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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein
Virology Journal, Volume 7, Article 6, Year 2010
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Description
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. © 2010 Müller et al; licensee BioMed Central Ltd.
Authors & Co-Authors
Müller, Marcel A.
Germany, Bonn
Universität Bonn
Germany, Berlin
Robert Koch Institute
van der Hoek, Lia M.
Netherlands, Amsterdam
Universiteit Van Amsterdam
Voss, Daniel
Germany, Berlin
Robert Koch Institute
Bader, Oliver
Germany, Berlin
Robert Koch Institute
Lehmann, Dörte
Germany, Berlin
Robert Koch Institute
Schulz, Axel Ronald
Germany, Berlin
Robert Koch Institute
Kallies, Stephan
Germany, Bonn
Universität Bonn
Suliman, Tasnim
South Africa, Bellville
University of the Western Cape
Fielding, Burtram Clinton
South Africa, Bellville
University of the Western Cape
Drosten, C.
Germany, Bonn
Universität Bonn
Niedrig, Matthias
Germany, Berlin
Robert Koch Institute
Statistics
Citations: 43
Authors: 11
Affiliations: 4
Identifiers
Doi:
10.1186/1743-422X-7-6
e-ISSN:
1743422X
Research Areas
Covid