Poxvirus Replication Remodels Host m⁶A Epitranscriptome to Advance Infection

Background Understanding how host gene regulation responds to viral infection is essential for developing effective antiviral strategies. Emerging evidence suggests that host transcripts undergo dynamic chemical modifications to counteract viral invasion. Conversely, viruses that rely on nuclear transcription exploit host RNA methyltransferases to enhance mRNA export and translation. Orthopoxviruses, however, complete their entire replication cycle in compartmentalized cytoplasmic “factories” utilizing enzymes encoded on the large double-stranded viral DNA genomes. The dynamic interplay between host and poxviral epitranscriptome remains poorly characterized. Results Using a temporally resolved model of Vaccinia virus (VV) infection, we investigated host-virus interactions through transcriptome and N6-methyladenosine (m⁶A) epitranscriptome whole genome sequencing. We found that host m⁶A modifications respond rapidly to VV infection, preceding the delayed transcriptional changes that emerge at later stages. These early m⁶A signatures included key innate immunity factors as well as host genes involved in transcriptional regulation, post-transcriptional modification, and protein ubiquitination. Functional assays validated two host factors with early m⁶A signature changes as essential for VV infection: the m⁶A reader YTHDF1, which exhibited a sharp gain of m⁶A modifications at 6 hours post infection, and FBXO31, a component of the SCF E3 ubiquitin ligase complex, which showed a consistent loss of m⁶A modifications. We further demonstrated that m⁶A gain on YTHDF1 increases its protein expression and is crucial for efficient VV replication. In addition, we identified previously unrecognized proviral roles of FBXO31 and the SCF E3 ligase complex in supporting VV infection. Conclusion Temporal profiling of the m⁶A epitranscriptome reveals how VV exploits host post-transcriptional regulatory pathways,specifically RNA m⁶A modification and protein ubiquitination. These findings highlight critical host factors co-opted during poxvirus infection and identify promising targets for therapeutic intervention.