The transmembrane domain of the adenovirus E3/19K protein acts as an ER retention signal and contributes to intracellular sequestration of MHC 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 MHC class-I (MHC-I) and 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 di-lysine motif in the cytoplasmic tail that confers retrieval from the Golgi 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 Kd. These were analysed for transport, cell-surface expression and impact on MHC-I/MICA/B down-regulation. Similar to the retrieval mutant, replacing the cytoplasmic tail of E3/19K allowed only limited transport of the chimera to the cell surface. Efficient cell-surface expression was only achieved 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 Kd 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 its ER-concentration. Remarkably, the ER-retrieval signal alone without E3/19K-TMD did not mediate efficient HLA-down regulation, 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 MIC-A/B-molecules

Structural analysis of the adenovirus type 2 E3/19K protein using mutagenesis and a panel of conformation-sensitive monoclonal antibodies

The E3/19K protein of human adenovirus type 2 (Ad2) was the first viral protein shown to interfere with antigen presentation. This 25 kDa transmembrane glycoprotein binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), thereby preventing transport of newly synthesized peptide–MHC complexes to the cell surface and consequently T cell recognition. Recent data suggest that E3/19K also sequesters MHC class I like ligands intracellularly to suppress natural killer (NK) cell recognition. While the mechanism of ER retention is well understood, the structure of E3/19K remains elusive. To further dissect the structural and antigenic topography of E3/19K we carried out site-directed mutagenesis and raised monoclonal antibodies (mAbs) against a recombinant version of Ad2 E3/19K comprising the lumenal domain followed by a C-terminal histidine tag. Using peptide scanning, the epitopes of three mAbs were mapped to different regions of the lumenal domain, comprising amino acids 3–13, 15–21 and 41–45, respectively. Interestingly, mAb 3F4 reacted only weakly with wild-type E3/19K, but showed drastically increased binding to mutant E3/19K molecules, e.g. those with disrupted disulfide bonds, suggesting that 3F4 can sense unfolding of the protein. MAb 10A2 binds to an epitope apparently buried within E3/19K while that of 3A9 is exposed. Secondary structure prediction suggests that the lumenal domain contains six β-strands and an α-helix adjacent to the transmembrane domain. Interestingly, all mAbs bind to non-structured loops. Using a large panel of E3/19K mutants the structural alterations of the mutations were determined. With this knowledge the panel of mAbs will be valuable tools to further dissect structure/function relationships of E3/19K regarding down regulation of MHC class I and MHC class I like molecules and its effect on both T cell and NK cell recognition

Sorting motifs in the cytoplasmic tail of the immunomodulatory E3/49K protein of species D adenoviruses modulate cell surface expression and ectodomain shedding.

The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved resulting in secretion of its large ectodomain (sec49K). Sec49K binds to CD45 on leukocytes, impairing activation and functions of NK cells and T cells. E3/49K is localized in the Golgi/trans-Golgi-network (TGN), early endosomes and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly-synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YxxΦ and LL that are important for binding the clathrin adaptor proteins AP-1 and AP-2 in vitro. Surprisingly, mutating the LL motif, either alone or together with YxxΦ, did not prevent proteolytic processing, but increased cell surface expression and secretion. Upon Brefeldin-A treatment cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix-metalloproteases

Characterization of E3/49K, a Novel, Highly Glycosylated E3 Protein of the Epidemic Keratoconjunctivitis-Causing Adenovirus Type 19a

The early transcription unit 3 (E3) of human adenoviruses (Ads) encodes proteins with various immunomodulatory functions. Ads from different subgenera differ considerably in their E3 coding capacity, suggesting that distinct sets of immunomodulatory E3 proteins may influence the disease pattern associated with different Ad subgenera. Interestingly, the E3 region of Ads classified in subgenus D, which are often isolated from AIDS patients and have the propensity to cause eye infections, contains a unique gene, named E3/49K, that may encode a protein with a calculated molecular weight of 48,984 that might be implicated in diseases caused by this subgenus. The 49K sequence predicts a highly glycosylated type I transmembrane protein with a short cytoplasmic tail containing two motifs, YXXΦ and LL, potentially involved in targeting the protein to endosomal or lysosomal compartments. Remarkably, the 49K protein is predicted to contain an unusual immunoglobulin-like fold. Here we have characterized the E3/49K protein of Ad type 19a, an Ad of subgenus D which causes epidemic keratoconjunctivitis. E3/49K was synthesized as an 80- to 100-kDa protein, which is unusually large for an E3 protein. In contrast to another early protein, E3/19K, the expression of E3/49K started early but continued throughout the infection cycle. Analysis of the 49K glycosylation revealed that the majority of 49K molecules contained only 12 of the predicted 14 N-glycans. Furthermore, we provide evidence that 49K is O-glycosylated. At steady state, E3/49K was localized in the Golgi-trans-Golgi network and in early endosomes. Interestingly, the 49K protein has a rather short half-life and seems to be proteolytically cleaved. A processing pattern similar to that in the early stages of infection is seen in transfected cells, constitutively expressing 49K in the absence of other Ad proteins. Together, our data provide the first biochemical and cell biological characterization of an unique E3 protein of subgenus D Ads

Tumor necrosis factor α stimulates expression of adenovirus early region 3 proteins: implications for viral persistence

Human adenovirus (Ad) can cause persistent infections in humans. Early region 3 (E3) of the virus appears to be implicated in this phenomenon. This transcription unit encodes proteins that interfere in various ways with host cell functions, including (i) cell-surface expression of histocompat- ibility class I antigens (HLA), (ih) cell-surface expression of the epidermal growth factor receptor (EGF-R), and (iii) the bio- logical activity of tumor necrosis factor a (TNF-a). We trans- fected the human cell line 293 with the entire E3 region of Ad2 and investigated the influence of the cytokines TNF-a and interferon y (IFN-y) on cell-surface expression of HLA class I and the EGF-R. Whereas IFN-y treatment induced expression of HLA to some extent but not that of the EGF-R, TNF-a treatment augmented the reduction of these cell-surface mol- ecules. Subsequent studies on the mechanism of this effect showed a TNF-a-dependent upregulation of E3 protein (E3/19K) and mRNA. The significance of this phenomenon was confirmed in infection experiments. A dramatic increase in the amount of E3/19K, even after short induction with low doses of TNF-o! could be demonstrated. The study provides evidence for an interaction between the immune system and Ad in which the virus takes advantage of an immune mediator to escape immunosurveillance of the host

The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections

Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon