies) according to the manufacturer's protocol. RNA was converted to cDNA by using RevertAid First Strand cDNA Synthesis Kit (Thermo) according to the manufacturer's protocol.
Relative transcript levels of selected cellular and genes were determined with gene-specific primers plus SYBR ® Premix Ex Taq™Ⅱ (Tli RNaseH Plus) (TaKaRa) by 7,500 fast real-time PCR system (Applied Biosystems). Primer sequences are listed in Table 1.
Relative expression levels were calculated using the ∆∆CT method after normalization to actin. Individual samples were assayed in duplication.

| Quantitative analysis of KSHV virions in supernatant
Viral DNA was collected and prepared from culture supernatant by using the AxyPrep™ Body Fluid Viral DNA/RNA Miniprep Kit (Axygen) according to the manufacturer's protocol and then was used to amplify the KSHV LANA gene by qPCR using primers in Table 1.
Relative expression levels were calculated using the ∆∆CT method after normalization to protrudin (protrudin plasmid was added to supernatant as a control). Individual samples were assayed in duplication.

| Luciferase reporter assay
Replication and transcription activator transactivation was quantified using the Dual-Luciferase Reporter Assay System from Promega.
Briefly, cells were transfected with indicated expression plasmids plus reporter plasmids. Cell lysates were prepared according to the manufacturer's protocol. Luciferase was measured on a GloMaxH-Multi Microplate Multimode Reader (Promega). Data were taken as a ratio of firefly/Renilla luciferase, and the results shown represent experiments performed in duplicate.

| ChIP assays
Cells were cross-linked with 1% formaldehyde (Thermo Scientific) for 10 min at room temperature. Glycine was added to a final concentration of 125 mM to stop the cross-linking reaction, and the samples were incubated for another 5 min at room temperature. After incubation at 65°C for 12 hr, the eluted solution was subjected to DNA gel extract kit (AXYGEN).
2 µl of 20 µl DNA solution was used as the template DNA for qPCR using primers specific for the A/T-rich region of ori-Lyt left and RTA promoter which were shown in Table 1.

| Statistical analyses
All data are represented by the mean ± standard error of at least two independent experiments. Student's t test was used for statistical significance of the differences between treatment groups. Statistical analysis was performed using analysis of variance at 5% (p < .05).

| GA inhibits cell growth and induces cell apoptosis in PEL cells
To determine the effects of GA on the PEL cells, a PEL cell line BCBL-1 (Nador et al., 1996)  As 200 μM GA caused most of the cell death, which was not conducive to the experiment. In the follow-up experiments, we chose 100 μM as the highest concentration.

| GA inhibits KSHV lytic reactivation in iSLK. rKSHV.219 cells
To determine whether GA functions in KSHV lytic replication, we  (Deng et al., 2007). Quantification of viral particles in the media revealed that cells treated with GA secreted fewer viral particles than those treated with DMSO ( Figure 2c).

| GA suppresses KSHV lytic reactivation in PEL cells
To determine whether GA functions in viral reactivation in other KSHV-infected cells, we treated BCBL-1 cells with 0, 20, 50, and 100 μM GA for 1 hr followed by treatment with NaB plus TPA to induce KSHV lytic reactivation.
RT-qPCR revealed that the mRNA levels of the lytic genes RTA, ORF59, ORF9, and ORF8.1 were significantly decreased in GAtreated cells in a dose-dependent manner (Figure 3a). Furthermore, the decreases in these lytic gene expressions corresponded to decreases in the quantity of viral particles secreted into the culture media ( Figure 3b).

F I G U R E 3 GA inhibits KSHV lytic reactivation in human herpesvirus 8 (HHV8)-harboring primary effusion lymphoma (PEL) cells. (a)
BCBL-1 cells were treated with GA at different concentrations for 1 hr followed by treatment with TPA plus NaB for 48 hr. mRNA levels of KSHV lytic genes RTA, ORF59, ORF9, and ORF8.1 were measured by RT-qPCR, with normalization to actin using the ΔΔCT method. (b) Viral DNA in the media was quantified using qPCR, with normalization to an added plasmid protrudin using the ΔΔCT method. Data are presented as means of two technical replicates (n = 2, group values are indicated by mean ± SEM; *p < .05; **p < .01)

| GA inhibits RTA transcriptional activities by modulating its binding to target gene promoters
Given RTA is the known trigger for KSHV lytic activation (Lukac et al., 1998;West & Wood, 2003), we next examined whether GA would directly regulate RTA transcriptional activity by reporter gene assays. Expression of RTA increased the activities of the luciferase reporters pGL3-Ori-Lyt-Luc and pGL4-RTA-Luc, but their activities were reduced when RTA transfected cells were treated with GA ( Figure 4a,b).
To evaluate the mechanism underlying GA-mediated inhibition of RTA transcription activity, we performed ChIP assays in HEK293T These results for reporter gene assay and ChIP together strongly suggest that the observed function of GA in regulating RTA's transcriptional activation of lytic genes results in some way to the modulation of RTA binding with its target gene promoters.

