Cordycepin is a novel chemical suppressor of Epstein-Barr virus replication

Abstract

Cordyceps species are known to produce numerous active components and are used for diverse medicinal purposes because of their varied physiological activities, including their ability to protect the liver from damage as well as their anticancer, antidepressant, anti-inflammatory, hypoglycemic, antimicrobial effects. Cordycepin, an adenosine derivative, differs from adenosine in that its ribose lacks an oxygen atom at the 3' position. Several research groups have reported that cordycepin has antiviral activity against several viruses including influenza virus, plant viruses, human immunodeficiency virus(HIV), murine leukemia virus, and Epstein-Barr virus (EBV). In this study, we identify the epigenetic mechanisms by which cordycepin exerts its anti-gammaherpesvirus effects. We show that cordycepin possesses antitumor and antiviral activity against gastric carcinoma and EBV, respectively. A comparison of the CD50 values of cordycepin and its analogs showed that the lack of a 2'-hydroxyl group in cordycepin was critical for its relatively potent cytotoxicity. Cordycepin treatment decreased the rate of early apoptosis in SNU719 cells by up to 64%, but increased late apoptosis/necrosis by up to 31%. Interestingly, cordycepin increased BCL7A methylation in SNU719 cells by up to 58% and decreased demethylation by up to 37%. Consistent with these changes in methylation, cordycepin treatment significantly downregulated most EBV genes tested. Under the same conditions, cordycepin significantly decreased the frequency of Q and F promoter usage, and H3K4me3 histone enrichment was significantly reduced at several important EBV genomic loci. Extracellular and intracellular EBV genome copy numbers were reduced by up to 55% and 30%, respectively, in response to 125 μM cordycepin treatment. Finally, cordycepin significantly suppressed the transfer of EBV from LCL-EBV-GFP to AGS cells, indicating that EBV infection of gastric epithelial cells was inhibited. These results suggest that cordycepin has antiviral and antitumor activities against gammaherpesviruses and host cells latently infected with virus.

Keywords: Cordycepin; Epstein–Barr virus; antiviral agent; gastric carcinoma.

Figure 1. Cytotoxicity of cordycepin

A cytotoxicity assay was performed using a cell counting assay (CCK-8 kit) to determine if cordycepin causes cytotoxicity. Cordycepin cytotoxic to SNU719 cells that latently infected by Epstein-Barr virus(EBV). The same methods were used to test the cytotoxicity of adenosine and its analogs. (A) Structures of cordycepin, adenosine, 2′-deoxyadenosine, and 2′,3′-dideoxyadenosine. (B) 50% cytotoxicity dose (CD₅₀) of cordycepin and its analogs in SNU719 cells. Each measurement was repeated three times. Averages and standard errors of measurements are displayed on the graphs.

Figure 2. Effect of cordycepin on apoptosis

Two apoptosis assays—Annexin V-FITC and caspase 3/7—were performed to analyze the effect of cordycepin on apoptosis in SNU719 cells. (A) The Annexin V-FITC apoptosis detection assay was performed in triple repeats. Early apoptosis was suppressed by cordycepin by up to 64%, but increased late apoptosis/necrosis by up to 31% in SNU719 cells treated with cordycepin for 48 h. SDW, blank treatment; Cordycepin, 125 μM cordycepin treatment. (B) The caspase 3/7 assay was conducted in triple repeats. Caspase 3 and 7 activities were gradually decreased in SNU719 cells in response to 100 and 200 μM cordycepin treatments. Numbers 0, 100, and 200 indicate the micromolar concentrations of the cordycepin treatments. P-values <0.05 (95% confidence) were considered statistically significant.

Figure 3. Effect of cordycepin on cellular membrane integrity

The Annexin V-FITC apoptosis detection assay was used to determine if cordycepin affects SNU719 membrane integrity. A n RT-qPCR assay was then performed to determine if cordycepin affects membrane protein transcription. (A) Effect of cordycepin on SNU719 cell membrane integrity, as determined using a Annexin V-FITC apoptosis detection assay. As expected, cordycepin significantly enhanced membrane integrity in SNU719 cells. SDW, blank; Cordycepin, 125 μM cordycepin treatment. (B) RT-qPCR was performed to quantify TLR (Toll-like receptor) transcripts (TLR2, TLR3, TLR4, TLR6, TLR7, and TLR9) localized to cytoplasmic or endosomal membranes. Cordycepin enhanced TLR4 and TLR3 transcription, but downregulated that of TLR7 and TLR9. TLR2 transcript was not detected by this assay. SDW, blank; Cordycepin, 125 μM cordycepin; NaB/TPA, NaB (3 mM)/TPA (20 ng/ml) treatments. P-values <0.05 (95% confidence) were considered statistically significant.

