DDX3X mutation and Epstein-Barr virus cooperate to induce R-loop-dependent oncogenesis

RNA helicase DDX3X is generally implicated in inflammasome activation and anti-viral responses. We characterize the common features of scattered DDX3X mutations in lymphoid cancers using molecular dynamics simulation and crystallization, thereby demonstrating their crucial role in Epstein-Barr virus (EBV) lytic gene-driven oncogenic processes. The DDX3X mutation is significantly related to impaired stimulator of interferon genes (STING)/ interferon regulatory factor 7 (IRF-7)/interferon (IFN)-α/β-mediated innate immunity, overexpression of EBV lytic gene BNLF2b, and increased formation of R-loops. In Ddx3x^449_450ET>DP conditional knockin transgenic mice, BNLF2b expression induces R-loop accumulation, genomic instability, and abnormal proliferation of CD3-/CD19-/NK1.1+ cells, thereby promoting malignant progression. The DNA-damaging agent etoposide enhances gamma-H2A histone family member X (γ-H2AX) co-localizing with R-loops, heightens genomic instability beyond cellular tolerance, and eventually triggers synthetic lethality in DDX3X^mutBNLF2b⁺ tumors. These findings provide a better understanding of the functional interaction of the DDX3X mutation with EBV to provoke R-loop-dependent oncogenesis, shedding light on the pathogenic mechanism of EBV and future therapeutic approaches targeting R-loops in diseases involving RNA helicase alterations.