Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding

• Published in: The Journal of biological chemistry 2016-03, Vol.291 (13), p.6796-6812
• Main Authors: Windheim, Mark, Höning, Stefan, Leppard, Keith N., Butler, Larissa, Seed, Christina, Ponnambalam, Sreenivasan, Burgert, Hans-Gerhard
• Format: Article
• Language: English
• Subjects: Adenoviridae - chemistry; Adenoviridae - immunology; Adenoviridae - pathogenicity; adenovirus; Adenovirus E3 proteins; Adenovirus E3 Proteins - chemistry; Adenovirus E3 Proteins - genetics; Adenovirus E3 Proteins - immunology; Amino Acid Motifs; Brefeldin A - pharmacology; CD45; Cell Biology; Cell Line, Tumor; Cell Membrane - immunology; Cell Membrane - virology; E3/49K; Endosomes - immunology; Endosomes - virology; Epidemic Keratoconjunctivitis; Epithelial Cells - drug effects; Epithelial Cells - immunology; Epithelial Cells - virology; Fibroblasts - drug effects; Fibroblasts - immunology; Fibroblasts - virology; Gene Expression; Gene Expression Regulation; Host-Pathogen Interactions - immunology; Humans; Immune evasion; Immunomodulation; intracellular trafficking; Jurkat Cells; Lysosomes - immunology; Lysosomes - virology; Molecular Sequence Data; Primary Cell Culture; protein sorting; Protein Structure, Tertiary; Proteolysis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - immunology; shedding; Signal Transduction; trans-Golgi Network - immunology; trans-Golgi Network - virology; Transfection; viral immunology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.684787

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 natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the 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 (where Φ represents a bulky hydrophobic amino acid) 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.