Figure 4. Effect of cordycepin on cell cycle

Effect of cordycepin on cell cycle progression was examined using PI staining and FACs analysis. Cordycepin (125 μM) did not arrest SNU719 cells at any phase in the cell cycle.

Figure 5. Effect of cordycepin on methylation

The effect of cordycepin on methylation was determined using Western blot analysis as well as methylation-specific PCR and RT-qPCR assays. (A) Western blot analysis was performed to determine if cordycepin affected DNMT1 and DNMT3a production in SNU719 cells treated with cordycepin. Beta-actin was used as loading and internal controls. These Western blot analysis showed that cordycepin did not affect DNMT1 levels, but did significantly induced DMNT3 expression. (B) Methylation-specific PCR was performed on cordycepin-treated (125 μM) SNU719 cells to determine if cordycepin affected methylation of BCL7A, a tumor suppressor gene. Compared to the mock treatment, 10 μM 5-aza-2′-deoxycytidine (DAC) treatment suppressed methylation by up to 35% and induced demethylation by up to 882% (left two panels). The methylation-specific PCR assay showed that cordycepin treatment (125 μM) induced BCL7A methylation by up to 58% and suppressed demethylation by up to 37% in SUN719 cells (right two panels). P-values <0.05 (95% confidence) were considered statistically significant. (C) An RT-PCR assay was performed to determine if cordycepin affected methylation of EBV genomic loci in SNU719 cells treated with cordycepin (125 μM). Methylation of EBV genomic loci near Cp/Wp promoters was not affected by cordycepin treatment, whereas that around Fp/Qp promoters was enhanced. Specifically, regions downstream of the Fp/Qp promoters were highly methylated in response to cordycepin treatment. Cp/Wp1 and Cp/Wp2 are EBV genomic loci upstream and downstream of Cp/Wp promoters, respectively. Fp/Qp1 and Fp/Qp2 are EBV genomic loci upstream and downstream of Fp/Qp promoters, respectively. M, methylation-specific primers; U, demethylation-specific primers.

Figure 6. Effect of cordycepin on signal transduction

A Cignal Finder reporter assay was performed to determine whether cordycepin-induced hypermethylation affects the expression of transcription factors that are known to interact with DNA methyltransferase (DNMT). An RT-qPCR assay was then performed to confirm the expression patterns of transcription factors that were upregulated or downregulated in the Cignal Finder reporter assay. (A) SNU719 cells were treated with cordycepin (125 μM) for 48 h and analyzed using an RT-qPCR assay. Transcription factors kwown not to interact with DNMT3 are Nrf2 (ARE reporter, antioxidant response pathway), YY1 (ERSE reporter, ER stress pathway), HIF-1a (HIF reporter, hypoxia pathway), and Elk1 (SRE reporter, MAPK/EK pathway) [37]. These factors are downregulated by cordycepin, but this effect was not statistically significant. In contrast, transcription factors known to interact with DNMT3 are ATF4 (AARE reporter, amino acid deprivation pathway), CREB1 (CRE reporter, cAMP/PKA pathway) and HNF4 (HNF4 reporter, HNF4 pathway) [37]. These factors are downregulated by cordycepin with statistical significance, especially the expression of ATF4 and HNF4. P-values are listed at the bottom of the graph. (B) SNU719 cells were treated with cordycepin (125 μM) for 48 h and analyzed using the Cignal Finder reporter assay. Reporters induced by cordycepin are shown in yellow, and those suppressed by cordycepin are shown in red. AARE, AR, CRE, GLI, HNF4, GAS, RBP-jK, NFAT, and STAT3 reporters were downregulated by cordycepin. ARE, p53, EGR1, ERSE, HIF, IRF1, KLF4, SRE, AP1, PR, SP1, VDR, and XRE reporters were upregulated by cordycepin. Each reporter assay was performed in triplicate.

