An epitranscriptomic map of RNA modifications on integrated HPV18 expressed from HeLa cells

Human papillomavirus(HPV)-driven cancers remain a major global health burden, and understanding how regulatory mechanisms fine-tune viral gene expression is critical for developing new therapeutic strategies. Here, we reanalyse publicly available data from the HeLa cell line to map the epitranscriptomic landscape of HPV18 transcripts. We combine Oxford Nanopore direct RNA-sequencing reads from both native RNAs and in-vitro transcribed controls to identify a robust set of m6A sites supported by multiple tools, which we validate using an orthogonal chemical‑profiling assay. We further show that the modification rates at many of these sites are reduced upon knockdown or inhibition of components of the m6A writer-complex, indicating their dependence on the host methyltransferase machinery. We also uncover a cluster of m6A marks upstream of the E6*I splice donor, which were strongly enriched in unspliced transcripts, implicating m6A in regulation of E6 intron retention. In contrast, we find no convincing evidence of m5C or pseudouridine in HPV18 transcripts. As the first high‑resolution epitranscriptomic map of HPV18, our study highlights a potential mechanistic link between m6A modification and viral RNA processing.