Reactivation of Epstein-Barr virus by a dual-responsive fluorescent EBNA1-targeting agent with Zn2+-chelating function

Abstract

Epstein-Barr nuclear antigen 1 (EBNA1) plays a vital role in the maintenance of the viral genome and is the only viral protein expressed in nearly all forms of Epstein-Barr virus (EBV) latency and EBV-associated diseases, including numerous cancer types. To our knowledge, no specific agent against EBV genes or proteins has been established to target EBV lytic reactivation. Here we report an EBNA1- and Zn²⁺-responsive probe (ZRL₅P₄) which alone could reactivate the EBV lytic cycle through specific disruption of EBNA1. We have utilized the Zn²⁺ chelator to further interfere with the higher order of EBNA1 self-association. The bioprobe ZRL₅P₄ can respond independently to its interactions with Zn²⁺ and EBNA1 with different fluorescence changes. It can selectively enter the nuclei of EBV-positive cells and disrupt the oligomerization and oriP-enhanced transactivation of EBNA1. ZRL₅P₄ can also specifically enhance Dicer1 and PML expression, molecular events which had been reported to occur after the depletion of EBNA1 expression. Importantly, we found that treatment with ZRL₅P₄ alone could reactivate EBV lytic induction by expressing the early and late EBV lytic genes/proteins. Lytic induction is likely mediated by disruption of EBNA1 oligomerization and the subsequent change of Dicer1 expression. Our probe ZRL₅P₄ is an EBV protein-specific agent that potently reactivates EBV from latency, leading to the shrinkage of EBV-positive tumors, and our study also suggests the association of EBNA1 oligomerization with the maintenance of EBV latency.

Keywords: EBNA1-targeting agent; EBV-specific lytic inducer; dual-responsive fluorescent EBV probe.

Fig. 1.

Chemical, fluorescent, and EBNA1-binding characteristics of the Zn²⁺ chelator EBNA1 probe ZRL₅P₄. (A) Chemical structures of L₂P₄ and ZRL₅P₄. (B) Schematic illustration of the emission response of ZRL₅P₄, binding to Zn²⁺ and EBNA1. (C) Representative conformations of ZRL₅P₄ and Zn²⁺-ZRL₅P₄ in the MD simulations. The calculated generalized Born (GB) and Poisson–Boltzmann (PB) values represent the binding free energy between EBNA1 and ZRL₅P₄ or Zn²⁺-ZRL₅P₄.

Fig. 2.

Dual-responsive emission of ZRL₅P₄ (2 µM; excitation 337 nm) toward Zn²⁺ and EBNA1. (A) Fluorescence spectral changes of ZRL₅P₄ in the presence of Zn²⁺ in aqueous solution (CH₃CN/0.05 M Hepes [pH 7.4], 50:50). (B) Fluorescence spectral changes of ZRL₅P₄ upon gradual addition of Zn²⁺ in aqueous solution (CH₃CN/0.05 M Hepes [pH 7.4], 50:50). (C) Solvation study of ZRL₅P₄ with a decrease of solvent polarity. Fluorescence spectral changes of ZRL₅P₄ in PBS upon addition of (D) EBNA1 and (E) HSA and upon addition of EBNA1 in the presence of (F) 1 equivalent Zn²⁺. (E, Inset) The selectivity of ZRL₅P₄ toward EBNA1 over HSA. (F, Inset) Interaction of ZRL₅P₄ with EBNA1 in the presence and absence of Zn²⁺. a.u., arbitrary units.

Fig. 3.

ZRL₅P₄ inhibits EBNA1 oligomerization and transactivation and cell viability. (A) Full- length EBNA1 self-association in the absence/presence of Zn²⁺ by ZRL₅/ZRL₅P₄/L₂P₄/ZRL₅ + L₂P₄; 0.1% DMSO serves as the solvent control. Long and short exposure images of the same blot are shown for comparison of various forms of EBNA1 self-association. Analysis of the effects of EBNA1 probes (ZRL₅P₄, L₂P₄) on the oriP-enhanced transactivation in EBV-positive (B) C666-1 and (C) NPC43 cells. The transactivation activities were detected by the oriP-Cp-luciferase reporter. EDTA and TPEN are the known chelators of Zn²⁺. (D–H) Cytotoxic activities of the EBNA1 probes ZRL₅P₂, ZRL₅P₄, and ZRL₅P₆ in the EBV-positive and -negative cell lines. Cytotoxicity of EBV-positive (D) NPC43 cells, (E) C666-1 cells, and (F) Raji cells (concentrations 1, 3, 5, 10, 15, and 20 µM) and EBV-negative (G) HK-1 cells and (H) HONE-1 cells (concentrations 1, 5, 10, 20, 50, and 100 µM) were measured by the MTT assay. Cells were treated with different probes and then incubated for 5 d to test their cytotoxicity (half of the medium was replaced every 4 d with fresh medium containing the appropriate concentration of the probes). Data are expressed as the means ± SD.

Fig. 4.

