Adenovirus early proteins E1A and E3-19K reveal separable regulatory states of the ER stress sensor IRE1a

The unfolded protein response (UPR) controls cellular homeostasis and virus infections. It is canonically initiated by dissociation of ER chaperone BiP/GRP78 from lumenal domains of transmembrane sensor proteins, including the central UPR sensor inositol-requiring enzyme 1 alpha (IRE1a). This results in IRE1a activation and splicing of X-box binding protein 1 (XBP1) mRNA, leading to transcriptional activation of XBP1 for cellular adaptation and sustained infection. Human adenovirus (AdV) preferentially activates the IRE1a-XBP1 pathway through the viral membrane protein E3-19K, yet the mechanisms underlying this selective activation remain unclear. Here, we show that AdV uses two distinct early proteins to dissociate BiP from IRE1a and activate IRE1a-mediated XBP1 splicing. The immediate early protein E1A forms a complex with the cytoplasmic domain of IRE1a, sufficient to dissociate BiP from IRE1a without inducing XBP1 splicing or UPR activation. In contrast, E3-19K is expressed downstream of E1A, associates with the lumenal domain of IRE1a, and induces XBP1 splicing without promoting BiP dissociation from IRE1a. Both proteins were detected in IRE1a-containing complexes, consistent with stepwise viral regulation. Together, our findings show that canonical IRE1a activation hallmarks can be mechanistically separated during viral infection, revealing distinct AdV-regulated IRE1a activation states with discrete upstream and downstream signaling outputs.