A novel adenovirus 19K/IX protein enhances infection by attenuating proteasomal degradation of tyrosine-ubiquitinated capsid protein IX

ABSTRACT 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 a newly identified alternatively spliced viral transcript, 19K/IX, derived from the E1B19K and pIX genes. We show that the 19K/IX protein binds to and stabilizes the minor capsid protein IX (pIX), thereby attenuating its proteasomal degradation and enhancing the production of infectious viral progeny. Mechanistically, we demonstrate that pIX degradation is mediated by non-canonical ubiquitination of the conserved tyrosine residues Y14 and Y49, which confer pIX subnuclear localization and partially mediate its interaction with the PSMC3 subunit of the 26S proteasome. Collectively, our study identifies 19K/IX as a novel regulator of HAdV-C5 infection and suggests that non-canonical tyrosine ubiquitination contributes to the regulation of capsid protein IX stability. IMPORTANCE Human adenoviruses (HAdVs) are clinically important pathogens and widely used as therapeutic tools. Because of their compact genomes, HAdVs use their genetic information very efficiently through alternative pre-mRNA processing. Here, we describe and characterize 19K/IX, a novel fusion protein generated by alternative splicing of two viral genes during infection. We demonstrate that 19K/IX stabilizes capsid protein IX by preventing its degradation, revealing a previously unrecognized strategy by which HAdVs ensure efficient production of new virus particles. These findings deepen our understanding of HAdV-C5 gene regulation and protein homeostasis and highlight the functional importance of non-canonical HAdV transcripts.