Dihydroartemisinin inhibits Epstein-Barr virus reactivation and replication targeting lytic proteins: insights for drug repurposing.

Epstein-Barr virus (EBV) is an oncogenic virus which is responsible for various malignant and non-malignant diseases that lead to about 200,000 deaths each year. Despite efforts, there are no FDA-approved drugs targeting EBV. Although EBV-associated malignancies are mostly driven by the latent stages of infection, reactivation of EBV plays a critical role in the transition from latency to lytic cycle, leading to viral replication and disease progression, and is primarily regulated by transactivator BZLF1. In this study, we integrated computational screening with experimental validation to identify repurposing drugs that inhibit EBV reactivation and replication. FDA-approved compounds predicted using in-house AI/ML-based model (Anti-EBV) and miRNA-seq and RNA-seq analyses were selected for further evaluation. Molecular docking against BZLF1, supported by in silico alanine scanning to identify critical DNA-binding residues, identified seven candidate drugs. Among these, an antimalarial drug, dihydroartemisinin (DHA), showed the strongest inhibitory activity in vitro, with an IC99 of 1 μM and an SI Index of 113.5. DHA reduced both EBV viral copy number and the expression of early and late lytic genes. Molecular docking, simulation, ChIP and IP studies demonstrated stable binding of DHA within the BZLF1 DNA-binding pocket, inhibiting the key residues involved in BZLF1 activation and DNA binding. Analysis at the gene level confirmed its inhibitory effect on EBV replication, while expression analysis at the transcriptional and protein levels, along with immunofluorescence analysis, indicated its inhibitory effect on EBV reactivation and virion production. These findings suggest DHA as a promising repurposing antiviral candidate targeting EBV lytic protein.