Isoform-Resolved Temporal Remodeling of Transcriptome Architecture in Monkeypox Virus

We reanalyzed previously generated replicated Oxford Nanopore direct cDNA sequencing data from six infection time points, together with cap analysis of gene expression and native direct RNA sequencing, to reconstruct full-length monkeypox virus transcripts and characterize their temporal regulation. A broad catalogue of candidate transcript models, including candidate 5′ poly(A)-associated structures, was filtered with independent 5′- and 3′-end evidence to define conservative analysis sets. Transcript-resolved profiles revealed a structured temporal program extending beyond gene-level abundance. Host-virus interaction, transcription, and nucleotide-metabolism genes contributed strongly early, whereas virion structure and morphogenesis genes dominated later. Transcription start and end site abundance profiles changed extensively over time, and many genes redistributed among alternative full-length transcript structures. Differential transcript usage was particularly prominent among host-virus interaction genes but depleted among virion structure and morphogenesis genes. Candidate 5′ poly(A)-associated transcripts also increased in relative contribution and showed locus-specific patterns. Building on our previous endpoint map, the replicated direct cDNA time course enabled temporal quantification of gene abundance, transcript-end dynamics, and isoform composition. Together, these results show that Monkeypox virus gene regulation involves coordinated remodeling of gene output, transcript boundaries, and full-length transcriptome architecture.