Integrative analysis of the m⁶A epitranscriptome in HeLa-expressed HPV18 early transcripts reveals its potential...

Human papillomavirus (HPV)-driven cancers remain a major global health burden, and understanding how posttranscriptional regulation shapes viral gene expression may inform new therapeutic strategies. Here, we reanalyzed independently generated public HeLa data sets to identify candidate RNA modification sites on integrated HPV18 transcripts expressed from the HeLa genome. Using Oxford Nanopore direct RNA sequencing of native RNAs and in vitro transcribed controls, together with GLORI, eTAM-seq and staged 4sU-GLORI data sets, we identified a set of candidate m⁶A sites on HPV18 early transcripts. m⁶A levels at a subset of these sites were reduced following perturbation of the m⁶A pathway, most clearly after METTL3 inhibition, WTAP knockdown, or FTO overexpression. We further show that the E6*I-proximal m⁶A site at position 224 is enriched on unspliced transcripts in direct RNA sequencing, as supported by several of the analyzed data sets. In contrast, we find no convincing evidence for m⁵C or pseudouridine within the HPV18 regions covered by these data sets. Together, our analyses provide a candidate map of RNA modifications on HeLa-expressed HPV18 early transcripts.