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 as well as non-malignant diseases and leads to about 200,000 deaths each year. Despite efforts, there are no FDA-approved drugs targeting EBV. 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 the transactivator BZLF1. In this study, we combined 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, led to the selection of seven candidate drugs. Among these, an antimalarial drug, dihydroartemisinin (DHA), showed the strongest inhibitory activity in vitro, with an IC99 of 1 {micro}M 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 and simulation 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 assembly. These findings suggest DHA as a promising repurposing antiviral candidate targeting EBV lytic proteins and offers an effective target-based therapeutic strategy. ImportanceThis study identifies a repurposed small-molecule inhibitor of EBV reactivation and replication. Here, we proposed target-based therapy, integrating computational and experimental approaches to target the EBV lytic transactivator BZLF1. Since early lytic EBV protein BZLF1 plays a critical role in viral reactivation and replication, inhibition of its activation and DNA-binding function represents a promising therapeutic approach to prevent EBV infection. Molecular docking and simulation studies revealed stable binding of DHA within the BZLF1 DNA-binding pocket. Furthermore, in vitro analyses demonstrated significant inhibition of viral gene copy number and reduced mRNA and protein levels of key lytic proteins. Thus, this study demonstrated DHA as a safe and effective repurposed therapeutic candidate against EBV infection.