The transmembrane domain of the adenovirus E3/19K protein acts as an endoplasmic reticulum retention signal and contributes to intracellular sequestration of major histocompatibility complex class I molecules.

The human adenovirus E3/19K protein is a type I transmembrane glycoprotein of the endoplasmic reticulum (ER) that abrogates cell surface transport of major histocompatibility complex class I (MHC-I) and MHC-I-related chain A and B (MICA/B) molecules. Previous data suggested that E3/19K comprises two functional modules: a luminal domain for interaction with MHC-I and MICA/B molecules and a dilysine motif in the cytoplasmic tail that confers retrieval from the Golgi apparatus back to the ER. This study was prompted by the unexpected phenotype of an E3/19K molecule that was largely retained intracellularly despite having a mutated ER retrieval motif. To identify additional structural determinants responsible for ER localization, chimeric molecules were generated containing the luminal E3/19K domain and the cytoplasmic and/or transmembrane domain (TMD) of the cell surface protein MHC-I K(d). These chimeras were analyzed for transport, cell surface expression, and impact on MHC-I and MICA/B downregulation. As with the retrieval mutant, replacement of the cytoplasmic tail of E3/19K allowed only limited transport of the chimera to the cell surface. Efficient cell surface expression was achieved only by additionally replacing the TMD of E3/19K with that of MHC-I, suggesting that the E3/19K TMD may confer static ER retention. This was verified by ER retention of an MHC-I K(d) molecule with the TMD replaced by that of E3/19K. Thus, we have identified the E3/19K TMD as a novel functional element that mediates static ER retention, thereby increasing the concentration of E3/19K in the ER. Remarkably, the ER retrieval signal alone, without the E3/19K TMD, did not mediate efficient HLA downregulation, even in the context of infection. This suggests that the TMD is required together with the ER retrieval function to ensure efficient ER localization and transport inhibition of MHC-I and MICA/B molecules.