The Human Adenovirus 2 Transcriptome: an Amazing Complexity of Alternatively Spliced mRNAs

We used the Nanopore long-read sequencing platform to demonstrate how amazingly complex the human adenovirus type 2 (Ad2) transcriptome is, with a flexible splicing machinery producing a range of novel mRNAs from both the early and late transcription units. In total, we report more than 900 alternatively spliced mRNAs produced from the Ad2 transcriptome, more than 850 of which are novel mRNAs. A surprising finding was that more than 50% of all E1A transcripts extended upstream of the previously defined transcriptional start site. The novel start sites mapped close to the inverted terminal repeat (ITR) and within the E1A enhancer region. We speculate that novel promoter- or enhancer-driven transcription, so-called eRNA transcription, is responsible for producing these novel mRNAs. Their existence was verified by a peptide in the Ad2 proteome that was unique to the E1A ITR mRNA. Although we show a high complexity of alternative splicing from most early and late regions, the E3 region was by far the most complex when expressed at late times of infection. More than 400 alternatively spliced mRNAs were observed in this region alone. These mRNAs included extended L4 mRNAs containing E3 and L5 sequences and readthrough mRNAs combining E3 and L5 sequences. Our findings demonstrate that the virus has a remarkable capacity to produce novel exon combinations, which gives the virus an evolutionary advantage, i.e., the ability to change the gene expression repertoire and protein production in an evolving environment. IMPORTANCE Work in the adenovirus system led to the groundbreaking discovery of RNA splicing and alternative RNA splicing in 1977. These mechanisms are essential in mammalian evolution, as they increase the coding capacity of a genome. Here, we used a long-read sequencing technology to characterize the complexity of human adenovirus pre-mRNA splicing in detail. It is mind-boggling that the viral genome, which houses only around 36,000 bp, not being much larger than a single cellular gene, generates more than 900 alternatively spliced mRNAs. Recently, adenoviruses have been used as the backbone in several promising severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines. Further improvement of adenovirus-based vaccines demands that the virus be modified into an innocent carrier of foreign genes. This requires a full understanding of the components that govern adenovirus replication and gene expression.