Human Coronavirus NL63 Open Reading Frame 3 encodes a virion-incorporated N-glycosylated membrane protein
| Autor: | Axel R. Schulz, Tasnim Suliman, Christian Drosten, Burtram C. Fielding, Oliver Bader, Marcel A. Müller, Daniel Voss, Lia van der Hoek, Dörte Lehmann, Matthias Niedrig, Stephan Kallies |
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| Přispěvatelé: | Faculteit der Geneeskunde, Amsterdam institute for Infection and Immunity, Medical Microbiology and Infection Prevention |
| Jazyk: | angličtina |
| Předmět: |
Models
Molecular viruses Blotting Western Golgi Apparatus medicine.disease_cause Endoplasmic Reticulum Models Biological Cell Line lcsh:Infectious and parasitic diseases Viral Matrix Proteins 03 medical and health sciences Endoglycosidase H Open Reading Frames Virology medicine Animals Humans lcsh:RC109-216 Peptide sequence 030304 developmental biology Coronavirus Glycoproteins chemistry.chemical_classification Viral Structural Proteins 0303 health sciences Viral matrix protein Microscopy Confocal biology 030306 microbiology Endoplasmic reticulum Research Virion virus diseases Molecular biology Macaca mulatta 3. Good health Cell biology Open reading frame Infectious Diseases chemistry Membrane protein Microscopy Fluorescence biology.protein Glycoprotein Lysosomes |
| Zdroj: | Virology Journal, 7(1). BioMed Central Virology Journal; Vol 7 Virology Journal Virology Journal, Vol 7, Iss 1, p 6 (2010) Virology journal, 7(1). BioMed Central |
| ISSN: | 1743-422X |
| DOI: | 10.1186/1743-422x-7-6 |
| Popis: | Background Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses. Results In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein. Conclusions This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein. |
| Databáze: | OpenAIRE |
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