Abstract

Background The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is a pivotal oncoprotein essential for maintaining viral latency, promoting cellular immortalization, and driving tumorigenesis in EBV-associated malignancies. Despite its central role in pathogenesis, current therapeutic strategies remain ineffective in targeting EBV latency or EBNA1-driven oncogenic processes. The DNA-binding domain (DBD) of EBNA1 is critical for its interaction with viral episomes and host chromatin, enabling genome maintenance and transcriptional regulation. Results Here, we present a novel, high-throughput yeast surface display-based screening platform to identify nanobodies with high specificity and affinity for the EBNA1-DBD. By leveraging a naive nanobody library and iterative complementarity-determining region (CDR) mutagenesis, we engineered nanobodies capable of disrupting the EBNA1-DNA interaction. Functional validation through in vitro assays and xenograft tumor models demonstrated that these nanobodies significantly inhibit the proliferation of EBV-positive tumor cells and suppress tumor growth in vivo. Epitope mapping analyses revealed four distinct binding sites on the EBNA1-DBD, with three epitopes localized near the DNA-binding interface, suggesting direct interference with EBNA1’s genomic functions. Conclusions This study not only establishes a robust screening methodology for targeting viral oncoproteins but also highlights the therapeutic potential of EBNA1-DBD-specific nanobodies in treating EBV-associated cancers. Graphical