Figure 7. Effects of cordycepin on EBV latent and lytic transcription

Effects of cordycepin on EBV gene transcription were determined by performing an RT-qPCR assay using cDNA from SNU719 treated with cordycepin. (A) cDNA synthesized from RNA isolated from cordycepin-treated SNU719 cells was analyzed using an RT-qPCR assay. The relative transcription levels of EBV latent and lytic genes was determined. Compared to mock treatment, cordycepin treatment increases only the transcription of the EBV non-coding RNA EBER. However, most EBV genes were significantly downregulated by cordycepin. Cp, Wp, and Qp are EBV promoters activated depending on the EBV latency type [17]. (B) cDNA synthesized from RNA isolated from SNU719 cells cotreated with cordycepin, sodium butyrate, and TPA was analyzed using an RT-qPCR assay. The levels of EBV latent and lytic genes transcription significantly increased. P-values <0.05 (95% confidence) were considered statistically significant. CTCF BSs, binding sites where CTCF are known to bind in the KSHV genome [22]. P-values are listed at the bottom of the graph.

Figure 8. Effect of cordycepin on EBV latency promoter selection

An RT-PCR assay was performed to determine if cordycepin affects the selection of EBV latency promoters. (A) As controls, KEM1 cells (specific to EBV type I) showed a high frequency of Qp and Fp promoter usage. KEM3 cells (specific to EBV type III) showed a high frequency of Cp/Wp promoter usage. (B) Cordycepin decreased frequencies of Qp and Fp promoter usage, but did not affect Cp/Wp promoter activity. (C) Combined treatment of cordycepin with HDAC inhibitors such as NaB and TPA did not reduce Qp and Fp promoter activities. Cp, Wp, Qp, and Fp are EBV promoters activated depending on the EBV latency type [17].

Figure 9. Effect of cordycepin on histone modification at EBV genomic loci

Chromatin immunoprecipitation (ChIP) was performed to analyze EBV histone modification patterns in SNU719 cells treated with cordycepin. Seven EBV genomic loci in the EBV genome—1300 (LMP2), 6926 (between BNRF1 and BCRF1), 11675 (between BCRF1 and BCRF2), 12450 (5′ UTR of BCRF2), 41126 (between BHLF1 and BHRF1), 51251 (BPLF1), and 83382 (EBNA3B)—were detected by RT-qPCR. Cordycepin reduces H3K4me3 histone enrichment at loci 1300, 12450, 41126, and 83382. Similarly, H3K9me3 histone enrichments at loci 1300, 12450, and 83382 were also a little reduced. These results indicate that cordycepin treatment results in histone modification at EBV genomic loci, consequently downregulating EBV genes.

Figure 10. Effect of cordycepin on EBV protein production

Protein levels were assessed by Western blot assay to determine the effect of cordycepin treatment on gammaherpesvirus translation in SNU719 cells. EBNA1 and LMP2A levels in SNU719 cells were greatly reduced, whereas BZLF1 expression was not changed by cordycepin treatment. Blank, Sterile distilled water(SDW); Cordycepin, 125 μM cordycepin treatment; NaB/TPA, NaB (1 mM)/TPA (1 ng/ml) treatment. β-actin was used as an internal control in the Western blot analysis.

Figure 11. Effect of cordycepin on EBV progeny production

Intracellular and extracellular EBV genome copy numbers were determined in SNU719 cells, following methods previously described. Intracellular (A) and extracellular (B) EBV genome copy numbers were significantly reduced by up to 55% and 30%, respectively, in response to 125 μM cordycepin treatment. SDW, blank; Cordycepin, 125 μM cordycepin treatment; NaB/TPA, NaB (1 mM)/TPA (1 ng/ml) treatment. Intracellular and extracellular are the relative intracellular (relative to GAPDH) and extracellular (relative to blank treatment) gammaherpesviral genome copy numbers, respectively. P-values <0.05 (95% confidence) were considered statistically significant.

Figure 12. Effect of cordycepin on EBV infection

A cell-to-cell coinfection assay was performed using LCL-EBV and AGS cells to determine whether cordycepin affects EBV infection. EBV transfers from LCL-EBV cells to AGS cells in the cell-to-cell coculture infection system [38]. As a donor, LCL-EBV-GFP cells (LCL-2266-36) were cocultured with AGS cells that had been seeded previously, and cordycepin treated was applied. (A) Cordycepin significantly suppresses the transfer of EBV from LCL-EBV-GFP cells to AGS cells, suggesting that EBV infection to gastric epithelial cells is significantly inhibited. (B) AGS cells infected with the EBV-GFP virus were attached and GFP-positive. P-values <0.05 (95% confidence) were considered statistically significant.