title: Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding.
abstract: 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-2in 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.
authors: From the School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom, the Institute of Biochemistry, Hannover Medical School, 30625 Hannover, Germany.
authors: Mark
authors: M
authors: Windheim
authors: the Institute for Biochemistry I and Center for Molecular Medicine Cologne, 50931 Cologne, Germany, and.
authors: Stefan
authors: S
authors: Höning
authors: From the School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
authors: Keith N
authors: KN
authors: Leppard
authors: From the School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
authors: Larissa
authors: L
authors: Butler
authors: From the School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
authors: Christina
authors: C
authors: Seed
authors: the School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors: Sreenivasan
authors: S
authors: Ponnambalam
authors: From the School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom, H-G.Burgert@warwick.ac.uk.
authors: Hans-Gerhard
authors: HG
authors: Burgert
mesh_terms: D000256:Adenoviridae / Q000737:chemistry / Q000276:immunology* / Q000472:pathogenicity; D017503:Adenovirus E3 Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D020816:Amino Acid Motifs; D020126:Brefeldin A / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000276:immunology* / Q000821:virology; D011992:Endosomes / Q000276:immunology / Q000821:virology; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology* / Q000821:virology; D005347:Fibroblasts / Q000187:drug effects / Q000276:immunology* / Q000821:virology; D015870:Gene Expression; D005786:Gene Expression Regulation; D054884:Host-Pathogen Interactions / Q000276:immunology; D006801:Humans; D056747:Immunomodulation; D019169:Jurkat Cells; D008247:Lysosomes / Q000276:immunology / Q000821:virology; D008969:Molecular Sequence Data; D061251:Primary Cell Culture; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction; D014162:Transfection; D021601:trans-Golgi Network / Q000276:immunology / Q000821:virology
chemical_list: D017503:Adenovirus E3 Proteins; D011994:Recombinant Proteins; D020126:Brefeldin A
keywords: Adenovirus E3 proteins; CD45; E3/49K; Epidemic Keratoconjunctivitis; Immune evasion; adenovirus; intracellular trafficking; protein sorting; shedding; viral immunology