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

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 proteinspecific 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.