Nuclear localization of the EBNA1 probes and their in vivo antitumor activities. (A–C) Two-photon fluorescence imaging of ZRL₅P₂, ZRL₅P₄, and ZRL₅P₆ in living EBV-positive (A) NPC43 cells and (B) C666-1 cells and EBV-negative (C) HONE-1 cells. ZRL₅P_n, signal emitted from the respective EBNA1 probe. DRAQ5 is a fluorescent dye used to label the cell nuclei of the living cells as indicated. (D) In vitro emission spectra (from confocal microscopy) of ZRL₅P₂, ZRL₅P₄, and ZRL₅P₆ in the nucleus of EBV-positive NPC43 and C666-1 and EBV-negative HONE-1 cells. Emission intensity was much greater for ZRL₅P₄ and ZRL₅P₆ in EBV-positive cells. (E) In vivo antitumor activity of ZRL₅P₂, ZRL₅P₄, and ZRL₅P₄. Mice transplanted with C666-1–derived tumors were treated twice weekly with 4 µg per injection of the probes for 18 d. Throughout the treatment period, tumor volumes were measured. At the experimental endpoint, tumors were excised. Data are expressed as the means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control (0.1% DMSO). (Scale bars, 10 mm.) (F) Representative photographs of tumors. (G) Representative H&E staining images of tumor sections derived from the above in vivo animal study. Cell necrosis (acellular areas indicated by asterisk) was observed in the tumor nodules treated with ZRL₅P₄ and ZRL₅P₆. T, adjacent area with tumor cells. Magnification, 400×. (Scale bars, 20 µm.) (H) Response of plasma EBV DNA levels in mice transplanted with C666-1 cells after the treatment of ZRL₅P₄. The circulating EBV DNA level of each mouse (DMSO #1, #2; ZRL₅P₄ #1, #2) is shown.

Fig. 5.

EBV lytic induction analysis of EBV-positive tumors and cells in response to the EBNA1 probes. (A) IHC analysis of lytic proteins Zta, BMRF1, and VCA-p18 in the transplanted C666-1–derived tumor tissues as described in Fig. 4 E–G. Representative results are shown. Nuclear and cytoplasmic staining of Zta, BMRF1, and VCA-p18 are observed in response to ZRL₅P₄. Insets (in the red boxes) are the enlarged images to indicate the cellular localization of the 3 proteins. Negative control staining images are included for the mouse antibodies (mAb) against Zta and BMRF1 and for the rat antibody (rAb) against VCA-p18. Magnification, 400×. (Scale bars, 20 µm.) (B) Representative images of immunofluorescent analysis of EBV lytic proteins, Zta, BMRF1, gp350/220, and VCA-p18 in HONE-1-EBV cells in response to 10 µM ZRL₅P₂ or ZRL₅P₄. TPA/NaB (20 ng/mL TPA, 3 mM NaB) serves as a positive control of lytic induction. The presence of these 4 lytic proteins is indicated by red signals. The nuclei were stained with DAPI. (Scale bars, 50 µm.) (C) Western blot analysis of Rta, BMRF1, and gp350/220 EBV lytic proteins in NPC43 cells cultured with or without 10 µM ZRL₅P₂ and ZRL₅P₄ for 7 d. Acetylated histone H3 (Ac-Histone H3) was also detected. β-actin serves as the loading control. (D) Gene expression analysis of EBV lytic genes, Zta, Rta, BMRF1, and VCA-p18 in NPC43, C666-1, and HONE-1-EBV cells cultured with or without 10 µM ZRL₅P₂, ZRL₅P₄, and L₂P₄, for 7 (NPC43 cells), 3 (C666-1 cells), or 5 (HONE-1-EBV) d. The gene expression was analyzed by qRT-PCR. The fold change of relative gene expression after each treatment was compared with the solvent control (DMSO). Data are expressed as the means ± CI.

Fig. 6.

Production of infectious EBV particle in response to ZRL₅P₄. The HONE-1-EBV cell line, which expresses GFP to indicate the presence of the EBV genome, was used. This cell line was treated with 10 µM ZRL₅P₂ or ZRL₅P₄ for 4 d, and the viral particles released in the culture medium were detected by the Raji cell assay. The culture medium was added to Raji cells for 3 d, and GFP expression reflects the reinfection by the HONE-1–released EBV particles. (A) Representative results are shown. The GFP signal was detected by ultraviolet light exposure, and the cell morphology was captured by phase-contrast light microscopy and the bright-field image was merged with the GFP image. Magnification, 400×. (Scale bars, 100 µm.) (B) Relative average viral titer in response to ZRL₅P₂ or ZRL₅P₄ was compared with the solvent control (DMSO). The Raji cell assay was performed in triplicate for each treatment. **P < 0.01, statistically significant difference. Data are expressed as the means ± SD. (C) Representative images of immunofluorescent analysis of Dicer1 and PML in HONE-1-EBV cells in response to 10 µM ZRL₅P₂ or ZRL₅P₄. The nuclei were counterstained with DAPI and indicated in blue. (Scale bars, 50 µm.) (D) Comparison of the number of Zta-positive cells after treatment with ZRL₅P₄ in the presence versus the absence of Dicer1. HONE-1-EBV and NPC43 were included. Gene silencing of Dicer1 was achieved by siRNA transfection. Zta was detected by immunofluorescent analysis. Control siRNA (siCTL) was used as a negative control. Relative percentage of Zta-positive was compared against the DMSO solvent control in the siRNA control cells. *P < 0.05.