Comprehensive analysis and validation of m6A-G4 colocalization and its regulatory role in Marek’s disease virus infection

N6-methyladenosine, the most prevalent post-transcriptional modification in eukaryotes, plays a critical role in regulating gene expression and disease pathogenesis. Concurrently, G-quadruplex structures are implicated in key biological processes, including the regulation of transcription and post-transcriptional events. Emerging evidence suggests that m6A modifications and G4 structures colocalize within viral genomes and human pre-mRNAs; however, their functional interplay remains poorly understood. Using existing MeRIP-seq data from Marek’s disease virus (MDV)-infected samples, we investigated the colocalization pattern and its potential role in viral infection. Our bioinformatic analyses revealed that the predicted G4 structures were predominantly two G-tetrad G-quadruplexes. Genes with m6A-G4 colocalization were significantly enriched in pathways related to immune response and tumorigenesis. Then we verified the presence of co-localization on the CCL4, which inhibited CCL4 expression. Co-localization also affected viral replication and regulated ICP4 expression. We systematically characterized the features of G4 structures co-localized with m6A modifications during MDV infection, investigated the role in viral replication and gene regulation, and provided experimental evidence for m6A-G4 co-localization. These findings shed new light on the epitranscriptomic regulatory mechanisms employed by an oncogenic virus and may inform the development of novel antiviral and anti-tumor therapeutic strategies.