Schubert, E.; Evangelopoulos, V. R.; Larsson, M.; Punga, T.

Human adenoviruses (HAdVs) are double-stranded DNA viruses that cause a wide range of diseases, including respiratory, ocular, and gastrointestinal infections. HAdVs rely extensively on alternative splicing to expand their coding capacity and regulate gene expression during infection. Using replication-competent epitope-tagged HAdV-C5 viruses, we investigated the function of the newly identified alternatively spliced viral transcript, 19K/IX, derived from E1B19K and pIX genes. We show that 19K/IX binds to and stabilizes the minor capsid protein IX (pIX), thereby preventing its proteasomal degradation and promoting the production of infectious viral progeny. Mechanistically, we demonstrate that pIX degradation is mediated by a non-canonical ubiquitination of conserved tyrosine residues, which also partially mediate pIX interaction with the PSMC3 subunit of the 26S proteasome. Collectively, these findings identify 19K/IX as a novel regulator of HAdV-C5 infection and suggest that non-canonical tyrosine ubiquitination may represent a mechanism by which HAdV-C5 modulates protein degradation during infection.