| D ISCUSS I ON
Gallic acid or 3, 4, 5-trihydroxybenzoic acid (CAS No 149-91-7) is one of the most abundant phenolic acids in the plant kingdom (Al Zahrani et al., 2020). Besides the extensive application in the food and pharmaceutical industries, there are diverse scientific reporters on biological and pharmacological activities of GA (Akbari, (2020); Kahkeshani et al., 2019). There are many reports about the effects of GA on antimicrobial and cancer cell growth (Rajamanickam et al., 2018;Zhang et al., 2019). However, the effects of GA on KSHV and KSHV-harboring cells have not been investigated.
In this study, we discovered that GA inhibits the proliferation of KSHV-harboring PEL cells by inducing cell apoptosis. GA can inhibit F I G U R E 4 GA regulates the transcriptional activity of KSHV RTA by modulating its binding to the promoters of its target genes. GA inhibits RTA transcriptional activities in reporter assays. HEK293T cells were transfected with the firefly luciferase reporter constructs pGL3-Ori-Lyt-Luc (a) or pGL4-RTA-Luc (b), a Renilla luciferase control plasmid, RTA, and treated with GA at different concentrations for reporter assay (n = 2, group values are indicated by mean ± SEM; *p < .05; **p < .01). GA inhibits RTA binding to the Ori-Lyt promoter (c) and the RTA promoter (d).
HEK293T cells were cotransfected with the reporter plasmid pOri-Lyt-Luc or pRTA-Luc, with RTA-Flag, and treated with GA at different concentrations. After 24 hr, the cells were harvested and subjected to ChIP assay using anti-Flag magnetic agarose beads. The quantities of Ori-Lyt promoter or RTA promoter DNA sequences in the precipitates were evaluated by qPCR using the ΔΔCT method. Data are presented as means of two technical replicates (n = 2, group values are indicated by mean ± SEM; *p < .05; **p < .01) the replication and reactivation of KSHV in KSHV-harboring cells.
Since KSHV is essential for KSHV-infected tumors survival (Godfrey et al., 2005), we propose that GA leads to KSHV-harboring cells death by inhibiting KSHV replication and reactivation. Through exploring the possible mechanism, we find that GA can inhibit RTA transcriptional activities by diminishing its binding to target gene promoters. These data indicate that GA may be a potential treatment for aggressive PEL and KSHV infection.
We have demonstrated that mRNA expression of lytic gene is suppressed by GA treatment in chemical reagents activated KSHVharboring cells; consistently, there were fewer viral particles in media from GA-treated cells (Figures 2 and 3). From the mechanistic point of view, GA can inhibit motility, adherence, and biofilm formation of some microbial (Borges et al., 2012;Shao et al., 2015). So, it will be interesting to determine whether GA has an effect on KSHV de novo infection. As GA can reduce virus production, our next step will be to explore whether it also affects the infection efficiency of these viruses.
In our study, GA inhibits RTA transcriptional activities by diminishing its binding to target gene promoters ( Figure 4). The binding of RTA and some target gene promoters is not directly, but at the same time, through the interaction with other host cell transcription factors (Liang & Ganem, 2004;Papp et al., 2019;Wang & Yuan, 2007). GA may affect the transcription activity of RTA by influencing other transcription factors. Thus, it will be fascinating to determine whether GA has an effect on these transcription factors which interact with RTA.
We determined that gallic acid inhibits the replication and reactivation of KSHV by repressing RTA transcriptional activities. Other herpesviruses also contain RTA or transcription factor similar to RTA (Walters et al., (2005); Walters et al., 2004;Goodwin et al., 2001), it will be interesting to determine whether GA can modulate other herpesviruses infection.
In conclusion, GA suppresses KSHV replication and reactivation, leading to apoptosis in KSHV-harboring cells. These finding suggests that GA can be a potential anti-KSHV drug candidate and may be considered as an effective treatment for KSHV-related tumors.