Mapping global transcript isoform landscapes of influenza A virus infection and interferon stimulation reveals RNA-binding protein networks associated with immune remodeling

Infected cells rapidly adapt their transcriptional profile to upregulate genes that limit pathogen replication and promote inflammation. Viruses also induce widespread changes in host gene expression, and both host and virus further remodel the transcriptome through targeted mRNA degradation, modulation of alternative splicing, and alterations at transcript 5 and 3 prime ends. The complexity of the response to infection makes it difficult to understand which transcriptional changes are driven by the virus and which reflect the host antiviral program. Short-read RNA sequencing has been the primary tool for studying these changes, but its inability to resolve full-length transcript isoforms limits isoform switching-based analyses, narrowing our understanding of how transcript usage shapes immunity. Here we profiled the host transcriptome in response to interferon-{beta} treatment or influenza A virus infection using both short- and long-read RNA sequencing analyzed with the SQANTI3 pipeline. Because isoform switching and transcript abundance change are distinct regulatory events, we focused our analysis on differential transcript usage to uncover a layer of transcriptome remodeling that standard expression analysis cannot detect. Pathway analysis revealed distinct regulons for genes undergoing transcriptional upregulation compared with those undergoing isoform switching. These data provide a rich resource for querying the isoform changes underlying shared and distinct transcriptional programs triggered by viral infection or innate immune stimulation. To identify putative mechanisms coordinating these stimulation-dependent isoform changes, we integrated 232 eCLIP datasets and found a small set of RNA-binding proteins localized at these isoform switches, including DDX3X. Comparing the responses to interferon and to virus highlighted RNA-binding proteins associated with infection-dependent immune remodeling. Altogether, these results demonstrate the value of hybrid sequencing for deciphering the post-transcriptional regulatory code active during the antiviral response.